Jennifer A. Locke

Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer.

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

Alterations in cholesterol regulation contribute to the production of intratumoral androgens during progression to castration‐resistant prostate cancer in a mouse xenograft model

Emerging evidence suggests that androgens and the androgen receptor (AR) are important mediators of castration‐resistant prostate cancer (CRPC) progression. Increased expression of several enzymes responsible for cholesterol synthesis and conversion into downstream androgens has been documented in human CRPC tumors in comparison to primary tumors. Based on these observations it is hypothesized that cholesterol and its overall regulation within the cell are altered, thus modifying precursor levels for de novo androgen synthesis within the castrate tumoral environment.

Insulin Increases De Novo Steroidogenesis in Prostate Cancer Cells

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.

Copy number alterations of c-MYC and PTEN are prognostic factors for relapse after prostate cancer radiotherapy

Despite the use of PSA, Gleason score, and T‐category as prognosticators in intermediate‐risk prostate cancer, 20–40% of patients will fail local therapy. In order to optimize treatment approaches for intermediate‐risk patients, additional genetic prognosticators are needed. Previous reports using array comparative genomic hybridization (aCGH) in radical prostatectomy cohorts suggested a combination of allelic loss of the PTEN gene on 10q and allelic gain of the c‐MYC gene on 8q were associated with metastatic disease. We tested whether copy number alterations (CNAs) in PTEN (allelic loss) and c‐MYC (allelic gain) were associated with biochemical relapse following modern‐era, image‐guided radiotherapy (mean dose 76.4 Gy). We used aCGH analyses validated by fluorescence in‐situ hybridization (FISH) of DNA was derived from frozen, pre‐treatment biopsies in 126 intermediate‐risk prostate cancer patients. Patients whose tumors had CNAs in both PTEN and c‐MYC had significantly increased genetic instability (percent genome alteration; PGA) compared to tumors with normal PTEN and c‐MYC status (p < 0.0001). We demonstrate that c‐MYC gain alone, or combined c‐MYC gain and PTEN loss, were increasingly prognostic for relapse on multivariable analyses (hazard ratios (HR) of 2.58/p = 0.005 and 3.21/p = 0.0004; respectively). Triaging patients by the use of CNAs within pre‐treatment biopsies may allow for better use of systemic therapies to target sub‐clinical metastases or locally recurrent disease and improve clinical outcomes. Cancer 2012.

Hsp27 Promotes Insulin-Like Growth Factor-I Survival Signaling in Prostate Cancer via p90Rsk-Dependent Phosphorylation and Inactivation of BAD

Hsp27 is highly expressed in castrate-resistant prostate cancer. Although its overexpression confers resistance to androgen ablation and chemotherapy, the mechanisms by which Hsp27 inhibits treatment-induced apoptosis are incompletely defined. Castrate-resistance often correlates with increased activity of autocrine and/or paracrine growth/survival stimulatory loops including the mitogen-activated protein kinase (MAPK) and Akt pathways and insulin-like growth factor (IGF) axis components. Because Hsp27 can be activated by both MAPK and Akt pathways, it is possible that interactions between IGF-I signaling and Hsp27 phosphoactivation function to promote castrate-resistant progression. Here, we report that Hsp27 expression and phosphorylation levels correlate with IGF-I signaling and castrate-resistant progression in human prostate cancer specimens and cell lines. IGF-I induces Hsp27 phosphorylation in a time- and dose-dependent manner via p90Rsk, which interacts directly with and phosphorylates Hsp27 in vitro and in vivo. Conversely, p90Rsk inhibition using short interfering RNA or a dominant negative mutant abolishes IGF-I-induced Hsp27 phosphorylation. Hsp27 overexpression increases IGF-I-induced phosphorylation of Erk, p90Rsk, and Akt. Conversely, Hsp27 knockdown abrogates IGF-I-induced phosphorylation of Erk, p90Rsk, and Akt, thereby destabilizing Bad/14-3-3 complexes and increasing apoptotic rates. These data elucidate the interactions between Hsp27 phosphorylation and the IGF-I receptor signaling pathway and support targeting Hsp27 as a therapeutic strategy for castrate-resistant prostate cancer.

Dissecting the association between metabolic syndrome and prostate cancer risk: analysis of a large clinical cohort.

BACKGROUND A biologic rationale exists for the association between metabolic syndrome (MetS) and prostate cancer (PCa). However, epidemiologic studies have been conflicting. OBJECTIVE To evaluate the association between MetS and the odds of PCa diagnosis in men referred for biopsy. DESIGN, SETTING, AND PARTICIPANTS Patients without prior PCa diagnosis undergoing prostate biopsy were identified from a large prostate biopsy cohort (in Toronto, Canada). The definition of MetS was based on the most recent interim joint consensus definition, requiring any three of five components (obesity, elevated blood pressure, diabetes or impaired fasting glucose, low high-density lipoprotein-cholesterol, and hypertriglyceridemia). Both the individual components of MetS and the cumulative number of MetS components were evaluated. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The outcomes were PCa detection overall, clinically significant PCa (CSPC; defined as any Gleason pattern ≥ 4, >50% involvement of a single biopsy core, or more than one of three total number of cores involved), and intermediate- or high-grade PCa (I-HGPC; Gleason 7-10). Tests for trend and multivariable logistic regression analyses were performed. RESULTS AND LIMITATIONS Of 2235 patients, 494 (22.1%) had MetS. No individual MetS component was independently associated with PCa. However, increasing number of MetS components was associated with higher PCa grade (p<0.001), as well as progressively higher odds of PCa outcomes (three or more; ie, MetS) compared with no MetS components: Odds ratios were 1.54 for PCa overall (95% confidence interval [CI], 1.17-2.04; p=0.002), 1.56 for CSPC (95% CI, 1.17-2.08; p=0.002), and 1.56 for I-HGPC (95% CI, 1.16-2.10; p=0.003) in multivariable analyses. The main limitation is the retrospective design. CONCLUSIONS Although the individual MetS components are not independently associated with PCa outcomes, MetS is significantly associated with higher odds of PCa diagnosis, CSPC, and I-HGPC. There is a biologic gradient between the number of MetS components and the risk of PCa, as well as cancer grade. PATIENT SUMMARY Metabolic syndrome is a collection of metabolic abnormalities that increases ones risk for heart disease. Our study shows that an increasing degree of metabolic abnormality is also associated with an increased risk of diagnosis of overall and aggressive prostate cancer.

Arachidonic acid activation of intratumoral steroid synthesis during prostate cancer progression to castration resistance.

De novo androgen synthesis and subsequent androgen receptor (AR) activation has recently been shown to contribute to castration‐resistant prostate cancer (CRPC) progression. Herein we provide evidence that fatty acids (FA) can trigger androgen synthesis within steroid starved prostate cancer (CaP) tumor cells.

Steroidogenesis inhibitors alter but do not eliminate androgen synthesis mechanisms during progression to castration-resistance in LNCaP prostate xenografts

In castration-resistant prostate cancer (CRPC) many androgen-regulated genes become re-expressed and tissue androgen levels increase despite low serum levels. We and others have recently reported that CRPC tumor cells can de novo synthesize androgens from adrenal steroid precursors or cholesterol and that high levels of progesterone exist in LNCaP tumors after castration serving perhaps as an intermediate in androgen synthesis. Herein, we compare androgen synthesis from [(3)H-progesterone] in the presence of specific steroidogenesis inhibitors and anti-androgens in steroid starved LNCaP cells and CRPC tumors. Similarly, we compare steroid profiles in LNCaP tumors at different stages of CRPC progression. Steroidogenesis inhibitors targeting CYP17A1 and SRD5A2 significantly altered but did not eliminate androgen synthesis from progesterone in steroid starved LNCaP cells and CRPC tumors. Upon exposure to inhibitors of steroidogenesis prostate cancer cells adapt gradually during CRPC progression to synthesize DHT in a compensatory manner through alternative feed-forward mechanisms. Furthermore, tumors obtained immediately after castration are significantly less efficient at metabolizing progesterone ( approximately 36%) and produce a different steroid profile to CRPC tumors. Optimal targeting of the androgen axis may be most effective when tumors are least efficient at synthesizing androgens. Confirmatory studies in humans are required to validate these findings.

NKX3.1 Haploinsufficiency Is Prognostic for Prostate Cancer Relapse following Surgery or Image-Guided Radiotherapy

Background: Despite the use of prostate specific antigen (PSA), Gleason-score, and T-category as prognostic factors, up to 40% of patients with intermediate-risk prostate cancer will fail radical prostatectomy or precision image-guided radiotherapy (IGRT). Additional genetic prognosticators are needed to triage these patients toward intensified combination therapy with novel targeted therapeutics. We tested the role of the NKX3.1 gene as a determinant of treatment outcome given its reported roles in tumor initiating cell (TIC) renewal, the DNA damage response, and cooperation with c-MYC during prostate cancer progression. Methods: Using high-resolution array comparative genomic hybridization (aCGH), we profiled the copy number alterations in TIC genes using tumor DNA from frozen needle biopsies derived from 126 intermediate-risk patients who underwent IGRT. These data were correlated to biochemical relapse-free rate (bRFR) by the Kaplan–Meier method and Cox proportional hazards models. Results: A screen of the aCGH-IGRT data for TIC genes showed frequent copy number alterations for NKX3.1, PSCA, and c-MYC. NKX3.1 haploinsufficiency was associated with increased genomic instability independent of PSA, T-category, and Gleason-score. After adjusting for clinical factors in a multivariate model, NKX3.1 haploinsufficiency was associated with bRFR when tested alone (HR = 3.05, 95% CI: 1.46–6.39, P = 0.0030) or when combined with c-MYC gain (HR = 3.88, 95% CI: 1.78–8.49, P = 0.00067). A similar association was observed for patients following radical prostatectomy with a public aCGH database. NKX3.1 status was associated with positive biopsies post-IGRT and increased clonogen radioresistance in vitro. Conclusions: Our results support the use of genomic predictors, such as NKX3.1 status, in needle biopsies for personalized approaches to prostate cancer management. Clin Cancer Res; 18(1); 308–16. ©2011 AACR.

A novel communication role for CYP17A1 in the progression of castration‐resistant prostate cancer

CYP17A1 is currently a target for total androgen blockade in advanced prostate cancer (CaP) patients. After castration, or removal of testicular androgens, CYP17A1 can act as a rate‐limiting enzyme in androgen synthesis from cholesterol or other adrenal precursors within the tumor microenvironment ultimately contributing to disease progression. Herein we provide evidence that CYP17A1 could also be a mediator of cell‐to‐cell communication within the CaP tumor microenvironment.