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Successful visualization of prostate cancer (PCa) tumor margins during surgery remains a major challenge. The visualization of these tumors during surgery via near infrared fluorescence (NIRF) imaging would greatly enhance surgical resection, minimizing tumor recurrence and improving outcome. Furthermore, chemotherapy is typically administered to patients after surgery to treat any missed tumor tissue around the surgical area, minimizing metastasis and increasing patient survival. For these reasons, a theranostics fluorescent nanoparticle could be developed to assist in the visualization of PCa tumor margins, while also delivering chemotherapeutic drug after surgery. Methods: Ferumoxytol (FMX) conjugated to the fluorescent dye and PCa targeting agent, heptamethine carbocyanine (HMC), yielded the HMC-FMX nanoprobe that was tested in vitro with various PCa cell lines and in vivo with both subcutaneous and orthotopic PCa mouse models. Visualization of these tumors via NIRF imaging after administration of HMC-FMX was performed. In addition, delivery of chemotherapeutic drug and their effect on tumor growth was also assessed. Results: HMC-FMX internalized into PCa cells, labeling these cells and PCa tumors in mice with near infrared fluorescence, facilitating tumor margin visualization. HMC-FMX was also able to deliver drugs to these tumors, reducing cell migration and slowing down tumor growth. Conclusion: HMC-FMX specifically targeted PCa tumors in mice allowing for the visualization of tumor margins by NIRF imaging. Furthermore, delivery of anticancer drugs by HMC-FMX effectively reduced prostate tumor growth and reduced cell migration in vitro. Thus, HMC-FMX can potentially translate into the clinic as a nanotheranostics agent for the intraoperative visualization of PCa tumor margins, and post-operative treatment of tumors with HMC-FMX loaded with anticancer drugs.

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.

Simple Summary An estimated ~25–30% of patients with advanced prostate cancer (PCa) develop the aggressive and lethal form of the disease known as treatment-induced neuroendocrine prostate cancer (t-NEPC). Owing to lack of treatment options, the identification of the underlying molecular mechanisms that propagate the t-NEPC phenotype is critical towards developing novel therapeutic strategies against advanced PCa. Further, the roles of extracellular vesicles (exosomes) and microRNAs—an increasingly recognized and key mode of propagation of the NEPC phenotype—remain elusive. Our studies reveal that TBX2 promotes SOX2- and N-MYC- driven t-NEPC through regulation of the intermediary factor—miR-200c-3p; and that TBX2/miR-200c-3p/SOX2/MYCN signaling can promote t-NEPC via both intracellular and exosome-mediated intercellular mechanisms. Abstract Deciphering the mechanisms that drive transdifferentiation to neuroendocrine prostate cancer (NEPC) is crucial to identifying novel therapeutic strategies against this lethal and aggressive subtype of advanced prostate cancer (PCa). Further, the role played by exosomal microRNAs (miRs) in mediating signaling mechanisms that propagate the NEPC phenotype remains largely elusive. The unbiased differential miR expression profiling of human PCa cells genetically modulated for TBX2 expression led to the identification of miR-200c-3p. Our findings have unraveled the TBX2/miR-200c-3p/SOX2/N-MYC signaling axis in NEPC transdifferentiation. Mechanistically, we found that: (1) TBX2 binds to the promoter and represses the expression of miR-200c-3p, a miR reported to be lost in castrate resistant prostate cancer (CRPC), and (2) the repression of miR-200c-3p results in the increased expression of its targets SOX2 and N-MYC. In addition, the rescue of mir-200c-3p in the context of TBX2 blockade revealed that miR-200c-3p is the critical intermediary effector in TBX2 regulation of SOX2 and N-MYC. Further, our studies show that in addition to the intracellular mode, TBX2/miR-200c-3p/SOX2/N-MYC signaling can promote NEPC transdifferentiation via exosome-mediated intercellular mechanism, an increasingly recognized and key mode of propagation of the NEPC phenotype.

Our understanding of neuroendocrine prostate cancer (NEPC) has assumed a new perspective in light of the recent advances in research. Although classical NEPC is rarely seen in the clinic, focal neuroendocrine trans-differentiation of prostate adenocarcinoma occurs in about 30% of advanced prostate cancer (PCa) cases, and represents a therapeutic challenge. Even though our knowledge of the mechanisms that mediate neuroendocrine differentiation (NED) is still evolving, the role of androgen deprivation therapy (ADT) as a key driver of this phenomenon is increasingly becoming evident. In this review, we discuss the molecular, cellular, and therapeutic mediators of NED, and emphasize the role of the tumor microenvironment (TME) in orchestrating the phenotype. Understanding the role of the TME in mediating NED could provide us with valuable insights into the plasticity associated with the phenotype, and reveal potential therapeutic targets against this aggressive form of PCa.

Purpose: A successful cancer surgery requires the complete removal of cancerous tissue, while also sparing as much healthy, non-cancerous tissue as possible. To achieve this, an accurate identification of tumor boundaries during surgery is critical, but intra-operative tumor visualization remains challenging. Fluorescence imaging is a promising method to improve tumor detection and delineate tumor boundaries during surgery, but the lack of stable, long-circulating, clinically-translatable fluorescent probes that can identify tumors with high signal-to-noise ratios and low background fluorescence signals have prevented its widespread application. Methods: We screened the optical properties of several fluorescent dyes before and after nanoprobe encapsulation, and then identified nanoprobes with quenched fluorescence that were re-activated upon dye release. The physical and biological properties of these nanoprobes leading to fluorescence activation were investigated in vitro. Further, the cancer imaging properties of both free dyes and nanoprobe-encapsulated dyes were compared in vivo. Results: A novel fluorescent nanoprobe was prepared by combining two FDA-approved agents commonly used in the clinic: Feraheme (FH) and indocyanine green (ICG). The resulting FH-entrapped ICG nanoprobe [FH(ICG)] displayed quenched fluorescence compared to other nanoprobes, and this quenched fluorescence was re-activated in acidic tumor microenvironment conditions (pH 6.8) and upon uptake into cancer cells. Finally, in vivo studies in a prostate cancer mouse model demonstrated that FH(ICG) treatments enhance long-term fluorescence signals in tumors compared to ICG treatments, allowing for fluorescence-guided tumor identification using clinically relevant fluorescence cameras. Conclusions: FH(ICG) nanoprobes were identified as fluorescent nanoprobes with beneficial fluorescence activation properties compared to other FH-entrapped dyes. The activatable nature of this nanoprobe allows for a low background fluorescence signal and high signal-to-noise ratio within a long-circulating nanoagent, which allows for long-term fluorescence signals from tumors that enabled their fluorescence-guided detection. This activatable nanoprobe offers tremendous potential as a clinically translatable image-guided cancer therapy modality that can be prepared in a clinical setting.

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.

// Manisha Tripathi 1, * , Srinivas Nandana 1, * , Sandrine Billet 1 , Karen A. Cavassani 1 , Rajeev Mishra 1 , Leland W.K. Chung 1 , Edwin M. Posadas 1 and Neil A. Bhowmick 1, 2 1 Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA 2 Department of Research, Greater Los Angeles Veterans Administration, Los Angeles, California 90048, USA * These authors have contributed equally to this work Correspondence to: Neil A. Bhowmick, email: neil.bhowmick@cshs.org Edwin M. Posadas, email: Edwin.Posadas@cshs.org Keywords: carcinoma associated fibroblasts, cabozantinib, metastasis, prostate cancer Received: May 30, 2017 Accepted: August 15, 2017 Published: September 23, 2017 ABSTRACT The tumor microenvironment (TME) is increasingly recognized as the arbiter of metastatic progression and drug resistance in advanced prostate cancer (PCa). Cabozantinib is a potent tyrosine kinase inhibitor (TKI) with reported biological activity in the PCa epithelia, but failed to provide an overall survival benefit in phase 3 clinical trials. However, the promising biologic efficacy of the drug in early trials warranted a better understanding of the mechanism of action, with the goal of improving patient selection for TKI-based therapy such as cabozantinib. We found a 100-fold lower cabozantinib IC 50 in macrophages, PCa associated fibroblasts, and bone marrow fibroblasts compared to PCa epithelia. In PCa mouse models, pre-treatment with cabozantinib potentiated osseous and visceral tumor engraftment, suggesting a pro-tumorigenic host response to the drug. We further found that the host effects of cabozantinib impacted bone turnover, but not necessarily tumor expansion. Cabozantinib affected M1 macrophage polarization in mice. Analogously, circulating monocytes from PCa patients treated with cabozantinib, demonstrated a striking correlation of monocyte reprograming with therapeutic bone responsivity, to support patient selection at early stages of treatment. Thus, a re-evaluation of TKI-based therapeutic strategies in PCa can be considered for suitable patient populations based on TME responses.

Juxtacrine signaling with the stromal compartment is important to the development of castrate resistance prostate cancer. We found that Notch signaling is upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs resulted in decreased Wnt signaling and decrease in expression of Notch pathway genes. Interestingly, androgen receptor antagonism resulted in increased Notch signaling and the upregulation of downstream Wnts. Further, generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling that are derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts of RBPJ-knockout mice that have a loss of Notch signaling demonstrated diminished tumorigenesis, compared with xenografts with the wild type fibroblasts. 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 caused increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together our results suggest that there is a stromal-derived feed-back - feed-forward signaling loop 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 potentially limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer. Citation Format: Manisha Tripathi, Srinivas Nandana, Jen-Ming Huang, Manabu Kato, Rajeev Mishra, Leland Chung, Li Xin, Neil Bhowmick. Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression [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 5164.

SummaryUnderstanding 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.

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.

BACKGROUND Matriptase, a type II transmembrane serine protease, has been linked to initiation and promotion of epidermal carcinogenesis in a murine model, suggesting that deregulation of its role in epithelia contributes to transformation. In human prostate cancer, matriptase expression correlates with progression. It is therefore of interest to determine how matriptase may contribute to epithelial neoplastic progression. One approach for studying this is to identify potential matriptase substrates involved in epithelial integrity and/or transformation like the extracellular matrix macromolecule, laminin-332 (Ln-332), which is found in the basement membrane of many epithelia, including prostate. Proteolytic processing of Ln-332 regulates cell motility of both normal and transformed cells, which has implications in cancer progression.

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 (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of YKL-40 (Chitinase-3-like 1; 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.

Purpose: The identification, functional status, distribution, and interactions of immune cells in prostate cancer (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 are known to 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 PCa patients exhibit a protumor 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 behaviors 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 (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of Chitinase-3-like 1 (CHI3L1, YKL-40) when compared with PCa-N patients and healthy-control individuals (HC). The only described receptor for CHI3L1, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in the human metastatic prostate cancer cell line, ARCaPM. Accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPM cells, and siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: Thus, we show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca J. Meza, David M. Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine A. Martins, Timothy R. Crother, Sungyong You, Cory M. Hogaboam, Neil Bhowmick, Edwin M. Posadas. Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients [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 5208.

5031 Background: The presence of VM in metastatic, castration-resistant prostate cancer (mCRPC) predicts poor survival. Cabozantinib (cabo) is a multi-kinase inhibitor that has clinical activity that did not improve survival in an unselected mCRPC population. Subgroup analyses suggested that the benefit may exist for patients (pts) with mCRPC-VM. The effect of cabo includes the tumor microenvironment, monocytes in particular, which in turn can alter tumor behavior. Methods: We conducted a single-arm study of cabo in men with mCRPC-VM. Pts received cabo 60 mg daily. Radiographs were used to assess response. Correlative blood samples were collected for the enumeration and characterization of circulating tumor cells using the NanoVelcro Assay and analysis of circulating monocytes by FACS. Results: A total of 17 pts enrolled with 16 evaluable for response. At 12 weeks, 19% experienced partial responses (PR), 44% stable disease (SD), and 38% progressive disease. The clinical benefit rate (PR+SD) at 12 weeks was 63%. Safety profile was consistent with previous reports. CTCs were detected in 80% of pts. NanoVelcro CTC counts showed reduction by week 8 in both PR+SD (88%) and PD (71%) groups with re-emergence at progression. Among pts with liver metastases, very-small-nuclear CTCs ( < 8.5 μm) were seen in 29% of pts with clinical benefit compared to 60% in non-benefiters. Analysis of monocyte polarization after initiation of therapy showed that reduction of M1 polarization was associated with improvement in bone pain and/or bone scan. Conclusions: In heavily-pretreated mCRPC-VM, cabo provided clinical benefit with acceptable toxicity. Circulating biomarkers related to both tumor and microenvironment may be useful in identifying patients who benefit from this type of therapeutic approach. Clinical trial information: NCT01834651.

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.

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.

Bone metastasis is a crucial turning point in the progression of PCa and is seen in > 90% of the lethal cases. Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human prostate cancer (PCa). TBX2, a transcription factor that belongs to the T-box family negatively regulates p21 cell cycle inhibitor and is known to play critical roles during embryonic development and cancer progression. Interestingly, a recent report found TBX2 over-expression in the bone metastases from patients with castrate resistant prostate cancer. We found TBX2 over-expression in human prostate cancer specimens and in aggressive androgen independent human PCa cell lines. Further, we found TBX2 over-expression in bone metastases in multiple xenograft mouse models of human PCa progression. Blocking endogenous TBX2 in PC3 and ARCaPM PCa cells through the use of a dominant negative construct resulted in decreased proliferation, colony formation and invasion in vitro. Utilizing various pre-clinical xenograft mouse models, we found that blocking endogenous TBX2 in PCa cells leads to decreased invasion and abrogation of bone and soft tissue metastasis in vivo. Further, utilizing the intra-tibial mouse model of growth in the bone microenvironment, we found that blocking endogenous TBX2 in PCa cells dramatically reduces bone remodeling. Additionally we found that TBX2 acts upstream of the canonical WNT (WNT3A) signaling in PCa and positively regulates its transcription by binding to its promoter. Further, rescue of WNT3A levels in PCa cells in the context of blocking endogenous TBX2 rescues the metastatic ability of the cells in vivo. Taken together, our findings highlight a novel and critical role of TBX2 in PCa progression, metastasis and the subsequent bone remodeling, and that TBX2 function in PCa is mediated through the canonical WNT signaling. Citation Format: Srinivas Nandana, Manisha Tripathi, Peng Duan, Gina Chu, Haiyen E. Zhau, Robert J. Matusik, Leland W.K. Chung. Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling. [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 4131.

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the β3 chain of Ln-332. N-terminal sequencing identified the cleavage site at β3 Arg245, in a sequence context (SQLR245↓LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.

The androgen receptor (AR) plays a central role in the development of castration-resistant prostate cancer (CRPC), which is a major challenge for hormone therapy of advanced/metastatic prostate cancer (PCa). Here, we demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally inactive AR for ubiquitination-dependent degradation, and this promotes the subsequent recruitment of p300-bound, transcriptionally active AR to a select group of AR target gene promoters/enhancers. Consequently, Siah2 promotes the expression of ∼13% of androgen-responsive genes involved in lipid metabolism, cell motility and proliferation. The Siah2 binding sites located within the AR ligand-binding domain are mutated in PCa, resulting in attenuation of Siah2-mediated regulation. Siah2 is required for growth of PCa cells under the androgen-deprivation condition in vitro and in vivo. Significantly, inhibition of Siah2 promotes PCa regression upon castration and Siah2 expression is markedly increased in human CRPCs. Collectively our findings identify a key role for Siah2 in CRPC through the selective regulation of AR transcriptional activity. Citation Format: Jianfei Qi, Manisha Tripathi, Natasha Sahgal, Ladan Fazil, Susan Ettinger, William J. Placzek, Giuseppina Claps, Leland W.K. Chung, David Bowtell, Martin Gleave, Neil Bhowmick, Ze'ev Ronai. The E3 ubiquitin ligase Siah2 regulates the androgen receptor activity and contributes to castration-resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5456. doi:10.1158/1538-7445.AM2013-5456 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human Prostate Cancer (PCa). TBX2, a transcription factor that belongs to the T-box family is known to play critical roles in the embryonic development of the organisms. TBX2 acts as a transcriptional repressor by negatively regulating p21 in humans and p19 ARF in mice. Further, TBX2 has been reported to interact with Retinoblastoma (Rb) and mediates its functional specificity. Previously, TBX2 was found to be over-expressed in BRCA1 and BRCA2 associated breast tumors and melanoma and pancreatic cancer cell lines. Interestingly, a recent report found that TBX2 expression is increased 10-fold at the mRNA level in an androgen-independent bone lesion from a human PCa patient when compared to a bone lesion from an androgen-responsive PCa patient. TBX2 was found to be over-expressed in more aggressive human PCa cell lines and that TBX2 expression correlates with BMP2 expression in these cell lines. Addition of BMP2 to androgen-responsive LAPC4 human prostate cancer cells robustly induces TBX2 expression. Androgen-independent PC3 cells, expressing wild-type TBX2, when infected with the dominant negative TBX2 retroviral construct (PC3-TBX2 DN), display decreased PTHrP, RANKL and SMAD 1,5,8. These results suggest that TBX2, a downstream BMP2 target, csignificantly controls cell signaling network in prostate cancer cells. In vivo, PC3-TBX2 DN cells tagged with firefly luciferase when injected intra-cardiacally in athymic mice lost their ability to metastasize to bone and soft tissues in mice when compared with the control vector infected cells. These results are supported by reduced invasive ability of the PC3-TBX2 DN tumors at the interface between tumor and kidney when grafted under the sub-renal capsule of the male athymic nude mice. Further, utilizing the intra-tibial mouse model, we have found that blocking endogenous TBX2 in PC3 cells significantly reduces their ability to grow in the bone microenvironment. Interestingly, blocking TBX2 does not affect tumor size suggesting that TBX2 is a metastasis suppressor gene. Further, the non-metastatic TrampC2 mouse prostate cancer cells stably over-expressing RANKL show increased Tbx2 expression and develop soft tissue metastases when injected intra-cardiacally in C56/Bl6 immune competent mice. These results suggest that Tbx2 and RANKL pathways cross-talk with each other in prostate cancer metastasis. In summary, we have found that blocking endogenous TBX2 levels in human PCa cells leads to decreased invasion, metastasis and growth in the bone microenvironment, and that TBX2 potentially mediates its effect through BMP2 and RANKL pathways. TBX2, therefore, is a tumor metastasis suppressor and therapeutic target for PCa metastasis. Citation Format: Srinivas Nandana, Manisha Tripathi, Murali Gururajan, Gina Chu, Robert J. Matusik, Leland W.K. Chung. Blocking endogenous TBX2 expression abrogates prostate cancer bone metastasis in a xenograft mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C23.