Extensive conformational and physical plasticity protects HER2-HER3 tumorigenic signaling.

Surface-targeting biotherapeutic agents have been successful in treating HER2-amplified cancers through immunostimulation or chemodelivery but have failed to produce effective inhibitors of constitutive HER2-HER3 signaling. We report an extensive structure-function analysis of this tumor driver, revealing complete uncoupling of intracellular signaling and tumorigenic function from regulation or constraints from their extracellular domains (ECDs). The canonical HER3 ECD conformational changes and exposure of the dimerization interface are nonessential, and the entire ECDs of HER2 and HER3 are redundant for tumorigenic signaling. Restricting the proximation of partner ECDs with bulk and steric clash through extremely disruptive receptor engineering leaves tumorigenic signaling unperturbed. This is likely due to considerable conformational flexibilities across the span of these receptor molecules and substantial undulations in the plane of the plasma membrane, none of which had been foreseen as impediments to targeting strategies. The massive overexpression of HER2 functionally and physically uncouples intracellular signaling from extracellular constraints.

Antibody drug conjugates for patients with breast cancer.

The receptor-based classification of breast cancer predicts its optimal therapy. Hormone Receptor (HR) positive breast cancer is treated with endocrine therapy, and HER2+ disease is treated with HER2-targeted therapy. Triple negative breast cancer (TNBC), defined as tumors lacking HR and HER2, represents an aggressive subtype of breast cancer associated with poor prognosis. Development of targeted therapy for this subtype has been challenging since TNBC usually lacks targetable genomic alterations. However, the advent of antibody drug conjugates (ADC) to target antigens overexpressed in breast cancer has opened the door to a new class of breast cancer therapeutics. In this review, we describe the current FDA-approved ADC therapies for breast cancer, including sacituzumab govitecan, as well as agents currently in advanced stages of investigation. In addition, we review the potential therapeutic application of ADCs across different breast cancer subtypes. In the future, therapeutic advances in ADCs targeting different antigens could redefine the current receptor-based classification of breast cancer.

The effects of trastuzumab on HER2-mediated cell signaling in CHO cells expressing human HER2.

BACKGROUND: Targeted therapy with trastuzumab has become a mainstay for HER2-positive breast cancer without a clear understanding of the mechanism of its action. While many mechanisms have been suggested for the action of trastuzumab, most of them are not substantiated by experimental data. It has been suggested that trastuzumab functions by inhibiting intracellular signaling initiated by HER2, however, the data are very controversial. A major issue is the different cellular background of various breast cancer cells lines used in these studies. Each breast cancer cell line has a unique expression profile of various HER receptors, which could significantly affect the effects of trastuzumab. METHODS: To overcome this problem, in this research we adopted a cell model that allow us to specifically examine the effects of trastuzumab on a single HER receptor without the influence of other HER receptors. Three CHO cell lines stably expressing only human EGFR (CHO-EGFR), HER2 (CHO-K6), or HER3 (CHO-HER3) were used. Various methods including cytotoxicity assay, immunoblotting, indirect immunofluorescence, cross linking, and antibody-dependent cellular cytotoxicity (ADCC) were employed in this research. RESULTS: We showed that trastuzumab did not bind EGFR and HER3, and thus did not affect the homodimerization and phosphorylation of EGFR and HER3. However, overexpression of HER2 in CHO cells, in the absence of other HER receptors, resulted in the homodimerization of HER2 and the phosphorylation of HER2 at all major pY residues. Trastuzumab bound to HER2 specifically and with high affinity. Trastuzumab inhibited neither the homodimerization of HER2, nor the phosphorylation of HER2 at most phosphotyrosine residues. Moreover, trastuzumab did not inhibit the phosphorylation of ERK and AKT in CHO-K6 cells, and did not inhibit the proliferation of CHO-K6 cells. However, trastuzumab induced strong ADCC in CHO-K6 cells. CONCLUSION: We concluded that, in the absence of other HER receptors, trastuzumab exerts its antitumor activity through the induction of ADCC, rather than the inhibition of HER2-homodimerization and phosphorylation.

Gonadotropin-Dependent Neuregulin-1 Signaling Regulates Female Rat Ovarian Granulosa Cell Survival.

Mammalian ovarian follicular development and maturation of an oocyte competent to be fertilized and develop into an embryo depends on tightly regulated, spatiotemporally orchestrated crosstalk among cell death, survival, and differentiation signals through extra- and intraovarian signals, as well as on a permissive ovarian follicular microenvironment. Neuregulin-1 (NRG1) is a member of the epidermal growth factor-like factor family that mediates its effects by binding to a member of the erythroblastoma (ErbB) family. Our experimental results suggest gonadotropins promote differential expression of NRG1 and erbB receptors in granulosa cells (GCs), and NRG1 in theca cells during follicular development, and promote NRG1 secretions in the follicular fluid (FF) of rat ovaries. During the estrous cycle of rat, NRG1 and erbB receptors are differentially expressed in GCs and correlate positively with serum gonadotropins and steroid hormones. Moreover, in vitro experimental studies suggest that the protein kinase C inhibitor staurosporine (STS) causes the physical destruction of GCs by the activation of caspase-3. Exogenous NRG1 treatment of GCs delayed onset of STS-induced apoptosis and inhibited cleaved caspase-3 expressions. Moreover, exogenous NRG1 treatment of GCs alters STS-induced death by maintaining the expression of ErbB2, ErbB3, pAkt, Bcl2, and BclxL proteins. Taken together, these studies demonstrate that NRG1 is gonadotropin dependent, differentially regulated in GCs and theca cells, and secreted in ovarian FF as an intracellular survival factor that may govern follicular maturation.

Neuregulin improves response to glucose tolerance test in control and diabetic rats.

Neuregulin (NRG) is an EGF-related growth factor that binds to the tyrosine kinase receptors ErbB3 and ErbB4, thus inducing tissue development and muscle glucose utilization during contraction. Here, we analyzed whether NRG has systemic effects regulating glycemia in control and type 2 diabetic rats. To this end, recombinant NRG (rNRG) was injected into Zucker diabetic fatty (ZDF) rats and their respective lean littermates 15 min before a glucose tolerance test (GTT) was performed. rNRG enhanced glucose tolerance without promoting the activation of the insulin receptor (IR) or insulin receptor substrates (IRS) in muscle and liver. However, in control rats, rNRG induced the phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3) in liver but not in muscle. In liver, rNRG increased ErbB3 tyrosine phosphorylation and its binding to phosphatidylinositol 3-kinase (PI3K), thus indicating that rNRG activates the ErbB3/PI3K/PKB signaling pathway. rNRG increased glycogen content in liver but not in muscle. rNRG also increased the content of fructose-2,6-bisphosphate (Fru-2,6-P2), an activator of hepatic glycolysis, and lactate in liver but not in muscle. Increases in lactate were abrogated by wortmannin, a PI3K inhibitor, in incubated hepatocytes. The liver of ZDF rats showed a reduced content of ErbB3 receptors, entailing a minor stimulation of the rNRG-induced PKB/GSK-3 cascade and resulting in unaltered hepatic glycogen content. Nonetheless, rNRG increased hepatic Fru-2,6-P2 and augmented lactate both in liver and in plasma of diabetic rats. As a whole, rNRG improved response to the GTT in both control and diabetic rats by enhancing hepatic glucose utilization.

ErbB receptors and their growth factor ligands in pediatric intestinal inflammation.

The ErbB tyrosine kinases (epidermal growth factor receptor (EGFR), ErbB2/HER2, ErbB3, and ErbB4) are cell surface growth factor receptors widely expressed in many developing mammalian tissues, including in the intestinal tract. Signaling elicited by these receptors promotes epithelial cell growth and survival, and ErbB ligands have been proposed as therapeutic agents for intestinal diseases of pediatric populations, including inflammatory bowel disease (IBD), necrotizing enterocolitis (NEC), and inflammation associated with total parenteral nutrition (TPN). Furthermore, emerging evidence points to reduced ErbB ligand expression and thus reduced ErbB activity in IBD, NEC, and TPN models. This review will discuss the current understanding of the role of ErbB receptors in the pathogenesis and potential treatment of pediatric intestinal inflammation, with focus on the altered signaling in disease and the molecular mechanisms by which exogenous ligands are protective.

Targeting receptor tyrosine kinases in HER2-negative breast cancer.

PURPOSE OF REVIEW: The targeting of receptor tyrosine kinases (RTKs) has been a major area for breast cancer therapy, exemplified by the targeting of HER2-amplified breast cancer. RECENT FINDINGS: We review the data on the activation of RTKs in HER2-negative breast cancer, and discuss the clinical translational challenge of identifying cancers that are reliant on a specific kinase for growth and survival. Substantial evidence suggests that subsets of breast cancer may be reliant on specific kinases, and that this could be exploited therapeutically. The heterogeneity of breast cancer, however, and the potential for adaptive switching between RTKs after inhibition of a single RTK, present challenges to targeting individual RTKs in the clinic SUMMARY: Targeting of RTKs in HER2-negative breast cancer presents a major therapeutic opportunity in breast cancer, although robust selection strategies will be required to identify cancers with activation of specific RTKs if this potential is to be realized.

Targeting HER proteins in cancer therapy and the role of the non-target HER3.

Members of the human epidermal growth factor receptor (HER) family have been of considerable interest in the cancer arena due to their potential to induce tumorigenesis when their signalling functions are deregulated. The constitutive activation of these proteins is seen in a number of different common cancer subtypes, and in particular EGFR and HER2 have become highly pursued targets for anti-cancer drug development. Clinical studies in a number of different cancers known to be driven by EGFR or HER2 show mixed results, and further mechanistic understanding of drug sensitivity and resistance is needed to realise the full potential of this treatment modality. Signalling in trans is a key feature of HER family signalling, and the activation of the PI3K/Akt pathway, so critically important in tumorigenesis, is driven predominantly through phosphorylation in trans of the kinase inactive member HER3. An increasing body of evidence shows that HER3 plays a critical role in EGFR- and HER2-driven tumours. In particular, HER3 lies upstream of a critically important tumorigenic signalling pathway with extensive ability for feedback and cross-talk signalling, and targeting approaches that fail to account for this important trans-target of EGFR and HER2 can be undermined by its resiliency and resourcefulness. Since HER3 is kinase inactive, it is not a direct target of kinase inhibitors and not presently an easily drugable target. This review presents the current evidence highlighting the role of HER3 in tumorigenesis and its role in mediating resistance to inhibitors of EGFR and HER2.

Inactivation of Akt by the epidermal growth factor receptor inhibitor erlotinib is mediated by HER-3 in pancreatic and colorectal tumor cell lines and contributes to erlotinib sensitivity.

Signaling through the receptor for epidermal growth factor receptor (EGFR) is frequently deregulated in solid tumors. Erlotinib (Tarceva, OSI-774, OSI Pharmaceuticals, Inc., Melville, NY) is a low molecular weight, orally bioavailable inhibitor of the EGFR that has been approved for both non-small cell lung cancer and pancreatic cancers. Previous studies have indicated that sensitivity to EGFR antagonists correlated with HER-3 signaling for non-small cell lung cancer. Herein, we have sought to understand the signaling pathways that mediate erlotinib sensitivity for pancreatic and colorectal cancers. In a panel of 12 pancreatic tumor cell lines, we find that EGFR is coexpressed with HER-3 in all cell lines sensitive to erlotinib but not in insensitive cell lines. Erlotinib can block HER-3 phosphorylation in these sensitive cell lines, suggesting that HER-3 is transactivated by EGFR. Knockdown of HER-3 in BxPC3, an erlotinib-sensitive pancreatic tumor cell line, results in inhibition of the phosphorylation for both Akt and S6 and is associated with a decrease in cell proliferation and reduced sensitivity to erlotinib. Therefore, EGFR transactivation of HER-3 mediates Akt signaling and can contribute to erlotinib sensitivity for pancreatic tumors. We extended our analysis to a panel of 13 colorectal tumor cell lines and find that, like pancreatic, HER-3 is coexpressed with EGFR in the most erlotinib-sensitive cell lines but not in erlotinib-insensitive cell lines. These studies suggest that HER-3 could be used as a biomarker to select patients who are most likely to respond to erlotinib therapy.

HER2 expression as a potential marker for response to therapy targeted to the EGFR.

Since human epidermal growth factor receptor 2 (HER2) is known to participate with the epidermal growth factor receptor (EGFR) in mitogenic signalling, we hypothesised that HER2 overexpression might indicate responsiveness to EGFR targeted therapies. MCF7 breast cancer cells transfected with the HER2 gene were subcloned to establish a set of genetically related cell lines expressing graded levels of HER2 by immunoblot analysis. The subcloned cell lines and parental MCF7 cells were characterised by their growth characteristics, and cell by cell patterns of EGFR, HER2 and HER3 expression as well as levels of phosphorylated mitogen-activated protein kinase (MAPK) and AKT by laser scanning cytometry (LSC). Growth inhibition assays were used to characterise response to EGFR targeted therapy, and to determine the relationship between therapeutic response and levels of tyrosine kinase expression. The levels of growth inhibition of AG1478 and of the AG1478-trastuzumab combinations were correlated with levels of HER2 expression among the different cell lines. Among EGFR, HER2 and HER3, HER2 overexpression was the best single predictive marker, but combinations of two markers provided additional predictive information.

Antagonists of growth hormone releasing hormone and bombesin inhibit the expression of EGF/HER receptor family in H-69 small cell lung carcinoma.

Effects of in vivo treatment with antagonists of growth hormone-releasing hormone (GHRH), JV-1-65 and MZ-J-7-110, and bombesin/gastrin-releasing peptide antagonist RC-3940-II, on the EGF receptor (EGFR) family, were investigated in H-69 SCLC. Tumors were analyzed by RT-PCR, immunoblotting and binding assays. Treatment with these analogs reduced the binding capacity of EGFR by 18-64%, and inhibited the mRNA expression for EGFR, HER-2 and -3 by 27-75.4, 17-26.3, and 13.8-46.6%, respectively. The antagonists also decreased the protein levels for EGFR by 21-34%, HER-2 by 36-68% and HER-3 by 43-49%. This is the first demonstration that antiproliferative effects of GHRH antagonists are associated with a downregulation of EGF/HER receptors.

Forskolin increases neuregulin receptors in human Schwann cells without increasing receptor mRNA.

Forskolin and heregulin synergistically drive human Schwann cell (HSC) proliferation in vitro, but the role of forskolin is not completely understood. To learn how forskolin might affect receptor levels in HSC cultured from adult nerve roots, we first studied expression and localization of HER2 and HER3 in intact roots, using Western blotting and light and electron microscopic immunocytochemistry. We then determined the effect of forskolin and heregulin on receptor expression in HSC cultured from nerve roots using Western blotting and RNase protection assays. HER2 and HER3 were expressed in nonmyelinating Schwann cells in roots and in cultured HSCs before exposure to forskolin. HER2, but not HER3, was also expressed in endoneurial fibroblasts and in cultured nerve root-derived fibroblasts. Treatment with forskolin for 24 h consistently increased HER2 and HER3 protein levels in HSCs but did not alter HER2 and HER3 mRNA levels. In addition, 24-h treatment with heregulin alone decreased HER2 and HER3 protein levels, an effect not previously described. When both heregulin and forskolin were present, HER2 and HER3 protein levels were similar to initial control values. The effect of forskolin on receptor levels was mimicked by dibutyryl-cAMP and receptor levels in both untreated and forskolin treated HSCs were decreased by treatment with the protein kinase A inhibitor H-89. Following pretreatment of HSCs with forskolin, increased receptor levels were correlated with increased rates of thymidine incorporation into HSCs. These results suggest that forskolin/heregulin synergy might derive, at least in part, from post-transcriptional effects leading to increased steady-state receptor levels.

Signal events: Cell signal transduction and its inhibition in cancer.

Signal transduction refers to communication processes used by regulatory molecules to mediate the essential cell processes of growth, differentiation, and survival. Signal transduction elements interact through complex biochemically related networks. Aberrations in signal transduction elements can lead to increased proliferative potential, sustained angiogenesis, tissue invasion and metastasis, and apoptosis inhibition. Most human neoplasms have aberrant signal transduction elements. Several compounds that target aberrant signal transduction elements, such as those in the ErbB family of tyrosine kinase receptors and mammalian target of rapamycin, are in development. To date, commercially available signal-transduction-targeting compounds include trastuzumab, a monoclonal antibody against the ErbB-2 receptor for the treatment of metastatic breast cancer overexpressing the ErbB-2 (HER-2) receptor, and gefitinib, an inhibitor of the ErbB-1 receptor tyrosine kinase that recently received regulatory approval for the treatment of patients with non-small cell lung cancer. In contrast to traditional cytotoxic treatments, although signal transduction inhibitors are capable of inducing tumor regression, particularly in malignancies that are principally driven by specific target aberrations, preclinical and early clinical investigations suggest that their predominant beneficial effects are growth inhibitory in nature; therefore, new clinical trial designs and evaluation end points may be required to ultimately assess their value. Prospective profiling of patients and tumors to determine treatment response is also essential to the success of these clinical trials. However, responsiveness to these novel therapies is dependent on a multitude of factors that ultimately determine the robustness and quality of the downstream response.

alpha-Tocopheryloxybutyric acid enhances necrotic cell death in breast cancer cells treated with chemotherapy agent.

The overexpression of HER-2 receptor contributes to malignant transformation of breast cancer cells. We have reported that alpha-tocopheryloxybutyric acid (TE), non-antioxidative vitamin E ether derivative inhibits the activation of HER-2 receptor. The present study was undertaken to estimate if TE could act as a useful anti-cancer agent against a breast cancer cell overexpressing HER-2 receptor (MDA-MB-453 cell line) in combination with a conventional chemotherapy agent, adriamycin (ADR). TE enhanced cytotoxic effect of ADR against the human breast cancer cell at low doses less than IC(50). The enhancing effect was mainly dependent on the elevation of necrotic-like cell death but not apoptotic cell death. In conjugation with this event, the inactivation of HER-2 receptor in the breast cancer cell was caused by the combination of TE with ADR. These results suggest that TE enhances necrotic-like cell death in the breast cancer cells and that the cell death relates to the inactivation of HER-2 receptor in the breast cancer cells.

Constitutive activation of the neuregulin-1/ErbB receptor signaling pathway is essential for the proliferation of a neoplastic Schwann cell line.

Neuregulin-1 (NRG-1) proteins promote Schwann cell survival, differentiation and proliferation during development. High levels of an NRG-like activity are also present in some human peripheral nerve sheath tumors, suggesting that NRG-1 isoforms may be involved in the development of these neoplasms. We examined the expression of NRG-1 and its receptors, the erbB membrane tyrosine kinases, in JS1 cells, a rapidly proliferating line derived from a chemically induced rat malignant peripheral nerve sheath tumor (MPNST). Relative to nontransformed Schwann cells, JS1 cells overexpress the NRG-1 receptor erbB3 and its erbB2 coreceptor; JS1 erbB2 transcripts show no evidence of the activating mutation commonly found in N-ethyl-N-nitrosourea-induced neoplasms. JS1 cells do not express the epidermal growth factor receptor (EGFR), a kinase implicated in the pathogenesis of a major subset of MPNSTs. JS1 cells also express mRNAs encoding multiple alpha and beta isoforms from the glial growth factor and sensory and motor neuron-derived factor NRG-1 subfamilies. Stimulation with NRG-1beta in the presence of forskolin produces a dose-dependent increase in JS1 DNA synthesis. Even in unstimulated JS1 cells, however, erbB2 and erbB3 are constitutively tyrosine phosphorylated. Reducing this constitutive phosphorylation with the specific erbB inhibitor PD158780 markedly impairs JS1 DNA synthesis. These observations support the hypothesis that NRG-1 isoforms and erbB kinases act in an autocrine and/or paracrine fashion to promote mitogenesis in JS1 cells. The absence of EGFR expression in JS1 cells suggests that constitutive activation of the NRG-1/erbB signaling pathway is an alternative means of inducing Schwann cell neoplasia.

Atypical expression of ErbB3 in myeloma cells: cross-talk between ErbB3 and the interferon-alpha signaling complex.

We have previously demonstrated that the responsiveness of multiple myeloma (MM) cells to interferon-alpha (IFN-alpha) stimulation is variable, with an atypical growth response displayed by some cells. Here we report the ability of IFN-alpha to induce tyrosine phosphorylation of a 180 kDa band in the KAS-6/1 MM cell line, which is growth responsive to IFN-alpha. Further characterization demonstrated that this band corresponds to ErbB3. To our knowledge, this is the first report of ErbB3 expression in a cell type of the hematopoietic lineage. Although ErbB receptors have been shown to crosscommunicate with various other receptors, our results show for the first time that the IFN-alpha receptor can crosscommunicate with ErbB3. To address the significance of these observations, we transfected ErbB3-negative DP-6 MM cells with ErbB3 and used siRNA to silence ErbB3 in the KAS-6/1 cell line. Although IFN-alpha transactivated ErbB3 in the DP-6 transfectants, it did not confer growth responsiveness to IFN-alpha. Interestingly, silencing ErbB3 expression in the KAS-6/1 cells decreased the overall growth response to IFN-alpha and to interleukin-6. These results suggest that ErbB3 expression alone does not uniquely confer IFN-alpha growth responsiveness, but instead may amplify proliferation rates in MM cells that have acquired atypical expression of this receptor.

Gab2 tyrosine phosphorylation by a pleckstrin homology domain-independent mechanism: role in epidermal growth factor-induced mitogenesis.

In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (DeltaPHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs): simple drugs with a complex mechanism of action?

A range of target-based agents for the treatment of solid tumors are in development. The epidermal growth factor receptor (EGFR) has been identified as a relevant target as it is involved in regulating several cellular functions important in the proliferation and survival of cancer cells, is commonly expressed at high levels in a range of tumors, and high expression is often related to poor prognosis. EGFR is a member of the ErbB family of receptors which also includes ErbB-2, ErbB-3, and ErbB-4. These receptors form dimers of the same type (homodimers) or with other family members (heterodimers), each combination resulting in different downstream effects. Some of the most advanced targeted agents in development are the EGFR tyrosine kinase inhibitors (EGFR-TKIs), of which ZD1839 ('Iressa') is an example. In Phase II monotherapy trials, oral ZD1839 was well tolerated and demonstrated clinically meaningful antitumor activity and symptom relief in pretreated patients with advanced NSCLC. Preclinical studies have suggested that the antitumor activity of ZD1839 does not depend on the level of EGFR expression. Furthermore, in addition to an effect on EGFR signaling, treatment with ZD1839 as well as with other quinazoline EGFR-TKIs, may also affect signaling of other ErbB family members. EGFR-TKIs have been shown in preclinical studies to increase the efficacy of cytotoxic drugs and Phase III trials of such combinations are ongoing. On the basis that different signal transduction pathways contribute to the control of tumor growth, future therapeutic approaches are likely to involve combination of different targeted agents.