TBX2 Drives Neuroendocrine Prostate Cancer through Exosome-Mediated Repression of miR-200c-3p.

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.

How Prostate Cancer Cells Use Strategy Instead of Brute Force to Achieve Metastasis.

Akin to many other cancers, metastasis is the predominant cause of lethality in prostate cancer (PCa). Research in the past decade or so has revealed that although metastatic manifestation is a multi-step and complex process that is orchestrated by distinct cellular and molecular mechanisms, the process in itself is an extremely inefficient one. It is now becoming increasingly evident that PCa cells employ a plethora of strategies to make the most of this inefficient process. These strategies include priming the metastatic sites ahead of colonization, devising ways to metastasize to specific organs, outsmarting the host defense surveillance, lying in a dormant state at the metastatic site for prolonged periods, and widespread reprogramming of the gene expression to suit their needs. Based on established, recent, and evolving lines of research, this review is an attempt to understand PCa metastasis from the perspective of military combat, wherein strategic maneuvering instead of brute force often plays a decisive role in the outcome.

Regulatory signaling network in the tumor microenvironment of prostate cancer bone and visceral organ metastases and the development of novel therapeutics.

This article describes cell signaling network of metastatic prostate cancer (PCa) to bone and visceral organs in the context of tumor microenvironment and for the development of novel therapeutics. The article focuses on our recent progress in the understanding of: 1) The plasticity and dynamics of tumor-stroma interaction; 2) The significance of epigenetic reprogramming in conferring cancer growth, invasion and metastasis; 3) New insights on altered junctional communication affecting PCa bone and brain metastases; 4) Novel strategies to overcome therapeutic resistance to hormonal antagonists and chemotherapy; 5) Genetic-based therapy to co-target tumor and bone stroma; 6) PCa-bone-immune cell interaction and TBX2-WNTprotein signaling in bone metastasis; 7) The roles of monoamine oxidase and reactive oxygen species in PCa growth and bone metastasis; and 8) Characterization of imprinting cluster of microRNA, in tumor-stroma interaction. This article provides new approaches and insights of PCa metastases with emphasis on basic science and potential for clinical translation. This article referenced the details of the various approaches and discoveries described herein in peer-reviewed publications. We dedicate this article in our fond memory of Dr. Donald S. Coffey who taught us the spirit of sharing and the importance of focusing basic science discoveries toward translational medicine.

Modulation of cabozantinib efficacy by the prostate tumor microenvironment.

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 IC50 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.

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.

SRC family kinase FYN promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer.

FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PC3 cells with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients.

Prostate cancer progression and metastasis: potential regulatory pathways for therapeutic targeting.

Skeletal metastasis in advanced prostate cancer (PCa) patients remains a significant cause of morbidity and mortality. Research utilizing animal models during the past decade has reached a consensus that PCa progression and distant metastasis can be tackled at the molecular level. Although there are a good number of models that have shown to facilitate the study of PCa initiation and progression at the primary site, models that mimic the distant dissemination of cancer cells, particularly bone metastasis, are scarce. Despite this limitation, the field has gleaned valuable knowledge on the underlying molecular mechanisms and pathways of PCa progression, including local invasion and distant metastasis, and has moved forward in developing the concepts of current therapeutic modalities. The purpose of this review is to put together recent work on pathways that are currently being targeted for therapy, as well as other prospective novel therapeutic targets to be developed in the future against metastatic and potentially lethal PCa in patients.

miR-409-3p/-5p promotes tumorigenesis, epithelial-to-mesenchymal transition, and bone metastasis of human prostate cancer.

PURPOSE: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. EXPERIMENTAL DESIGN: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. RESULTS: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle-treated cells. CONCLUSION: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer.

Hepsin cooperates with MYC in the progression of adenocarcinoma in a prostate cancer mouse model.

BACKGROUND: Hepsin is a cell surface protease that is over-expressed in more than 90% of human prostate cancer cases. The previously developed Probasin-hepsin/Large Probasin-T antigen (PB-hepsin/LPB-Tag) bigenic mouse model of prostate cancer demonstrates that hepsin promotes primary tumors that are a mixture of adenocarcinoma and neuroendocrine (NE) lesions, and metastases that are NE in nature. However, since the majority of human prostate tumors are adenocarcinomas, the contribution of hepsin in the progression of adenocarcinoma requires further investigation. METHODS: We crossed the PB-hepsin mice with PB-Hi-myc transgenic mouse model of prostate adenocarcinoma and characterized the tumor progression in the resulting PB-hepsin/PB-Hi-myc bigenic mice. RESULTS: We report that PB-hepsin/PB-Hi-myc bigenic mice develop invasive adenocarcinoma at 4.5 months. Further, histological analysis of the 12- to 17-month-old mice revealed that the PB-hepsin/PB-Hi-myc model develops a higher grade adenocarcinoma compared with age-matched tumors expressing only PB-Hi-myc. Consistent with targeting hepsin to the prostate, the PB-hepsin/PB-Hi-myc tumors showed higher hepsin expression as compared to the age-matched myc tumors. Furthermore, endogenous expression of hepsin increased in the PB-Hi-myc mice as the tumors progressed. CONCLUSIONS: Although we did not detect any metastases from the prostates in either the PB-hepsin/PB-Hi-myc or the PB-Hi-myc mice, our data suggests that hepsin and myc cooperate during the progression to high-grade prostatic adenocarcinoma.

Candidate metastasis suppressor genes uncovered by array comparative genomic hybridization in a mouse allograft model of prostate cancer.

BACKGROUND: The purpose of this study was to identify candidate metastasis suppressor genes from a mouse allograft model of prostate cancer (NE-10). This allograft model originally developed metastases by twelve weeks after implantation in male athymic nude mice, but lost the ability to metastasize after a number of in vivo passages. We performed high resolution array comparative genomic hybridization on the metastasizing and non-metastasizing allografts to identify chromosome imbalances that differed between the two groups of tumors. RESULTS: This analysis uncovered a deletion on chromosome 2 that differed between the metastasizing and non-metastasizing tumors. Bioinformatics filters were employed to mine this region of the genome for candidate metastasis suppressor genes. Of the 146 known genes that reside within the region of interest on mouse chromosome 2, four candidate metastasis suppressor genes (Slc27a2, Mall, Snrpb, and Rassf2) were identified. Quantitative expression analysis confirmed decreased expression of these genes in the metastasizing compared to non-metastasizing tumors. CONCLUSION: This study presents combined genomics and bioinformatics approaches for identifying potential metastasis suppressor genes. The genes identified here are candidates for further studies to determine their functional role in inhibiting metastases in the NE-10 allograft model and human prostate cancer.

Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression.

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 beta3 chain of Ln-332. N-terminal sequencing identified the cleavage site at beta3 Arg(245), in a sequence context (SQLR(245) 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.

Prostate epithelial cell fate.

Androgen receptor (AR) within prostatic mesenchymal cells, with the absence of AR in the epithelium, is still sufficient to induce prostate development. AR in the luminal epithelium is required to express the secretory markers associated with differentiation. Nkx3.1 is expressed in the epithelium in early prostatic embryonic development and expression is maintained in the adult. Induction of the mouse prostate gland by the embryonic mesenchymal cells results in the organization of a sparse basal layer below the luminal epithelium with rare neuroendocrine cells that are interdispersed within this basal layer. The human prostate shows similar glandular organization; however, the basal layer is continuous. The strong inductive nature of embryonic prostatic and bladder mesenchymal cells is demonstrated in grafts where embryonic stem (ES) cells are induced to differentiate and organize as a prostate and bladder, respectively. Further, the ES cells can be driven by the correct embryonic mesenchymal cells to form epithelium that differentiates into secretory prostate glands and differentiated bladders that produce uroplakin. This requires the ES cells to mature into endoderm that gives rise to differentiated epithelium. This process is control by transcription factors in both the inductive mesenchymal cells (AR) and the responding epithelium (FoxA1 and Nkx3.1) that allows for organ development and differentiation. In this review, we explore a molecular mechanism where the pattern of transcription factor expression controls cell determination, where the cell is assigned a developmental fate and subsequently cell differentiation, and where the assigned cell now emerges with it's own unique character.

PATE gene clusters code for multiple, secreted TFP/Ly-6/uPAR proteins that are expressed in reproductive and neuron-rich tissues and possess neuromodulatory activity.

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATE-like genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region of the sperm acrosome and at high levels on malignant prostatic epithelial cells. The syntenic mouse Pate-like locus encompasses 14 active genes coding for secreted proteins, which are all, except for Pate-P and Pate-Q, expressed primarily in prostate and/or testis. Pate-P and Pate-Q are expressed solely in placental tissue. Castration up-regulates prostate expression of mouse Pate-B and Pate-E, whereas testosterone ablates this induced expression. The sequence similarity between TFP/Ly-6/uPAR proteins that modulate activity of nicotinic acetylcholine receptors and the PATE (Pate)-like proteins stimulated us to see whether these proteins possess analogous activity. Pharmacological studies showed significant modulation of the nicotinic acetylcholines by the PATE-B, Pate-C, and Pate-P proteins. In concert with these findings, certain PATE (Pate)-like genes were extensively expressed in neuron-rich tissues. Taken together, our findings indicate that in addition to participation of the PATE (Pate)-like genes in functions related to fertility and reproduction, some of them likely act as important modulators of neural transmission.

Rescue of TNFalpha-inhibited neuronal cells by IGF-1 involves Akt and c-Jun N-terminal kinases.

Proinflammatory cytokines, especially tumor necrosis factor alpha (TNFalpha), is a pleiotropic mediator of a diverse array of physiologic and neurologic functions and is upregulated during various inflammatory and neurodegenerative diseases. A common survival response during such situations is the increased expression of the hormone insulin-like growth factor 1 (IGF-1). Although it was thought previously that the mechanisms of TNFalpha and IGF-1 action were unrelated, it has been shown that low doses of TNFalpha can inhibit the survival effects of IGF-1 in mouse cerebellar granule neurons. We used a neuronal cell line SH-SY5Y, which underwent apoptosis in response to TNFalpha and this process could be reversed substantially by IGF-1. Crosstalk between signaling pathways of these two factors was found at various points downstream of their signal transduction. To determine the mechanisms of IGF-1-mediated rescue, we looked at the MAP kinases, which are known to be involved in IGF-1 as well as TNFalpha signaling. The c-Jun N-terminal kinase pathway, which is known normally to promote cell death, was found to actually promote survival of TNFalpha-mediated cell death. Inhibiting the c-Jun survival pathway completely reversed the rescue mediated by IGF-1. In addition, the Akt pathway played an equally important role in this rescue.