Phosphorylation Of ErbB2 Research Articles

Abstract A133: Activating ERBB4 mutations in non-small cell lung cancer

Abstract Genes encoding the ErbB receptor tyrosine kinases (EGFR/ERBB1, ERBB2, ERBB3, and ERBB4) are key regulators of cellular proliferation, survival, and differentiation, and thus represent potent proto-oncogenes. In particular, mutations or copy number variations of EGFR or ERBB2 are present in human malignancies and serve as predictive markers for targeted therapies. Recent efforts to comprehensively characterize the mutational landscape of human cancers have identified frequent somatic mutations in ERBB4 in various cancer types, such as non-small cell lung cancer (NSCLC), melanoma, and colorectal cancer. However, the significance of mutated ERBB4 in cancer remains elusive. Here, we have functionally characterized nine ERBB4 mutations previously identified in lung adenocarcinoma. Four out of the nine mutations, Y285C, D595V, D931Y and K935I, were found to be activating, increasing both basal and ligand-induced ErbB4 phosphorylation. According to structural analysis, the four activating mutations were located at critical positions at the dimerization interfaces of the ErbB4 extracellular (Y285C, D595V) and kinase (D931Y and K935I) domains. Consistently, the mutations enhanced ErbB4 dimerization and increased the trans activation in ErbB4 homodimers and ErbB4/ErbB2 heterodimers. The expression of the activating ERBB4 mutants promoted survival of NIH 3T3 cells in the absence of serum. Interestingly, serum starvation of NIH 3T3 cells expressing the ERBB4 mutants only moderately increased the phosphorylation of canonical ErbB signaling pathway effectors Erk1/2 and Akt as compared to wild-type ERBB4. In contrast, the mutations clearly enhanced the proteolytic release of signaling-competent ErbB4 intracellular domain. These results suggest the presence of activating, oncogenic mutations of ERBB4 in non-small cell lung cancer. Citation Format: Kari J. Kurppa, Konstantin Denessiouk, Mark S. Johnson, Klaus Elenius. Activating ERBB4 mutations in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A133.

HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo.

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.

Neuregulin 1 improves glucose tolerance in adult and old rats

AimStudies both in vitro and ex vivo of rodent skeletal muscle have highlighted the potential involvement of neuregulin 1 (NRG1) in glucose metabolism regulation, yet nothing is known of the role of NRG1 in systemic glucose homoeostasis. For this reason, it was hypothesized that systemic delivery of NRG1 might improve glucose tolerance and that the effect might be age-dependent. MethodsGlucose tolerance tests were performed in 6-month-old (adult) and 22-month-old (old) male Wistar rats 15min after a single injection of either NRG1 (50μg/kg) or saline (controls). Skeletal muscle and liver samples were also collected 30min after the acute NRG1 or saline treatment, while the phosphorylation status of ErbB receptors and AKT was assessed by Western blotting. ResultsAcute NRG1 treatment decreased the glycaemic response to an oral glucose load in both adult and old rats. NRG1 injection did not activate ErbB receptors in skeletal muscle, whereas phosphorylation of ErbB3 and AKT was markedly increased in the liver of NRG1-treated adult and old rats compared with controls. ConclusionThis study shows that NRG1 has a possible glucose-lowering effect in the liver and via an ErbB3/AKT signaling pathway. This NRG1 effect is also maintained in old rats, suggesting that the NRG1/ErbB signaling pathway might represent a promising therapeutic target in insulin resistance states.

ErbB3-ErbB2 Complexes as a Therapeutic Target in a Subset of Wild-type BRAF/NRAS Cutaneous Melanomas.

The treatment options remain limited for patients with melanoma who are wild-type for both BRAF and NRAS (WT/WT). We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand neuregulin-1 (NRG1). In WT/WT melanoma cells displaying high levels of phospho-ErbB3, knockdown of NRG1 reduced cell viability and was associated with decreased phosphorylation of ErbB3, its coreceptor ErbB2, and its downstream target, AKT. Similar effects were observed by targeting ErbB3 with either siRNAs or the neutralizing ErbB3 monoclonal antibodies huHER3-8 and NG33. In addition, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro, and xenograft growth in vivo. Pertuzumab also potentiated the effects of MEK inhibitor on WT/WT melanoma growth in vitro and in vivo. These findings demonstrate that targeting ErbB3-ErbB2 signaling in a cohort of WT/WT melanomas leads to tumor growth reduction. Together, these studies support the rationale to target the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of cutaneous melanomas.

Abstract LB-015: VAMP2-NRG1 fusion gene is a novel oncogenic driver of non-small cell lung adenocarcinoma

Abstract Neuregulin 1 (NRG1) has been discovered as the tail moiety of fusion genes with several distinct partner head genes in lung cancers. These fusion genes activate ERBB2/ERBB3 receptor-mediated cell signaling and thereby function as oncogenic drivers. We have carried out whole-transcriptome sequencing of 100 non-small cell lung carcinoma (NSCLC) tumors and isolated a novel fusion gene consisting of Vesicle-Associated Membrane Protein 2 (VAMP2) and NRG1. RT-PCR and genomic DNA analysis were used to demonstrate inter-chromosomal translocation. Immunoblotting and soft agar assays were used to examine stimulating activity of the fusion gene through ERBB2/ERBB3 signaling pathway. The most highly expressed splice variant of VAMP2-NRG1 fusion gene was shown to be membrane-bound and display EGF-like domain of NRG1 extracellularly. VAMP2-NRG1 promotes anchorage-independent colony formation of H1568 lung adenocarcinoma cells. Ectopic expression of the fusion gene stimulates phosphorylation of ERBB2 and ERBB3 as well as down-stream targets, AKT and ERK, confirming activation of the signaling pathway. VAMP2-NRG1 is a novel oncogenic fusion gene representing a new addition to the list of NRG1 fusion genes which together may form an important diagnostic and clinical category of lung adenocarcinoma cases. Citation Format: Yeonjoo Jung, Seunghui Yong, Pora Kim, Hee-Young Lee, Yeonhwa Jung, Juhee Keum, Suyeon Kim, Sanghyuk Lee, Jhingook Kim, Jaesang Kim. VAMP2-NRG1 fusion gene is a novel oncogenic driver of non-small cell lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-015. doi:10.1158/1538-7445.AM2015-LB-015

Abstract 139: Activating ERBB4 mutations in non-small cell lung cancer

Abstract Genes encoding the ErbB receptor tyrosine kinases (EGFR/ERBB1, ERBB2, ERBB3, and ERBB4) are key regulators of cellular proliferation, survival, and differentiation, and thus represent potent proto-oncogenes. In particular, mutations or copy number variations of EGFR or ERBB2 are present in human malignancies and serve as predictive markers for targeted therapies. Recent efforts to comprehensively characterize the mutational landscape of human cancers have identified frequent somatic mutations in ERBB4 in various cancer types, such as non-small cell lung cancer (NSCLC), melanoma, and colorectal cancer. However, the significance of mutated ERBB4 in cancer remains elusive. Here, we have functionally characterized nine ERBB4 mutations previously identified in lung adenocarcinoma. Four out of the nine mutations, Y285C, D595V, D931Y and K935I, were found to be activating, increasing both basal and ligand-induced ErbB4 phosphorylation. According to structural analysis, the four activating mutations were located at critical positions at the dimerization interfaces of the ErbB4 extracellular (Y285C, D595V) and kinase (D931Y and K935I) domains. Consistently, the mutations enhanced ErbB4 dimerization and increased the trans activation in ErbB4 homodimers and ErbB4/ErbB2 heterodimers. The expression of the activating ERBB4 mutants promoted survival of NIH 3T3 cells in the absence of serum. Interestingly, serum starvation of NIH 3T3 cells expressing the ERBB4 mutants only moderately increased the phosphorylation of canonical ErbB signaling pathway effectors Erk1/2 and Akt as compared to wild-type ERBB4. In contrast, the mutations clearly enhanced the proteolytic release of signaling-competent ErbB4 intracellular domain. These results suggest the presence of activating, oncogenic mutations of ERBB4 in non-small cell lung cancer. Citation Format: Kari J. Kurppa, Konstantin Denessiouk, Mark S. Johnson, Klaus Elenius. Activating ERBB4 mutations in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 139. doi:10.1158/1538-7445.AM2015-139

Neuregulin 1 Improves Glucose Tolerance in db/db Mice.

In vitro experiments using rodent skeletal muscle cells suggest that neuregulin 1 (NRG1) is involved in glucose metabolism regulation, although no study has evaluated the role of NRG1 in systemic glucose homeostasis. The purpose of this study was to investigate the effect of chronic and acute NRG1 treatment on glucose homeostasis in db/db mice. To this aim, glucose tolerance tests were performed in 8-week-old male db/db mice after treatment with NRG1 (50μg.kg-1) or saline 3 times per week for 8 weeks. In other experiments, glucose tolerance and pyruvate tolerance tests were performed in db/db mice 15 minutes after a single NRG1 (50μg.kg-1) or saline injection. Liver, adipose tissue, hypothalamus and skeletal muscle were also collected 30 minutes after acute NRG1 (50μg.kg-1) or saline treatment, and the phosphorylation status of the ERBB receptors, AKT (on Ser473) and FOXO1 (on Ser256) was assessed by western blotting. Chronic treatment (8 weeks) with NRG1 improved glucose tolerance in db/db mice. Acute treatment also lowered glycemia and insulinemia during glucose or pyruvate tolerance tests. NRG1 acute injection induced activation of ERBB3 receptors and phosphorylation of AKT and FOXO1 only in liver. Altogether, this study shows that acute and chronic NRG1 treatments improve glucose tolerance in db/db mice. This effect could be mediated through inhibition of hepatic gluconeogenesis.

Abstract A09: ErbB3/ErbB2 complexes as a therapeutic target in a subset of wild-type BRAF/NRAS cutaneous melanomas

Abstract The treatment options remain poor for melanoma patients who are wild-type for both BRAF and NRAS (WT/WT). ErbB3 regulates melanocyte homeostasis and elevated ErbB3 expression in malignant melanomas correlates with reduced patient survival; however, the effect of selectively targeting ErbB3 in melanoma remains poorly characterized. We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand, neuregulin-1 (NRG1). In phospho-ErbB3-high WT/WT cell lines, knockdown of NRG1 reduced cell viability that was associated with decreased phosphorylation of ErbB3, its co-receptor ErbB2 and its downstream target, AKT. Analogous effects were obtained targeting ErbB3 with either small interfering RNAs or the neutralizing ErbB3 monoclonal antibodies, huHER3-8 and NG33. Additionally, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro and xenograft growth in vivo. Our study demonstrates that targeting ErbB3-ErbB2 signaling in a cohort WT/WT melanomas leads to tumor growth reduction. These findings are supportive of targeting the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of malignant cutaneous melanoma. Citation Format: Claudia Capparelli, Sheera Rosenbaum, Lisa D. Berman-Booty, Nadège Gaborit, Tingting Zhan, Michael A. Davies, Yulius Y. Setiady, Yosef Yarden, Andrew E. Aplin. ErbB3/ErbB2 complexes as a therapeutic target in a subset of wild-type BRAF/NRAS cutaneous melanomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A09.

VAMP2–NRG1 Fusion Gene is a Novel Oncogenic Driver of Non–Small-Cell Lung Adenocarcinoma

Neuregulin 1 (NRG1) has been discovered as the tail moiety of fusion genes with several distinct partner head genes in lung cancers. These fusion genes activate ERBB2/ERBB3 receptor-mediated cell signaling and thereby function as oncogenic drivers. We have carried out whole-transcriptome sequencing of 100 non-small-cell lung carcinoma tumors and isolated a novel fusion gene consisting of vesicle-associated membrane protein 2 (VAMP2) and NRG1. Reverse transcription-polymerase chain reaction and genomic DNA analysis were used to demonstrate interchromosomal translocation. Immunoblotting and soft agar assays were used to examine stimulating activity of the fusion gene through ERBB2/ERBB3 signaling pathway. The most highly expressed splice variant of VAMP2-NRG1 fusion gene was shown to be membrane bound and display EGF-like domain of NRG1 extracellularly. VAMP2-NRG1 promotes anchorage-independent colony formation of H1568 lung adenocarcinoma cells. Ectopic expression of the fusion gene stimulates phosphorylation of ERBB2 and ERBB3 as well as downstream targets, AKT and ERK, confirming activation of the signaling pathway. VAMP2-NRG1 is a novel oncogenic fusion gene representing a new addition to the list of NRG1 fusion genes, which together may form an important diagnostic and clinical category of lung adenocarcinoma cases.

Abstract B37: Mechanisms of acquired resistance to the PI3K inhibitor GDC-0941 in breast cancer cell lines

Abstract Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K is a promising therapeutic target for cancer. There are several PI3K inhibitors in clinical trials and the current study was intended to investigate preclinical mechanisms of acquired resistance to GDC-0941, a class I selective PI3K inhibitor. GDC-0941 resistant pools and clones were generated in both EVSA-T and HCC-1954 breast cancer cell lines by treating cells with increasing concentrations of drug. We found that resistant clones from both lines demonstrated marked increase of downstream PI3K pathway signaling and upregulation of the MAPK pathway, through two different mechanisms. In HCC-1954, autocrine signaling to EGFR was observed in the resistant clones and resulted in increased dependency on the downstream factor c-Myc. EVSA-T clones with acquired resistance to GDC-0941 had elevated HER2 copy number and protein expression levels. A corresponding increase in phosphorylation of HER2 binding partner ErbB3 was also discovered in the EVSA-T resistance cells. In both cases sensitivity to PI3K inhibition was restored by blocking the activated upstream receptor tyrosine kinase, EGFR for HCC-1954 and HER2 for EVSA-T. Additionally both models were re-sensitized to GDC-0941 by blocking downstream signaling of the MAPK pathway. These preclinical data may provide rationale for combination therapy with PI3K inhibitors in the clinic. Citation Format: Kyle A. Edgar, Ling Hwu, Kimberly Walter, Mark Lackner, Lori S. Friedman, Jeffrey J. Wallin. Mechanisms of acquired resistance to the PI3K inhibitor GDC-0941 in breast cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B37.

Abstract A52: Extracellular matrix regulation of ErbB3 in a subset of wild-type BRAF/NRAS melanoma

Abstract While the incidence of melanoma increases each year, treatment options for melanoma patients wild-type for both BRAF and NRAS (WT-WT) remain limited. The tumor microenvironment has been highly implicated in the promotion of tumor growth and proliferation; however, its precise role in melanoma is not well understood. Extracellular matrix (ECM) proteins within the tumor microenvironment have been shown to regulate many cellular pathways, including the activation of several growth factor receptors. The receptor ErbB3 is involved in the regulation of melanocyte homeostasis, and elevated ErbB3 expression in malignant melanomas correlates with reduced patient survival. In our study we demonstrate that, in a subgroup of WT-WT cells, an interaction between melanoma cells and ECM proteins within the tumor microenvironment promotes the activation of the NRG1/ErbB3 pathway, leading to phosphorylation of the downstream target AKT and increased cell growth. Furthermore, knockdown of NRG1 reduced cell viability, which was associated with decreased phosphorylation of ErbB3, its co-receptor ErbB2 and AKT. Analogous effects were obtained targeting ErbB3 with either small interfering RNAs or the neutralizing ErbB3 monoclonal antibodies, huHER-8 and NG33. Additionally, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro and xenograft growth in vivo. These findings are supportive of the crucial role of the ECM in regulating tumor growth through the activation of ErbB3 signaling, and establish a basis for targeting the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of malignant cutaneous melanoma. Citation Format: Sheera Rosenbaum, Claudia Capparelli, Lisa D. Berman-Booty, Nadège Gaborit, Tingting Zhan, Michael A. Davies, Yulius Y. Setiady, Yosef Yarden, Andrew E. Aplin. Extracellular matrix regulation of ErbB3 in a subset of wild-type BRAF/NRAS melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A52.

The dual targeting of EGFR and ErbB2 with the inhibitor Lapatinib corrects high glucose-induced apoptosis and vascular dysfunction by opposing multiple diabetes-induced signaling changes

The epidermal growth factor receptors, EGFR and EGFR2 (ErbB2), appear important mediators of diabetes-induced vascular dysfunction. We investigated whether targeted dual inhibition of EGFR and ErbB2 with Lapatinib would be effective in treating diabetes-induced vascular dysfunction in a rat model of type 1 diabetes. In streptozotocin-induced diabetes, chronic 4-week oral or acute, ex vivo, administration of Lapatinib prevented the development of vascular dysfunction as indicated by the attenuation of the hyper-reactivity of the diabetic mesenteric vascular bed (MVB) to norephinephrine without correcting hyperglycemia. Chronic in vivo or acute ex vivo Lapatinib treatment also significantly attenuated diabetes-induced increases in phosphorylation of EGFR, ErbB2, ERK1/2, AKT, ROCK2 and IkB-alpha as well as normalized the reduced levels of phosphorylated FOXO3A, and eNOS (Ser1177) in the diabetic MVB. Similar results were observed in vascular smooth muscle cells (VSMCs) cultured in high glucose (25 mM) treated with Lapatinib or small interfering RNA (siRNA) targeting the ErbB2 receptor. Lapatinib also prevented high glucose-induced apoptosis in VSMC. Thus, Lapatinib corrects hyperglycemia-induced apoptosis and vascular dysfunction with concomitant reversal of diabetes or high glucose-induced signaling changes in EGFR/ErbB2 and downstream signaling pathways implying that targeted dual inhibition of EGFR/ErbB2 might be an effective vasculoprotective treatment strategy in diabetic patients.

Activating ERBB4 mutations in non-small cell lung cancer.

Recent efforts to comprehensively characterize the mutational landscape of non-small cell lung cancer have identified frequent mutations in the receptor tyrosine kinase ERBB4. However, the significance of mutated ERBB4 in non-small cell lung cancer remains elusive. Here, we have functionally characterized nine ERBB4 mutations previously identified in lung adenocarcinoma. Four out of the nine mutations, Y285C, D595V, D931Y and K935I, were found to be activating, increasing both basal and ligand-induced ErbB4 phosphorylation. According to structural analysis, the four activating mutations were located at critical positions at the dimerization interfaces of the ErbB4 extracellular (Y285C and D595V) and kinase (D931Y and K935I) domains. Consistently, the mutations enhanced ErbB4 dimerization and increased the trans activation in ErbB4 homodimers and ErbB4-ErbB2 heterodimers. The expression of the activating ERBB4 mutants promoted survival of NIH 3T3 cells in the absence of serum. Interestingly, serum starvation of NIH 3T3 cells expressing the ERBB4 mutants only moderately increased the phosphorylation of canonical ErbB signaling pathway effectors Erk1/2 and Akt as compared with wild-type ERBB4. In contrast, the mutations clearly enhanced the proteolytic release of signaling-competent ErbB4 intracellular domain. These results suggest the presence of activating driver mutations of ERBB4 in non-small cell lung cancer.

Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats.

Some studies suggest that neuregulin 1 (NRG1) could be involved in the regulation of skeletal muscle energy metabolism in rodents. Here we assessed whether unbalanced diet is associated with alterations of the NRG1 signalling pathway and whether exercise and diet might restore NRG1 signalling in skeletal muscle of obese rats. We show that diet-induced obesity does not impair NRG1 signalling in rat skeletal muscle. We also report that endurance training and a well-balanced diet activate the NRG1 signalling in skeletal muscle of obese rats, possibly via a new mechanism mediated by the protease ADAM17. These results suggest that some beneficial effects of physical activity and diet in obese rats could be partly explained by stimulation of the NRG1 signalling pathway. Some studies suggest that the signalling pathway of neuregulin 1 (NRG1), a protein involved in the regulation of skeletal muscle metabolism, could be altered by nutritional and exercise interventions. We hypothesized that diet-induced obesity could lead to alterations of the NRG1 signalling pathway and that chronic exercise could improve NRG1 signalling in rat skeletal muscle. To test this hypothesis, male Wistar rats received a high fat/high sucrose (HF/HS) diet for 16weeks. At the end of this period, NRG1 and ErbB expression/activity in skeletal muscle was assessed. The obese rats then continued the HF/HS diet or were switched to a well-balanced diet. Moreover, in both groups, half of the animals also performed low intensity treadmill exercise training. After another 8weeks, NRG1 and ErbB expression/activity in skeletal muscle were tested again. The 16week HF/HS diet induced obesity, but did not significantly affect the NRG1/ErbB signalling pathway in rat skeletal muscle. Conversely, after the switch to a well-balanced diet, NRG1 cleavage ratio and ErbB4 amount were increased. Chronic exercise training also promoted NRG1 cleavage, resulting in increased ErbB4 phosphorylation. This result was associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats.

Docosahexaenoic Acid Downregulates EGF-Induced Urokinase Plasminogen Activator and Matrix Metalloproteinase 9 Expression by Inactivating EGFR/ErbB2 Signaling in SK-BR3 Breast Cancer Cells

Urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) play crucial roles in tumor metastasis. Despite the well-known anticancer role of docosa-hexaenoic acid (DHA), its specific effect on ErbB2-mediated breast cancer metastasis is not fully clarified. In this study, we investigated the effect of DHA on epidermal growth factor (EGF)-induced uPA and MMP-9 activity, expression and cell invasion in SK-BR3 breast cancer cells and the possible mechanisms involved. The results showed that EGF (40 ng/ml) induced uPA and MMP-9 mRNA and protein expression, enzyme activity, and 100 μM DHA significantly inhibited EGF-induced uPA and MMP-9 mRNA, protein expression, enzyme activity, cell migration, and cell invasion. EGF increased protein expression and phosphorylation of EGF receptor (EGFR) and ErbB2 as well as of JNK2, ERK1/2, and Akt, and these changes were attenuated by DHA pretreatment. AG1478, an inhibitor of EGFR, also attenuated EGF-induced activation of EGFR, JNK2, ERK1/2, and Akt. Knocked down ErbB2 expression resulted in a similar inhibition of uPA and MMP-9 expression as noted by DHA and AG1478. Taken together, these results suggest that suppression of EGF-induced metastasis by DHA is likely through an inhibition of EGFR and ErbB2 protein expression and the downstream target uPA and MMP-9 activation in SK-BR3 human breast cancer cells.

Abstract P6-07-04: Targeting ErbB2 with human PEPD

Abstract ErbB2, also known as Her2 or Neu, belongs to the ErbB family of plasma membrane-bound receptor tyrosine kinases, which also include ErbB1, ErbB3 and ErbB4. ErbB2 is best known for its involvement in human breast cancer. ErbB2 gene amplification occurs in ∼20% of breast cancer, and ErbB2 amplification or overexpression is a strong predictor of poor disease prognosis. ErbB2-targeted therapies, particularly humanized monoclonal antibody trastuzumab (Ttzm) in combination with chemotherapy, have shown considerable clinical efficacy. However, primary and secondary resistance remains a clinical challenge, and Ttzm, produced in mammalian cells, is very expensive. We have found that human prolidase, also known as peptidase D (PEPD) among several other names, binds to ErbB2 with high affinity (Kd = ∼7 nM) and binds as a homodimer (493 amino acids per subunit) to subdomain 3 in the extracellular domain of ErbB2. Each monomer of PEPD binds to one copy of ErbB2. However, PEPD is a weak ErbB1 binder (Kd = ∼5 μM) and does not bind to ErbB3 or ErbB4. PEPD is the first-ever natural ligand of ErbB2, and unlike the other ligands of ErbB receptors, it is devoid of an EGF motif. PEPD has been long known to hydrolyze dipeptides with proline or hydroxylproline at the carboxy terminus, but the dipeptidase activity of PEPD is not involved in ErbB2/ErbB1 modulation. In cells overexpressing ErbB2, where both activated dimers and inactive monomers of ErbB2 exist, as ErbB2 overexpression causes spontaneous dimerization, auto-tyrosine phosphorylation and recruitment and activation of downstream signals, PEPD rapidly binds to ErbB2 homodimers (<∼10 min) and silences the existing ErbB2-SRC signaling, a key oncogenic pathway of ErbB2, by disrupting SRC association with ErbB2. In contrast, PEPD binds to ErbB2 monomers relatively slowly (>∼30 min), but this binding causes ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. PEPD binding to ErbB2 subsequently causes pronounced ErbB2 depletion, resulting from its internalization and degradation. PEPD also strongly inhibits the DNA synthesis, anchorage-independent growth and invasion of cells that overexpress ErbB2, but has no effect on cells without overexpression of ErbB2. In fact, cells become sensitized to inhibition by PEPD upon achieving stable ErbB2 overexpression. Thus, the overall impact of PEPD on ErbB2 is inhibitory, and PEPD targets cells addicted to ErbB2. In ErbB2-overexpressing cells, at equimolar concentrations, PEPD was more effective than Ttzm in driving ErbB2 depletion, but is weaker than Ttzm in stimulating ErbB2 phosphorylation. In mouse tumor models, PEPD administered by intraperitoneal injection (Monday, Wednesday, Friday) at 0.2-2 mg/kg body weight strongly inhibited the growth of ErbB2-overexpressing tumors, but had no impact on tumors without ErbB2 overexpression, and the PEPD-treated mice showed no adverse effects. Given that the findings described above were made using human PEPD generated in bacteria, there is a distinct possibility that recombinant human PEPD may be a low cost alternative to Ttzm. Further investigation of the antitumor activity of PEPD and its modulation of ErbB2 signaling is warranted. Citation Format: Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. Targeting ErbB2 with human PEPD [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-04.

Validation of an in vitro model of erbB2(+) cancer cell redirection.

Overexpression of the oncoprotein erbB2/HER2 is present in 20-30% of breast cancer patients and inversely correlates with patient survival. Reports have demonstrated the deterministic power of the mammary microenvironment where the normal mammary microenvironment redirects cells of non-mammary origin or tumor-derived cells to adopt a mammary phenotype in an in vivo model. This phenomenon is termed tumor cell redirection. Tumor-derived cells that overexpress the erbB2 oncoprotein lose their tumor-forming capacity in this model. In this model, phosphorylation of erbB2 is attenuated thus reducing the tumor cell's tumor-forming potential. In this report, we describe our results using an in vitro model based on the in vivo model mentioned previously. Tumor-derived cells are mixed in predetermined ratios with normal mammary epithelial cells prior to seeding in vitro. In this in vitro model, the tumor-derived cells are redirected as determined by attenuated phosphorylation of the receptor and reduced sphere and colony formation. These results match those observed in the in vivo model. This in vitro model will allow expanded experimental options in the future to determine additional aspects of tumor cell redirection that can be translated to other types of cancer.

OCAM regulates embryonic spinal cord stem cell proliferation by modulating ErbB2 receptor.

The proliferation and differentiation of neural stem cells are tightly controlled by intrinsic and extrinsic cues. Cell adhesion molecules are increasingly recognized as regulators of these processes. Here we report the expression of the olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) during mouse spinal cord development and in neural stem cells cultured as neurospheres. OCAM is also weakly expressed in the dormant adult stem cell niche around the central canal and is overexpressed after spinal cord injury. Both transmembrane (TM) and glycosylphosphatidylinositol (GPI)-linked isoforms are present in neurospheres. Electron microscopy and internalisation experiments revealed a dynamic trafficking of OCAM between the membrane and intracellular compartments. After differentiation, OCAM remains in neurons and oligodendrocytes whereas no expression is detected in astrocytes. Using OCAM knockout (KO) mice, we found that mutant spinal cord stem cells showed an increased proliferation and self-renewal rates although no effect on differentiation was observed. This effect was reversed by lentivirus-mediated re-introduction of OCAM. Mechanistically, we identified the ErbB2/Neu/HER2 protein as being implicated in the enhanced proliferation of mutant cells. ErbB2 protein expression and phosphorylation level were significantly increased in KO cells whereas no difference was observed at the mRNA level. Overexpression of ErbB2 in wild-type and mutant cells also increased their growth while reintroduction of OCAM in mutant cells reduced the level of phosphorylated ErbB2. These results indicate that OCAM exerts a posttranscriptional control on the ErbB2 signalling in spinal cord stem cells. This study adds further support for considering cell adhesion molecules as regulators of the ErbB signalling.

ERBB3-independent activation of the PI3K pathway in EGFR-mutant lung adenocarcinomas.

ERBB3, a member of the EGFR family of receptor tyrosine kinases, has been implicated in activation of the PI3K pathway in human lung adenocarcinomas driven by EGFR mutations. We investigated the contribution of ERBB3 to the initiation, progression, and therapeutic response of EGFR-induced lung adenocarcinomas using tetracycline- and tamoxifen-inducible transgenic mouse models. Deletion of Erbb3 at the time of induction of mutant EGFR had no effect on tumorigenesis, demonstrating that ERBB3 is not required to initiate tumorigenesis. Tumors that developed in the absence of ERBB3 remained sensitive to EGFR tyrosine kinase inhibitors and retained activation of the PI3K-AKT pathway. Interestingly, acute loss of Erbb3 suppressed further growth of established EGFR(L858R)-mediated lung tumors. Four weeks after deletion of Erbb3, the tumors exhibited phosphorylation of EGFR, of the adaptor proteins GAB1 and GAB2, and of the downstream signaling molecules AKT and ERK, suggesting that alternative signaling pathways could compensate for loss of Erbb3. Similar to our observations with mouse tumors, we found that GAB adaptor proteins play a role in ERBB3-independent activation of the PI3K pathway by mutant EGFR in EGFR-mutant human cell lines. Finally, in such cell lines, increased levels of phosphorylation of ERBB2 or MET were associated with reduced sensitivity to acute loss of ERBB3, suggesting remarkable plasticity in the signaling pathways regulated by mutant EGFR with important therapeutic implications.

Abstract B38: Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition

Abstract Trastuzumab is a humanized recombinant monoclonal antibody directed against extracellular region of ErbB2 and used for the treatment of different ErbB2-positive cancers. Based on literature reports and our clinical data from trastuzumab neoadjuvant studies, increased ErbB2-Y1248 phosphorylation is associated with a better response to trastuzumab in patient samples. Also, in trastuzumab-sensitive cell lines (SKBR3 and BT474), increased basal level and trastuzumab-mediated increase in ErbB2-Y1248 phosphorylation was readily detectable compared to trastuzumab-resistant cell line model (JIMT1). The mechanisms of trastuzumab-induced ErbB2-Y1248 phosphorylation and the role of ErbB2-Y1248 phosphorylation in trastuzumab sensitivity/resistance are still incompletely understood. We demonstrate that trastuzumab activates ErbB2 kinase activity in trastuzumab-sensitive cells, which may further contribute to trastuzumab-mediated ErbB2-Y1248 phosphorylation. However, blocking ErbB1/ErbB2 kinase activity with Lapatinib, a small molecule tyrosine kinase inhibitor only partially reduced trastuzumab-mediated ErbB2-Y1248 phosphorylation. This led to the discovery of a previously unappreciated relationship between trastuzumab and Csk-homologous kinase (CHK), a non-receptor tyrosine kinase also known as MATK. We establish that trastuzumab treatment promotes interaction between ErbB2 and CHK in trastuzumab-sensitive but not in trastuzumab-resistant cells despite similar CHK expression levels in both types of cells. Mimicking trastuzumab, CHK overexpression also induces ErbB2-Y1248 phosphorylation, Akt downregulation and cell growth inhibition and further stimulated trastuzumab-mediated reduction in total ErbB2 levels in trastuzumab sensitive cells. Also, CHK overexpression combined with trastuzumab exerts an additive effect on cell growth inhibition in trastuzumab-sensitive but not in trastuzumab-resistant cells. Understanding of the molecular mechanisms of trastuzumab mediated ErbB2 phosphorylation changes may suggest novel therapeutic approaches in the treatment of trastuzumab resistant cancers. Disclaimer: The information presented in this article reflects the views of the authors and does not represent the policy of the U.S. Food and Drug Administration. Citation Format: Milos Dokmanovic, Yun Wu, Yi Shen, Jieqing Chen, Dianne S. Hirsch, Wen Jin Wu. Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B38.