Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.     

NRG1 / ERBB3 signaling in melanocyte development and melanoma: inhibition of differentiation and promotion of proliferation

Neuregulin (NRG) signaling through the receptor tyrosine kinase, ERBB3, is required for embryonic development, and dysregulated signaling has been associated with cancer progression. Here, we show that NRG1/ERBB3 signaling inhibits melanocyte (MC) maturation and promotes undifferentiated, migratory and proliferative cellular characteristics. Embryonic analyses demonstrated that initial MC specification and distribution were not dependent on ERBB3 signaling. However NRG1/ERBB3 signaling was both necessary and sufficient to inhibit differentiation of later stages of MC development in culture. Analysis of tissue arrays of human melanoma samples suggests that ERBB3 signaling may also contribute to metastatic progression of melanoma as ERBB3 was phosphorylated in primary tumors compared with nevi or metastatic lesions. Neuregulin 1‐treated MCs demonstrated increased proliferation and invasion and altered morphology concomitant with decreased levels of differentiation genes, increased levels of proliferation genes and altered levels of melanoma progression and metastases genes. ERBB3 activation in primary melanomas suggests that NRG1/ERBB3 signaling may contribute to the progression of melanoma from benign nevi to malignancies. We propose that targeting ERBB3 activation and downstream genes identified in this study may provide novel therapeutic interventions for malignant melanoma.     

Geldanamycin selectively targets the nascent form of ERBB3 for degradation

Heat shock protein 90 (HSP90) targets a broad spectrum of client proteins with divergent modes of interaction and consequences. The homologous epidermal growth factor receptor (EGFR) and ERBB2 receptors as well as kinase-deficient mutants thereof differ in their requirement for HSP90 in the nascent versus mature state of the receptor. Specific features of the kinase domain have been implicated for the selective association of HSP90 with mature ERBB2. We evaluated the role of HSP90 for the homologous ERBB3 receptor. ERBB3 is naturally kinase deficient, a central mediator in cell survival and stress response and the primary dimerization partner for ERBB2 in signaling. Cellular studies indicate that, similar to EGFR, the geldanamycin (GA) sensitivity of ERBB3 and HSP90 binding resides in the nascent state and is dependent on the presence of the kinase domain of ERBB3. Furthermore, despite its intrinsic lack of kinase activity and in contrast to the reported GA sensitivity of mature and kinase-deficient EGFR, the GA sensitivity of the nascent state of ERBB3 appears to be exclusive. Geldanamycin disrupts the interaction of ERBB3 and HSP90 and inhibits ERBB3 maturation at an early stage of synthesis, prior to export from the ER. Studies with a photo-convertible fusion protein of ERBB3 suggest geldanamycin sensitivity at a later stage in maturation, possibly through the putative role of HSP90 in structural proofreading.     

Neuregulin1 signaling promotes dendritic spine growth through kalirin

The biological functions of the neuregulin 1 (NRG1) and ERBB4 genes have received much recent attention due to several studies showing associations between these genes and schizophrenia. Moreover, reduced forebrain dendritic spine density is a consistent feature of schizophrenia. It is thus important to understand the mechanisms whereby NRG1 and erbB4 modulate spine morphogenesis. Here, we show that long‐term incubation with NRG1 increases both spine size and density in cortical pyramidal neurons. NRG1 also enhances the content of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors in spines. Knockdown of ERBB4 expression prevented the effects of NRG1 on spine size, but not on spine density. The effects of NRG1 and erbB4 on spines were mediated by the RacGEF kalirin, a well‐characterized regulator of dendritic spines. Finally, we show that environmental enrichment, known to promote spine growth, robustly enhances the levels of erbB4 protein in the forebrain. These findings provide a mechanistic link between NRG1 signaling and spine morphogenesis.

     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffinembedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p<0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

The Oncogene HER2/neu (ERBB2) Requires the Hypoxia-inducible Factor HIF-1 for Mammary Tumor Growth and Anoikis Resistance

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.     

Schizophrenia is not associated with the ERBB3 gene in a Han Chinese population sample: Results from case-control and family-based studies

ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), encoding a receptor of neuregulin-1 (NRG1), has been considered a functional candidate gene for schizophrenia susceptibility. In order to investigate a relationship between ERBB3 gene and schizophrenia in the Chinese population, case-control and family-based studies were carried out in 470 cases matched by controls, and in 532 family trios. Our results failed to show any evidence of significant association between the ERBB3 rs2292238 polymorphism and schizophrenia.     

Allicin suppresses growth and metastasis of gastric carcinoma: the key role of microRNA-383-5p-mediated inhibition of ERBB4 signaling

ABSTRACT

Allicin is a natural product suppressing the progression of gastric carcinoma (GC). In the current study, the mechanism underlying the anti-GC effect of allicin was explored by focusing on the role of miR-383-5p/ERBB4 signaling. Two GC cell lines were treated with allicin and the effects on viability, apoptosis, migration, invasion, and miR-383-5p/ERBB4 activity in the cells were assessed. The interaction between allicin and miR-383-5p was further explored by inhibiting the miR-383-5p level. Allicin suppressed cell viability and induced apoptosis in both GC cell lines. The compound also inhibited migration and invasion of GC cells, which was associated with the up-regulation miR-383-5p and down-regulation of ERBB4. The inhibition of miR-383-5p by specific inhibitor blocked the anti-GC effect of allicin. Our results demonstrated that allicin contributed to the suppressed growth and metastasis potentials in GC cell lines. The effect was accompanied by an increased level of miR-383-5p and subsequent inhibition of ERBB4.     

Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

AKT signaling is modulated by a complex network of regulatory proteins and is commonly deregulated in cancer. Here, we present a dual mechanism of AKT regulation by the ERBB receptor feedback inhibitor 1 (ERRFI1). We show that in cells expressing high levels of EGFR, ERRF1 inhibits growth and enhances responses to chemotherapy. This is mediated in part through the negative regulation of AKT signaling by direct ERRFI1‐dependent inhibition of EGFR. In cells expressing low levels of EGFR, ERRFI1 positively modulates AKT signaling by interfering with the interaction of the inactivating phosphatase PHLPP with AKT, thereby promoting cell growth and chemotherapy desensitization. These observations broaden our understanding of chemotherapy response and have important implications for the selection of targeted therapies in a cell context‐dependent manner. EGFR inhibition can only sensitize EGFR‐high cells for chemotherapy, while AKT inhibition increases chemosensitivity in EGFR‐low cells. By understanding these mechanisms, we can take advantage of the cellular context to individualize antineoplastic therapy. Finally, our data also suggest targeting of EFFRI1 in EGFR‐low cancer as a promising therapeutic approach.     

Reactive oxygen species regulate ERBB2 and ERBB3 expression via miR‐199a/125b and DNA methylation

Overexpression of ERBB2 or ERBB3 is associated with cancer development and poor prognosis. In this study, we show that reactive oxygen species (ROS) induce both ERBB2 and ERBB3 expression in vitro and in vivo. We also identify that miR‐199a and miR‐125b target ERBB2 and/or ERBB3 in ovarian cancer cells, and demonstrate that ROS inhibit miR‐199a and miR‐125b expression through increasing the promoter methylation of the miR‐199a and miR‐125b genes by DNA methyltransferase 1. These findings reveal that ERBB2 and ERBB3 expression is regulated by ROS via miR‐199a and miR‐125b downregulation and DNA hypermethylation.     

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.     

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.     

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long‐term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK‐2206 in combination with paclitaxel and carboplatin. Although single agent MK‐2206 inhibited phospho‐AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK‐2206 with paclitaxel and carboplatin was cytotoxic in long‐term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase‐dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti‐oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.     

HER2+ Cancer Cell Dependence on PI3K vs. MAPK Signaling Axes Is Determined by Expression of EGFR,ERBB3 and CDKN1B

Understanding the molecular pathways by which oncogenes drive cancerous cell growth, and how dependence on such pathways varies between tumors could be highly valuable for the design of anti-cancer treatment strategies. In this work we study how dependence upon the canonical PI3K and MAPK cascades varies across HER2+ cancers, and define biomarkers predictive of pathway dependencies. A panel of 18 HER2+ (ERBB2-amplified) cell lines representing a variety of indications was used to characterize the functional and molecular diversity within this oncogene-defined cancer. PI3K and MAPK-pathway dependencies were quantified by measuring in vitro cell growth responses to combinations of AKT (MK2206) and MEK (GSK1120212; trametinib) inhibitors, in the presence and absence of the ERBB3 ligand heregulin (NRG1). A combination of three protein measurements comprising the receptors EGFR, ERBB3 (HER3), and the cyclin-dependent kinase inhibitor p27 (CDKN1B) was found to accurately predict dependence on PI3K/AKT vs. MAPK/ERK signaling axes. Notably, this multivariate classifier outperformed the more intuitive and clinically employed metrics, such as expression of phospho-AKT and phospho-ERK, and PI3K pathway mutations (PIK3CA, PTEN, and PIK3R1). In both cell lines and primary patient samples, we observed consistent expression patterns of these biomarkers varies by cancer indication, such that ERBB3 and CDKN1B expression are relatively high in breast tumors while EGFR expression is relatively high in other indications. The predictability of the three protein biomarkers for differentiating PI3K/AKT vs. MAPK dependence in HER2+ cancers was confirmed using external datasets (Project Achilles and GDSC), again out-performing clinically used genetic markers. Measurement of this minimal set of three protein biomarkers could thus inform treatment, and predict mechanisms of drug resistance in HER2+ cancers. More generally, our results show a single oncogenic transformation can have differing effects on cell signaling and growth, contingent upon the molecular and cellular context.     

Contribution of PGAP3 co-amplified and co-overexpressed with ERBB2 at 17q12 involved poor prognosis in gastric cancer

The locus at 17q12 erb-b2 receptor tyrosine kinase 2 (ERBB2) has been heavily amplificated and overexpressed in gastric cancer (GC), but it remains to be elucidated about the clinical significance of the co-amplification and co-overexpression of PGAP3 gene located around ERBB2 in GC. The profile of PGAP3 and ERBB2 in four GC cell lines and tissue microarrays containing 418 primary GC tissues was assessed to investigate the co-overexpression and clinical significance of the co-amplified genes, and to evaluate the impact of the co-amplified genes on the malignancy of GC. Co-amplification of PGAP3 and ERBB2 accompanied with co-overexpression was observed in a haploid chromosome 17 of NCI-N87 cells with double minutes (DMs). PGAP3 and ERBB2 were overexpressed and positively correlated in 418 GC patients. Co-overexpression of the PGAP3 and ERBB2 was correlated with T stage, TNM stage, tumour size, intestinal histological type and poor survival proportion in 141 GC patients. In vitro, knockdown of the endogenous PGAP3 or ERBB2 decreased cell proliferation and invasion, increased G1 phase accumulation and induced apoptosis in NCI-N87 cells. Furthermore, combined silencing of PGAP3 and ERBB2 showed an additive effect on resisting proliferation of NCI-N87 cells compared with targeting ERBB2 or PGAP3 alone. Taken together, the co-overexpression of PGAP3 and ERBB2 may be crucial due to its significant correlation with clinicopathological factors of GC. Haploid gain of PGAP3 co-amplified with ERBB2 is sufficient to facilitate the malignancy and progression of GC cells in a synergistic way.     

Presenilin-dependent gamma-secretase processing regulates multiple ERBB4/HER4 activities

Transmembrane receptors typically transmit cellular signals following growth factor stimulation by coupling to and activating downstream signaling cascades. Reports of proteolytic processing of cell surface receptors to release an intracellular domain (ICD) has raised the possibility of novel signaling mechanisms directly mediated by the receptor ICD. The receptor tyrosine kinase ERBB4/HER4 (referred to here as ERBB4) undergoes sequential processing by tumor necrosis factor-alpha converting enzyme and presenilin-dependent gamma-secretase to release the ERBB4 ICD (4ICD). Our recent data suggests that regulation of gene expression by the ERBB4 nuclear protein and the proapoptotic activity of ERBB4 involves the gamma-secretase release of 4ICD. To determine the role gamma-secretase processing plays in ERBB4 signaling, we generated an ERBB4 allele with the transmembrane residue substitution V673I (ERBB4-V673I). We demonstrate that ERBB4-V673I fails to undergo processing by gamma-secretase but retains normal cell surface signaling activity. In contrast to wild-type ERBB4, however, ERBB4-V673I was excluded from the nuclei of transfected cells and failed to activate STAT5A stimulation of the beta-casein promoter. These results support the contention that gamma-secretase processing of ERBB4 is necessary to release a functional 4ICD nuclear protein which directly regulates gene expression. We also demonstrate that 4ICD failed to accumulate within mitochondria of ERBB4-V673I transfected cells and the potent proapoptotic activity of ERBB4 was completely abolished in cells expressing ERBB4-V673I. Our results provide the first formal demonstration that proteolytic processing of ERBB4 is a critical event regulating multiple receptor signaling activities.