Manisha Tripathi

The E3 Ubiquitin Ligase Siah2 Contributes to Castration-Resistant Prostate Cancer by Regulation of Androgen Receptor Transcriptional Activity

Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.

Understanding the role of stromal fibroblasts in cancer progression

The major cellular components of tumor microenvironment, referred to as the cancer stroma, are composed of cancer-associated fibroblasts that support tumor epithelial growth, invasion and therapeutic resistance. Thus when we speak of developing therapies that address tumor heterogeneity it is not only a matter of different mutations within the tumor epithelia. While individual mutations in the stromal compartment are controversial, the heterogeneity in fibroblastic population in a single tumor is not up for debate. Cooperative interaction among heterotypic fibroblasts and tumor cells contribute to cancer progression. Therefore to tackle solid tumors, we need to understand its complex microenvironment. Here we review some seminal developments in the field of tumor microenvironment, mainly focusing on cancer-associated fibroblast.

Bone Metastasis of Prostate Cancer Can Be Therapeutically Targeted at the TBX2–WNT Signaling Axis

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer. TBX2, a T-box family transcription factor that negatively regulates cell-cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here, we report that TBX2 is overexpressed in human prostate cancer specimens and bone metastases from xenograft mouse models of human prostate cancer. Blocking endogenous TBX2 expression in PC3 and ARCaPM prostate cancer cell models using a dominant-negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro Blocking endogenous TBX2 in human prostate cancer mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in prostate cancer cells dramatically reduced bone-colonizing capability through reduced tumor cell growth and bone remodeling in an intratibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in prostate cancer cells with endogenously blocked TBX2 partially restored the TBX2-induced prostate cancer metastatic capability in mice. Conversely, WNT3A-neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2-induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in prostate cancer patients. Cancer Res; 77(6); 1331-44. ©2017 AACR.

Histone deacetylase inhibitors mediate DNA damage repair in ameliorating hemorrhagic cystitis

Hemorrhagic cystitis is an inflammatory and ulcerative bladder condition associated with systemic chemotherapeutics, like cyclophosphomide. Earlier, we reported reactive oxygen species resulting from cyclophosphamide metabolite, acrolein, causes global methylation followed by silencing of DNA damage repair genes. Ogg1 (8-oxoguanine DNA glycosylase) is one such silenced base excision repair enzyme that can restore DNA integrity. The accumulation of DNA damage results in subsequent inflammation associated with pyroptotic death of bladder smooth muscle cells. We hypothesized that reversing inflammasome-induced imprinting in the bladder smooth muscle could prevent the inflammatory phenotype. Elevated recruitment of Dnmt1 and Dnmt3b to the Ogg1 promoter in acrolein treated bladder muscle cells was validated by the pattern of CpG methylation revealed by bisulfite sequencing. Knockout of Ogg1 in detrusor cells resulted in accumulation of reactive oxygen mediated 8-Oxo-dG and spontaneous pyroptotic signaling. Histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), restored Ogg1 expression in cells treated with acrolein and mice treated with cyclophosphamide superior to the standard of care, mesna or nicotinamide-induced DNA demethylation. SAHA restored cyclophosphamide-induced bladder pathology to that of untreated control mice. The observed epigenetic imprinting induced by inflammation suggests a new therapeutic target for the treatment of hemorrhagic cystitis.

Heterogeneous cancer-associated fibroblast population potentiates neuroendocrine differentiation and castrate resistance in a CD105-dependent manner.

Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression and resistance to androgen signaling deprivation therapy (ADT). CAF subjected to extended passaging, compared to low passage CAF, were found to lose tumor expansion potential and heterogeneity. Cell surface endoglin (CD105), known to be expressed on proliferative endothelia and mesenchymal stem cells, was diminished in high passage CAF. RNA-sequencing revealed SFRP1 to be distinctly expressed by tumor-inductive CAF, which was further demonstrated to occur in a CD105-dependent manner. Moreover, ADT resulted in further expansion of the CD105+ fibroblastic population and downstream SFRP1 in 3-dimensional cultures and patient-derived xenograft tissues. In patients, CD105+ fibroblasts were found to circumscribe epithelia with neuroendocrine differentiation. CAF-derived SFRP1, driven by CD105 signaling, was necessary and sufficient to induce prostate cancer neuroendocrine differentiation in a paracrine manner. A partially humanized CD105 neutralizing antibody, TRC105, inhibited fibroblastic SFRP1 expression and epithelial neuroendocrine differentiation. In a novel synthetic lethality paradigm, we found that simultaneously targeting the epithelia and its microenvironment with ADT and TRC105, respectively, reduced castrate-resistant tumor progression, in a model where either ADT or TRC105 alone had little effect.

Stromal epigenetic alterations drive metabolic and neuroendocrine prostate cancer reprogramming

Prostate cancer is an androgen-dependent disease subject to interactions between the tumor epithelium and its microenvironment. Here, we found that epigenetic changes in prostatic cancer-associated fibroblasts (CAF) initiated a cascade of stromal-epithelial interactions. This facilitated lethal prostate cancer growth and development of resistance to androgen signaling deprivation therapy (ADT). We identified a Ras inhibitor, RASAL3, as epigenetically silenced in human prostatic CAF, leading to oncogenic Ras activity driving macropinocytosis-mediated glutamine synthesis. Interestingly, ADT further promoted RASAL3 epigenetic silencing and glutamine secretion by prostatic fibroblasts. In an orthotopic xenograft model, subsequent inhibition of macropinocytosis and glutamine transport resulted in antitumor effects. Stromal glutamine served as a source of energy through anaplerosis and as a mediator of neuroendocrine differentiation for prostate adenocarcinoma. Antagonizing the uptake of glutamine restored sensitivity to ADT in a castration-resistant xenograft model. In validating these findings, we found that prostate cancer patients on ADT with therapeutic resistance had elevated blood glutamine levels compared with those with therapeutically responsive disease (odds ratio = 7.451, P = 0.02). Identification of epigenetic regulation of Ras activity in prostatic CAF revealed RASAL3 as a sensor for metabolic and neuroendocrine reprogramming in prostate cancer patients failing ADT.

Abstract B086: Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients

Purpose: The identification, functional status, distribution, and interactions of immune cells in PCa patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a tumor-promoting phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behavior of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (RD CHI3L1) when compared with PCa-N patients and healthy controls (HC), classified as individuals with no known history of cancer. The only described receptor for YKL-40, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in ARCaPm cells; accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPm. In addition, siRNA directed against this receptor in ARCaPm cells caused a significant reduction in the invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: We show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of YKL-40, and YKL-40 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca Meza, David Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine Martins, Timothy R. Crother, Cory M. Hogaboam, Sungyong You, Neil Bhowmick, Edwin Posadas. Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B086.

Circulating monocytes from prostate cancer patients promote invasion and motility of epithelial cells

Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. We tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor‐derived signals.

Abstract LB-274: Microenvironment mediates the efficacy of Cabozantinib in prostate cancer

Therapeutic targeting of the epithelial compartment alone in prostate cancer has resulted in unfavorable outcomes, including the development of resistance. Cabozantinib (XL184), a small molecule inhibitor of tyrosine kinases including c-MET and VEGFR2 has been shown to decrease tumor growth and metastasis. Our working rationale is that therapeutic strategies that target the prostate tumor microenvironment would have improved outcomes compared with those that solely target the cancer epithelia. Cabozantinib is demonstrated to have a role in inhibiting osteoclast and osteoblast differentiation and thereby limit prostate cancer (PCa) bone lesions, as determined by technetium-99 bone scan. However, the ultimate survival benefit of castrate resistant prostate cancer patients treated with cabozantinib has not born out in recent clinical trials for PCa patients with bone metastasis. In this study, utilizing various pre-clinical xenograft mouse models, prostate epithelial cell lines and human and mouse prostate fibroblasts, we investigated the effect of cabozantinib on prostate tumor microenvironment and delineated the cellular targets of cabozantnib. We found that cabozantinib affects prostate stromal cells at a significantly lower concentration compared with epithelial cells in vitro. We found that human cancer associated fibroblasts pre-treated with cabozantinib induced a significantly increased tumor growth in the intra-tibial mouse model of growth in the bone microenvironment. Further, in a novel and surprising discovery, we found that cabozantinib alters the immune microenvironment by reducing the M1 macrophages. However, cabozantinib depleted both basal and luminal epithelial progenitor cells in vivo. Taken together, we found that cabozantnib can have a tumor potentiating role by limiting M1 macrophages, yet the down regulation of carcinoma associated fibroblasts and associated epithelial progenitors could suggest interesting combination therapies. A lower dose of cabozantnib with traditional anti-proliferative therapies, like taxanes or radiation is supported by the pre-clinical experiments. Citation Format: Manisha Tripathi, Srinivas Nandana, Sandrine Billet, Edwin M. Posadas, Leland W.K. Chung, Neil A. Bhowmick. Microenvironment mediates the efficacy of Cabozantinib in prostate cancer. [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 LB-274.

Abstract PR06: Reciprocal prostate cancer signaling with its microenvironment mediates castrate-resistant disease progression

Our lab has previously reported that the mouse model knocked out for TGF-beta receptor type II in fibroblasts results in castrate resistance of prostate tissues, mediated by paracrine Wnt signaling. We found that antagonizing androgen signaling leads to Wnt ligand expression by prostatic fibroblasts, interestingly dependent on cell-cell contact. This led us to hypothesize that a juxtacrine action within the stromal compartment was important to the development of castrate resistance. Building upon this observation, we found that Notch signaling was upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs, using DAPT, resulted in decrease in Wnt2b and decreased expression of Notch target genes. Similarly, castration or androgen receptor antagonism (i.e. bicalutamide or enzalutamide) resulted in increased Notch signaling and downstream Wnts 2 and 2b up regulation. The further generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling, derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts having a loss of Notch signaling, from RBPJ-knockout mice, had diminished tumorigenesis, compared to xenografts with the wild type fibroblasts counterpart. In trying to determine the mediator of Wnt-associated paracrine prostate epithelial expansion, we found downstream YAP activation in the RV1 cells. In a reciprocal epithelial-to-fibroblast communication we found that the RV1 cells treated with conditioned media from NICD fibroblasts showed increased IL-6 expression. Conversely, conditioned media from prostate cancer cells increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together there is a stromal-derived signaling cascade involving Notch and Wnt ligands, that activates YAP in the epithelia for paracrine IL-6-dependent Notch activity back in the stromal fibroblasts. Critically, disruption of this cross-talk in the fibroblasts can limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer. This abstract is also presented as Poster A44. Citation Format: Manisha Tripathi, Srinivas Nandana, Jen-Ming Huang, Manabu Kato, Leland W. K. Chung, Rajeev Mishra, Li Xin, Neil Bhowmick. Reciprocal prostate cancer signaling with its microenvironment mediates castrate-resistant disease progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR06.