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// Joseph H. Taube 1, 3, 4, * , Nathalie Sphyris 1, * , Kelsey S. Johnson 3 , Keighley N. Reisenauer 3 , Taylor A. Nesbit 3 , Robiya Joseph 1 , Geraldine V. Vijay 1 , Tapasree R. Sarkar 1, 5 , Neeraja A. Bhangre 1 , Joon Jin Song 6 , Jeffrey T. Chang 7, 8 , Min Gyu Lee 9, 10 , Rama Soundararajan 1 and Sendurai A. Mani 1, 2 1 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Metastasis Research Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Biology, Baylor University, Waco, Texas, USA 4 Institute of Biomedical Sciences, Baylor University, Waco, Texas, USA 5 Department of Integrative Bioscience, Texas A & M University, College Station, Texas, USA 6 Depatment of Statistical Science, Baylor University, Waco, Texas, USA 7 Center for Clinical and Translational Sciences, The University of Texas Health Science Center at Houston, Texas, USA 8 Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Texas, USA 9 Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 10 Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA * Co-first authors Correspondence to: Sendurai A. Mani, email: smani@mdanderson.org Joseph H. Taube, email: Joseph_Taube@baylor.edu Keywords: epithelial-mesenchymal transition, mesenchymal-epithelial transition, KDM6A, bivalent genes, GSK-J4 Received: July 20, 2016 Accepted: April 26, 2017 Published: July 10, 2017 ABSTRACT The deposition of the activating H3K4me3 and repressive H3K27me3 histone modifications within the same promoter, forming a so-called bivalent domain, maintains gene expression in a repressed but transcription-ready state. We recently reported a significantly increased incidence of bivalency following an epithelial-mesenchymal transition (EMT), a process associated with the initiation of the metastatic cascade. The reverse process, known as the mesenchymal-epithelial transition (MET), is necessary for efficient colonization. Here, we identify numerous genes associated with differentiation, proliferation and intercellular adhesion that are repressed through the acquisition of bivalency during EMT, and re-expressed following MET. The majority of EMT-associated bivalent domains arise through H3K27me3 deposition at H3K4me3-marked promoters. Accordingly, we show that the expression of the H3K27me3-demethylase KDM6A is reduced in cells that have undergone EMT, stem-like subpopulations of mammary cell lines and stem cell-enriched triple-negative breast cancers. Importantly, KDM6A levels are restored following MET, concomitant with CDH1 /E-cadherin reactivation through H3K27me3 removal. Moreover, inhibition of KDM6A, using the H3K27me3-demethylase inhibitor GSK-J4, prevents the re-expression of bivalent genes during MET. Our findings implicate KDM6A in the resolution of bivalency accompanying MET, and suggest KDM6A inhibition as a viable strategy to suppress metastasis formation in breast cancer.

Identifying bioenergetics that facilitate the epithelial to mesenchymal transition (EMT) in breast cancer cells may uncover targets to treat incurable metastatic disease. Metastasis is the number one cause of cancer-related deaths, therefore, it is urgent to identify new treatment strategies to prevent the initiation of metastasis. To characterize the bioenergetics of EMT, we compared metabolic activities and gene expression in cells induced to differentiate into the mesenchymal state with their epithelial counterparts. We found that levels of GLS2, which encodes a glutaminase, are inversely associated with EMT. GLS2 down-regulation was correlated with reduced mitochondrial activity and glutamine independence even in low-glucose conditions. Restoration of GLS2 expression in GLS2-negative breast cancer cells rescued mitochondrial activity, enhanced glutamine utilization, and inhibited stem-cell properties. Additionally, inhibition of expression of the transcription factor FOXC2, a critical regulator of EMT in GLS2-negative cells, restored GLS2 expression and glutamine utilization. Furthermore, in breast cancer patients, high GLS2 expression is associated with improved survival. These findings suggest that epithelial cancer cells rely on glutamine and that cells induced to undergo EMT become glutamine independent. Moreover, the inhibition of EMT leads to a GLS2-directed metabolic shift in mesenchymal cancer cells, which may make these cells susceptible to chemotherapies.

Identifying bioenergetics that facilitate the epithelial to mesenchymal transition (EMT) in breast cancer cells may uncover targets to treat incurable metastatic disease. Metastasis is the number one cause of cancer-related deaths; therefore, it is urgent to identify new treatment strategies to prevent the initiation of metastasis. To characterize the bioenergetics of EMT, we compared metabolic activities and gene expression in cells induced to differentiate into the mesenchymal state with their epithelial counterparts. We found that levels of GLS2, which encodes a glutaminase, are inversely associated with EMT. GLS2 down-regulation was correlated with reduced mitochondrial activity and glutamine independence even in low-glucose conditions. Restoration of GLS2 expression in GLS2-negative breast cancer cells rescued mitochondrial activity, enhanced glutamine utilization, and inhibited stem-cell properties. Additionally, inhibition of expression of the transcription factor FOXC2, a critical regulator of EMT in GLS2-negative cells, restored GLS2 expression and glutamine utilization. Furthermore, in breast cancer patients, high GLS2 expression is associated with improved survival. These findings suggest that epithelial cancer cells rely on glutamine and that cells induced to undergo EMT become glutamine independent. Moreover, the inhibition of EMT leads to a GLS2-directed metabolic shift in mesenchymal cancer cells, which may make these cells susceptible to chemotherapies.

Metastatic breast cancer is the most dreadful malignant disease that accounts for the majority of cancer-related deaths worldwide among women. A number of studies have shown that the tumor microenvironment (TME) plays a crucial role in regulating metastasis. It is therefore imperative to understand the dynamic interactions between cancer cells and their microenvironment to examine the molecular interaction and to effectively target cancer cells. TME comprises a variety of cells including immune cells which can influence tumor survival, growth and metastasis. Tumor-infiltrating lymphocytes (TILs), in particular, the CD8 T lymphocytes, has emerged as a promising prognostic marker for immunotherapy in a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and CD8 T lymphocytes and its impact on metastatic traits in breast cancer is still inconclusive. Platelets are crucial components of the tumor microenvironment that are known to modulate tumor promotion and metastasis. The contribution of platelets and platelet secreted molecules are also carefully examined in metastasis of various cancers. The primary objective of this study is to investigate the role of CD8 T lymphocytes and platelets in breast tumor progression using isogenic tumor lines that form identical primary tumors but differ in their ability to develop metastasis. Citation Format: Robiya Joseph, Rama Soundararajan, Suhas Vasaikar, Fei Yang, Sevinj Isgandarova, Lin Tian, Monika Haemmerle, Barbara Mino, Tieling Zhou, Geraldine Vidhya Raja, Esmeralda Ramirez Pena, Petra Den Hollander, Neeraja Bhangre, Crystal Shin, Melisa Martinez, Jaime Rodriguez Canales, Jeffrey Chang, Anil Sood, Ignacio Ivan Wistuba, Don L. Gibbons, Jeffrey M. Rosen, Ghanashyam Acharya, Navin Varadarajan, Xiang H. Zhang, Sendurai A. Mani. Regulation of metastasis by CD8 T lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3761.

Prostate cancer (PCa) accounts for approximately 26,730 deaths each year in the United States. The tremendous heterogeneity observed among prostate cancers contributes to the enormous clinical challenge. Among these tumors, variant Neuroendocrine Prostate Cancers (NEPC) account for up to a third of prostate cancer-related deaths. These tumors are enriched with cancer cells with stem-cell properties (CSCs). Prostate CSCs lack the expression of androgen-receptor (AR), and are hence resistant to AR inhibition. In addition, CSCs are capable of sustaining tumor relapse and metastasis. We previously demonstrated that aberrant induction of epithelial-mesenchymal transition (EMT) in differentiated prostate epithelial cells could result in the activation of CSC features, including ADT resistance and NEPC attributes. Using multiple prostate cancer cell lines as well as patient-derived xenograft samples, we demonstrated a reciprocal relationship between the EMT/CSC phenotype and expression of AR, and also observed a vital role for the central EMT regulator FOXC2 (Forkhead transcription factor C2) in the generation/maintenance of AR-negative CSCs, which are in turn responsible for the formation of NEPCs. We therefore sought to identify conserved EMT-signaling pathways necessary for EMT and neuroendocrine trans-differentiation in PCa cells, and investigate if targeting EMT-promoting pathways renders CSC-enriched PCa cells sensitive to ADT. We demonstrate that FOXC2 is a direct target of p38MAPK (a pleiotropic kinase), and that the p38-FOXC2 signaling cascade is indispensable for NEPC attributes and associated stem-cell functions. Targeting the p38 pathway using a systemic small-molecule inhibitor in animal models in vivo induced epithelial differentiation in NEPCs and indeed rendered them sensitive to androgen-deprivation therapy using enzalutamide, via loss of FOXC2 function. This study therefore highlights a novel (and targetable) signaling pathway that fosters CSC-associated drug resistance in NEPC, and a pioneering treatment potential for patients with NEPC. Acknowledgments: We thank Dr. Nupam Mahajan (Moffitt Cancer Center, University of South Florida, Tampa), Dr. Ignacio Wistuba, Dr. Jaime Rodriguez-Canales, Dr. Sue-Hwa Lin (Department of Translational Molecular Pathology, UT MDACC, Houston) and Dr. Dean Tang (Department of Molecular Carcinogenesis, MDACC, Smithville) for their contributions. We thank the UTMDACC Prostate Cancer SPORE for financial support (RS, SM). Citation Format: Rama Soundararajan, Anurag Paranjape, Robiya Joseph, Neeraja Bhangre, Ana Aparicio, Sankar Maity, Christopher Logothetis, Jeffrey Chang, Sendurai Mani. Modulating a cancer stem cell-specific signaling pathway to reverse the course of neuroendocrine prostate cancer [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 A065.

Prostate cancer cells depend on androgens for growth and survival. While androgen-deprivation-therapy (ADT) results in regression of the tumor bulk, in many cases, the cancers recur in aggressive androgen-independent forms that are also resistant to standard-of-care therapies. The prevailing hypothesis is that pre-existing androgen-independent prostate cancer (PCa) stem-cells in the primary tumor are selectively enriched following therapy. However, we have previously shown that induction of epithelial-mesenchymal-transition (EMT) in differentiated breast epithelial cells can result in the generation of cells with stem-cell properties. In the current study, we investigated if induction of EMT in PCa promotes stem-cell features and androgen-receptor (AR) regulation, and if targeting EMT-promoting pathways renders PCa stem-cells sensitive to ADT. We found that LNCaP-PSAlow cells, which were previously shown to represent the PCa stem-cell population, exhibit EMT properties. Induction of EMT through over-expression of EMT-inducing transcription factors or with TGFβ1 treatment, significantly stimulated stem-cell properties in the androgen-sensitive LNCaP cells, whereas inhibition of EMT in the androgen-insensitive DU145 cells using targeted shRNA or a specific TGFβ1-signaling inhibitor, resulted in markedly reduced stemness. We observed that FOXC2 expression consistently correlated with induction of EMT and androgen-insensitivity in PCa cells. Our study demonstrates that targeting EMT-inducing molecules and signaling pathways could represent a tangible approach to inhibiting the generation and maintenance of therapy-resistant PCa stem-cells, that are the prime harbingers of tumor recurrence. Citation Format: Anurag N. Paranjape, Rama Soundararajan, Steven J. Werden, Robiya Joseph, Joseph H. Taube, Neeraja Bhangre, Hui Liu, Jaime Rodriguez-Canales, Ignacio I. Wistuba, Jeffrey T. Chang, Dean G. Tang, Nupam Mahajan, Kiran Mahajan, Naoyuki Miura, Sendurai A. Mani. Delineating the role of epithelial-mesenchymal transition in the generation and maintenance of prostate cancer stem cells. [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 4065. doi:10.1158/1538-7445.AM2015-4065