Tyrosine Phosphorylation Of ErbB2 Research Articles

Abstract A19: Discovery and characterization of selective and nonselective inhibitors of ErbB4 signaling: Putative targeted melanoma therapeutics

Abstract Introduction: Gain-of-function mutations in the gene that encodes the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma samples. Melanoma cell lines that harbor some of these mutations are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent melanomas. Our drug discovery approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist/antagonist at ErbB4. NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and competitively antagonizes agonist stimulation of ErbB4-dependent cell proliferation. ErbB4 partial agonists that function as antagonists at ErbB4 may hold promise as targeted therapeutics for ErbB4-dependent melanomas. Experimental Procedures: Automated, high-throughput phospho-ErbB4 sandwich ELISA and ErbB4-dependent proliferation assays were developed and deployed to identify 19 small-molecule compounds that stimulate ErbB4 tyrosine phosphorylation, yet inhibit ErbB4-dependent cell proliferation. These compounds were prioritized on the basis of their potency, efficacy, and specificity for inhibition of ErbB4-dependent proliferation. Traditional immunoblotting approaches specific to candidate proteins have been deployed to identify targets for hit molecules of interest. Data: The single high-priority candidate (AU-05) inhibits agonist-induced ErbB4-dependent cellular proliferation with an IC50 of 6.8 uM, but inhibits IL3-dependent proliferation with an IC50 of ~2100 uM, suggesting that AU-05 possesses specificity for ErbB4-dependent proliferation. In contrast, AU-39 potently inhibits both ErbB4-dependent cell proliferation with an IC50 of 1.05 nM and IL3-dependent cellular proliferation with an IC50 of 2.61 nM. We have hypothesized that AU-39 targets the PI3K/Akt signaling pathway, which lies downstream of both ErbB4 and the IL3 receptor. We are testing this hypothesis in part by examining the effects of AU-39 on agonist-induced ErbB4 and IL3 receptor coupling to phosphorylation of Akt and other components of the PI3K/Akt pathway. Conclusions: AU-05 is a starting point for the development of specific ErbB4 inhibitors, whereas AU-39 is a candidate inhibitor of the PI3K/Akt signaling pathway. The inhibition of ErbB4-dependent cellular proliferation by these molecules justifies further development of both molecules. Citation Format: Lauren M. Lucas, Richard L. Cullum, Jared I. Senfeld, Laura J. Cook, Mackenzie H. Harris, David Z. Kaufmann, Cristina C. Rael, Darby C. Taylor, John T. Piazza, Logan T. Neel, Ram B. Gupta, Allan E. David, David J. Riese II. Discovery and characterization of selective and nonselective inhibitors of ErbB4 signaling: Putative targeted melanoma therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A19.

The PTN-PTPRZ signal activates the AFAP1L2-dependent PI3K-AKT pathway for oligodendrocyte differentiation: Targeted inactivation of PTPRZ activity in mice.

Protein tyrosine phosphatase receptor type Z (PTPRZ) maintains oligodendrocyte precursor cells (OPCs) in an undifferentiated state. The inhibition of PTPase by its ligand pleiotrophin (PTN) promotes OPC differentiation; however, the substrate molecules of PTPRZ involved in the differentiation have not yet been elucidated in detail. We herein demonstrated that the tyrosine phosphorylation of AFAP1L2, paxillin, ERBB4, GIT1, p190RhoGAP, and NYAP2 was enhanced in OPC-like OL1 cells by a treatment with PTN. AFAP1L2, an adaptor protein involved in the PI3K-AKT pathway, exhibited the strongest response to PTN. PTPRZ dephosphorylated AFAP1L2 at tyrosine residues in vitro and in HEK293T cells. In OL1 cells, the knockdown of AFAP1L2 or application of a PI3K inhibitor suppressed cell differentiation as well as the PTN-induced phosphorylation of AKT and mTOR. We generated a knock-in mouse harboring a catalytically inactive Cys to Ser (CS) mutation in the PTPase domain. The phosphorylation levels of AFAP1L2, AKT, and mTOR were higher, and the expression of oligodendrocyte markers, including myelin basic protein (MBP) and myelin regulatory factor (MYRF), was stronger in CS knock-in brains than in wild-type brains on postnatal day 10; however, these differences mostly disappeared in the adult stage. Adult CS knock-in mice exhibited earlier remyelination after cuprizone-induced demyelination through the accelerated differentiation of OPCs. These phenotypes in CS knock-in mice were similar to those in Ptprz-deficient mice. Therefore, we conclude that the PTN-PTPRZ signal stimulates OPC differentiation partly by enhancing the tyrosine phosphorylation of AFAP1L2 in order to activate the PI3K-AKT pathway.

Protein kinase C mediated internalization of ErbB2 is independent of clathrin, ubiquitination and Hsp90 dissociation

Overexpression of ErbB2 is frequent in cancer and understanding the mechanisms which regulate its expression is important. ErbB2 is considered endocytosis resistant. It has no identified ligand, but upon heterodimerization it is a potent mediator of proliferative signaling. A recent study established a role for protein kinase C (PKC) in internalization and recycling of ErbB2. We have now further investigated the molecular mechanisms involved in PKC-mediated downregulation of ErbB2. We confirm that PMA-induced PKC activation causes ErbB2 internalization, but while the Hsp90 inhibitor 17-AAG induced ErbB2 degradation, PMA had no such effect. When combined with 17-AAG, PMA had additive effect on ErbB2 internalization indicating that Hsp90 inhibition and PKC activation induce internalization by alternative mechanisms. We confirm that while 17-AAG-induced internalization was clathrin-mediated, PMA-induced internalization was clathrin independent. This difference may be explained by while both 17-AAG and PMA reduced the constitutive tyrosine phosphorylation of ErbB2, only 17-AAG induced Hsp90 dissociation, Hsp70 recruitment and ubiquitination of ErbB2. Importantly, since PMA induced internalization of ErbB2, but not dissociation of Hsp90, Hsp90 does not per se retain ErbB2 at the plasma membrane. The morphology of the compartment into which receptors are sorted upon PKC activation has not previously been identified. By immuno-electron microscopy, we show that PMA sorts ErbB2 into a complex tubulovesicular or cisternal organelle resembling a previously described endocytic recycling compartment.

Abstract A159: Characterization of putative ErbB4 antagonists: targeted melanoma drug discovery

Abstract Introduction: Gain-of-function mutations in the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma cell lines that are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent tumors. Our drug discovery approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist/antagonist at ErbB4. NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and competitively antagonizes agonist stimulation of ErbB4-dependent cell proliferation. ErbB4 partial agonists that function as antagonists at ErbB4 may hold promise as targeted therapeutics for ErbB4-dependent melanomas. Experimental Procedures: An automated phospho-ErbB4 sandwich ELISA and automated proliferation assays were developed and deployed to identify small-molecule compounds that are likely to function as ErbB4 partial agonists/antagonists. Hit molecules were then further evaluated for ErbB4 specificity and antiproliferative mechanism(s) of action. To examine specificity for ErbB4, we tested whether the hit molecules inhibit ErbB4-dependent and -independent proliferation of the same cell line. ErbB4 specificity was also evaluated by determining whether the hit molecules exert effects on any of the other three members of the ErbB family of receptor tyrosine kinases: EGFR, ErbB2, and ErbB3. The mechanism(s) of action for antiproliferative effects were investigated by determining whether the hit molecules inhibited known effectors that couple ErbB4 to cellular proliferation, whether they degraded ErbB4, and whether the inhibitory effects induced by the molecules were reversible. Results: High-throughput screening (HTS) strategies have been utilized to identify 20 small-molecule compounds that stimulate ErbB4 tyrosine phosphorylation, fail to stimulate ErbB4-dependent cellular proliferation, and inhibit agonist-induced ErbB4-dependent cellular proliferation. Efforts to determine whether these hits are specific for ErbB4 and by what mechanism they induce antiproliferative effects are under way and will be reported. Structures of these small-molecule ErbB4 partial agonists/antagonists may be reported, pending submission of a provisional patent application. Conclusions: Using validated HTS methodologies for identifying ErbB4 partial agonists that function as ErbB4 antagonists has led to the identification of 20 hits. Molecules that are specific for ErbB4 hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors. Citation Format: Richard L. Cullum, Lauren M. Lucas, John T. Piazza, Jared I. Senfeld, Logan T. Neel, Ram B. Gupta, Allan E. David, David J. Riese. Characterization of putative ErbB4 antagonists: targeted melanoma drug discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A159.

Nectin-like molecule-4/cell adhesion molecule 4 inhibits the ligand-induced dimerization of ErbB3 with ErbB2

The ligand-induced dimerization of cell surface single-transmembrane receptors is essential for their activation. However, physiological molecules that inhibit their dimerization and activation have not been identified. ErbB3 dimerizes with ErbB2 upon binding of heregulin (HRG) to ErbB3, causing the ErbB2-catalyzed tyrosine phosphorylation of ErbB3, which leads to the activation of the signalling pathways for cell movement and survival. Genetic disorders of this receptor cause tumorigenesis and metastasis of cancers. We show here that nectin-like molecule-4/cell adhesion molecule 4, known to serve as a tumour suppressor, interacts with ErbB3 in the absence of HRG and inhibits the HRG-induced dimerization of ErbB3 with ErbB2 and its activation. The third immunoglobulin-like domain of nectin-like molecule-4 cis-interacts with the extracellular domain 3 of ErbB3. We describe here a novel regulatory mechanism for the activation and signalling of cell surface single-transmembrane receptors.

Abstract 1245: Screening methodologies for the discovery of small molecule melanoma therapeutics targeted at the ErbB4 receptor tyrosine kinase

Abstract Introduction: Gain-of-function mutations in the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma cell lines that are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent tumors. Consequently, we have developed high-throughput screening assays to identify small molecule ErbB4 antagonists that may hold promise as targeted melanoma therapeutics. Our approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist at ErbB4. NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and competitively antagonizes agonist stimulation of ErbB4-dependent cell proliferation. Experimental procedures: Therefore, we have developed three high-throughput screening assays to identify ErbB4 partial agonists that function as antagonists. The primary screen identifies molecules that stimulate ErbB4 tyrosine phosphorylation. The secondary screen identifies molecules that stimulate or fail to stimulate ErbB4-dependent cell proliferation. The tertiary screen identifies molecules that antagonize agonist stimulation of ErbB4-dependent cell proliferation. Results: A phospho-ErbB4 sandwich ELISA assay identifies molecules that stimulate ErbB4 tyrosine phosphorylation with high sensitivity and fidelity (Z’ >0.5). IL3-independence assays in conjunction with MTT assays using a cell line that displays ErbB4-dependent cell proliferation distinguish between molecules that stimulate and fail to stimulate ErbB4-dependent proliferation (Z’>0.5) and identify molecules that antagonize agonist stimulation of ErbB4 dependent proliferation. These assays have been used to identify small molecules that stimulate ErbB4 tyrosine phosphorylation. Efforts to determine whether these hits function as ErbB4 full agonists or partial agonists (antagonists) are underway and will be reported. Structures of these small molecule ErbB4 full and partial agonists may be reported, pending submission of a provisional patent application. Conclusions: We have validated an HTS strategy for identifying ErbB4 partial agonists that function as ErbB4 antagonists and deployment of that strategy has led to the identification of several hits (~20 from the primary screen). Such molecules may hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors. Citation Format: Richard L. Cullum, John T. Piazza, Jared I. Senfeld, Logan T. Neel, Ram B. Gupta, Allan E. David, David J. Riese. Screening methodologies for the discovery of small molecule melanoma therapeutics targeted at the ErbB4 receptor tyrosine kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2017-1245

Neuregulin-1 positively modulates glial response and improves neurological recovery following traumatic spinal cord injury.

Spinal cord injury (SCI) results in glial activation and neuroinflammation, which play pivotal roles in the secondary injury mechanisms with both pro- and antiregeneration effects. Presently, little is known about the endogenous molecular mechanisms that regulate glial functions in the injured spinal cord. We previously reported that the expression of neuregulin-1 (Nrg-1) is acutely and chronically declined following traumatic SCI. Here, we investigated the potential ramifications of Nrg-1 dysregulation on glial and immune cell reactivity following SCI. Using complementary in vitro approaches and a clinically-relevant model of severe compressive SCI in rats, we demonstrate that immediate delivery of Nrg-1 (500 ng/day) after injury enhances a neuroprotective phenotype in inflammatory cells associated with increased interleukin-10 and arginase-1 expression. We also found a decrease in proinflammatory factors including IL-1β, TNF-α, matrix metalloproteinases (MMP-2 and 9) and nitric oxide after injury. In addition, Nrg-1 modulates astrogliosis and scar formation by reducing inhibitory chondroitin sulfate proteoglycans after SCI. Mechanistically, Nrg-1 effects on activated glia are mediated through ErbB2 tyrosine phosphorylation in an ErbB2/3 heterodimer complex. Furthermore, Nrg-1 exerts its effects through downregulation of MyD88, a downstream adaptor of Toll-like receptors, and increased phosphorylation of Erk1/2 and STAT3. Nrg-1 treatment with the therapeutic dosage of 1.5 μg/day significantly improves tissue preservation and functional recovery following SCI. Our findings for the first time provide novel insights into the role and mechanisms of Nrg-1 in acute SCI and suggest a positive immunomodulatory role for Nrg-1 that can harness the beneficial properties of activated glia and inflammatory cells in recovery following SCI.

Feedback control of ErbB2 via ERK-mediated phosphorylation of a conserved threonine in the juxtamembrane domain.

Tyrosine kinase activity of the asymmetric EGFR homodimer is negatively regulated via ERK-mediated phosphorylation of Thr-669 in the juxtamembrane domain. In the present study, we investigated in human breast cancer cells whether a similar mechanism plays a role in the feedback regulation of the ErbB2/ErbB3 heterodimer, the most potent ErbB receptor dimer. Constitutive tyrosine phosphorylation of ErbB2 and ErbB3 was significantly decreased in phorbol ester- and growth factor-treated BT-474 and MDA-MB-453 cells. In contrast to the decreased tyrosine phosphorylation, Phos-tag Western blot analysis revealed that TPA induced phosphorylation of ErbB2 in an ERK-dependent manner. The target threonine residue corresponding to EGFR Thr-669 and the surrounding residues are highly conserved in ErbB2, but not in ErbB3. Therefore, we demonstrated ERK-mediated phosphorylation of ErbB2 at Thr-677 by generating phospho-specific monoclonal antibodies. Moreover, treatment with trametinib and SCH772984, inhibitors of the MEK-ERK pathway, and substitution of Thr-677 to alanine impaired the feedback inhibition of ErbB2 and ErbB3. These results demonstrated that ERK-mediated phosphorylation of the conserved threonine is a common mechanism for the negative feedback control of active ErbB receptor dimers.

Abstract 1353: Validation and implementation of screens for partial agonists and antagonists of the ErbB4/HER4 receptor tyrosine kinase: Targeted melanona drug discovery

Abstract Introduction: Gain-of-function mutations in the gene encoding the ErbB4 (HER4) receptor tyrosine kinase have been detected in melanoma cell lines and these cell lines are dependent on ErbB4 expression for proliferation. Consequently, ErbB4 antagonists hold promise as targeted melanoma therapeutics and we have sought to identify small molecule ErbB4 antagonists. Our approach is based on the observation that the Q43L mutant of the ErbB4 peptide growth factor agonist Neuregulin 2beta (NRG2b) functions as a partial agonist at ErbB4; NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4 coupling to cell proliferation, antagonizes stimulation of cell proliferation by ErbB4 full agonists, and causes ErbB4 down-regulation. Experimental procedures: We have developed a high-throughput screening (HTS) strategy that can be used to identify ErbB4 antagonists. The primary screen identifies molecules that stimulate ErbB4 tyrosine phosphorylation. The secondary screen distinguishes between molecules that stimulate and fail to stimulate ErbB4-dependent proliferation. The tertiary screen identifies molecules that antagonize agonist stimulation of ErbB4-dependent proliferation. Results: An phospho-ErbB4 sandwich ELISA assay identifies molecules that stimulate ErbB4 tyrosine phosphorylation with high sensitivity and fidelity (Z’ >0.5). MTT assays using a cell line that displays ErbB4-dependent proliferation distinguish between molecules that stimulate and fail to stimulate ErbB4-dependent proliferation (Z’>0.5) and identify molecules that antagonize agonist stimulation of ErbB4 dependent proliferation (Z’>0.5). These assays have been used to identify small molecules that stimulate ErbB4 tyrosine phosphorylation. Efforts to determine whether these hits function as ErbB4 full agonists or partial agonists (antagonists) are underway and will be reported. Structures of these small molecule ErbB4 full and partial agonists may be reported, pending submission of a provisional patent application. Conclusions: We have validated an HTS strategy for identifying ErbB4 partial agonists that function as ErbB4 antagonists and deployment of that strategy has led to the identification of several hits. Such molecules may hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors. Citation Format: Richard Cullum, Allan David, David J. Riese. Validation and implementation of screens for partial agonists and antagonists of the ErbB4/HER4 receptor tyrosine kinase: Targeted melanona drug discovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1353.

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.

Loss of PTPN12 Stimulates Progression of ErbB2-Dependent Breast Cancer by Enhancing Cell Survival, Migration, and Epithelial-to-Mesenchymal Transition.

PTPN12 is a cytoplasmic protein tyrosine phosphatase (PTP) reported to be a tumor suppressor in breast cancer, through its capacity to dephosphorylate oncogenic receptor protein tyrosine kinases (PTKs), such as ErbB2. However, the precise molecular and cellular impact of PTPN12 deficiency in breast cancer progression remains to be fully clarified. Here, we addressed this issue by examining the effect of PTPN12 deficiency on breast cancer progression in vivo, in a mouse model of ErbB2-dependent breast cancer using a conditional PTPN12-deficient mouse. Our studies showed that lack of PTPN12 in breast epithelial cells accelerated breast cancer development and lung metastases in vivo. PTPN12-deficient breast cancer cells displayed enhanced tyrosine phosphorylation of the adaptor Cas, the adaptor paxillin, and the kinase Pyk2. They exhibited no detectable increase in ErbB2 tyrosine phosphorylation. PTPN12-deficient cells were more resistant to anoikis and had augmented migratory and invasive properties. Enhanced migration was corrected by inhibiting Pyk2. PTPN12-deficient breast cancer cells also acquired partial features of epithelial-to-mesenchymal transition (EMT), a feature of more aggressive forms of breast cancer. Hence, loss of PTPN12 promoted tumor progression in a mouse model of breast cancer, supporting the notion that PTPN12 is a tumor suppressor in human breast cancer. This function was related to the ability of PTPN12 to suppress cell survival, migration, invasiveness, and EMT and to inhibit tyrosine phosphorylation of Cas, Pyk2, and paxillin. These findings enhance our understanding of the role and mechanism of action of PTPN12 in the control of breast cancer progression.

ErbB-2 signaling plays a critical role in regulating androgen-sensitive and castration-resistant androgen receptor-positive prostate cancer cells

While androgen deprivation therapy (ADT) reduces tumor burden, autocrine growth factor loops such as human epidermal growth factor receptor 2 (HER2/ErbB-2/neu) have been proposed to contribute to prostate cancer (PCa) survival and relapse. However, the role of ErbB-2 in regulating androgen-sensitive (AS) and castration-resistant (CR) cell proliferation remains unclear. Here, we determined the role of ErbB-2 in PCa progression and survival under steroid-reduced conditions using two independent PCa cell progression models. In AR-positive androgen-independent (AI) PCa cells that exhibit the CR phenotype, ErbB-2 was constitutively activated, compared to corresponding AS PCa cells. In AS LNCaP C-33 cells, androgen-induced ErbB-2 activation through ERK1/2 mediates PCa cell proliferation. Further, the ErbB-2-specific but not EGFR-specific inhibitor suppresses basal and androgen-stimulated cell proliferation and also blocks ERK1/2 activation. ErbB-2 ectopic expression and cPAcP siRNA transfection of LNCaP C-33 cells each increases ErbB-2 tyrosine phosphorylation, correlating with increased AI PSA secretion and cell proliferation. Conversely, trapping ErbB-2 by transfected endoplasmic reticulum-targeting ScFv5R expression vector abolished DHT-induced LNCaP C-33 cell growth. Moreover, inhibition of ErbB-2 but not EGFR in AI LNCaP C-81 and MDA PCa2b-AI PCa cells significantly abolished AI cell growth. In contrast to androgens via ErbB-2/ERK1/2 signaling in AS PCa cells, the inhibition of ErbB-2 abrogated AI cell proliferation by inhibiting the cell survival protein Akt in those AI cells. These results suggest that ErbB-2 is a prominent player in mediating the ligand-dependent and -independent activation of AR in AS and AI/CR PCa cells respectively for PCa progression and survival.

Abstract B17: A high-throughput screening process for the discovery of melanoma chemotherapeutics targeted at the ErbB4 receptor tyrosine kinase

Abstract Introduction. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, a family that also includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Gain-of-function ErbB4 mutants are found in a significant fraction of melanoma cell lines, suggesting that ErbB4 is a melanoma oncogene. However, there is a paucity of specific inhibitors for probing ErbB4 function or for treating ErbB4-dependent tumors. Consequently, we are developing high-throughput screening (HTS) approaches to address this void. Experimental Strategy. The Q43L mutant of the naturally-occurring ErbB4 agonist Neuregulin 2beta (NRG2beta) functions as a partial agonist at ErbB4; NRG2beta/Q43L stimulates ErbB4 tyrosine phosphorylation, but does not stimulate ErbB4 coupling to cell proliferation and competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. Therefore, we are developing three high-throughput assays to identify ErbB4 partial agonists that function as antagonists. (1) The primary screen will utilize a sandwich enzyme-linked immunosorbent assay (ELISA) to identify molecules that stimulate ErbB4 tyrosine phosphorylation. (2) The secondary screen will use an MTT proliferation assay to identify molecules that fail to stimulate ErbB4 coupling to cell proliferation. (3) An MTT proliferation assay will be used in the tertiary screen to identify molecules that inhibit agonist-induced ErbB4 coupling to cell proliferation. Molecules that stimulate ErbB4 tyrosine phosphorylation, do not stimulate ErbB4 coupling to cell proliferation, and inhibit agonist-induced ErbB4 coupling to proliferation are highly likely to function as ErbB4 antagonists. Results. (1) A high-throughput ELISA assay reproducibly detects stimulation of ErbB4 tyrosine phosphorylation by the ErbB4 full agonist NRG2beta (Z' = 0.730) and the ErbB4 antagonist (partial agonist) NRG2beta/Q43L (Z' = 0.664). (2) A high-throughput MTT assay reproducibly detects stimulation of ErbB4 coupling to cell proliferation by the ErbB4 full agonist NRG1beta (Z' = 0.236; 10 nM NRG1beta versus unstimulated). (3) A high-throughput MTT assay detects the difference between stimulation of ErbB4 coupling to cell proliferation by 2 nM NRG1beta and 10 nM NRG1beta (Z' = 0.246), suggesting that 80% inhibition of the activity of 10 nM NRG1beta at ErbB4 can be detected using this assay. (4) Initial screening of small molecule libraries using the primary screen has yielded more than 20 hits (potential novel small-molecule ErbB4 ligands). These hits are being validated and subjected to secondary and tertiary screens. Conclusions. We have validated HTS approaches for the identification of small molecule ErbB4 partial agonists and antagonists. The initial implementation of these approaches has yielded promising results. Citation Format: Richard L. Cullum, Ram B. Gupta, David J. Riese, II. A high-throughput screening process for the discovery of melanoma chemotherapeutics targeted at the ErbB4 receptor tyrosine kinase. [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 B17.

AKT3 regulates ErbB2, ErbB3 and estrogen receptor α expression and contributes to endocrine therapy resistance of ErbB2+ breast tumor cells from Balb-neuT mice

ErbB2+ breast cancer is an aggressive breast cancer subtype generally associated with lower estrogen receptor alpha (ERα) expression and more aggressive tumor behavior compared to ERα+/ErbB2− breast cancer. The ErbB2+ phenotype is associated with resistance to endocrine therapy, e.g. the selective estrogen receptor modulator Tamoxifen. However, the mechanisms underlying endocrine resistance are not fully understood. Here, we investigated the impact of AKT signaling and distinct functional roles of AKT isoforms in ErbB2+ breast cancer from Balb-neuT mice. AKT isoform specific in vitro kinase assays revealed that AKT3 is activated in Balb-neuT breast tumors in comparison to normal murine breast tissue. Knock-down of AKT3, but not of AKT1 or AKT2, led to reduced expression and tyrosine-phosphorylation of ErbB2 and ErbB3 in Balb-neuT-derived mammary tumor cells. In contrast, expression of ERα was strongly up-regulated and phosphorylation of the AKT substrate Foxo3a which regulates ERα transcription was decreased in AKT3 knockdown cells. These data suggest that ERα expression is down regulated via AKT3/Foxo3a signaling in ErbB2+ breast cancer cells. Furthermore, up-regulation of ERα after depletion of AKT3 resulted in a significant increase in Tamoxifen responsiveness of Balb-neuT-derived mammary tumor cells. In addition, Tamoxifen resistant human breast cancer cell lines showed increased AKT3 expression and activity in comparison to Tamoxifen responsive MCF-7 cells. Finally, by AKT isoform specific in vitro kinase assays of human breast cancer samples, AKT3 activity was detected in ErbB2+ and triple negative tumors but not in ERα+ breast cancer. Our data indicate that AKT3 regulates the expression of ErbB2, ErbB3 and ERα and demonstrate that down-regulation of activated AKT3 can sensitize ErbB2+ breast cancer cells for treatment with Tamoxifen. Therefore, AKT3 targeting might be a new promising strategy for therapy of ErbB2+/ERα− breast cancer and might further increase the responsiveness to an endocrine therapy approach.

P66Shc longevity protein regulates the proliferation of human ovarian cancer cells

p66Shc functions as a longevity protein in murine and exhibits oxidase activity in regulating diverse biological activities. In this study, we investigated the role of p66Shc protein in regulating ovarian cancer (OCa) cell proliferation. Among three cell lines examined, the slowest growing OVCAR-3 cells have the lowest level of p66Shc protein. Transient transfection with p66Shc cDNA expression vector in OVCAR-3 cells increases cell proliferation. Conversely, knock-down of p66Shc by shRNA in rapidly growing SKOV-3 cells results in decreased cell growth. In estrogen (E2)-treated CaOV-3 cells, elevated p66Shc protein level correlates with ROS level, ErbB-2 and ERK/MAPK activation, and cell proliferation. Further, the E2-stimulated proliferation of CaOV-3 cells was blocked by antioxidants and ErbB-2 inhibitor. Additionally, in E2-stimulated cells, the tartrate-sensitive, but not the tartrate-resistant, phosphatase activity decreases; concurrently, the tyrosine phosphorylation of ErbB-2 increases. Conversely, inhibition of phosphatase activity by L(+)-tartrate treatment increases p66Shc protein level, ErbB-2 tyrosine phosphorylation, ERK/MAPK activation, and cell growth. Further, inhibition of the ERK/MAPK pathway by PD98059 blocks E2-induced ERK/MAPK activation and cell proliferation in CaOV-3 cells. Moreover, immunohistochemical analyses showed that the p66Shc protein level was significantly higher in cancerous cells than in noncancerous cells in archival OCa tissues (n = 76; P = 0.00037). These data collectively indicate that p66Shc protein plays a critical role in up-regulating OCa progression.

Reduction of the ST6 β-Galactosamide α-2,6-Sialyltransferase 1 (ST6GAL1)-catalyzed Sialylation of Nectin-like Molecule 2/Cell Adhesion Molecule 1 and Enhancement of ErbB2/ErbB3 Signaling by MicroRNA-199a

Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 β-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.

The Q43L mutant of neuregulin 2β is a pan-ErbB receptor antagonist

The ErbB4 receptor tyrosine kinase possesses both tumour suppressor and oncogenic activities. Thus pharmacological agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2β (neuregulin 2β) stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus in the present paper we hypothesize that NRG2β/Q43L may be an ErbB4 antagonist. NRG2β/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2β/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2β. In addition, NRG2β stimulation of cell proliferation requires PI3K (phosphoinositide 3-kinase) activity and NRG2β stimulates greater Akt phosphorylation than does NRG2β/Q43L. Moreover, EGFR [EGF (epidermal growth factor) receptor] kinase activity (but not that of ErbB4) is critical for coupling ErbB4 to proliferation. Experiments utilizing ErbB4 splicing isoforms and mutants suggest that NRG2β and NRG2β/Q43L may differentially stimulate ErbB4 coupling to the transcriptional co-regulator YAP (Yes-associated protein). Finally, NRG2β/Q43L competitively antagonizes agonist stimulation of EGFR and ErbB2/ErbB3, indicating that NRG2β/Q43L is a pan-ErbB antagonist. Thus we postulate that NRG2β/Q43L and other antagonistic ligands stimulate ErbB tyrosine phosphorylation on a set of residues distinct from that stimulated by agonists, thus suggesting a novel mechanism of ErbB receptor regulation. Moreover, NRG2β/Q43L and related ligand-based antagonists establish a paradigm for the discovery of anti-ErbB therapeutics.

Leptin stimulates phosphoinositide 3-kinase in the vascular wall: role of ErbB receptors

Phosphoinositide 3-kinase (PI3K) phosphorylates phosphatidylinositol 4,5-diphosphate to phosphatidylinositol 3,4,5-triphosphate which then activates multiple intracellular signaling proteins such as protein kinase B (Akt), glycogen synthase kinase-3β, mammalian target of rapamycin (mTOR), etc. PI3K is involved in the regulation of vascular smooth muscle cell contractility, migration and proliferation - processes crucial for the pathogenesis of hypertension, atherosclerosis and restenosis. Increased leptin concentration in the metabolic syndrome contributes to the pathogenesis of cardiovascular diseases, but the effect of leptin on vascular smooth muscle cells is controversial. We examined the effect of experimentally induced hyperleptinemia on PI3K activity in the rat aortic media in adult male Wistar rats. Leptin, administered for 10 days in increasing dose (from 0.05 to 0.25 mg/kg every 12 hours), increased PI3K activity in the aortic media about 5-fold. The effect of leptin was markedly attenuated by epidermal growth factor receptor (EGFR) inhibitor, AG1478, as well as by the ErbB2 receptor inhibitor, AG825. In addition, leptin increased tyrosine phosphorylation of ErbB2, which was abolished by either AG1478 or AG825. These results indicate that hyperleptinemia increases the activity of vascular ErbB2 receptor in EGFR-dependent manner. In addition, both EGFR and ErbB2 contribute to PI3K stimulation by leptin. Activation of ErbB2 and PI3K may contribute to detrimental effects of leptin on vascular contractility and remodeling and may be involved, at least in part, in the relationship between metabolic syndrome and increased risk of vascular restenosis after angioplasty. Adipobiology 2011; 3: 53-59.

ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

ErbB4 is a member of the ErbB family of receptor tyrosine kinases. This family includes ErbB2 (HER2/Neu), a validated therapeutic target in breast cancer. Several studies indicate that ErbB4 functions as a tumor suppressor in breast cancer, whereas others indicate that ErbB4 functions as an oncogene. Here the authors explore the context in which ErbB4 functions as an oncogene. Silencing expression of either ErbB2 or ErbB4 in breast tumor cell lines results in reduced stimulation of anchorage independence and cell motility by the ErbB4 agonist neuregulin 2β. ErbB2 tyrosine kinase activity, but not ErbB4 tyrosine kinase activity, is required for neuregulin 2β to stimulate cell proliferation. Moreover, sites of ErbB4 tyrosine phosphorylation, but not sites of ErbB2 tyrosine phosphorylation, are required for neuregulin 2β to couple to cell proliferation. These data suggest that targeting ErbB2 expression or tyrosine kinase activity may be effective in treating ErbB4-dependent breast tumors, even those tumors that lack ErbB2 overexpression.