Publications

Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B⁺ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B⁺ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.

Conventional methods for humanizing animal-derived antibodies involve grafting their complementarity-determining regions onto homologous human framework regions. However, this process can substantially lower antibody stability and antigen-binding affinity, and requires iterative mutational fine-tuning to recover the original antibody properties. Here we report a computational method for the systematic grafting of animal complementarity-determining regions onto thousands of human frameworks. The method, which we named CUMAb (for computational human antibody design; available at http://CUMAb.weizmann.ac.il), starts from an experimental or model antibody structure and uses Rosetta atomistic simulations to select designs by energy and structural integrity. CUMAb-designed humanized versions of five antibodies exhibited similar affinities to those of the parental animal antibodies, with some designs showing marked improvement in stability. We also show that (1) non-homologous frameworks are often preferred to highest-homology frameworks, and (2) several CUMAb designs that differ by dozens of mutations and that use different human frameworks are functionally equivalent.

Intratumoral heterogeneity impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By isolating single clones from the resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and potentially shared sensitivities for repression of protein kinase C. Our in vitro findings are consistent with tumor-heterogeneity that is observed in breast cancer patients thus highlighting the need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.

The proteasome is responsible for removal of ubiquitinated proteins. Although several aspects of its regulation (e.g., assembly, composition, and post-translational modifications) have been unraveled, studying its adaptive compartmentalization in response to stress is just starting to emerge. We found that following amino acid starvation, the proteasome is translocated from its large nuclear pool to the cytoplasma response regulated by newly identified mTOR-agonistic amino acidsTyr, Trp, and Phe (YWF). YWF relay their signal upstream of mTOR through Sestrin3 by disrupting its interaction with the GATOR2 complex. The triad activates mTOR toward its downstream substrates p62 and transcription factor EB (TFEB), affecting both proteasomal and autophagic activities. Proteasome translocation stimulates cytosolic proteolysis which replenishes amino acids, thus enabling cell survival. In contrast, nuclear sequestration of the proteasome following mTOR activation by YWF inhibits this proteolytic adaptive mechanism, leading to cell death, which establishes this newly identified pathway as a key stress-coping mechanism.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2−/−) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2−/− mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2−/− mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

In this article the acknowledgements of Efrat Shema are omitted and should have read E.S is supported by the Emerson Collective and the Israel Cancer Research Fund, and is an incumbent of the Lisa and Jeffrey Aronin Family Career Development chair. The original article has been corrected.

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a \u201cbasal-like\u201d state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed \u201cbasal-like\u201d genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.

Background: Antitumor therapies targeting HER1/EGFR and HER2, such as monoclonal antibodies (MAbs) and tyrosine-kinase inhibitors (TKIs), have demonstrated a significant clinical benefit, but the emergence of resistance limits long-term efficacy. While secondary HER1 mutations confer tolerance to TKI, compensatory upregulation of HER2 drives resistance to anti-HER1 MAbs, which identifies MAb combinations targeting both receptors as an attractive therapeutic strategy. Nevertheless, toxicity hampers the clinical validation of this approach. Alternatively, cancer vaccines may induce antibodies directed against several antigens with less concern about induced toxicity. Methods: Polyclonal antibodies (PAbs) targeting HER1 and HER2 were induced in mice or rabbits through immunization. Recognition of different epitopes on targets by PAbs was validated by phage-display technology. Receptor downregulation was evaluated by flow cytometry, immunofluorescence, and Western blot. MTT assays assessed cytotoxicity, while the antitumor effect of PAbs was assayed in nude mice. Results: PAbs promoted degradation of HER1 and HER2 regarding clinical MAbs or their combinations. As a result, inhibition of cytotoxicity on tumor cell lines was improved, even in the presence of oncogenic mutations in HER1, as well as in cetuximab-insensitive cells. Accordingly, the antitumor effect of vaccination-induced PAbs was observed in lung tumor lines representative of sensitivity or resistance to HER1 targeting therapies. Conclusions: Immunization against HER1 and HER2 receptors offers an alternative to passive administration of combinations of MAbs, since vaccination-induced PAbs promote the downregulation of both receptors and they have a higher impact on the survival of tumor cells.

Missense mutations in the p53 tumor suppressor abound in human cancer. Common (\u201chotspot\u201d) mutations endow mutant p53 (mutp53) proteins with oncogenic gain of function (GOF), including enhanced cell migration and invasiveness, favoring cancer progression. GOF is usually attributed to transcriptional effects of mutp53. To elucidate transcription-independent effects of mutp53, we characterized the protein interactome of the p53R273H mutant in cells derived from pancreatic ductal adenocarcinoma (PDAC), where p53R273H is the most frequent p53 mutant. We now report that p53R273H, but not the p53R175H hotspot mutant, interacts with SQSTM1/p62 and promotes cancer cell migration and invasion in a p62-dependent manner. Mechanistically, the p53R273H-p62 axis drives the proteasomal degradation of several cell junctionassociated proteins, including the gap junction protein Connexin 43, facilitating scattered cell migration. Concordantly, down-regulation of Connexin 43 augments PDAC cell migration, while its forced overexpression blunts the promigratory effect of the p53R273H-p62 axis. These findings define a mechanism of mutp53 GOF.

Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Treatment options for TNBC patients are limited and further insights into disease aetiology are needed to develop better therapeutic approaches. microRNAs ability to regulate multiple targets could hold a promising discovery approach to pathways relevant for TNBC aggressiveness. Thus, we address the role of miRNAs in controlling three signalling pathways relevant to the biology of TNBC, and their downstream phenotypes. Methods: To identify miRNAs regulating WNT/β-catenin, c-Met, and integrin signalling pathways, we performed a high-throughput targeted proteomic approach, investigating the effect of 800 miRNAs on the expression of 62 proteins in the MDA-MB-231 TNBC cell line. We then developed a novel network analysis, Pathway Coregulatory (PC) score, to detect miRNAs regulating these three pathways. Using in vitro assays for cell growth, migration, apoptosis, and stem-cell content, we validated the function of candidate miRNAs. Bioinformatic analyses using BC patients datasets were employed to assess expression of miRNAs as well as their pathological relevance in TNBC patients. Results: We identified six candidate miRNAs coordinately regulating the three signalling pathways. Quantifying cell growth of three TNBC cell lines upon miRNA gain-of-function experiments, we characterised miR-193b as a strong and consistent repressor of proliferation. Importantly, the effects of miR-193b were stronger than chemical inhibition of the individual pathways. We further demonstrated that miR-193b induced apoptosis, repressed migration, and regulated stem-cell markers in MDA-MB-231 cells. Furthermore, miR-193b expression was the lowest in patients classified as TNBC or Basal compared to other subtypes. Gene Set Enrichment Analysis showed that miR-193b expression was significantly associated with reduced activity of WNT/β-catenin and c-Met signalling pathways in TNBC patients. Conclusions: Integrating miRNA-mediated effects and protein functions on networks, we show that miRNAs predominantly act in a coordinated fashion to activate or repress connected signalling pathways responsible for metastatic traits in TNBC. We further demonstrate that our top candidate, miR-193b, regulates these phenotypes to an extent stronger than individual pathway inhibition, thus emphasizing that its effect on TNBC aggressiveness is mediated by the coordinated repression of these functionally interconnected pathways.

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometrybased proteomic approach and analyzed a breast cancer cohort of 113 formalinfixed paraffinembedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumoradjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of posttreatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ERpositive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy.

The epidermal growth factor receptor (EGFR) has served as the founding member of the large family of growth factor receptors harboring intrinsic tyrosine kinase function. High abundance of EGFR and large internal deletions are frequently observed in brain tumors, whereas point mutations and small insertions within the kinase domain are common in lung cancer. For these reasons EGFR and its preferred heterodimer partner, HER2/ERBB2, became popular targets of anticancer therapies. Nevertheless, EGFR research keeps revealing unexpected observations, which are re-viewed herein. Once activated by a ligand, EGFR initiates a timedependent series of molecular switches comprising downregulation of a large cohort of microRNAs, upregulation of newly syn-thesized mRNAs, and covalent protein modifications, collectively controlling phenotypedetermin-ing genes. In addition to microRNAs, long noncoding RNAs and circular RNAs play critical roles in EGFR signaling. Along with driver mutations, EGFR drives metastasis in many ways. Paracrine loops comprising tumor and stromal cells enable EGFR to fuel invasion across tissue barriers, survival of clusters of circulating tumor cells, as well as colonization of distant organs. We conclude by listing all clinically approved anticancer drugs targeting either EGFR or HER2. Because emergence of drug resistance is nearly inevitable, we discuss the major evasion mechanisms.

Cancer is initiated largely by specific cohorts of genetic aberrations, which are generated by mutagens and often mimic active growth factor receptors, or downstream effectors. Once initiated cells outgrow and attract blood vessels, a multi-step process, called metastasis, disseminates cancer cells primarily through vascular routes. The major steps of the metastatic cascade comprise intravasation into blood vessels, circulation as single or collectives of cells, and eventual colonization of distant organs. Herein, we consider metastasis as a multi-step process that seized principles and molecular players employed by physiological processes, such as tissue regeneration and migration of neural crest progenitors. Our discussion contrasts the irreversible nature of mutagenesis, which establishes primary tumors, and the reversible epigenetic processes (e.g. epithelialmesenchymal transition) underlying the establishment of micro-metastases and secondary tumors. Interestingly, analyses of sequencing data from untreated metastases inferred depletion of putative driver mutations among metastases, in line with the pivotal role played by growth factors and epigenetic processes in metastasis. Conceivably, driver mutations may not confer the same advantage in the microenvironment of the primary tumor and of the colonization site, hence phenotypic plasticity rather than rigid cellular states hardwired by mutations becomes advantageous during metastasis. We review the latest reported examples of growth factors harnessed by the metastatic cascade, with the goal of identifying opportunities for anti-metastasis interventions. In summary, because the overwhelming majority of cancer-associated deaths are caused by metastatic disease, understanding the complexity of metastasis, especially the roles played by growth factors, is vital for preventing, diagnosing and treating metastasis.

Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.

Cancer immunotherapy focuses on inhibitors of checkpoint proteins, such as programmed death ligand 1 (PD-L1). Unlike RAS-mutated lung cancers, EGFR mutant tumors have a generally low response to immunotherapy. Because treatment outcomes vary by EGFR allele, intrinsic and microenvironmental factors may be involved. Among all non-immunological signaling pathways surveyed in patients datasets, EGFR signaling is best associated with high PD-L1. Correspondingly, active EGFRs stabilize PD-L1 transcripts and depletion of PD-L1 severely inhibits EGFR-driven tumorigenicity and metastasis in mice. The underlying mechanisms involve the recruitment of phospholipase C-γ1 (PLC-γ1) to a cytoplasmic motif of PD-L1, which enhances PLC-γ1 activation by EGFR. Once stimulated, PLC-γ1 activates calcium flux, Rho GTPases, and protein kinase C, collectively promoting an aggressive phenotype. Anti-PD-L1 antibodies can inhibit these intrinsic functions of PD-L1. Our results portray PD-L1 as a molecular amplifier of EGFR signaling and improve the understanding of the resistance of EGFR⁺ tumors to immunotherapy.

Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anticancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first, second, and thirdgeneration drugs. To explore alternatives to such whackamole strategies, we simulated in patientderived xenograft models the situation of patients receiving firstline TKIs. Monotherapies comprising approved firstline TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drugdrug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistanceconferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing nextgeneration TKIs.

Tumor chemosensitivity testing plays a pivotal role in the optimal selection of chemotherapeutic regimens for cancer patients in a personalized manner. High-throughput drug screening approaches have been developed but they failed to take into account intratumor heterogeneity and therefore only provided limited predictive power of therapeutic response to individual cancer patients. Single cancer cell drug sensitivity testing (SCC-DST) has been recently developed to evaluate the variable sensitivity of single cells to different anti-tumor drugs. In this review, we discuss how SCC-DST overcomes the obstacles of traditional drug screening methodologies. We outline critical procedures of SCC-DST responsible for single-cell generation and sorting, cell-drug encapsulation on a microfluidic chip and detection of cell-drug interactions. In SCC-DST, droplet-based microfluidics is emerging as an important platform that integrated various assays and analyses for drug susceptibility tests for individual patients. With the advancement of technology, both fluorescence imaging and label-free analysis have been used for detecting single cell-drug interactions. We also discuss the feasibility of integrating SCC-DST with single-cell RNA sequencing to unravel the mechanisms leading to drug resistance, and utilizing artificial intelligence to facilitate the analysis of various omics data in the evaluation of drug susceptibility. SCC-DST is setting the stage for better drug selection for individual cancer patients in the era of precision medicine.

ERBB2 is a ligand-less tyrosine kinase receptor expressed at very low levels in normal tissues; when overexpressed, it is involved in malignant transformation and tumorigenesis in several carcinomas. In cancer cells, ERBB2 represents the preferred partner of other members of the ERBB receptor family, leading to stronger oncogenic signals, by promoting both ERK and AKT activation. The identification of the specific signaling downstream of ERBB2 has been impaired by the lack of a ligand and of an efficient way to selectively activate the receptor. In this paper, we found that antibodies (Abs) targeting different epitopes on the ERBB2 extracellular domain foster the activation of ERBB2 homodimers, and surprisingly induce a unique cytostatic signaling cascade promoting an ERK-dependent ERBB2-Thr(701) phosphorylation, leading to AKT de-phosphorylation, via PP2A Ser/Thr phosphatases. Furthermore, the immunophilin Cyclophilin A plays a crucial role in this pathway, acting as a negative modulator of AKT de-phosphorylation, possibly by competing with Ser/Thr phosphatases for binding to AKT. Altogether, our data show that Ab recognizing ERBB2 extracellular domain function as receptor agonists, promoting ERBB2 homodimer activation, leading to an anti-proliferative signaling. Thus, the ultimate outcome of ERBB2 activity might depend on the dimerization status: pro-oncogenic in the hetero-, and anti-oncogenic in the homo-dimeric form.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFR(T)(790M) m and EGFR(C)(797S). It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFR(T)(790M) and EGFR(C)(797S) tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFR(T)(790M) and EGFR(C)(797S) mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR's transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

Circular RNAs (circRNAs) comprise an emerging new class of endogenous RNAs expressed abundantly by the eukaryotic transcriptome. They are characterized by a covalently closed loop structure, resulting in RNA molecules that are more stable than linear RNAs. A growing number of studies indicate that circRNAs play critical roles in human diseases and show great potential as biomarkers and therapeutic targets. The molecular events determined by circRNA activity, the circRNA code, involve other types of noncoding RNA molecules, primarily microRNAs, long noncoding RNAs, and RNA-binding proteins. Herein, we mainly focus on the circRNA-microRNA code, showing how this relationship impacts the regulation of gene expression in cancer. The emerging roles for circRNAs in oncogenic pathways highlight new perspectives for the detailed molecular dissection of cancer pathogenesis and, at the same time, offer new opportunities to design innovative therapeutic strategies. Here, we review recent research advancements in understanding the biogenesis, molecular functions, and significance of circRNAs in cancer diagnosis and treatment.

Since the approval of the first monoclonal antibody (mAb), rituximab, for hematological malignancies, almost 30 additional mAbs have been approved in oncology. Despite remarkable advances, relatively weak responses and resistance to antibody monotherapy remain major open issue. Overcoming resistance might require combinations of drugs blocking both the major target and the emerging secondary target. We review clinically approved combinations of antibodies and either cytotoxic regimens (chemotherapy and irradiation) or kinase inhibitors. Thereafter, we focus on the most promising and currently very active arena that combines mAbs inhibiting immune checkpoints or growth factor receptors. Clinically approved and experimental oligoclonal mixtures of mAbs targeting different antigens (hetero-combinations) or different epitopes of the same antigen (homo-combinations) are described. Effective oligoclonal mixtures of antibodies that mimic the polyclonal immune response will likely become a mainstay of cancer therapy.

Cetuximab (CTX) is a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), commonly used to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, objective remissions occur only in a minority of patients and are of short duration, with a population of cells surviving the treatment and eventually enabling CTX resistance. Our previous study on CRC xenopatients associated poor response to CTX with increased abundance of a set of pro-inflammatory cytokines, including the interleukins IL-1A, IL-1B and IL-8. Stemming from these observations, our current work aimed to assess the role of IL-1 pathway activity in CTX resistance. We employed a recombinant decoy TRAP IL-1, a soluble protein combining the human immunoglobulin Fc portion linked to the extracellular region of the IL-1-receptor (IL-1R1), able to sequester IL-1 directly from the medium. We generated stable clones expressing and secreting a functional TRAP IL-1 into the culture medium. Our results show that IL-1R1 inhibition leads to a decreased cell proliferation and a dampened MAPK and AKT axes. Moreover, CRC patients not responding to CTX blockage displayed higher levels of IL-1R1 than responsive subjects, and abundant IL-1R1 is predictive of survival in patient datasets specifically for the consensus molecular subtype 1 (CMS1). We conclude that IL-1R1 abundance may represent a therapeutic marker for patients who become refractory to monoclonal antibody therapy, while inhibition of IL-1R1 by TRAP IL-1 may offer a novel therapeutic strategy.

Within the tumor microenvironment, cancer cells coexist with noncancerous adjacent cells that constitute the tumor microenvironment and impact tumor growth through diverse mechanisms. In particular, cancer-associated fibroblasts (CAFs) promote tumor progression in multiple ways. Earlier studies have revealed that in normal fibroblasts (NFs), p53 plays a cell nonautonomous tumor-suppressive role to restrict tumor growth. We now wished to investigate the role of p53 in CAFs. Remarkably, we found that the transcriptional program supported by p53 is altered substantially in CAFs relative to NFs. In agreement, the p53-dependent secretome is also altered in CAFs. This transcriptional rewiring renders p53 a significant contributor to the distinct intrinsic features of CAFs, as well as promotes tumor cell migration and invasion in culture. Concordantly, the ability of CAFs to promote tumor growth in mice is greatly compromised by depletion of their endogenous p53. Furthermore, cocultivation of NFs with cancer cells renders their p53-dependent transcriptome partially more similar to that of CAFs. Our findings raise the intriguing possibility that tumor progression may entail a nonmutational conversion ("education") of stromal p53, from tumor suppressive to tumor supportive.

MicroRNAs (miRNAs) might be considered both predictors and players of cancer development. The aim of the present report was to investigate whether many years before the diagnosis of breast cancer miRNA expression is already disregulated. In order to test this hypothesis, we compared miRNAs extracted from leukocytes in healthy women who later developed breast cancer and in women who remain healthy during the whole 15-year follow-up time. Accordantly, we used a casecontrol study design nested in the hOrmone and Diet in the ETiology of breast cancer (ORDET) prospective cohort study addressing the possibility that miRNAs can serve as both early biomarkers and components of the hormonal etiological pathways leading to breast cancer development in premenopausal women. We compared leukocyte miRNA profiles of 191 incident premenopausal breast cancer cases and profiles of 191 women who remained healthy over a follow-up period of 20 years. The analysis identified 20 differentially expressed miRNAs in women candidate to develop breast cancer versus control women. The upregulated miRNAs, miR-513-a-5p, miR-513b-5p and miR-513c-5p were among the most significantly deregulated miRNAs. In multivariate analysis, miR-513a-5p upregulation was directly and statistically significant associated with breast cancer risk (OR = 1.69; 95% CI 1.082.64; P = 0.0293). In addition, the upregulation of miR-513-a-5p displayed the strongest direct association with serum progesterone and testosterone levels. The experimental data corroborated the inhibitory function of miR-513a-5p on progesterone receptor expression confirming that progesterone receptor is a target of miR-513a-5p. The identification of upregulated miR-513a-5p with its oncogenic potential further validates the use of miRNAs as long-term biomarker of breast cancer risk

Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3. (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.

While the epidermal growth factor receptor (EGFR) is an established regulator of skin development and homeostasis, the functions of the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue have only recently been examined. Previously reported, skin-specific deletion of each of these receptors in mice resulted in similar defects in keratinocyte proliferation and migration, resulting in impaired wound healing and tumorigenesis. Because both ERBB2 and ERBB3 are targets for treating an array of cancer types, it is important to examine the consequences of receptor inhibition in human keratinocytes. Here, we employed the CRISPR/Cas9 technology to generate HaCaT cells (an established human keratinocyte cell line) lacking ERBB2 or ERBB3. HaCaT clones lacking ERBB2 or ERBB3 showed comparable reductions in cell proliferation as assessed by BrdU staining. Apoptosis, in contrast, was reduced in ERBB3-deficient HaCaT cells only. Assessment of cell migration using a wound healing (scratch) assay showed that the closure of the wound gaps was completed by 48 h in mock and in ERBB3 knockout clones. In contrast, this process was considerably delayed in ERBB2 knockout clones, and a complete closure of the gap in the latter cells did not occur before 72 h. In conclusion, both ERBB2 and ERBB3 are essential for normal proliferation of skin keratinocytes, but in contrast to ERBB3, ERBB2 is essential for migration of human keratinocytes. These observations might bear significance to patient adverse effects of therapeutic agents targeting ERBB2 and ERBB3.

Breast cancer subtypes display distinct biological traits that influence their clinical behavior and response to therapy. Recent studies have highlighted the importance of chromatin structure regulators in tumorigenesis. The RNF20-RNF40 E3 ubiquitin ligase complex monoubiquitylates histone H2B to generate H2Bub1, while the deubiquitinase (DUB) USP44 can remove this modification. We found that RNF20 and RNF40 expression and global H2Bub1 are relatively low, and USP44 expression is relatively high, in basal-like breast tumors compared with luminal tumors. Consistent with a tumor-suppressive role, silencing of RNF20 in basal-like breast cancer cells increased their proliferation and migration, and their tumorigenicity and metastatic capacity, partly through upregulation of inflammatory cytokines. In contrast, in luminal breast cancer cells, RNF20 silencing reduced proliferation, migration and tumorigenic and metastatic capacity, and compromised estrogen receptor transcriptional activity, indicating a tumor-promoting role. Notably, the effects of USP44 silencing on proliferation and migration in both cancer subtypes were opposite to those of RNF20 silencing. Hence, RNF20 and H2Bub1 have contrasting roles in distinct breast cancer subtypes, through differential regulation of key transcriptional programs underpinning the distinctive traits of each subtype.

A relatively well-understood multistep process enables mutation-bearing cells to form primary tumours, which later use the circulation system to colonize new locations and form metastases. However, in which way the emerging abundance of different non-coding RNAs supports tumour progression is poorly understood. Here, we review new lines of evidence linking long and short types of non-coding RNAs to signalling pathways activated in the course of cancer progression by growth factors and by the tumour micro-environment. Resolving the new dimension of non-coding RNAs in oncogenesis will probably translate to earlier detection of cancer and improved therapeutic strategies.

Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A, IL1B and IL8, which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro. This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting.

The European Society for Medical Oncology (ESMO) and the American Society of Clinical Oncology (ASCO) are publishing a new edition of the ESMO/ASCO Global Curriculum (GC) thanks to contribution of 64 ESMO-appointed and 32 ASCO-appointed authors. First published in 2004 and updated in 2010, the GC edition 2016 answers to the need for updated recommendations for the training of physicians in medical oncology by defining the standard to be fulfilled to qualify as medical oncologists. At times of internationalisation of healthcare and increased mobility of patients and physicians, the GC aims to provide state-of-the-art cancer care to all patients wherever they live. Recent progress in the field of cancer research has indeed resulted in diagnostic and therapeutic innovations such as targeted therapies as a standard therapeutic approach or personalised cancer medicine apart from the revival of immunotherapy, requiring specialised training for medical oncology trainees. Thus, several new chapters on technical contents such as molecular pathology, translational research or molecular imaging and on conceptual attitudes towards human principles like genetic counselling or survivorship have been integrated in the GC. The GC edition 2016 consists of 12 sections with 17 subsections, 44 chapters and 35 subchapters, respectively. Besides renewal in its contents, the GC underwent a principal formal change taking into consideration modern didactic principles. It is presented in a template-based format that subcategorises the detailed outcome requirements into learning objectives, awareness, knowledge and skills. Consecutive steps will be those of harmonising and implementing teaching and assessment strategies.

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECMassociated pathologies.

The epidermal growth factor receptor (EGFR) and a coreceptor denoted HER2/ERBB2 are frequently overexpressed or mutated in solid tumors, such as carcinomas and gliomas. In line with driver roles, cancer drugs intercepting EGFR or HER2 currently outnumber therapies targeting other hubs of signal transduction. To explain the roles for EGFR and HER2 as prime drivers and targets, we take lessons from invertebrates and refer to homeostatic regulation of several mammalian tissues. The model we infer ascribes to the EGFR-HER2 module pivotal functions in rapid clonal expansion of progenitors called transient amplifying cells (TACs). Accordingly, TACs of tumors suffer from replication stress, and hence accumulate mutations. In addition, several lines of evidence propose that in response to EGF and related mitogens, TACs might undergo dedifferentiation into tissue stem cells, which might enable entry of oncogenic mutations into the stem cell compartment. According to this view, antibodies or kinase inhibitors targeting EGFR-HER2 effectively retard some solid tumors because they arrest mutation-enriched TACs and possibly inhibit their dedifferentiation. Deeper understanding of the EGFR-HER2 module and relations between cancer stem cells and TACs will enhance our ability to control a broad spectrum of human malignancies.

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes.

Tyrosine-specific and other protein kinases are embedded in signaling networks critical for progression of tumors of all types. Hence, kinase inhibitors have nucleated a major arm of personalized cancer therapy. Unfortunately, almost all kinase inhibitors evoke resistance within a year or two, due to secondary mutations, and other alterations within the targeted kinase, or due to emergence of feedback regulatory loops that compensate for extinguished kinases. We review clinically approved kinase inhibitors and the emergence of resistance in leukemia, melanoma, lung and breast tumors, and draw parallel lines in terms of secondary mutations and compensatory mechanisms. Currently emerging are pharmacological strategies able to circumvent resistance and re-sensitize patients to therapeutic treatments. They include second and third generation inhibitors that overcome new mutations, novel drug combinations that simultaneously block the primary oncogenic pathway and compensatory routes, as well as monoclonal antibodies. Deeper understanding of biological signaling networks and their responses to perturbations will aid in the development of effective therapies for patients with cancer.

Growth factors of the epidermal growth factor (EGF)/neuregulin family are involved in tumor progression and, accordingly, antibodies that intercept a cognate receptor, epidermal growth factor receptor (EGFR)/ERBB1, or a co-receptor, HER2, have been approved for cancer therapy. Although they might improve safety and delay onset of chemoresistance, no anti-ligand antibodies have been clinically approved. To identify suitable ligands, we surveyed fluids from ovarian and lung cancer patients and found that amphiregulin (AREG) is the most abundant and generalized ligand secreted by advanced tumors. AREG is a low affinity EGFR ligand, which is upregulated following treatment with chemotherapeutic drugs. Because AREG depletion retarded growth of xenografted ovarian tumors in mice, we generated a neutralizing monoclonal anti-AREG antibody. The antibody inhibited growth of ovarian cancer xenografts and strongly enhanced chemotherapy efficacy. Taken together, these results raise the possibility that AREG and other low- or high-affinity binders of EGFR might serve as potential targets for cancer therapy.

Cancer progression depends on stepwise accumulation of oncogenic mutations and a select group of growth factors essential for tumor growth, metastasis and angiogenesis. Agents blocking the epidermal growth factor receptor (EGFR, also called HER1 and ERBB1) and the co-receptor called HER2/ERBB2 have been approved over the last decade as anti-cancer drugs. Because the catalytically defective member of the family, HER3/ERBB3, plays critical roles in emergence of resistance of carcinomas to various drugs, current efforts focus on antibodies and other anti-HER3/ERBB3 agents, which we review herein with an emphasis on drug combinations and some unique biochemical features of HER3/ERBB3.

We present a system of nonlinear ordinary differential equations used to quantify the complex dynamics of the interactions between tumor growth, vasculature generation, and antiangiogenic treatment. The primary dataset consists of longitudinal tumor size measurements (1,371 total observations) in 105 colorectal tumor-bearing mice. Mice received single or combination administration of sunitinib, an antiangiogenic agent, and/or irinotecan, a cytotoxic agent. Depending on the dataset, parameter estimation was performed either using a mixed-effect approach or by nonlinear least squares. Through a log-likelihood ratio test, we conclude that there is a potential synergistic interaction between sunitinib when administered in combination with irinotecan in preclinical settings. Model simulations were then compared to data from a follow-up preclinical experiment. We conclude that the model has predictive value in identifying the therapeutic window in which the timing between the administrations of these two drugs is most effective.

Although antibodies against EGFR and HER2 are used to treat cancer, only some patients respond and resistance often emerges. Jacobsen and colleagues present in this issue experimental evidence favoring replacement of the currently applied monoclonal antibodies with oligoclonal mixtures of six synergistic antibodies, simultaneously engaging EGFR, HER2, and also HER3.

Tumor progression requires cancer cell proliferation, migration, invasion, and attraction of blood and lymph vessels. These processes are tightly regulated by growth factors and their intracellular signaling pathways, which culminate in transcriptional programs. Hence, oncogenic mutations often capture growth factor signaling, and drugs able to intercept the underlying biochemical routes might retard cancer spread. Along with messenger RNAs, microRNAs play regulatory roles in growth factor signaling and in tumor progression. Because growth factors regulate abundance of certain microRNAs and the latter modulate the abundance of proteins necessary for growth factor signaling, the two classes of molecules form a dense web of interactions, which are dominated by a few recurring modules. We review specific examples of the alliance formed by growth factors and microRNAs and refer primarily to the epidermal growth factor (EGF) pathway. Clinical applications of the crosstalk between microRNAs and growth factors are described, including relevance to cancer therapy and to emergence of resistance to specific drugs.

Despite initial responses to targeted kinase inhibitors, lung cancer patients presenting with primary epidermal growth factor receptor (EGFR) mutations acquire resistance, often due to a second-site mutation (T790M). However, clinical trials found no survival benefits in patients treated with a monoclonal antibody (mAb) to EGFR that should block activation of the mutated receptor and thus bypass resistance to molecules that target the catalytic or ATP-binding site. Using cell lines with the T790M mutation, we discovered that prolonged exposure to mAbs against only the EGFR triggered network rewiring by (i) stimulating the extracellular signal-regulated kinase (ERK) pathway; (ii) inducing the transcription of HER2 (human epidermal growth factor receptor 2) and HER3, which encode other members of the EGFR family, and the gene encoding HGF, which is the ligand for the receptor tyrosine kinase MET; and (iii) stimulating the interaction between MET and HER3, which promoted MET activity. Supplementing the EGFR-specific mAb with those targeting HER2 and HER3 suppressed these compensatory feedback loops in cultured lung cancer cells. The triple mAb combination targeting all three receptors prevented the activation of ERK, accelerated the degradation of the receptors, inhibited the proliferation of tumor cells but not of normal cells, and markedly reduced the growth of tumors in mice xenografted with cells that were resistant to combined treatment with erlotinib and the single function-blocking EGFR mAb. These findings uncovered feedback loops that enable resistance to treatment paradigms that use a single antibody and indicate a new strategy for the treatment of lung cancer patients.

Dissemination of primary tumor cells depends on migratory and invasive attributes. Here, we identify Navigator-3 (NAV3), a gene frequently mutated or deleted in human tumors, as a regulator of epithelial migration and invasion. Following induction by growth factors, NAV3 localizes to the plus ends of microtubules and enhances their polarized growth. Accordingly, NAV3 depletion trimmed microtubule growth, prolonged growth factor signaling, prevented apoptosis and enhanced random cell migration. Mathematical modeling suggested that NAV3-depleted cells acquire an advantage in terms of the way they explore their environment. In animal models, silencing NAV3 increased metastasis, whereas ectopic expression of the wild-type form, unlike expression of two, relatively unstable oncogenic mutants from human tumors, inhibited metastasis. Congruently, analyses of > 2,500 breast and lung cancer patients associated low NAV3 with shorter survival. We propose that NAV3 inhibits breast cancer progression by regulating microtubule dynamics, biasing directionally persistent rather than random migration, and inhibiting locomotion of initiated cells.

ERBB: The receptor network ERBB receptors (also called HER receptors) are composed of an extra-cellular domain (comprising subdomains IIV), a single transmembrane portion, and a large intra-cellular domain comprising a short juxtamembrane portion, a bilobular tyrosine kinase domain and a carboxyl-terminal tail. The principal ERBB receptor activation mechanism involves ligand binding, which activates the kinase domains of receptor homo- and heterodimers. Notably, the ligandless ERBB2 and the kinase-dead ERBB3 are non-autonomous, yet confer potent signaling upon heterodimerization. Activated kinase domains then phosphorylate tyrosine residues located in the cytoplasmic receptor's portion, which serve as docking sites for proteins containing phosphotyrosine-binding or Src homology-2 domains (Figure 10.1). These signaling effectors and adaptor proteins link activated receptors directly or indirectly to canonical intra-cellular pathways, depicted in Figure 10.2, as well as to the endocytic, desensitizing machinery. Although there is considerable overlap amongst the individual ERBB receptors with regards to the recruited signaling effectors and adaptor proteins, the stoichiometry of recruited adaptors varies, and some pathways are unique to individual receptors (1,2). Moreover, many tyrosine residues can bind several adaptors and effectors, which, in turn, can act as molecular scaffolds. For instance, phosphorylated tyrosine residues 1068 and 1086 of the EGFR recruit the adaptor protein Grb2, which can bind both positive (Sos) and negative (e.g. Cbl, Ship, Socs, Sprouty, Ack1) regulators of EGFR signaling. Further fine-tuning of receptor activity and connectivity is achieved by phosphorylation of cytoplasmic ERBB receptor residues by intra-cellular kinases (e.g. Src phosphorylates EGFR on multiple residues, including tyrosine 845, which then serves as a novel docking site for STAT5b (3)). ERBB signaling may both activate and undergo activation by several heterologous receptors (e.g. the HGF-receptor MET) through multiple mechanisms, including formation of signaling-competent receptor heteromers, receptor transmodulation, and by transcriptional induction of heterologous ligands and receptors.

Aptamers are an emerging class of molecules in cancer therapy. They can be easily synthesized and are considered cost-effective drug candidates. In this book chapter we describe the selection and characterization of DNA aptamers specific to the human epidermal growth factor receptor 2 (ERBB2/HER2), an oncogenic tyrosine kinase. First, a DNA aptamer library is applied and ERBB2-specific aptamers are selected using SELEX. Binders are subcloned into a pGEM-T vector, sequenced, and characterized using biochemical and cell biological techniques. By multimerizing the selected ERBB2 aptamers, it might be possible to significantly increase their avidity. For example, we could show that a trimeric ERBB2-specific aptamer could efficiently internalize membranal ERBB2. Furthermore, the receptor assembled in cytoplasmic puncta and was finally degraded by the lysosome. In addition, the selected, trimeric aptamer inhibited proliferation in an XTT assay in comparison to a control sequence. Aptamers selected using the protocol we describe might exert anticancer effect. In our example of a trimeric anti-HER2 aptamer, we could report that a human gastric xenograft mouse model demonstrated pharmacological value of the selected aptamer in vivo. This chapter should enable the interested reader to replicate selection of DNA aptamers specific to oncogenic cell surface. We would like to particularly emphasize some experimental approaches which were used to further characterize selected aptamer sequences, upon SELEX selection. For instance, we included several blotting techniques, antiproliferative assays of aptamers in vitro, and describe the handling of an in vivo human xenograft mouse model.

In metazoa, inductive cell-to-cell interactions play crucial roles, both in embryonic development and in homeostasis. Many such interactions are mediated by RTKs and their soluble or surface-bound ligand growth factors. Type 1 RTKs, also called the ERBB or HER family, comprises four proteins, which bind growth factors of the EGF and neuregulin families. The four ERBB/HER proteins are crucial not only for the determination of several epithelial, glial, muscle, and neuronal lineages, but they are also key players in pathological processes, such as malignant transformation. Multiple mechanisms underlay the driver or supportive functions of ERBB/HER proteins in tumors, and they include receptor overexpression, receptors point mutations, and internal deletions, as well as autocrine loops, meaning that a cell initiates its own proliferation through ligand secretion. Although all ERBB/HER receptors share similar domain architecture, ERBB2/HER2 binds with no known ligand and ERBB3/HER3 harbors a barely active kinase domain. Like other RTKs, ERBB/HER proteins signal downstream through the ERK/MAPK and PI3K/AKT pathways. However, ERBB/HER proteins are characterized by extensive receptor-receptor interactions, with ERBB2/HER2 functioning as a preferred heterodimer partner. In recent years, the ability of ERBB/HER proteins to redirect signals by means of network plasticity is emerging as an important way developed by tumors to evade pharmacological interceptors aimed at specific nodes of the network.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

Receptor tyrosine kinases, along with G protein-coupled receptors and the group of cytokine receptors, transmit a great majority of extracellular cues to the cytoplasm and nucleus of target cells. Here we focus on one subgroup of receptor tyrosine kinases, whose prototype is the epidermal growth factor receptor (EGFR). Due to ligand-induced homo- and heterodimerization by EGFR (also called ERBB1) and other family members, extracellular signals are processed by a layered signaling network, which generates a complex, time-dependent output. Mass-action models well describe the emergent behavior of the network, but their establishment requires detailed experimental data. For example, mass-action models incorporate feedback regulatory loops and explain ligand-specific rewiring of the network, as well as the emergence of ultrasensitivity. Other computational models are employed when the volume of experimental data is limited. Both mass-action models and the more abstractive models help uncover fragile nodes amenable for therapeutic intervention. Likewise, co-option of networks robustness by disease states might be modeled and help understand sensitivity, as well as resistance, to drugs targeting signal transduction by the ERBB and related networks.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2- breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

Tumor initiation and progression are the outcomes of a stepwise accumulation of genetic alterations. Among these, gene amplification and aberrant expression of oncogenic proteins, as well as deletion or inactivation of tumor suppressor genes, represent hallmark steps. Mounting evidence collected over the last few years has identified different populations of non-coding RNAs as major players in tumor suppression in almost all cancer types. Elucidating the diverse molecular mechanisms underlying the roles of non-coding RNAs in tumor progression might provide illuminating insights, potentially able to assist improved diagnosis, better staging and effective treatments of human cancers. Here we focus on several groups of tumor suppressor microRNAs, whose downregulation exerts a profound oncologic impact and might be harnessed for the benefit of cancer patients.

Epigen is the latest addition to the mammalian family of EGFR ligands. Epigen was initially identified as a novel expressed sequence tag with homology to the EGF family by high throughput sequencing of a mouse keratinocyte complementary DNA library, and received its name for its ability to act as an epithelial mitogen. In vitro studies attributed to epigen several unique features, such as persistent and potent biological actions involving low affinity receptor binding, as well as sub-maximal receptor activation and inactivation. Similarly to the other EGFR ligands, the expression of epigen is up-regulated by hormones and in certain cancer types. While the biological functions of epigen remain to be uncovered, it appears to play a role in epidermal structures, such as the mammary gland and the sebaceous gland. The latter organ, in particular, was greatly enlarged in transgenic mice overexpressing epigen. Interestingly, mice lacking epigen develop and grow normally, probably due to functional compensation by other EGFR ligands. Future studies are likely to reveal the biological roles of the unique receptor binding properties of epigen, as well as its potential harnessing during disease.

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR's positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR's feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

Endocytosis entails selective packaging of cell-surface proteins, such as receptors for cytokines and adhesion components, in cytoplasmic vesicles (endosomes). The series of sorting events that determines the fate of internalized proteins, either degradation in lysosomes or recycling back to the plasma membrane, relies on intrinsic sequence motifs, posttranslational modifications (e.g., phosphorylation and ubiquitination), and transient assemblies of both Rab GTPases and phosphoinositide-binding proteins. This multicomponent process is enhanced and skewed in cancer cells; we review mechanisms enabling both major drivers of cancer, p53 and Ras, to bias recycling of integrins and receptor tyrosine kinases (RTKs). Likewise, cadherins and other junctional proteins of cancer cells are constantly removed from the cell surface, thereby disrupting tissue polarity and instigating motile phenotypes. Mutant forms of RTKs able to evade Cbl-mediated ubiquitination, along with overexpression of the wild-type forms and a variety of defective feedback regulatory loops, are frequently detected in tumors. Finally, we describe pharmacological attempts to harness the peculiar endocytic system of cancer, in favor of effective patient treatment.

Hypoxia has been long-time acknowledged as major cancer-promoting microenvironment. In such an energy-restrictive condition, post-transcriptional mechanisms gain importance over the energy-expensive gene transcription machinery. Here we show that the onset of hypoxia-induced cancer stem cell features requires the beta-catenin-dependent post-transcriptional up-regulation of CA9 and SNAI2 gene expression. In response to hypoxia, beta-catenin moves from the plasma membrane to the cytoplasm where it binds and stabilizes SNAI2 and CA9 mRNAs, in cooperation with the mRNA stabilizing protein HuR. We also provide evidence that the post-transcriptional activity of cytoplasmic beta-catenin operates under normoxia in basal-like/triple-negative breast cancer cells, where the betacatenin knockdown suppresses the stem cell phenotype in vitro and tumor growth in vivo. In such cells, we unravel the generalized involvement of the beta-catenin-driven machinery in the stabilization of EGF-induced mRNAs, including the cancer stem cell regulator IL6. Our study highlights the crucial role of post-transcriptional mechanisms in the maintenance/acquisition of cancer stem cell features and suggests that the hindrance of cytoplasmic beta-catenin function may represent an unprecedented strategy for targeting breast cancer stem/basal-like cells.

Contemporary microRNA research has led to significant advances in our understanding of the process of tumorigenesis. MicroRNAs participate in different events of a cancer cell's life, through their ability to target hundreds of putative transcripts involved in almost every cellular function, including cell cycle, apoptosis, and differentiation. The relevance of these small molecules is even more evident in light of the emerging linkage between their expression and both prognosis and clinical outcome of many types of human cancers. This identifies microRNAs as potential therapeutic modifiers of cancer phenotypes. From this perspective, we overview here the miR-10b locus and its involvement in cancer, focusing on its role in the establishment (miR-10b*) and spreading (miR-10b) of breast cancer. We conclude that targeting the locus of microRNA 10b holds great potential for cancer treatment.

Monoclonal antibodies targeting specific surface antigens of cancer cells are rapidly becoming the main stay drugs in specific diseases, such as lymphoma and breast cancer. Therapeutic antibodies almost invariably induce endocytosis of their antigens, and this attribute is already harnessed as a strategy to deliver cytotoxic payloads into cancer cells. The therapeutic potential, however, extends to direct antitumor activity of naked (unconjugated) antibodies, but the contribution of antibody-induced endocytosis to antitumor effects is variable and remains largely unclear. Interestingly, mixtures of monoclonal antibodies, each engaging a distinct epitope of the same antigen, synergistically induce receptor degradation and correspondingly collaborate in tumor inhibition. Here we describe several examples of therapeutic and experimental antibodies, with an emphasis on growth factor receptors and the possibility that future immunotherapy will employ specific antibody combinations, which robustly strip tumors of their most essential receptors.

Collective migration is an important cellular trait, which is intensely studied by both basic and translational researchers. Investigation of the underlying mechanisms necessitates high-throughput assays and computational algorithms capable of generating reproducible quantitative measurements of cell migration. We present a desktop tool that can be used easily by any researcher, to quantify both fluorescent and phase-contrast images produced in the course of commonly used gap closure ("scratch," "wound healing") collective migration assays. The software has a simple graphical interface that allows the user to tune the relevant parameters and process large numbers of images (or movies). The output contains segmented images and the numerical values inferred from them, allowing easy quantitative analysis of the results.

Unlike the well-characterized checkpoints of the cell cycle, which establish commitment to cell division, signaling pathways and gene expression programs that commit cells to migration are incompletely understood. Apparently, several molecular switches are activated in response to an extracellular cue, such as the epidermal growth factor (EGF), and they simultaneously confer distinct features of an integrated motile phenotype. Here we review such early (transcription-independent) and late switches, in light of a novel ERK-ERF-EGR1 switch we recently reported in the FASEB Journal. The study employed human mammary cells and two stimuli: EGF, which induced mammary cell migration, and serum factors, which stimulated cell growth. By contrasting the underlying pathways we unveiled a cascade that allows the active form of the ERK mitogen-activated protein kinase (MAPK) cascade to export the ERF repressor from the nucleus, thereby permitting tightly balanced stimulation of an EGR1-centered gene expression program.

MicroRNAs (miRs) function primarily as post-transcriptional negative regulators of gene expression through binding to their mRNA targets. Reliable prediction of a miR's targets is a considerable bioinformatic challenge of great importance for inferring the miR's function. Sequence-based prediction algorithms have high false-positive rates, are not in agreement, and are not biological context specific. Here we introduce CoSMic (Context-Specific MicroRNA analysis), an algorithm that combines sequence-based prediction with miR and mRNA expression data. CoSMic differs from existing methods-it identifies miRs that play active roles in the specific biological system of interest and predicts with less false positives their functional targets. We applied CoSMic to search for miRs that regulate the migratory response of human mammary cells to epidermal growth factor (EGF) stimulation. Several such miRs, whose putative targets were significantly enriched by migration processes were identified. We tested three of these miRs experimentally, and showed that they indeed affected the migratory phenotype; we also tested three negative controls. In comparison to other algorithms CoSMic indeed filters out false positives and allows improved identification of context-specific targets. CoSMic can greatly facilitate miR research in general and, in particular, advance our understanding of individual miRs' function in a specific context.

Once stimulated, the epidermal growth factor receptor (EGFR) undergoes self-phosphorylation, which, on the one hand, instigates signaling cascades, and on the other hand, recruits CBL ubiquitin ligases, which mark EGFRs for degradation. Using RNA interference screens, we identified a deubiquitinating enzyme, Cezanne-1, that opposes receptor degradation and enhances EGFR signaling. These functions require the catalytic-and ubiquitin-binding domains of Cezanne-1, and they involve physical interactions and transphosphorylation of Cezanne-1 by EGFR. In line with the ability of Cezanne-1 to augment EGF-induced growth and migration signals, the enzyme is overexpressed in breast cancer. Congruently, the corresponding gene is amplified in approximately one third of mammary tumors, and high transcript levels predict an aggressive disease course. In conclusion, deubiquitination by Cezanne-1 curtails degradation of growth factor receptors, thereby promotes oncogenic growth signals.

Although it is broadly agreed that the improved treatment of patients with cancer will depend on a deeper molecular understanding of the underlying pathogenesis, only a few examples are already available. This Timeline article focuses on the ERBB (also known as HER) network of receptor tyrosine kinases (RTKs), which exemplifies how a constant dialogue between basic research and medical oncology can translate into both a sustained pipeline of novel drugs and ways to overcome acquired treatment resistance in patients. We track the key early discoveries that linked this RTK family to oncogenesis, the course of pioneering clinical research and their merger into a systems-biology framework that is likely to inspire further generations of effective therapeutic strategies.

The signaling pathways that commit cells to migration are incompletely understood. We employed human mammary cells and two stimuli: epidermal growth factor (EGF), which induced cellular migration, and serum factors, which stimulated cell growth. In addition to strong activation of ERK by EGF, and AKT by serum, early transcription remarkably differed: while EGF induced early growth response-1 (EGR1), and this was required for migration, serum induced c-Fos and FosB to enhance proliferation. We demonstrate that induction of EGR1 involves ERK-mediated down-regulation of microRNA-191 and phosphorylation of the ETS2 repressor factor (ERF) repressor, which subsequently leaves the nucleus. Unexpectedly, knockdown of ERF inhibited migration, which implies migratory roles for exported ERF molecules. On the other hand, chromatin immunoprecipitation identified a subset of direct EGR1 targets, including EGR1 autostimulation and SERPINB2, whose transcription is essential for EGF-induced cell migration. In summary, EGR1 and the EGF-ERK-ERF axis emerge from our study as major drivers of growth factor-induced mammary cell migration.

Mammalian cells are constantly exposed to multiple mitogens and, hence, have developed machineries that help them ignore fortuitous signals. In a recent report in Molecular Cell, we highlighted the molecular details of such a noise-reduction filter, including roles for EGR1, AKT, and p53. Brief exposure to a mitogen drives formation of inhibitory p53-chromatin complexes, which are disabled only if the growth factor is still present several hours later. We propose that this "consistency test" prevents repeated division cycles of normal cells but might become defective in most cancer cells.

Stringent regulation of biochemical signalling pathways involves feedback and feedforward loops, which underlie robust cellular responses to external stimuli. Regulation occurs in all horizontal layers of signalling networks, primarily by proteins that mediate internalization of receptor-ligand complexes, dephosphorylation of kinases and their substrates, as well as transcriptional repression. Recent studies have unveiled the role of miRNAs (microRNAs), post-transcriptional regulators that control mRNA stability, as key modulators of signal propagation. By acting as genetic switches or fine-tuners, miRNAs can directly and multiply regulate cellular outcomes in response to diverse extracellular signals. Conversely, signalling networks temporally control stability, biogenesis and abundance of miRNAs, by regulating layers of the miRNA biogenesis pathway. In the present mini-review, we use a set of examples to illustrate the extensive interdependence between miRNAs and signalling networks.

The erbb-2 gene receptor is often over-expressed in human cancer and its overexpression is accompanied by worse prognosis. Targeting erbb-2 gene with antibodies is an effective approach to curtail the progression of erbb-2 gene-expressing cancer types. Two monoclonal antibodies, L-26 and N-12, previously generated in our laboratory, have shown effective tumor inhibition in mice, especially when used in combination. Here, we describe novel peptide mimics of erbb-2 gene protein epitopes, also called mimotopes, that were selected from a constraint random 12-mer peptide phage library, specific for the antibodies L-26 and N-12. Initial sequencing analyses revealed little sequence conservation among the peptide mimotopes, and no sequence homology with the erbb-2 gene protein. However, computational analyses of the two groups of peptides, specific for L-26 and N-12, suggested different epitopes on the erbb-2 gene extracellular domain. In vitro assays showed that the phage displayed peptide mimotopes were specific to their respective antibodies. Selected cyclic peptide mimotopes, but not their corresponding linear equivalents, were able to inhibit binding of the antibodies L-26 and N-12 to the surface of erbb-2 gene-expressing cancer cells in a concentration-dependent manner. In line with this observation, phage-displayed cyclic peptides successfully competed in vitro with recombinant erbb-2 gene protein for binding to their respective antibodies L-26 or N-12. Consistent with the antibody inhibition experiments, we detected specific anti-erbb-2 gene antibodies following vaccination with KLH-coupled cyclic peptides but not with multiple antigenic linear peptides. Potentially, the selected peptides could serve as a starting point for the development of a vaccine against erbb-2 gene over-expressing cancer.

Monoclonal antibodies (mAbs) to HER2 are currently used to treat breast cancer, but low clinical efficacy, along with primary and acquired resistance to therapy, commonly limit clinical applications. We previously reported that combinations of antibodies directed at non-overlapping epitopes of HER2 are endowed with enhanced antitumor effects, probably due to accelerated receptor degradation. Here, we extend these observations to three-dimensional mammary cell models, and compare the effects of single mAbs with the effects of antibody combinations. Collectively, our in vitro assays and computational image analyses indicate that combining mAbs against different epitopes of HER2 better inhibits invasive growth. Importantly, while growth factors are able to reduce intraluminal apoptosis and induce an invasive phenotype, combinations of mAbs better than single mAbs can reverse the growth factor-induced phenotypes of HER2-overexpressing spheroids. In conclusion, our studies propose that mAb combinations negate the biological effects of growth factors on invasive growth of HER2-overexpressing cells. Hence, combining mAbs offers a therapeutic strategy, potentially able to enhance clinical efficacy of existing antireceptor immunotherapeutics.

The understanding of cellular signaling pathways in malignant tumors is an important aspect of cancer research and modern targeted therapy strategies. Growth factors and their receptors in particular are critical to modern cancer therapy research, because these factors control all phases of tumor development and metastasis. Most importantly, growth factors are responsible for cell survival under cytotoxic drugs and radiotherapy. These growth factor signaling pathways are composed of complex networks that have adapted to efficiently respond to certain disturbances, such as a single agent that targets one aspect of the pathway. Meanwhile, multiple insults to the pathway, such as combination therapy regimens, are known to be effective in shutting down these pathways and, consequently, killing the tumor cell. Research is currently under way to find new ways to exploit fragile aspects of oncogenic networks, such as uncommon, multiple perturbations that target essential hubs through immunotherapy, combinations of antibodies, heat shock protein inhibitors, or novel drug combinations. Complex growth factor signaling networks and novel methods to shut down these networks are described within a framework of engineering and mathematical concepts.

Ligand-induced dimerization of the epidermal growth factor receptor (ErbB-1/EGFR) involves conformational changes that expose an extracellular dimerization interface. Subsequent alterations within the cytoplasmic kinase domain, which culminate in tyrosine phosphorylation, are less understood. Our study addressed this question by using two strategies: a chimeric receptor approach employed ErbB-3, whose defective kinase domain was replaced by the respective part of EGFR. The implanted full-length kinase, unlike its subdomains, conferred dimerization and catalysis. The data infer that the kinase function of EGFR is restrained by the carboxyl tail; once grafted distally to the ectopic tail of ErbB-3, the kinase domain acquires quasi-dimerization and activation. In an attempt to alternatively refold the cytoplasmic tail, our other approach employed kinase inhibitors. Biophysical measurements and covalent cross-linking analyses showed that inhibitors targeting the active conformation of EGFR, in contrast to a compound recognizing the inactive conformation, induce quasi-dimers in a manner similar to the chimeric ErbB-3 molecule. Collectively, these observations unveil kinase domain-mediated quasi-dimers, which are regulated by an autoinhibitory carboxyl tail. On the basis of these observations, we propose that quasi-dimers precede formation of ligand-induced, fully active dimers, which are stabilized by both extracellular and intracellular receptor-receptor interactions.-Bublil, E. M., Pines, G., Patel, G., Fruhwirth, G., Ng, T., Yosef Yarden. Kinase-mediated quasi-dimers of EGFR. FASEB J. 24, 4744-4755 (2010). www.fasebj.org

A myriad of cellular processes instigated by growth factors are mediated by cell surface-associated receptor tyrosine kinases (RTKs). Subsequent downstream activation of signaling cascades, as well as their crosstalk, endows specificity in terms of the phenotypic outcome, e.g., cellular proliferation, migration, or differentiation. Such signaling diversity is exemplified by the ability of the epidermal growth factor receptor (EGFR) to stimulate different MAPK cascades, especially the ERK1/2 cascade. It has been shown that the ability of the ERK1/2 cascade to specify cell fate, such as cell migration, is dependent on signal duration governed by feedback control. Here we focus on one experimental system, MCF10A human mammary cells, and a phenotypic outcome of cell migration. We present methods to identify key components of underlying cascades and their effects on the migratory phenotype. We focus on profiling activation of signaling modules, as well as transcriptional regulation, emphasizing the high-throughput potential of such approaches.

Growth factors are implicated in several processes essential for cancer progression. Specifically, growth factors that bind to ErbB family receptors have been implicated in cell proliferation and in resistance of solid tumors to chemotherapy. We quantified ligand secretion by several human cancer cell lines, and generated mAbs against two ligands, namely TGF-alpha and heparin-binding EGF-like growth factor. These growth factors are frequently secreted by pancreatic tumor cell lines, including BxPC3 cells. The monoclonal antibodies were tested for their antigen specificity and ability to inhibit growth of BxPC3 cells in vitro. Combining the two antibodies resulted in enhanced inhibition of BxPC3 cell growth, both in vitro and in tumor-bearing animals. Hence, we combined the two antibodies with gemcitabine, an effective chemotherapeutic drug commonly used to treat pancreatic cancer patients. Because treatment with a combination of two monoclonal antibodies enhanced the ability of chemotherapy to inhibit BxPC3 tumors in mice, we propose a general cancer therapeutic strategy that entails profiling the repertoire of growth factors secreted by a tumor, and combining with chemotherapy several antibodies capable of blocking autocrine ligands.

The EGF-receptor is frequently mutated in a large variety of tumors. Here we review the most frequent mutations and conclude that they commonly enhance the intrinsic tyrosine kinase activity, or they represent loss-of-function of suppressive regulatory domains. Interestingly, the constitutive activity of mutant receptors translates to downstream pathways, which are subtly different from those stimulated by the wild-type receptor. Cancer drugs intercepting EGFR signaling have already entered clinical application. Both kinase inhibitors specific to EGFR, and monoclonal antibodies to the receptor are described, along with experimental approaches targeting the HSP90 chaperone. Deeper understanding of signaling pathways downstream to mutant receptors will likely improve the outcome of current EGFR-targeted therapies, as well as help develop new drugs and combinations.

Several cancer drugs intercept the ErbB family receptors EGFR (ErbB-1) and HER2 (ErbB-2). However, the therapeutic value of targeting ErbB-3 has been less clear. A report in this issue of Cancer Cell by Sheng et al. renews hopes that intercepting ErbB-3-mediated autocrine loops bears potential for treatment of ovarian cancer.

Despite extensive diagnostic and therapeutic efforts, breast cancer remains the second leading cause of female cancer mortality in affluent countries. Because receptor tyrosine kinases of the Epidermal Growth Factor Receptor [EGFR, also ErbB] family are potent gatekeepers of cell fate decisions, their aberrations rank among the most frequent oncogenic insults in breast cancers. Although monoclonal antibodies and kinase inhibitors that intercept ErbB signalling can effectively inhibit progression of mammary tumours, their efficacy remains confined to a specific subset of patients. This review highlights not only the oncogenic derangements of the ErbB network in breast cancer including growth factor or receptor over-expression, but also additional loss or gain of multiple negative and positive network regulators, which override physiological mechanisms of systems control embedded in the ErbB network. We further envisage strategies to specifically target tumours at their unique network hubs, to circumvent resistance to ErbB-targeting agents and focus on network fragility exposed by oncogenic perturbations or, conversely, by targeted therapies.

Background: The epidermal growth factor (EGF) stimulates rapid tyrosine phosphorylation of the EGF receptor (EGFR). This event precedes signaling from both the plasma membrane and from endosomes, and it is essential for recruitment of a ubiquitin ligase, CBL, that sorts activated receptors to endosomes and degradation. Because hyperphosphorylation of EGFR is involved in oncogenic pathways, we performed an unbiased screen of small interfering RNA (siRNA) oligonucleotides targeting all human tyrosine phosphatases. Results: We report the identification of PTPRK and PTPRJ (density-enhanced phosphatase-1 [DEP-1]) as EGFR-targeting phosphatases. DEP-1 is a tumor suppressor that dephosphorylates and thereby stabilizes EGFR by hampering its ability to associate with the CBL-GRB2 ubiquitin ligase complex. DEP-1 silencing enhanced tyrosine phosphorylation of endosomal EGFRs and, accordingly, increased cell proliferation. In line with functional interactions, EGFR and DEP-1 form physical associations, and EGFR phosphorylates a substrate-trapping mutant of DEP-1. Interestingly, the interactions of DEP-1 and EGFR are followed by physical segregation: whereas EGFR undergoes endocytosis, DEP-1 remains confined to the cell surface. Conclusions: EGFR and DEP-1 physically interact at the cell surface and maintain bidirectional enzyme-substrate interactions, which are relevant to their respective oncogenic and tumor-suppressive functions. These observations highlight the emerging roles of vesicular trafficking in malignant processes.

Aberrant endocytosis, vesicle targeting, and receptor recycling represent emerging hallmarks of cancer. In this issue of the JCI, Zhang and colleagues demonstrate that RAB-coupling protein (RCP; also known as RAB11FIP1) is a "driver" of the 8p11-12 amplicon in human breast cancer and mouse xenograft models of mammary carcinogenesis (see the related article beginning on page 2171). Their finding that RAB GTPase function enables genomic amplification to confer aggressiveness to mammary tumors adds significantly to the body of evidence supporting pivotal roles for receptor trafficking in the proliferation and metastasis of cancer.

Clathrin-mediated endocytosis sorts for degradation of more than 50 different growth factor receptors capable of relaying growth and differentiation signals by means of their cytoplasm-facing, intrinsic tyrosine kinase activity. The kinetics and alternative routings of receptor endocytosis critically regulate growth factor signaling, which underscores the importance of understanding mechanisms underlying fail-safe operation (robustness) and fidelity of the pathway. Like other robust systems, a layered hub-centric network controls receptor endocytosis. Characteristically, the modular hubs (e.g., AP2-Eps15 and Hrs) contain a membrane-anchoring lipid-binding domain, an ubiquitin-binding module, which recruits ubiquitinylated cargo, and a machinery enabling homo-assembly. Scheduled hub transitions, as well as cascades of Rab family guanosine triphosphatases and membrane bending machineries, define points of commitment to vesicle budding, thereby securing unidirectional trafficking. System's bistability permits stimulation by a growth factor, which oscillates a series of switches based on posttranslational protein modifications (i.e., phosphorylation, ubiquitinylation and neddylation), as well as transient low-affinity/ high-avidity protein assemblies. Cbl family ubiquitin ligases, along with a set of phosphotyrosine-binding adaptors (e.g., Grb2), integrate receptor endocytosis into the densely wired networks of signal transduction pathways, which are involved in health and disease.

Once engaged by soluble or matrix-anchored ligands, cell surface proteins are commonly sorted to lysosomal degradation through several endocytic pathways. Defective vesicular trafficking of growth factor receptors, as well as unbalanced recycling of integrin- and cadherin-based adhesion complexes, has emerged in the past 5 years as a multifaceted hallmark of malignant cells. In line with the cooperative nature of endocytic machineries, multiple oncogenic alterations underlie defective endocytosis, such as altered ubiquitylation (Cbl and Nedd4 ubiquitin ligases, for example), altered cytoskeletal interactions and alterations to Rab family members. Pharmaceutical interception of the propensity of tumour cells to derail their signalling and their adhesion receptors may constitute a novel target for cancer therapy.

Receptor tyrosine kinases (RTKs) are transmembrane allosteric enzymes: binding of ligand growth factors to their ectodomains stimulates a cytoplasm-facing tyrosine kinase activity, which initiates a plethora of cellular processes. The enormous complexity of RTK signalling, along with rich involvement in pathologies (e.g. cancer and diabetes), motivated the establishment of the international, multi-disciplinary RTK consortium (http://www.rtkconsort.org/) in 2005. In collaboration with the British Society for Proteome Research and the European Bioinformatics Institute, the Consortium held on July 23rd and 24th a Workshop on Proteomics and Phosphoproteomics of RTK Signalling Networks (Hinxton Hall Conference Centre, Cambridge, UK). As highlighted below, systems control (a layered web of regulatory loops summarised in Fig.1) emerged throughout the workshop as a common theme of many presentations.

Several distinct mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are associated with non-small cell lung cancer, but mechanisms underlying their oncogenic potential are incompletely understood. Although normally ligand-induced kinase activation targets EGFR to Cbl-mediated receptor ubiquitinylation and subsequent degradation in lysosomes, we report that certain EGFR mutants escape this regulation. Defective endocytosis characterizes a deletion mutant of EGFR, as well as a point mutant (L858R-EGFR), whose association with c-Cbl and ubiquitinylation are impaired. Our data raise the possibility that refractoriness of L858R-EGFR to downregulation is due to enhanced heterodimerization with the oncogene product HER2, which leads to persistent stimulation.

Keywords: SQUAMOUS-CELL CARCINOMA; FACTOR RECEPTOR; BREAST-CANCER; LUNG-CANCER; CETUXIMAB; ACTIVATION; MECHANISM; NECK; HEAD

Ten years after the first clinical application of Rituximab, an anti-CD20 recombinant monoclonal antibody, immunotherapy has become common practice in oncology wards. Thanks to the great diversity of the immune system and the powerful methodology of genetic engineering, the pharmacologic potential of antibody-based therapy is far from exhaustion. The recent application of Trastuzumab, an antibody to a receptor tyrosine kinase, in adjuvant breast cancer therapy marks the beginning of a new phase in cancer treatment. Here we discuss molecular mechanisms of antibody-based therapy, the emerging ability to target minimal disease and the therapeutic potential of combining antibodies with other modalities.

The human EGF receptor (HER) 2 receptor tyrosine kinase is a survival factor for human cardiomyocytes, and its inhibition may explain the increased incidence of cardiomyopathy associated with the anti-HER2 monoclonal antibody trastuzumab (Genentech, South San Francisco, CA), particularly in patients with prior exposure to cardiotoxic chemotherapies e.g., anthracyclines. Here, we show that GW2974 (HER2/EGF receptor tyrosine kinase inhibitor), but not trastuzumab, activates AMP-activated protein kinase (AMPK), initiating a metabolic stress response in human cardiomyocytes that protects against TNFα-induced cell death. GW2974 stimulates calcium dependent fatty acid oxidation in vitro and in the myocardium of GW2974-treated rodents. Calcium chelation or siRNA-targeted AMPK knockdown blocks GW2974 induced fatty acid oxidation. In addition, inhibition of AMPK by a specific inhibitor resulted in increased killing of cardiomyocytes. Elucidating the effects of HER2-targeted therapies on AMPK may predict for risk of cardiomyopathy and provide a novel HER2-targeted strategy designed to protect myocardium from the pro-apoptotic effects of pro-inflammatory cytokines released in response to cardiac injury by chemotherapy or acute ischemia.

The ErbB receptor tyrosine kinases evolved as key regulatory entities enabling the extracellular milieu to communicate with the intracellular machinery to bring forth the appropriate biological response in an ever-changing environment. Since its discovery, many aspects of the ErbB family have been deciphered, with emphasis on aberration of signaling in human diseases. However, only now, with the availability of the atomic coordinates of these receptors, can we construct a comprehensive model of the mechanisms underlying ligand-induced receptor dimerization and subsequent tyrosine kinase activation. Furthermore, the recent introduction of new high-throughput screening methodologies, combined with the materialization of a systems biology perspective, reveals an overwhelming network complexity, enabling robust signaling and evolvability. This knowledge is likely to impact our view of diseases as system perturbations and resistance to ErbB-targeted therapeutics as manifestations of robustness.

Robust biological signaling networks evolved, through gene duplications, from simple, relatively fragile cascades. Architectural features such as layered configuration, branching and modularity, as well as functional characteristics (e.g., feedback control circuits), enable fail-safe performance in the face of internal and external perturbations. These universal features are exemplified here using the receptor tyrosine kinase (RTK) family. The RTK module is richly mutated and overexpressed in human malignancies, and pharmaceutical interception of its signaling effectively retards growth of specific tumors. Therapy-induced interception of RTK-signaling pathways and the common evolvement of drug resistance are respectively considered here as manifestations of fragility and plasticity of robust networks. The systems perspective we present views pathologies as hijackers of biological robustness and offers ways for identifying fragile hubs, as well as strategies to overcome drug resistance.

As the complex network of signalling pathways involved in regulating tumor cell growth and survival is unraveled, tractable targets for therapeutic drug development have been identified. Questions regarding the best approach towards developing these targeted therapies remain [eg, what is the best strategy for (i) selecting doses, (ii) identifying target patient populations for clinical trials, and (iii) designing combination therapies based on scientific rationale]. Since these agents exert biological and clinical effects that are generally distinct from traditional cytotoxic agents, a different paradigm for their development has been suggested. Here we will discuss how incorporation of biomaker analysis in early-phase clinical trials can provide valuable information to guide further development of targeted therapies.

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation.Wediscuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.

Clinical and experimental data suggest that ErbB-4, a member of the epidermal growth factor receptor family, may have a role in cancer progression and response to treatment. We found recently, using a retrospective clinical analysis, that expression of ErbB-4 receptor is correlated with metastatic potential and patient survival in non-small-cell lung cancer (NSCLC). The purpose of this work was to correlate the expression of the ErbB-4 and lung cancer cells growth in vitro and in vivo and to determine the therapeutic potential of a monoclonal antibody to ErbB-4 against lung cancer. For this aim, we ectopically expressed ErbB-4 in a human NSCLC cell line that did not express the ErbB-4 protein. Overexpression of ErbB-4 produced a constitutively activated ErbB-4 receptor. The transfected ErbB-4 positive clones showed an increased cell proliferation in vitro and in vivo in comparison with parental ErbB-4 negative cells and with the cells transfected by neomycin-resistant gene. A monoclonal antibody to ErbB-4 showed both an inhibitory effect on growth rate and an increasing apoptotic rate in the cells expressing ErbB-4. The results of the current study provide evidence that ErbB-4 plays a significant role in human lung cancer and may serve as a molecular target for anticancer therapy.

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFκB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

ErbB2, a member of the EGF receptor family of tyrosine kinases is overexpressed on many tumor cells of epithelial origin and is the molecular target of trastuzumab (Herceptin), the first humanized antibody used in the therapy of solid tumors. Trastuzumab, which is thought to act, at least in part, by downregulating ErbB2 expression is only effective in ∼30-40% of ErbB2 positive breast tumors. Geldanamycin and its derivative 17-AAG are potential antitumor agents capable of downregulating client proteins of Hsp90, including ErbB2. To investigate the ability of 17-AAG to downregulate ErbB2 in trastuzumab resistant breast cancer cells and the possibility of 17-AAG and trastuzumab potentiating each other's effect, the recently established trastuzumab resistant breast cancer cell line, JIMT-1 was compared to the known trastuzumab sensitive SKBR-3 line. Baseline and stimulus-evoked dimerization and activation levels of ErbB2, and the effects of trastuzumab and 17-AAG alone and in combination on cell proliferation and apoptosis, as well as on ErbB2 expression and phosphorylation have been measured. Baseline activation and amenability to activation and downregulation by trastuzumab was much lower in the resistant line. However, 17-AAG enhanced ErbB2 homodimerization after 5-10 min of treatment in both cell lines, and decreased proliferation with an IC ₅₀ of 70 nM for SKBR-3 and 10 nM for JIMT-1. Thus, 17-AAG may be a useful drug in trastuzumab resistant ErbB2 overexpressing tumors. The antiproliferative effect of 17-AAG was positively correlated with phosphorylation and downregulation of ErbB2 and was dominated by apoptosis, although, especially at higher doses, necrosis was also present. Interestingly, IC₅₀ values for ErbB2 downregulation and phosphorylation, in the 30-40 nM range, were not significantly different for the two cell lines. This observation and the negative correlation between resting ErbB2 levels and the antiproliferative effect of 17-AAG may indicate that activation of ErbB2 to some extent could counteract the overall cytostatic effect, especially at higher levels of ErbB2 expression. The usual therapeutic dose of trastuzumab did not change the IC₅₀ of 17-AAG on the proliferation of either cell line, but nevertheless decreased overall ErbB2 phosphorylation and at low doses of 17-AAG further decreased cell growth in the sensitive SKBR-3, thus trastuzumab may be a good combination partner to counteract undesired activating effects of 17-AAG.

Sprouty proteins are evolutionarily conserved negative feedback regulators of multiple receptor tyrosine kinases. Mammalian versions of these proteins differentially regulate signaling induced by the fibroblast and the epidermal growth factors (FGF and EGF, respectively). Herein we show that, although both growth factors elevate expression of Sprouty-2, FGF- and not EGF-induced activation of the Erk/MAPK pathway is inhibited by Sprouty-2. Attenuation of FGF-signaling is accompanied by the induction of Sprouty-2 phosphorylation on the amino-terminal as well as carboxyl-terminal tyrosine residues, which are less effectively modified upon EGF treatment. Mutagenesis of carboxyl-terminal tyrosines, especially a newly identified phosphorylation site, tyrosine 227, impaired the inhibitory activity of Sprouty-2. These results attribute a novel role for carboxyl-terminal tyrosine residues and yet unidentified phosphotyrosine-binding proteins in the differential regulation of Sprouty-2 activity.

mAbs to receptor tyrosine kinases such as EGF receptor ErbB-1 and HER2/ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Ab-receptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.

Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cbl-mediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

Keywords: Oncology; Immunology; Medicine, Research & Experimental

Kekkon proteins negatively regulate the epidermal growth factor receptor (EGFR) during oogenesis in Drosophila. Their structural relative in mammals, LRIG1, is a transmembrane protein whose inactivation in rodents promotes skin hyperplasia, suggesting involvement in EGFR regulation. We report upregulation of LRIG1 transcript and protein upon EGF stimulation, and physical association of the encoded protein with the four EGFR orthologs of mammals. Upregulation of LRIG1 is followed by enhanced ubiquitylation and degradation of EGFR. The underlying mechanism involves recruitment of c-Cbl, an E3 ubiquitin ligase that simultaneously ubiquitylates EGFR and LRIG1 and sorts them for degradation. We conclude that LRIG1 evolved in mammals as a feedback negative attenuator of signaling by receptor tyrosine kinases.

The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.

Growth factors and their transmembrane receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis, including multiple stages of cancer. The amplitude and kinetics of signaling by growth factor receptors are determined by an endocytic process, which sorts activated, autophosphorylated recetors to degradation in lysosomes. Recent studies uncovered the role of protein ubiquitylation in vesicular trafficking of growth factor receptors. Decoration of ligand-activated receptors by multiple monomeric ubiquitins distinguishes this degradative route from the proteasome-mediated pathway, which involves polymeric chains of ubiquitin. Although receptor ubiquitylation occurs at the cell surface, its major role is to sort internalized receptors to the lumen of the multivesicular body, en route to the lysosome. The ubiquitin ligases that control this late sorting event belong to the Cbl family of RING finger adaptors, which bind specific phosphotyrosine residues in the receptors upon activation by ligand. Another group of E3 ubiquitin ligases, the Nedd4 family, regulates the initial sorting event, which targets receptors to clathrin-coated regions of the plasma membrane. This step entails ubiquitin-dependent assembly of a clathrin-binding complex of adaptors such as epsins, which share ubiquitin-interacting motifs. The concerted action of both ubiquitin-binding adaptors of membrane coats and E3 ligases, as well as their regulation by protein phosphorylation and ubiquitylation, ensure robust endocytosis of growth factor receptors. Genetic defects and virus-mediated manipulations of the endocytic pathway divert receptors to a default recycling pathway, thereby enabling unrestrained signaling characteristic to transformed cells.

ErbB-2/HER2 drives epithelial malignancies by forming heterodimers with growth factor receptors. The primordial invertebrate receptor is sorted to the basolateral epithelial surface by binding of the PDZ domain of Lin-7 to the receptor's tail. We show that all four human ErbBs are basolaterally expressed, even when the tail motif is absent. Mutagenesis of hLin-7 unveiled a second domain, KID, that binds to the kinase region of ErbBs. The PDZ interaction mediates stabilization of ErbB-2 at the basolateral surface. On the other hand, binding of KID is involved in initial delivery to the basolateral surface, and in its absence, unprocessed ErbB-2 molecules are diverted to the apical surface. Hence, distinct domains of Lin-7 regulate receptor delivery to and maintenance at the basolateral surface of epithelia.

Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.

Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.

Cancer cells depend on multiple, locally produced growth factors. Signaling by growth factors entails phosphorylation events, and its termination is determined primarily by endocytosis of growth factor receptor complexes. One group of growth factor receptors frequently implicated in human cancer is the ErbB family of receptor tyrosine kinases. By using ErbB as a prototype, here we review the role of protein ubiquitylation in the process that terminates signaling. Specifically, we concentrate on several adaptor proteins, including c-Cbl and Hgs, to elucidate the complexity of receptor sorting for degradation. Detailed understanding of ubiquitylation control on receptor desensitization may lead to better ways to diagnose and eradicate cancer.

The neuregulin (NRG)/epidermal growth factor (EGF) family of growth factors consists of several ligands that specifically activate four erbB receptor-tyrosine kinases, namely erbB-1 (EGF-R), erbB-2 (neu), erbB-3, and erbB-4. We have previously shown that islet morphogenesis is impaired and β-cell differentiation delayed in mice lacking functional EGF-R [EGF-R (-/-)]. The present study aims to clarify which erbB ligands are important for islet development. Pancreatic expression of EGF, TGF-α, heparin-binding EGF, betacellulin (BTC), and NRG-4 was detected as early as embryonic d 13 (E13). Effects of these ligands were studied in E12.5 pancreatic explant cultures grown for 5 d ex vivo. None of the growth factors affected the ratio of endocrine to exocrine cells. However, significant effects within the endocrine cell populations were induced by EGF, BTC, and NRG-4. β-Cell development was augmented by BTC, whereas the development of somatostatin-expressing δ-cells was stimulated by NRG-4. Both ligands decreased the numbers of glucagon-containing α-cells. The effect of BTC was abolished in the EGF-R (-/-) mice. A soluble erbB-4 binding fusion protein totally inhibited the effects of NRG-4 but not of BTC. Neutralization of endogenous NRG-4 activity in the model system effectively inhibited δ-cell development, indicating that this erbB4-ligand is an essential factor for delineation of the somatostatin-producing δ-cells. Our results suggest that ligands of the EGF-R/erbB-1 and erbB-4 receptors regulate the lineage determination of islet cells during pancreatic development. BTC, acting through EGF-R/erbB-1, is important for the differentiation of β-cells. This could be applied in the targeted differentiation of stem cells into insulin-producing cells.

Just as important as starting cellular signalling pathways is switching them off again. It seems that the Cbl protein has a dual function in accelerating the degradation of certain signalling molecules.

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, here we report that defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction.

Cbl proteins function as ubiquitin protein ligases for the activated epidermal growth factor receptor and, thus, negatively regulate its activity. Here we show that Cbl-b is ubiquitinated and degraded upon activation of the receptor. Epidermal growth factor (EGF)-induced Cbl-b degradation requires intact RING finger and tyrosine kinase binding domains and requires binding of the Cbl-b protein to the activated EGF receptor (EGFR), Degradation of both the EGFR and the Cbl-b protein is blocked by lysosomal and proteasomal inhibitors, Other components of the EGFR-signaling complex (i.e. Grb2 and Shc) are also degraded in an EGF-induced Cbl-b-dependent fashion. Our results suggest that the ubiquitin protein ligase function of Cbl-b is regulated by coordinated degradation of the Cbl-b protein along with its substrate. Furthermore, the data demonstrate that Cbl-b mediates degradation of multiple proteins in the EGFR-signaling complex.

When epidermal growth factor and its relatives bind the ErbB family of receptors, they trigger a rich network of signalling pathways, culminating in responses ranging from cell division to death, motility to adhesion. The network is often dysregulated in cancer and lends credence to the mantra that molecular understanding yields clinical benefit: over 25, 000 women with breast cancer have now been treated with trastuzumab (Herceptin®), a recombinant antibody designed to block the receptor ErbB2. Likewise, small-molecule enzyme inhibitors and monoclonal antibodies to ErbB1 are in advanced phases of clinical testing. What can this pathway teach us about translating basic science into clinical use?

The anti-cancer agent paclitaxel (Taxol) stabilizes microtubules leading to G2/M cell cycle arrest and apoptotic cell death. In order to analyse the molecular mechanisms of Taxol-induced cytotoxicity, we studied the involvement of mitogen-activated protein kinases (MAPK) ERK and p38 as well as the p53 pathways in Taxol-induced apoptosis, The human breast carcinoma cell line MCF7 and its derivatives, MCF7/HER-2 and MDD2, sere used in the study. We found that Taxol treatment strongly activated ERK, p38 MAP kinase and p53 in MAP kinase MCF9 cells prior to apoptosis, PD98059 or SB203580, specific inhibitors of ERK and p38 kinase activities, significantly decreased apoptosis, leaving the surviving cells arrested in G2/M. These inhibitors did not significantly affect Taxol-induced alterations in the cell cycle regulatory proteins Rb, p53, p11/Waf1 and Cdk-2, In addition, inactivation of p53 did not affect cellular sensitivity to Taxol killing. However, cells with inactivated p53, unlike cells harboring wild type p53, failed to arrest in G2/M after treatment with Taxol and continued to divide or go into apoptosis, Our data show that both ERK and p38 MAP kinase cascades are essential for apoptotic response to Taxol-induced cellular killing and are independent of p53 activity, However, p53 may serve as a survival factor in breast carcinoma cells treated with Taxol by blocking cells in G2/M phase of the cell cycle.

Human epidermal growth factor receptors (HER/erbB) constitute a family of four cell surface receptors involved in transmission of signals controlling normal cell growth and differentiation. A range of growth factors serve as ligands, but none is specific for the HER2 receptor. HER receptors exist as both monomers and dimers, either homo- or heterodimers. Ligand binding to HER1, HER3 or HER4 induces rapid receptor dimerization, with a marked preference for HER2 as a dimer partner. Moreover, HER2-containing heterodimers generate intracellular signals that are significantly stronger than signals emanating from other HER combinations. In normal cells, few HER2 molecules exist at the cell surface, so few heterodimers are formed and growth signals are relatively weak and controllable. When HER2 is overexpressed multiple HER2 heterodimers are formed and cell signaling is stronger, resulting in enhanced responsiveness to growth factors and malignant growth. This explains why HER2 overexpression is an indicator of poor prognosis in breast tumors and may be predictive of response to treatment. HER2 is a highly specific and promising target for new breast cancer treatments. The recombinant human anti-HER2 monoclonal antibody (rhuMAb-HER2, trastuzumab, Herceptin) induces rapid removal of HER2 from the cell surface, thereby reducing its availability to heterodimers and reducing oncogenicity.

Overexpression of ErbB2, a receptor-like tyrosine kinase, is shared by several types of human carcinomas. In breast tumors the extent of overexpression has a prognostic value, thus identifying the oncoprotein as a target for therapeutic strategies. Already, antibodies to ErbB2 are used in combination with chemotherapy in the treatment of metastasizing breast cancer. The mechanisms underlying the oncogenic action of ErbB2 involve a complex network in which ErbB2 acts as a ligand-less signaling subunit of three other receptors that directly bind a large repertoire of stroma-derived growth factors. The major partners of ErbB2 in carcinomas are ErbB1 (also called EGFR) and ErbB3, a kinase-defective receptor whose potent mitogenic action is activated in the context of heterodimeric complexes. Why ErbB2-containing heterodimers are relatively oncopotent is a function of a number of processes. Apparently, these heterodimers evade normal inactivation processes, by decreasing the rate of ligand dissociation, internalizing relatively slowly and avoiding the degradative pathway by returning to the cell surface. On the other hand, the heterodimers strongly recruit survival and mitogenic pathways such as the mitogen-activated protein kinases and the phosphatidylinositol 3-kinase. Hyper-activated signaling through the ErbB-signaling network results in dysregulation of the cell cycle homeostatic machinery, with upregulation of active cyclin-D/CDK complexes. Recent data indicate that cell cycle regulators are also linked to chemoresistance in ErbB2-dependent breast carcinoma. Together with D-type cyclins, it seems that the CDK inhibitor p21^Waf1 plays an important role in evasion from apoptosis. These recent findings herald a preliminary understanding of the output layer which connects elevated ErbB-signaling to oncogenesis and chemoresistance.

A rodent oncogenic mutant of the Neu receptor tyrosine kinase is a useful experimental model because over-expression of the respective receptor, namely HER2/ErbB-2, in human malignancies is associated with relatively aggressive diseases. Here we show that the oncogenic form of Neu is constitutively associated with the product of the c-cbl proto-oncogene and is part of a large complex that includes the phosphoinositide 3-kinase and Shc. Ectopic expression of c-Cbl, a ubiquitin-protein isopeptide ligase specific to activated tyrosine kinases, causes rapid removal of Neu from the cell surface and severely reduces signaling downstream of oncogenic Neu. c-Cbl-induced down-regulation of Neu involves covalent attachment of ubiquitin molecules and requires the carboxyl-terminal domain of Neu. The negative effect of c-Cbl is antagonized by v-Cbl, a virus-encoded oncogenic truncated form of c-Cbl. In an in vivo model, infection of a Neu-transformed neuroblastoma with a c-Cbl-encoding retrovirus caused enhanced down-regulation of Neu and correlated with tumor retardation. Our results implicate c-Cbl in negative regulation of Neu and offer a potential target for treatment of HER2/ErbB-2-positive human malignancies.

Keywords: Endocrinology & Metabolism

Overexpression of HER-2/ErbB-2, a homologue of the epidermal growth factor receptor, is associated with poor prognosis, and an ErbB-2-specific antibody is therapeutic when administered to patients with metastatic breast cancer. To understand the mechanism underlying immunotherapy, we concentrated on antibody- and epidermal growth factor-induced degradation of ErbB-2. We show that enhanced degradation is preceded by poly-ubiquitination of ErbB-2. This process necessitates recruitment of the c-Cbl ubiquitin ligase to tyrosine 1112 of ErbB-2. Consequently, mutagenesis of this site retards antibody-induced degradation. Thus, the therapeutic potential of certain antibodies may be due to their ability to direct ErbB-2 to a c-Cbl-regulated proteolytic pathway.

HER2/ErbB-2 belongs to a family of four receptors that bind growth factors as dimers and transmit cellular signals. The ErbB-2 signaling unit shares functional characteristics with other modules whose function is essential for morphogenesis of epithelial organs, including the mammary gland. However, unlike other receptors, ErbB-2 binds no known growth factor ligand with high affinity, and its oncogenic potential is exceptionally high. Biochemical and genetic lines of evidence imply that ErbB-2 is a unique receptor: by serving as a preferred heterodimeric partner of the other ErbB receptors, it enhances and prolongs cell-to-cell signals. ErbB-2-containing heterodimers are long-lived and their signals are relatively potent because the rate of ligand dissociation is decelerated by ErbB-2, and their rate of endocytosis is relatively slow. Apparently, all ErbB ligands are bivalent molecules, whose low affinity site prefers ErbB-2. Hence, overexpression of ErbB-2 in epithelial tumor cells biases formation of heterodimers, leading to enhanced responsiveness to stromal growth factors and, eventually, to oncogenic transformation. Consequently, removal of ErbB-2 from the cell surface or inhibition of its intrinsic enzymatic activity may reduce oncogenicity. Indeed, anti-ErbB-2 antibodies that can effectively internalize the oncoprotein are therapeutically beneficial. We conclude that ErbB-2 developed as a master regulator of a signaling network essential for normal physiology. However, ErbB-2 opportunistically is exploited by oncogenic mechanisms. This understanding may prove useful for developing clinical strategies to inhibit ErbB-mediated cancer.

Neuregulin can trigger morphogenetic signals in cells both in vivo and in culture through the activation of receptors from the ErbB family. We have ectopically expressed various ErbB-receptors in 32D myeloid cells lacking endogenous ErbB-proteins, and in CHO cells, which express only ErbB-2. We show here that activation of ErbB-3/ErbB-2 heterodimeric receptors triggers PI3-kinase-dependent lamellipodia formation and spreading, while individual ErbB-receptor homodimers as well as ErbB3/ErbB-1 heterodimers are much less effective. CHO cells expressing ErB-3/ErbB-2 together with N-cadherin, an adhesion receptor, form epithelioid colonies. Neuregulin activates cell motility leading to transition of these colonies into ring-shaped multicellular arrays, similar to those induced by neuregulin in epithelial cells of different types. This process requires both PI3-kinase and MAP kinase kinase activity and depends on coordinated changes in the actin- and microtubule-based cytoskeleton. Transactivation of ErbB-2 is not sufficient for the activation of cell motility and ring formation, and the C-terminal domain of ErbB-3 bearing the docking sites for the p85 subunit of PI3-kinase is essential for these morphogenetic effects. Thus, ErbB-3 in conjunction with ErbB-2 mediates, via its C-terminal domain, cytoskeletal and adhesion alterations which activate cell spreading and motility, leading to the formation of complex structures such as multicellular rings.

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal- regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both downregulation and ubiquitination of EG;FR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.

Signaling by the epidermal growth factor (EGF) family and the neuregulin group of ligands is mediated by four ErbB receptor tyrosine kinases, that form homo- and heterodimeric complexes. Paradoxically, the neuregulin receptor ErbB-3 is devoid of catalytic activity, but its heterodimerization with other ErbBs, particularly the ligand-less ErbB-2 oncoprotein of carcinomas, reconstitutes superior mitogenic and transforming activities. To understand the underlying mechanism we constructed a chimeric EGF-receptor (ErbB-1) whose autophosphorylation C-terminal domain was replaced by the corresponding portion of ErbB-3. Consistent with the possibility that this domain recruits a relatively potent signaling pathway(s), the mitogenic signals generated by the recombinant fusion protein were superior to those generated by ErbB-1 homodimers and comparable to the proliferative activity of ErbB-2/ErbB-3 heterodimers. Upon ligand binding, the chimeric receptor recruited an ErbB-3-specific repertoire of signaling proteins, including Shc and the phosphatidylinositol 3-kinase, but excluding the ErbB-1-specific substrate, phospholipase Cγ1. Unlike ErbB-1, which is destined to lysosomal degradation through a mechanism that includes recruitment of c-Cbl and receptor poly-ubiquitination, the C-terminal tail of ErbB-3 shunted the chimeric protein to the ErbB-3-characteristic recycling pathway. These observations attribute the mitogenic superiority of ErbB-3 to its C-terminal tail and imply that the flanking kinase domain has lost catalytic activity in order to restrain the relatively potent signaling capability of the C-terminus.

The erbB-2/HER2 oncogene is overexpressed in a significant fraction of human carcinomas of the breast, ovary, and lung in a manner that correlates with poor prognosis. Although the encoded protein resembles several receptors for growth factors, no high affinity ligand of ErbB-2 has so far been fully characterized. However, several lines of evidence have raised the possibility that ErbB-2 can augment signal transduction initiated by binding of certain growth factors to their direct receptors. Here, we contrasted these two models of ErbB-2 function: First, examination of a large series of epidermal growth factor (EGF)-like ligands and neuregulins, including virus-encoded ligands as well as related motifs derived from the precursor of EGF, failed to detect interactions with ErbB-2 when this protein was singly expressed. Second, by using antibodies that block inter-ErbB interactions and cells devoid of surface ErbB-2, we learned that signaling by all ligands examined, except those derived from the precursor of EGF, was enhanced by the oncoprotein. These results imply that ErbB-2 evolved as a shared receptor subunit of all ErbB-specific growth factors. Thus, oncogenicity of ErbB-2 in human epithelia may not rely on the existence of a specific ligand but rather on its ability to act as a coreceptor for multiple stroma-derived growth factors.

Neu differentiation factors (NDFs), or neuregulins, are epidermal growth factor-like growth factors which bind to two tyrosine kinase receptors, ErbB- 3 and ErbB-4. The transcription of several genes is regulated by neuregulins, including genes encoding specific subunits of the acetylcholine receptor at the neuromuscular junction. Here, we have examined the promoter of the acetylcholine receptor ε subunit and delineated a minimal CA-rich sequence which mediates transcriptional activation by NDF (NDF-response element [NRE]). Using gel mobility shift analysis with an NRE oligonucleotide, we detected two complexes that are induced by treatment with neuregulin and other growth factors and identified Sp1, a constitutively expressed zinc finger phosphoprotein, as a component of one of these complexes. Phosphatase treatment, two-dimensional gel electrophoresis, and an in-gel kinase assay indicated that Sp1 is phosphorylated by a 60-kDa kinase in response to NDF- induced signals. Moreover, Sp1 seems to act downstream of all members of the ErbB family and thus may funnel the signaling of the ErbB network into the nucleus.

Cell-to-cell communication is an important facet of every biological system. Unlike metabolic pathways (e.g. glycolysis) that successively modify biological compounds to yield product molecules, signal transduction pathways are communication circuits that process information, rather than molecules, and culminate in the determination of cell fate. Some major steps involved in generation, transfer, processing and decoding of signals carried by various growth factors and cytokines have been elucidated and their wiring uncovered. Along with the realization that signaling pathways are primarily linear and vertical (from membrane to nucleus), examples of lateral interactions allowing crosstalk between vertical axes as well as feedback. regulation, are accumulating. Other emerging issues include the highly compartmentalized nature of intracellular signaling and the consequent role of protein translocation, the essential balance between signal amplification and stringent control, and the wealth of protein motifs acting as adaptor modules. Here we use the family of ErbB receptor tyrosine kinases and their EGF/neuregulin-type ligands to exemplify combinatorial signaling and its role in both inductive morphogenesis and epithelial cancer. Modeling the ErbB pathway as a layered neural-like network may help in clarifying many aspects of this and other interconnected and elaborate biological systems.

Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin.

Keywords: Oncology

The ErbB signaling module consists of four receptor tyrosine kinases and several dozen ligands that activate specific homo- and heterodimeric complexes of ErbB proteins. Combinatorial ligand/receptor/effector interactions allow large potential for signal diversification. Here we addressed the possibility that turn-off mechanisms enhance the diversification potential. Concentrating on ErbB-1 and two of its ligands, epidermal growth factor (EGF) and transforming growth factor α (TGF-α), and the Neu differentiation factor (NDF/neuregulin) and one of its receptors, ErbB-3, we show that ligand binding variably accelerates endocytosis of the respective ligand-receptor complex. However, unlike the EGF-activated ErbB- 1, which is destined primarily to degradation in lysosomes, NDF and TGF-α direct their receptors to recycling, probably because these ligands dissociate from their receptors earlier along the endocytic pathway. In the case of NDF, structural, as well as biochemical, analyses imply that ligand degradation occurs at a relatively late endosomal stage. Attenuation of receptor down-regulation by this mechanism apparently confers to both NDF and TGF-α more potent and prolonged signaling activity. In conclusion, alternative endocytic trafficking of ligand-ErbB complexes may tune and diversify signal transduction by EGF family ligands.

The ErbB-1 receptor tyrosine kinase binds to six different growth factors, whose prototype is the epidermal growth factor (EGF). Two homologous epithelial receptors, ErbB-3 and ErbB-4, bind all isoforms of another family of growth factors, the Neu differentiation factors (NDFs/neuregulins). The fourth member of the ErbB family, ErbB-2, acts as the preferred heterodimeric partner of ligand-occupied complexes of the three other ErbB proteins. Here we report that at high concentrations, EGF can induce cell growth and differentiation in the absence of ErbB-1. This function is shared by betacellulin, but not by three other ligands, including the transforming growth factor a (TGFα). The functional receptor was identified as a heterodimer between ErbB-3 and ErbB-2, a previously identified oncogenic complex. When singly expressed, neither ErbB-3 nor ErbB-2 can mediate signaling by EGF. In addition, when co-expressed, blocking either receptor by using site-specific antibodies inhibited EGF and betacellulin activities, indicating strict cooperativity between ErbB-3 and ErbB-2. Through analysis of chimeras between EGF and TGFα, we identified the middle portion of EGF (loop B) as the site that enables activation of ErbB-2/ErbB-3. In conclusion, cooperative and promiscuous binding of stroma-derived growth factors by the epithelium-expressed ErbB-2/ErbB-3 heterodimer may be significant to cancer development. The mechanistic implications of our results for a model that attributes receptor dimerization to ligand bivalency, as well as to a recently proposed mechanism of secondary dimerization, are discussed.

Substantial evidence exists supporting direct roles for ErbB-2/neu and Src kinase activation in breast cancer. The Csk homologous kinase (CHK) is a recently identified tyrosine kinase which, like Csk, phosphorylates the C- terminal tyrosine of Src kinases, resulting in inactivation of these enzymes. Recently, we observed that CHK is associated with the ErbB-2/neu receptor upon heregulin stimulation of breast cancer cells. Here, we report that CHK expression was observed in 70 out of 80 primary breast cancer specimens but not in normal breast tissues (0/19). Confocal microscopy analysis revealed colocalization of CHK with ErbB-2 in these primary specimens (6/6). In addition, we observed that the cytoplasmic domain of the ErbB-2/neu receptor is sufficient for its interaction with the CHK(SH2) domain. Phosphopeptide inhibition of the in vitro interaction of CHK(SH2) or native CHK with ErbB- 2/neu, as well as site-directed mutagenesis of ErbB-2/neu, indicated that CHK(SH2) binds to Tyr¹²⁵³ of ErbB-2/neu. Interestingly, autophosphorylation at this site confers oncogenicity to this receptor. Moreover, CHK was able to down-regulate ErbB-2/neu-activated Src kinases. Overexpression of CHK in MCF-7 breast cancer cells markedly inhibited cell growth and proliferative response to heregulin as well as decreased colony formation in soft agar. These studies indicate that CHK binds, via its SH2 domain, to Tyr¹²⁵³ of the activated ErbB-2/neu and down-regulates the ErbB-2/neu-mediated activation of Src kinases, thereby inhibiting breast cancer cell growth. These data strongly suggest that CHK is a novel negative growth regulator in human breast cancer.

The recently isolated second family of neuregulins, NRG2, shares its primary receptors, ErbB-3 and ErbB-4, and induction of mammary cell differentiation with NRG1 isoforms, suggesting functional redundancy of the two growth factor families. To address this possibility, we analyzed receptor specificity of NRGs by using an engineered cellular system. The activity of isoform-specific but partly overlapping patterns of specificities that collectively activate all eight ligand-stimulatable ErbB dimers was revealed. Specifically, NRG2-β, like NRG1-α, emerges as a narrow-specificity ligand, whereas NRG2-α is a pan-ErbB ligand that binds with different affinities to all receptor combinations, including those containing ErbB-1, but excluding homodimers of ErbB-2. The latter protein, however, displayed cooperativity with the direct NRG receptors. Apparently, signaling by all NRGs is funneled through the mitogen-activated protein kinase (MAPK). However, the duration and potency of MAPK activation depend on the identity of the stimulatory ligand-receptor ternary complex. We conclude that the NRG-ErbB network represents a complex and nonredundant machinery developed for fine-tuning of signal transduction.Errata: Volume 18, no. 10, p. 60906101, 1998. Page 6090, article byline, line 2: The names should read as shown above.Author Correction: Volume 18, no. 10, p. 60906101, 1998: The two isoforms of neuregulin 2 (NRG2), NRG2-a and NRG2-b, were accidentally mislabeled. The names should therefore be exchanged throughout the paper, except in Fig. 9.8695

The ErbB/HER family of transmembrane receptor tyrosine kinases includes four members that bind more than two dozens ligands sharing an epidermal growth factor- (EGF)³ like motif. This family plays a pivotal role in cell lineage determination in a variety of tissues, including mesenchyme-epithelial inductive processes and the interactions between neurons and muscle, glia and Schwann cells. Certain ligands and receptors of the family, especially the ErbB-2 receptor tyrosine kinase, contribute to a relatively virulent phenotype of some human tumors; most notable are carcinomas of secretory epithelia. This large variety of biological signals is generated through a combinatorial network of signal transduction in which different ErbB ligands are apparently capable of stabilizing discrete homo- and heterodimeric receptor complexes, each coupled to a specific set of cytoplasmic signaling proteins. Because each receptor is unique in terms of catalytic activity, cellular routing and transmodulation, the resulting network allows not only an enormous potential for signal diversification but also fine tuning and stringent control of cellular functions. ErbB-2 emerges as a master coordinator of the network, prolonging and amplifying signaling by decelerating the dissociation rates of its heterologous ligands. Thus, the tumorigenic action of ErbB-2 may be attributed to its ability to act as a shared signaling subunit, rather than by functioning as a bone fide receptor.

ErbB-2 is an orphan receptor that belongs to a family of tyrosine kinase receptors for either epidermal growth factor (EGF) or Neu differentiation factor (NDF/neuregulin). Because overexpression of the erbB-2 proto-oncogene is frequently associated with an aggressive clinical course of certain human adenocarcinomas, the encoded protein is an attractive target for immunotherapy. Indeed, certain monoclonal antibodies (mAbs) to ErbB-2 effectively inhibit tumor growth in animal models and in clinical trials, but the underlying mechanism is incompletely understood. To study this question, we generated a large battery of mAbs to ErbB-2, that were classified epitopically. Whereas most antibodies stimulated tyrosine phosphorylation of ErbB-2, their anti-tumor effect correlated with its accelerated endocytic degradation. One group of tumor-inhibitory mAbs (Class II mAbs) was elicited by the most antigenic site of ErbB-2, and inhibited in trans binding of NDF and EGF to their direct receptors. The inhibitory effect was due to acceleration of ligand dissociation, and it resulted in the reduction of the ability of ErbB-2 to transactivate the mitogenic signals of NDF and EGF. These results identify two potential mechanisms of antibody-induced therapy: acceleration of ErbB-2 endocytosis by homodimerization and blocking of heterodimerization between ErbB-2 and growth factor receptors.

The ErbB family includes two receptors, ErbB-1 and ErbB-3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB-2. Unlike ErbB-1 and ErbB-2, the intrinsic tyrosine kinase of ErbB-3 is catalytically impaired. By using interleukin-3-dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB-3 is devoid of any biological activity but both ErbB-1 and ErbB-2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB-3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity, Inter-receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB-3-containing complexes, especially the ErbB-2/ErbB-3 heterodimer, are more active than ErbB-1 complexes, Nevertheless, ErbB-1 signaling displays dominance over ErbB-3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen-activated protein kinases ERK and c-Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins, It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase-defective ErbB-3.

The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB- 2), and two receptors for the Neu differentiation factor (NDF/heregulin), namely: ErbB-3 and ErhB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB-3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB-3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase- defective ErbB-3, probably with ErbB-2, is essential for transmission of NDF signals through ErbB-3.

The neu/erbB-2/HER-2 proto-oncogene is amplified and/or overexpressed in up to 30% of mammary carcinomas and has been variably correlated with poor prognosis. The signaling activity of the encoded receptor tyrosine kinase is regulated by interactions with other type 1 receptors and their ligands. We have used a novel approach, phosphorylation-sensitive anti-Neu antibodies, to quantify signaling by Neu and epidermal growth factor receptor in a panel o f frozen sections of mammary carcinoma specimens. We also determined the relationship of Neu, phosphorylated Neu (and epidermal growth factor receptor), and phosphotyrosine to the expression of Neu-related receptors (epidermal growth factor receptor, HER-3, and HER-4) and to prognostic factors (estrogen and progesterone receptor). We found that tyrosine phosphorylation of Neu (and hence signaling activity) is highly variable among mammary carcinomas. Neu and HER-4 were associated with divergent correlates, suggesting that they have profoundly different biological activities. These results have implications for etiology of mammary carcinoma, for clinical evaluation of mammary carcinoma patients, and for development of Neu-targeted therapeutic strategies.

Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

Previously we reported that neu differentiation factor (NDF)/heregulin (HRG) elevates tyrosine phosphorylation of its receptors erbB-3, erbB-4, and erbB-2 (through heterodimer formation). We also showed that both NDF/HRG and antibodies to erbB-2 can arrest growth and induce differentiation in breast cancer cells. In this study, we report on the mechanism of NDF/HRG-induced cellular effects. We show that NDF/HRG and antibodies to erbB-2 receptors up-regulate expression of p53 by stabilizing the protein. This is accompanied by upregulation of the p53 inducible gene, p21(CIP1/WAF1), in a variety of cell lines: MCF7 and their derivatives (MCF7/HER2, MN1 and MCF-7-puro), ZR75T and LnCap cells. The induction of p21 is further enhanced when cells are treated with both NDF/HRG and DNA-damaging chemotherapeutic agents (i.e. doxorubicin). The NDF/HRG mediated induction of p21 is dependent on wild-type p53, as it fails to occur in cells expressing dominant negative p53 (MDD2). Furthermore, p21 induction is capable of inactivating cdk2 complexes as measured by Histone H1 phosphorylation assays. Finally, we show that in primary cultures of breast and other cancers, p21 is significantly induced in response to NDF/HRG treatment. Collectively, these observations suggest that the mechanism of breast cancer cell growth inhibition and differentiation via erbB receptors activation is through a p53-mediated pathway.

Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reliculoendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [³H]cholesteryl ether ([³H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio (-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [³H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.

Keywords: GROWTH-FACTOR RECEPTOR; STEM-CELL FACTOR; EXTRACELLULAR DOMAIN; INSULIN-RECEPTOR; BINDING-SITE; TYROSINE PHOSPHORYLATION; SIGNAL-TRANSDUCTION; ERYTHROPOIETIN RECEPTOR; MONOCLONAL-ANTIBODIES; TRANSMEMBRANE DOMAIN

Amplification and overexpression of the erbB-2/neu protooncogene are frequently associated with aggressive clinical course of certain human adenocarcinomas, and therefore the encoded surface glycoprotein is considered a candidate target for immunotherapy. We previously generated a series of anti-ErbB-2 monoclonal antibodies (mAbs) that either accelerate or inhibit the tumorigenic growth of erbB-2-transformed murine fibroblasts. The present study extended this observation to a human tumor cell line grown as xenografts in athymic mice and addressed the biochemical differences between the two classes of mAbs. We show that the inhibitory effect is dominant in an antibody mixture, and it depends on antibody bivalency. By using radiolabeled mAbs we found that all of three tumor-inhibitory mAbs became rapidly inaccessible to acid treatment when incubated with tumor cells. However, a tumor-stimulatory mAb remained accessible to extracellular treatments, indicating that it did not undergo endocytosis. In addition, intracellular fragments of the inhibitory mAbs, but not of the stimulatory mAb, were observed. Electron microscopy of colloidal gold-antibody conjugates confirmed the absence of endocytosis of the stimulatory mAb but detected endocytic vesicles containing an inhibitory mAb. We conclude that acceleration of cell growth by ErbB-2 correlates with cell surface localization, whereas inhibition of tumor growth is associated with an intrinsic ability of anti- ErbB-2 mAbs to induce endocytosis. These conclusions are relevant to the selection of optimal mAbs for immunotherapy and may have implications for the mechanism of cellular transformation by an overexpressed erbB-2 gene.

The neu proto-oncogene product, p185(neu) (HER2, c-ErbB-2), encodes a cell-surface tyrosine kinase receptor with high oncogenic potential, which correlates with increased tyrosine kinase activity and a rapid receptor internalization rate. To investigate the interactions and signal(s) leading to the endocytosis of Neu receptors, we employed lateral mobility and internalization studies. Fluorescence photobleaching recovery measurements revealed that activation of Neu receptors (induced by mutation or by agonistic antibodies) markedly reduced their mobile fractions. To elucidate the signals involved, other mutants, all carrying a constitutively dimerizing oncogenic mutation, were analyzed. A kinase-negative mutant and a mutant lacking all cytoplasmic tyrosine phosphorylation consensus sequences exhibited high mobile fractions, similar to nonactivated Neu. Retention of a single tyrosine autophosphorylation site (Tyr-1253) out of the five known such sites was sufficient to immobilize a large fraction of the receptor. For all mutants, internalization correlated with receptor immobilization and was blocked by treatments that interfere with coated pit structure, indicating that the immobilization is due to interactions with coated pits. This was supported by the coimmunoprecipitation of α-adaptin only with the constitutively activated Neu mutants. We conclude that activated Neu receptors become stably associated with coated pits via plasma membrane adaptor complexes (AP-2). Efficient Neu receptor endocytosis requires activation, a functional kinase domain, and at least one tyrosine autophosphorylation site.

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosinekinase family of receptors. We have found that MadinDarby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mousederived cDNA encoding FGF receptor1 (FGFR1) in MDCK cells resulted in the acquisition of a fibroblastlike morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGFinduced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR1 expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR1transfected cells with bFGF resulted in the loss of transepithelial resitance. Monolayers of parental MDCK cells did not lose their transepithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their transepithelial resistance, indicating that the effects on the transepithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of transepithelial resistance is separable from the morphological transition. © 1995 WileyLiss, Inc.

The HC11 mouse mammary epithelial cell line has proven to be a valuable in vitro model to study the roles of peptide factors and hormones involved in the growth and differentiation of mammary cells. Treatment of HC11 cells with the lactogenic hormones, dexamethasone, insulin, and PRL (DIP), leads to cellular differentiation and production of the milk protein beta-casein. We have analyzed the effects of Neu differentiation factor (NDF)/heregulin, a newly described activating ligand for erbB-2 and other members of the epidermal growth factor (EGF) receptor family, on cell growth and the expression of milk proteins in HC11 cells. In these cells, NDF induces tyrosine phosphorylation of erbB-2 and erbB-3. Both NDF and EGF stimulate HC11 cell proliferation and promote the responsiveness of HC11 cells to lactogenic hormones. NDF induces the expression of a 22-kilodalton milk protein. This protein is up-regulated by other factors, including dexamethasone, EGF, and basic fibroblast growth factor, and is controlled in a manner distinct from that of beta-casein. Like EGF, NDF inhibits the DIP-induced expression of beta-casein at the level of transcription. The inhibition is due to the negative effect of NDF on the activation of mammary gland factor (MGF/Stat5), a member of the Stat family of transcription factors, which is essential for beta-casein gene expression.

The histological hallmarks for the diagnosis of medullary breast cancer are circumscription, syncytial architecture, diffuse inflammatory infiltrate, and highly atypical nuclei. The biological and prognostic implication is a lower propensity to metastasize. We studied 19 medullary carcinomas for expression of the intercellular adhesion molecule-1 and lymphocyte-function-associated antigen-1, Neu differentiation factor, tumor necrosis factor-α, and the expression of HER-2/ neu, HER-4, and HER-3 receptors. Our study revealed that all of the 19 medullary carcinomas expressed the intercellular adhesion molecule-1 and lymphocyte function associated antigen. Eighteen of 19 cancers expressed Neu differentiation factor and tumor necrosis factor-α. All medullary cancers expressed the HER-2/neu receptor, however, in the majority of the cases, the staining was confined to the cytoplasm. Only 4 of 12 cancers expressed HER-4 and none of the eight medullary cancers tested expressed HER-3. By comparison, in a control group of infiltrating ductal carcinomas, expression of intercellular adhesion molecule-1, lymphocyte function associated antigen-1, and Neu differentiation factor was positive in about 25 to 30% of the cases, HER-4 was expressed in 75% and HER-3 in 95% of the cases. Taken together, our observations suggest that the expression of intercellular adhesion molecule-1, lymphocyte function associated antigen, Neu differentiation factor, and tumor necrosis factor-α as factors that may affect the special morphology and the biological behavior that characterizes medullary carcinomas.

The Heregulin (HGL) gene, encoding a ligand for a member of the ERBB receptor family, is located at 8p 12p22, in or close to a region frequently amplified in breast carcinoma. Amplification of HGL was detected in three of 83 (3.6%) cases of breast tumors. No overexpression of the gene was observed in the amplified tumors. This and the low incidence of amplification suggest that HGL is not the key gene of the 8p 12 amplification but may be used as a marker of large amplification units.

The Neu proto-oncogene (also called ErbB-2 and HER-2) encodes a tyrosine kinase transmembrane receptor homologous to the epidermal growth factor receptor (EGF-R). Overexpression, a point-mutation, and co-expression with EGF-R activate the oncogenic potential of the Neu protein by permanent coupling to signal transducing pathways. The search for ligands that elevate tyrosine phosphorylation of Neu led to the discovery of a 44-kDa glycoprotein that acts either as a differentiation factor or as a mitogen for mammary tumor cells. This protein, termed Neu differentiation factor (NDF), is derived from a transmembrane precursor that contains an EGF-like motif and an immunoglobulin-like domain. Alternative splicing generates a dozen NDF-related proteins that are expressed in a variety of mesenchymal and neuronal tissues. This unprecedented multiplicity raises the possibility that different isoforms fulfill distinct biological roles.

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms α and β), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-α2 is the predominant form in mesenchymal cells, whereas pro-NDF-β1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its β-type C terminus displays higher affinity than α-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue- specific functions.

The transforming potential of the Neu/ErbB-2 receptor tyrosine kinase undergoes inactivation by deletion of the non-catalytic C-terminal tail, which contains five autophosphorylation sites. To determine which site is essential for oncogenicity, we tailed the C-terminally-deleted mutant with individual autophosphorylation sites. Complete restoration of the transforming action in vitro and in vivo was conferred by a stretch of 12 amino acids that contained the most C-terminal tyrosine autophosphorylation site (Y1253). Reconstitution of transformation was specific to this amino acid sequence because none of the other autophosphorylation sites, when grafted individually, caused transformation, and replacement of the tyrosine with a phenylalanine residue significantly reduced the oncogenic potential of both the full-length and the tailed proteins. When present alone the most C-terminal sequence enabled coupling to a biochemical pathway that includes Ras, MAP kinase and transactivation of Jun. These results indicate that the multiplicity of autophosphorylation sites on a receptor tyrosine kinase is not essential for transformability, and implicate the MAP kinase pathway in transduction of the oncogenic signal of Neu/ErbB-2.

Transmembrane receptor tyrosine kinases that bind to growth factors transmit signals that are essential to growth and differentiation. These receptors can be classified into groups based on their structure. One group implicated in the pathogenesis of breast cancer contains receptors belonging to the erbB family. This group includes the epidermal growth factor receptors, the HER-2/neu (erbB-2), HER-3, and HER-4. Despite the structural similarity of these receptors, HER-2/neu, HER-4, and HER-3 do not bind to any ligand of the epidermal growth factor receptor. However, a 44-kD glycoprotein called neu differentiation factor (neu differentiation factor/heregulin) has been isolated. This ligand phosphorylates the HER-2/neu receptor and binds directly to HER-4 and HER-3. The abundance of erbB receptors and their ligands in breast cancers points to their functional importance in the pathogenesis and biological behavior of breast cancers. Furthermore, these receptors and ligands may hold a promise for targeted therapy for breast cancer in the future.

Transmembrane receptor tyrosine kinases that bind to peptide factors transmit essential growth and differentiation signals. A growing list of orphan receptors, of which some are oncogenic, holds the promise that many unknown ligands may be discovered by tracking the corresponding surface molecules. The neu gene (also called erbB2 and HER2) encodes such a receptor tyrosine kinase whose oncogenic potential is released in the developing rodent nervous system through a point mutation. Amplification and overexpression of neu are thought to contribute to malignancy of certain human adenocarcinomas. The search for soluble factors that interact with the Neu receptor led to the discovery of a 44 kDa glycoprotein that induces phenotypic differentiation of cultured mammary tumor cells to growtharrested and milkproducing cells. The Neu differentiation factor (NDF or heregulin), however, also acts as a mitogen for epithelial, Schwann and glial cells. Multiple forms of the factor are produced by alternative splicing and their expression is confined predominantly to the central and to the peripheral nervous systems. One identified neuronal function of this family of polypeptides is to control the formation of neuromuscular junctions, but their physiological role in secretory epithelia is still unknown. Other open questions relate to the transmembrane topology of various precursors, the identity of a putative coreceptor, the possible existence of additional ligands of Neu and the functional significance of the interaction between Neu and at least three highly related receptor tyrosine kinases.

Previous studies in vivo and in vitro show that KIT kinase promotes normal melanocyte development and growth. However, the role of the KIT proto-oncogene in neoplastic melanocytes is not certain. We therefore examined KIT expression and function in human melanomas. Our results show that KIT mRNA was expressed in 12 of 28 melanoma cell lines (∼40%), mainly in those originating from pigmented tumors. Surprisingly, activation of KIT with mast cell growth factor (MGF) in melanoma cells produced biological responses opposite to those elicited in normal melanocytes. MGF inhibited rather than stimulated the growth of metastatic melanoma cell lines. The opposite effects may be due to aberrant signal transduction by KIT in melanoma cells in response to MGF. The in vitro inhibition of melanoma cells by MGF suggests that growth in vivo of this tumor is not promoted by KIT kinase activation, but rather that transformed melanocytes might regress when MGF is expressed in their immediate environment.

THE PURPOSE OF this study was to determine if the expression of receptor tyrosine kinases would distinguish astrocytosis from astrocytoma. Because the expression of this family of receptor proteins is greater in higher-grade tumors, a graded series of both reactive and neoplastic astrocytic lesions in humans was evaluated. The reactive processes included both mild gliosis and severe (intense) gliosis. The two immunocytochemically detected membrane receptor proteins, p145 and p185, are those encoded by the kit and neu genes, respectively. Semiquantitative assessments were made independently for the frequency and intensity of astrocytic immunostaining together with corollary immunocytochemical staining to detect glial fibrillary acidic protein. It was found that both mild gliosis and low-grade astrocytomas only minimally express these receptors. In contrast, severe gliosis and high-grade neoplasms consistently express these receptors at much higher levels. In both astrocytosis and astrocytomas, a cell with abundant perikaryal cytoplasm is most likely to be immunoreactive, often with dense reaction product concentrated at the periphery of the somal cytoplasm. The extent and pattern of immunoreactivity in high-grade astrocytomas preclude the interpretation that all immunoreactive cells were merely reactive astrocytes. We conclude that the expression of these receptors per se does not differentiate astrocytic neoplasia from reactive astrocytosis. Second, we conclude that immunocytochemically detectable levels of neu or kit receptor expression is not constitutive in normal astrocytes and in some stages of astrocytic neoplasia. On the basis of these findings, we speculate that some neoplastic astrocytes maintain and manifest the capacity to respond to transitory exogenous stimuli, as do reactive astrocytes. This reaction could include the elaboration of receptor tyrosine kinases. Alternatively, even if the function of these receptors in gliosis differs from that in gliomas, the common expression could still reflect an \u201cactive\u201d state shared by astrocytes in these two processes.

The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.

The Neu/HER-2 receptor tyrosine kinase is overexpressed in some types of human adenocarcinomas, including tumors of the breast and the ovary. A 44 kDa glycoprotein that elevates tyrosine phosphorylation of Neu has been isolated and named Neu differentiation factor (NDF), or heregulin. Here we show that NDF affects tyrosine phosphorylation of Neu in human tumor cells of breast, colon and neuronal origin, but not in ovarian cells that overexpress the receptor. By using monoclonal antibodies (mAbs) to Neu, we found that the ovarian receptor is immunologically and biochemically similar to the mammary p185neu. Nevertheless, unlike breast-derived Neu, the ovarian protein did not display covalent cross-linking to radiolabeled NDF, and was devoid of ligand-induced association with phosphatidylinositol 3'-kinase. Direct binding analysis showed that NDF binds with high affinity (K(d) approximately 10(-9) M) to mammary cells, but its weak association with ovarian cells is probably mediated by heparin-like molecules. Similar to the endogenous receptor, the ectopically overexpressed Neu of mammary cells, but not of ovarian and fibroblastic cells, exhibited elevated levels of NDF-induced phosphorylation and covalent cross-linking of the radiolabeled factor. Taken together, our results imply that NDF binding to cells requires both Neu and an additional cellular component, whose identity is still unknown, but its tissue distribution is more restricted than the expression of the neu gene.