Sarah Truong

Therapy-induced developmental reprogramming of prostate cancer cells and acquired therapy resistance

Treatment-induced neuroendocrine transdifferentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR+/PSA+ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem medium. Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells and tumor-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or return back to an AR+ prostate-like state. Once returned, the AR+ cells were resistant to androgen signaling inhibition. Acute androgen deprivation or anti-androgen treatment in serum-containing medium led to the transient appearance of a sub-population of cells with similar characteristics. Finally, a 132 gene signature derived from reprogrammed PCa cell lines distinguished tumors from PCa patients with adverse outcomes. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumors from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumor behaviors.

Determinants of Gli2 co-activation of wildtype and naturally truncated androgen receptors

Gli2, a transcription factor in the Hedgehog pathway, is overexpressed in castrate‐resistant prostate cancer (PCa). Previously we showed that Gli2 overexpression increased transcriptional activity of androgen receptor (AR) and conferred androgen growth‐independence to normally growth‐dependent PCa cells. Here we localized the regions of AR‐Gli2 protein interaction and determined the domains within Gli2 needed for AR co‐activation.

Non-canonical activation of hedgehog in prostate cancer cells mediated by the interaction of transcriptionally active androgen receptor proteins with Gli3

Hedgehog (Hh) is an oncogenic signaling pathway that regulates the activity of Gli transcription factors. Canonical Hh is a Smoothened- (Smo-) driven process that alters the post-translational processing of Gli2/Gli3 proteins. Though evidence supports a role for Gli action in prostate cancer (PCa) cell growth and progression, there is little indication that Smo is involved. Here we describe a non-canonical means for activation of Gli transcription in PCa cells mediated by the binding of transcriptionally-active androgen receptors (ARs) to Gli3. Androgens stimulated reporter expression from a Gli-dependent promoter in a variety of AR + PCa cells and this activity was suppressed by an anti-androgen, Enz, or by AR knockdown. Androgens also upregulated expression of endogenous Gli-dependent genes. This activity was associated with increased intranuclear binding of Gli3 to AR that was antagonized by Enz. Fine mapping of the AR binding domain on Gli2 showed that AR recognizes the Gli protein processing domain (PPD) in the C-terminus. Mutations in the arginine-/serine repeat elements of the Gli2 PPD involved in phosphorylation and ubiquitinylation blocked the binding to AR. β-TrCP, a ubiquitin ligase that recognizes the Gli PPD, competed with AR for binding to this site. AR binding to Gli3 suppressed its proteolytic processing to the Gli3 repressor form (Gli3R) whereas AR knockdown increased Gli3R. Both full-length and truncated ARs were able to activate Gli transcription. Finally, we found that an ARbinding decoy polypeptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR. Exogenous overexpression of this decoy suppressed Gli transcriptional activity in PCa cells. Collectively, this work identifies a novel pathway for non-canonical activation of Hh signaling in PCa cells and identifies a means for interference that may have clinical relevance for PCa patients.

Paracrine sonic hedgehog signaling contributes significantly to acquired steroidogenesis in the prostate tumor microenvironment.

Despite the substantial benefit of androgen deprivation therapy (ADT) for metastatic prostate cancer, patients often progress to castration‐resistant disease (CRPC) that is more difficult to treat. CRPC is associated with renewed androgen receptor activity in tumor cells and restoration of tumor androgen levels through acquired intratumoral steroidogenesis (AIS). Although prostate cancer (PCa) cells have been shown to have steroidogenic capability in vitro, we previously found that benign prostate stromal cells (PrSCs) can also synthesize testosterone (T) from an adrenal precursor, DHEA, when stimulated with a hedgehog (Hh) pathway agonist, SAG. Here, we show exposure of PrSCs to a different Smoothened (Smo) agonist, Ag1.5, or to conditioned medium from sonic hedgehog overexpressing LNCaP cells induces steroidogenic enzyme expression in PrSCs and significantly increases production of T and its precursor steroids in a Smo‐dependent manner from 22‐OH‐cholesterol substrate. Hh agonist‐/ligand‐treated PrSCs produced androgens at a rate similar to or greater than that of PCa cell lines. Likewise, primary bone marrow stromal cells became more steroidogenic and produced T under the influence of Smo agonist. Treatment of mice bearing LNCaP xenografts with a Smo antagonist, TAK‐441, delayed the onset of CRPC after castration and substantially reduced androgen levels in residual tumors. These outcomes support the idea that stromal cells in ADT‐treated primary or metastatic prostate tumors can contribute to AIS as a consequence of a paracrine Hh signaling microenvironment. As such, Smo antagonists may be useful for targeting prostate tumor stromal cell‐derived AIS and delaying the onset of CRPC after ADT.

PD33-01 NON-CANONICAL ACTIVATION OF HEDGEHOG SIGNALING IN PROSTATE CANCER CELLS MEDIATED BY THE BINDING OF TRANSCRIPTIONALLY ACTIVE ANDROGEN RECEPTORS TO GLI TRANSCRIPTION FACTORS

hospitals with excess readmission rates for certain targeted conditions. We aim to evaluate the effect of this program on targeted and nontargeted surgical conditions. METHODS: We used a 20% Medicare sample to identify readmissions following targeted (total hip arthroplasty, total knee arthroplasty) and non-targeted (cystectomy, abdominal aortic aneurysm repair, colectomy, lung resection) procedures from 2006 to 2014. Multivariable logistic regression was used to calculate adjusted readmission rates. Changes in hospital level readmission rates were analyzed for three distinct time periods (Pre, Measurement, Penalty) corresponding to the HRRP implementation timeline, using an interrupted time series approach. RESULTS: We identified 538,293 targeted and 165,432 nontargeted procedures performed at 2,779 hospitals. There was a significant decrease in the odds of readmission for all procedures, except cystectomy (Table) which also had the highest readmission rate at 27%. Prior to the policy, the readmission rate for non-targeted procedures was decreasing faster than that of targeted procedures (Figure). However, this trend reversed during the Measurement period (difference in slope for targeted to non-targeted -0.10 [95% Confidence Interval -0.16 to -0.044]). Neither group had a significant change during the Penalty period. CONCLUSIONS: While the HRRP effectively decreased readmissions for targeted surgery, there was no spillover benefit for nontargeted procedures. To decrease the burden of readmission after cystectomy, future efforts should focus on identifying the interventions that resulted in readmission reduction for targeted procedures and evaluating their effectiveness in this population.

Abstract 4492: Androgen receptor affects GLI activity by promoting transcriptionally active states of GLI upon androgen stimulation

Introduction: While “castration” therapies benefit metastatic prostate cancer (PCa) patients, their effects are transient and progression to castration resistant disease (CRPC) remains a vexing clinical problem. Despite low levels of circulating androgens in treated patients, CRPC typically remains dependent on androgen/androgen receptor (AR) signaling for further growth. Previously we showed that Gli protein binding to AR co-activates AR transcriptional activity and supports androgen-independent growth. Here we show evidence that the interaction of AR with Gli proteins, particularly Gli3, allows for a “non-canonical” activation of Hedgehog signaling that contributes to PCa cell growth and progression to CRPC. Methods: Androgen growth-dependent (LNCaP) or -independent (LNCaP-AI, LN95) PCa cells were transfected with a Gli-luciferase (Gli-luc) reporter and were treated with R1881, enzalutamide (Enz), or a combination. Luciferase activity was measured. R1-D567 (harboring a genomic deletion of AR) served as a negative control. Knockdown of AR-FL with siRNA or overexpression of a small decoy peptide from the Gli2 AR-binding domain (Gli-DP) was also performed in above cells. Western blot and real-time PCR confirmed both AR and Gli target gene/protein expressions in LNCaP and LNCaP-AI cells (-/+ R1881). Sonic hedgehog (Shh) ligand-responsive 3T3-E1 cells were transiently expressed with AR-FL to confirm the involvement of canonical Shh-Gli activation pathway in AR co-activation of Gli transcription. Co-immunoprecipitation (co-IP) was performed to verify the blocking efficiency of Gli-DP in AR-Gli3 interaction in LN95 cells and AR-overexpressing 293FT. Results: Androgen (R1881) treatment strongly induced Gli transcription activity in AR+ PCa cells while Enz reversed this effect; AR-FL-specific knockdown or exogenous Gli-DP expression suppressed androgen-induced Gli reporter expression as well as expression of endogenous Gli-responsive genes. R1881 treatment promoted Gli2/3 protein processing to transcriptionally active states (Gli3-FullLength and Gli2-Active) and enhanced the expression of Gli-target genes, Gli1/Ptch1, at protein and/or mRNA levels. Co-IP revealed that exogenous Gli-DP blocked the interaction between AR and transcriptionally active Gli3-FL in PCa cells. Overexpression of AR in 3T3-E1 cells confirmed that AR interaction with Gli stabilizes the active Gli3-FL form by suppressing degradation of Gli3 in a manner independent of the canonical Shh-Gli activation pathway. Conclusions: Our findings further identify the importance of AR-Gli interaction in progression of PCa to CRPC and shows that cooperative binding of AR to Gli proteins enhances both AR and Hedgehog signaling in PCa cells. Interference with the AR-Gli interaction using a decoy peptide provides a means to suppress AR activation of Hedgehog in PCa cells. Citation Format: Sarah Truong, Na Li, Mannan Nouri, Ralph Buttyan. Androgen receptor affects GLI activity by promoting transcriptionally active states of GLI upon androgen stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2017-4492

Abstract 5574: Gli2 protein and the AR operational switch to the castration resistant prostate cancer transcription program

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Gli proteins are transcription factors that mediate Hedgehog (HH) signaling in cells. Previously, we reported that Gli2 binds to the androgen receptor (AR) protein in prostate cancer (PCa) cells and further upregulates expression of genes under AR control. This “co-activator” effect allows the growth of Gli2 overexpressing LNCaP cells in androgen-depleted medium, mimicking the behavior of castrate resistant variants of PCa. We report here our success in localizing the specific binding domains between Gli2 and AR proteins. Outcomes of GST-pulldown and co-immunoprecipitation studies showed that Gli2 binds to AR via a limited domain (aa628-aa897) within the C-terminal region and this recognizes the tau5/AF5 domain (aa 392-558) within the N-terminal region of the AR protein. Because tau5/AF5 is conserved in truncated (splice variant) ARs, we also showed that Gli2 recognizes and co-activates the transcriptional activity of a truncated variant AR (AR-V7). To further define Gli2 regulatory functions on AR transcriptional activity in CRPC, we overexpressed Gli2ΔN (a constitutive-active form of Gli2) in LNCaP-AI (androgen-independent) cells and examined its effects on AR target gene expressions. Surprisingly, we found that Gli2Δ overexpression coincided with significant downregulated expression of genes considered “traditional” AR targets (i.e., KLK3 and KLK2), while increasing the expression of M-phase check-point genes, such as UBE2C, CDK1 and CDC20. In contrast, treatment of unmodified LNCaP-AI cells with the Gli-inhibitor, GANT61, significantly increased expression of KLK2 and KLK3 while downregulating UBE2C, CDC20 and CDK1. Our outcomes now imply that Gli2 differentially affects the AR transcriptional program in androgen-dependent (LNCaP) vs androgen-independent (LNCaP-AI) cells. In androgen-dependent cells, Gli2 co-activates expression of the traditional AR target genes while suppressing expression of genes involved in the CRPC transcriptional program whereas in androgen-independent cells, Gli2 co-activates expression of CRPC program genes while downregulating expression of traditional AR target genes. This dichotomy suggests that Gli2 participates in the decisions through which AR transcriptional activity is redirected in castration resistant disease. Supported by the DOD PCRP (W81XH-10-1-0493, to RB) Citation Format: Na Li, Ralph Buttyan, Sarah Truong, Yue Yu, Mengqian Chen. Gli2 protein and the AR operational switch to the castration resistant prostate cancer transcription program. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5574. doi:10.1158/1538-7445.AM2014-5574