Background Prostate cancer development involves various mechanisms, which are poorly understood but pointing to epithelial mesenchymal transition (EMT) as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, acts as tumor suppressor in prostate cancer but the role of ABI1 in EMT is not clear. Methods To investigate the molecular mechanism by which loss of ABI1 contributes to tumor progression, we disrupted the ABI1 gene in the benign prostate epithelial RWPE-1 cell line and determined its phenotype. Levels of ABI1 expression in prostate organoid tumor cell lines was evaluated by Western blotting and RNA sequencing. ABI1 expression and its association with prostate tumor grade was evaluated in a TMA cohort of 505 patients and metastatic cell lines. Results Low ABI1 expression is associated with biochemical recurrence, metastasis and death (p = 0.038). Moreover, ABI1 expression was significantly decreased in Gleason pattern 5 vs. pattern 4 (p = 0.0025) and 3 (p = 0.0012), indicating an association between low ABI1 expression and highly invasive prostate tumors. Disruption of ABI1 gene in RWPE-1 cell line resulted in gain of an invasive phenotype, which was characterized by a loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 spheroids. Through RNA sequencing and protein expression analysis, we discovered that ABI1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of ABI1. Furthermore, an increase in STAT3 phosphorylation upon ABI1 inactivation and the evidence of a high-affinity interaction between the FYN SH2 domain and ABI1 pY421 support a model in which ABI1 acts as a gatekeeper of non-canonical WNT-EMT pathway activation downstream of the FZD2 receptor. Conclusions ABI1 controls prostate tumor progression and epithelial plasticity through regulation of EMT-WNT pathway. Here we discovered that ABI1 inhibits EMT through suppressing FYN-STAT3 activation downstream from non-canonical WNT signaling thus providing a novel mechanism of prostate tumor suppression.

280 Background: Prostate cancer is characterized by heterogeneity of mechanisms which are poorly understood but pointing to epithelial plasticity as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, is proposed to act as tumor suppressor in prostate cancer, but the mechanism of tumor progression due to Abi1 loss is not clear. Methods: To address Abi1’s role in prostate cancer we used CRISPR-based gene editing and retroviral expression to manipulate Abi1 levels in prostate cancer cell lines. Levels of Abi1 expression in prostate organoid tumor cell lines were evaluated by Western blotting and/or RNA sequencing. Association of Abi1 loss with tumor grade was evaluated by immunohistochemistry. Results: Abi1 expression is downregulated in tumor organoid cell lines from metastatic bone and lymph node biopsies. Moreover, low Abi1 expression is associated with high-grade prostate tumors (GG3 or higher, p < 0.001). Disruption of Abi1 gene in a benign prostate epithelial cell line RWPE-1 resulted in a gain of invasive phenotype, which is characterized by loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 Abi1 KO spheroids. Through RNA sequencing and protein expression analysis we discovered that Abi1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of Abi1. Furthermore, increase in STAT3 phosphorylation upon Abi1 inactivation and evidence for high affinity interaction of FYN-SH2 domain with Abi1 pY421 support the model that Abi1 acts as a gatekeeper of the non-canonical WNT-EMT pathway activation downstream from FZD2 receptor. The gene expression profile of Abi1-EMT-WNT pathway overlaps with the reported gene signature of high-risk prostate tumors. Conclusions: Abi1 contributes to prostate cancer progression and epithelial plasticity through regulation of EMT-WNT pathways. Understanding of Abi1’s role may provide more mechanistic understanding of prostate cancer tumor progression.

Breast cancer is the most commonly diagnosed non-cutaneous cancer in American women and is estimated to cause 40,000 deaths this year. Despite standard of care, breast cancer patients often relapse after a few years of treatment thus highlighting the need for new molecular targets for improved management of metastatic disease. Abelson interactor 1 (Abi1) is an adaptor protein associated with the WAVE (Wiscott-Aldrich syndrome protein family verprolin homologous) regulatory complex and Arp2/3 (Actin-related proteins 2 and 3)-mediated actin cytoskeleton remodeling. Our analysis of human tumor data indicates that Abi1 is frequently upregulated in invasive breast cancers, is associated with poor survival, and may promote an aggressive breast tumor phenotype. Abi1 has been shown to positively regulate breast cancer cell proliferation, motility, division, and invasion in vitro however its exact role in vivo remains unknown. We therefore hypothesize that Abi1 promotes breast tumor progression and metastasis in a mouse breast cancer model. Using the well-characterized Polyoma Middle T (PyMT) breast cancer model, we conditionally deleted Abi1 from the mammary epithelium and determined the effects on tumor growth kinetics and WAVE complex protein levels. Interestingly, our studies show delayed tumor growth only in mice with heterozygous deletion of Abi1 while homozygote KO mice show relatively unchanged tumor growth. Western blot analyses of Abi1 KO breast tumors show concomitant loss of WAVE complex proteins supporting our previous findings that WAVE complex integrity is dependent on Abi1. Our data also show significant upregulation of Abi2 only in homozygous Abi1 KO mice, suggesting a potential compensatory role for Abi2 that may support primary tumor growth in absence of Abi1. Most interestingly, Abi1 null PyMT mice show significantly reduced incidence of lung metastasis, supporting our hypothesis that Abi1 promotes metastasis of breast cancer cells. In summary, Abi1 loss abrogates lung metastasis in PyMT mice however primary tumor growth remains largely unaffected, possibly due to a compensatory mechanism by a related protein, Abi2. This work will establish the biochemical dynamics between members of the Abi protein family and WAVE complex to improve targeted treatments in aggressive human breast cancer. [Supported by NCI grant R01-CA161018 and Carol M. Baldwin Breast Cancer Research Fund of CNY] Citation Format: Angelina Regua, Isabelle Bichindaritz, Tiffany Caza, Julie R. White, Alexander Nappi, Claudia Mondragon, Mira Krendel, Gennady Bratslavsky, Abirami Sivapiragasam, Leszek Kotula. Abi1 positively regulates lung metastasis of aggressive breast cancer in PyMT mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 108.

American Cancer Society predicts over 160,000 new prostate cancer cases in 2017, which amounts to about 20 percent of all cancer diagnoses in men. Despite several treatment options being available, poor prognosis of high-risk and metastasis remains a major concern for prostate cancer (PCa) patients. Therefore, it is important to understand molecular mechanisms of PCa progression to aggressive disease. Our previous research identified Abelson interactor 1 (Abi1), a member of WAVE complex, as a tumor suppressor in human prostate. Not only did we find mutations and deletions in the gene in prostate tumor patient samples, but WAVE complex levels were also found to be inversely correlated to disease recurrence and castration resistance. Moreover, Abi1 is downregulated in majority of prostate cancer cell lines, and patient organoid cultures derived from metastatic bone and lymph node biopsies. These data point to the clinical significance of defining Abi1-dependent tumor suppression mechanisms. To understand these mechanisms we generated CRISPR-mediated Abi1 KO in the RWPE-1 non-tumor human prostate cell line. When plated in 3D cultures in matrigel, we observed striking phenotypic differences in the appearance and behavior of the Abi1 KO spheroids compared to the parental cells. While the parental RWPE-1 grew into spherical organoids with tight cell-cell boundaries, the Abi1 KO cells grew as loose irregular-shaped spheres, with many cells migrating out of the organoids. Hence, the loss of Abi1 in these non-tumor cells lead to gain of an invasive phenotype. Western Blots of Abi1-KO cells demonstrated concurrent disruption of WAVE complex and deregulation of WAVE complex protein levels. Moreover, cell-cell adhesion proteins such as E-cadherin and β-catenin showed modest decrease and/or a substantial loss of membrane localization. Rescue experiments of Abi1 re-expression in the KO cells reversed the phenotype indicating that the observed phenotype is Abi1-dependent. Analyses of RNA sequencing of cells obtained from 3D cultures demonstrated differences in expression signatures characteristic of altered cell-cell and cell-matrix adhesion pathways and indicated upregulation of pathways associated with the invasive phenotype of cancer cells. We propose that Abi1 loss and WAVE complex deregulation represents key mediators of invasive phenotype in PCa. [Supported by NCI R01 CA161018 and NYS Department of Health Prostate Cancer Hypothesis Development RFA #1410200115] Citation Format: Disharee Das, Ming Chen, Claudia Mondragon, Dawn Post, Alexander Nappi, Heidi Hehnly, Anita Hryniewicz-Jankowska, Pier Paolo Pandolfi, Gennady Bratslavsky, Leszek Kotula. Modeling of Abi1 loss using spheroid cultures to investigate mechanisms of prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1403.