Unraveling the underlying mechanisms of tumor growth and metastasis is critical to developing curative strategies against castration-resistant prostate cancer (CRPC). Recent findings demonstrate that tumor-recruited adipose stem cells (ASCs) can promote tumor growth, especially in obese patients. Unlike normal counterparts, our preliminary data showed that factors secreted by PC cells induce phenotypic genotypic changes in patient-derived ASCs (pASCs) and subvert them to undergo neoplastic transformation. Microvesicles (MVs) (50-200 nm) from PC cells (C4-2B and PC-3), but not normal prostate epithelial cells (RWPE1), primed tumor-tropic pASCs to form neoplastic lesions with cytogenetic aberrations reminiscent of the molecular features of prostate tumors and mesenchymal-to-epithelial transition (MET). The oncogenic 'reprogramming'of pASCs was associated with the transfer of a number of oncogenic miRNAs (miR-125b, miR-130b, miR-155) and oncogenic mRNAs (H-Ras and K-Ras), which in turn down-regulated several key tumor suppressors (TP53, PDCD4 and Lats2) in pASCs. Our data deciphered previously uncharacterized roles for tumor-derived extracellular RNAs (exRNAs) in promoting tumor growth via the release and uptake of MVs by the recipient pASCs. Cancer patient's sera contain high levels of circulating MVs in comparison to normal subjects;thus it is possible to speculate the transfer of oncogenic exRNA cargo by the tumor-derived MVs enable neoplastic transformation of pASCs in cancer patients. Accordingly, targeting MVs biogenesis and release by tumor cells and/or uptake by pASCs, rather than individual exRNAs, would be more efficacious in abrogating the transfer and tumor development by multiple oncogenic exRNAs in pASCs. We therefore hypothesize that a high throughput screening (HTS) of clinically approved compounds will enable us to select lead agents that suppress the biogenesis and release of MVs from tumor cells and/or the uptake of MVs by pASCs. We will corroborate our hypothesis by utilizing milestone-driven experiments in cell culture models and by proof-of-concept pre-clinical studies in animal models of PC. The UH2 (phase-I) will identify compounds that potently abrogate the biogenesis, release and/or uptake of MVs in vitro and in vivo. The UH3 (phase-II) will further validate the in vivo anti-tumor efficacy of these pharmacological leads and will support more rigorous milestone-driven pre-clinical studies in animal models. The following specific aims, to be executes through two phases, corroborate our hypothesis: Phase-I (UH2) (1) Demonstrate if circulating MVs procured from CRPC patients harbor oncogenic exRNAs and induce neoplastic transformation of pASCs. (2) Optimize HTS assays to identify lead compounds which inhibit the release of MVs by tumor cells, or their uptake by recipient pASCs, or reduce their oncogenic miRNA/RNA load. (3) Establish if lead compounds inhibit tumor cell release and/or uptake of MVs by pASCs in vivo. Phase II (UH3) (1) Examine the efficacy of lead compounds in inhibiting ASC-derived tumor development in vivo. (2) Clinical applicability of the lead compounds from the NCATS library. Although MVs have been implicated in cancer progression (neovacularization), their role in neoplastic transformation of stem cells in cancer patients has not been investigated. Our demonstration of tumor cell derived MVs in transfer of extracellular onco-RNAs and transformation of patient procured ASCs is novel and lend credence to their potential role in outgrowth and/or progression of metastatic disease in cancer patients. Accordingly, the proposed work is innovative, because it capitalizes not only in underpinning discovery of new functional roles for MV-mediated onco-miRs and onco-mRNAs, but also in identifying new lead therapeutic compounds to circumvent PC progression. By establishing preventive and/or therapeutic intervention strategies, the outcome of the proposed studies is expected to exert a positive impact on the clinical management of advanced PC.

Public Health Relevance

Unraveling the underlying mechanisms of tumor growth and metastasis is critical to developing curative strategies against castration-resistant prostate cancer (CRPC). Our in vitro and in vivo preliminary data (Figs 1- 6) provide ccompelling evidence that tumor-derived MVs is a major underlying molecular culprit in neoplastic transformation of patient-derived adipose stem cells (pASCs). Specifically, the transfer of oncogenic exRNAs (miRNAs/mRNAs) by tumor cell-derived MVs to pASCs cause formation of prostate tumors that are marked with genetic instability, neovasculature and mesenchymal-to-epithelial transition (MET). Our data deciphered previously uncharacterized roles for tumor-derived extracellular RNAs (exRNAs) in promoting tumor growth via the release and uptake of MVs by the recipient pASCs. Cancer patient's sera contain high levels of circulating MVs in comparison to normal subjects;thus it is possible to speculate the transfer of oncogenic exRNA cargo by the tumor-derived MVs enable neoplastic transformation of pASCs in cancer patients. Accordingly, targeting MVs biogenesis and release by tumor cells and/or uptake by pASCs, rather than individual exRNAs, would be more efficacious in abrogating the transfer and tumor development by multiple oncogenic exRNAs in pASCs. We therefore hypothesize that a high throughput screening (HTS) of clinically approved compounds will enable us to select lead agents that suppress the biogenesis and release of MVs from tumor cells and/or the uptake of MVs by pASCs. Over the past decade, High Throughput Screening (HTS) emerged as a way to screen a large number of compound libraries against multiple targets while simultaneously reducing development and operating costs. Indeed, several scientific advances have driven the need for improved drug discovery screening technology through HTS. HTS can be considered the process in which batches of compounds are tested for binding or biological activity against target molecules. Today, many pharmaceutical companies are screening 100,000- 300,000 or more compounds per screen to produce approximately 100-300 hits. The invention of automated plate-handling robotics and robust miniaturized assays, primarily through 384-well and higher density plates, has made HTS a reality. The ultimate goal of the proposed research is to exploit this technology to identify quality leads with the desirable drug like properties tat would effectively inhibit release of MVs, and consequently oncogenic exRNAs, by tumor cells and/or uptake by pASCs. The MV targeting drugs are expected to reduce or circumvent tumor burden and progression in cancer patients. During phase I (UH2) of the proposed research, the cell-, and animal based initial HTS screenings are expected to identify hits (10-20) that inhibit biogenesis/secretion of MVs by prostate tumor cells and/or uptake of MVs by the recipient cells (pASCs). During Phase 2 (UH3) further validation studies will be conducted to examine and validate if inhibition of MVs release and/or uptake by the lead compounds is associated with tumor formation by pASCs in vivo. Additionally, in phase 2 of the project, safety, tolerability, phamacokinetics and pharmacodynamics of the lead compounds will be assessed for clinical utility in humans.

Agency
National Institute of Health (NIH)
Institute
National Center for Advancing Translational Sciences (NCATS)
Type
Exploratory/Developmental Cooperative Agreement Phase I (UH2)
Project #
5UH2TR000928-02
Application #
8711591
Study Section
Special Emphasis Panel (ZRG1-OBT-Z (50))
Program Officer
Tagle, Danilo A
Project Start
2013-08-01
Project End
2016-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
2
Fiscal Year
2014
Total Cost
$425,691
Indirect Cost
$132,246
Name
Tulane University
Department
Urology
Type
Schools of Medicine
DUNS #
053785812
City
New Orleans
State
LA
Country
United States
Zip Code
70118