oa Stem Cell-Based Selective Delivery of Alpha Keto Reductases for Therapeutic Targeting of Residual Androgens in Metastatic Prostate Cancer

Introduction: The de novo synthesis and “intracrine” production of androgens by prostate tumors provides a significant survival advantage leading to the outgrowth of castration resistant tumors. Currently, targeting residual androgens in metastatic prostate cancer (PC) microenvironment is the toughest challenge in the clinical management of advanced PC patients treated with androgen ablation therapy (AAT), as they are implicated for development of castration-resistant PC (CRPC). Our previous studies (Stem Cells, 2014) demonstrate that PC-derived adipose derived stem cells (pASCs) promote tumor growth and undergo PC mimicry through neoplastic reprogramming. The present study examines the role of pASCs in synthesizing and producing and rogens and its potential in supporting growth of hormone-dependent PC cells under castrate conditions in vitro and in vivo. Further more, the study attempts to identify key players among the and rogen-metabolizing enzymes (AMEs) in the backdoor pathway for targeting and degradation of androgen synthesis in the prostate tumor microenvironment, by both in vitro and in vivo assays. Methods: Based on an IRB approved protocol, the pASCs were isolated from fatt issues procured from PC patients undergoing radical prostatectomies. The purity of the pASC isolates was validated by FACS analysis and differentiation assays. The ability of pASCs exposed to PC-derived conditioned media (CM) to produce androgens was monitored by expression of AMEs (PCR) and androgen release (EIAkit). The ability of pASC CM to support growth of androgen-dependent LNCaP cells was assessed by MTT assay and growth of LNCaP tumors co-transplanted with pASCs in castrated mice in vivo. Normal donor-derived adipose derived stem cells (nASCs) were enriched for tumor tropicity by a transwell system. Next, we subcloned in a GFP bicistronic IRES lentivirus construct (pLVX-IRES-ZsGreen1) the rat androgen hydrolyzing enzyme 3α-hydroxysteroid dehydrogenase (Type I, 3α-HSD), as known as AKRIC14, in-frame with IL-2SS sequence at the N-terminus. R1C14. The ILL2SS sequencec enables the AKR1C14 by the transduced cells. Upon transduction, the enriched nASC population was monitored for the transgene expression and release by qRT-PCR, immunoblotting and EIA kit. The efficacy and functional activity of the recombinant enzyme and the nASC-released ANR1C14 enzyme were monitored by MTT, LDH and luciferase reporterassays in the androgen-dependent LNCaP cells in vitro. The tumor-homing potential of the GFP-AKRC14 expressing nASCs infused by IV route was validated by their engraftment in LNCaP tumor-bearing mice. To examine the efficacy of secreted enzyme to hydrolyze androgens andinduce tumor regression in vivo, the GFP-AKR1C14 transduced nASCs were injected in mice bearing LNCaP tumors and tumor volumes were measured weekly. Results: We demonstrate here in that unlike normal adipose-tissue derived stem cells (nASCs), the PC cell microenvironment subverts tumor-tropic PC patients' derived ASCs (pASCs) to synthesize and produce androgens in the tumor microenvironment. The pASC-mediated hormone production was sufficient to support the growth of androgen-dependent LNCaP PC cells in vitro under hormone-deprivation conditions as well as in castrated nude mice. Comparative analysis of gene expression of AMEs in microdissected cells versus the adjacent normal tissue revealed AKR1C4, an alpha keto reductase, to be the most promising candidate for targeting the backdoor pathway in dihydrotestesterone (DHT) synthesis. Type 1 3α-HSD enzyme has been shown to readily oxidize testosterone to Δ4-androstene-3, 17-dione in a substrate dependent reaction. The recombinant AKR1C4 induced cytotoxicity and apoptosis in androgen-dependent PC cells in vitro. Next, using a GFP bicistronic IRES lentivirus construct, we demonstrate that the genetically engineered tumor-tropic nASCs enable expression and secretion of AKR1C14. The results showed that the engineered nASCs are capable of expressing the enzyme, which in turn catabolizes androgens in vitro and induce apoptosis in androgen-dependent LNCaP cells. The AKR1C14 transduced nASCs successfully engrafted in and reduced growth of LNCaP tumors in nude mice. Conclusions: Together, our data demonstrates that selective delivery of alpha keto reductases by nASCs could eliminate residual androgens and further paves the way for their potential use in combination with AAT for treatment of CRPC.