Platelet-Synthesized Testosterone in Men with Prostate Cancer Induces Androgen Receptor Signaling

Abstract

Platelets have been long postulated to play a critical role in the pathogenesis of prostate cancer, although relatively little is known regarding the precise mechanisms involved. Androgen deprivation therapy (ADT) for prostate cancer eventually fails with relapse occurring in the form of castration-resistant prostate cancer (CRPC). CRPC tumors typically overexpress androgen receptor (AR), demonstrating continued dependence upon AR signaling. Platelets have been previously demonstrated to contain androgens, and we sought to explore the contribution of platelet-derived androgens in CRPC. In this study, we examined the role of platelet-derived androgens in vitro using platelets from men with CRPC, men with high-risk prostate cancer, and healthy male donors. A series of in vitro assays was performed to elucidate the impact of platelet-derived androgens on androgen-sensitive prostate tumor cells. By examining platelet-derived androgen effects on AR signaling in prostate tumor cells, we found that platelets, from men with CRPC and on ADT, strongly induce AR target genes and tumor cell proliferation. Moreover, we show a fully intact testosterone (T) biosynthetic pathway within platelets from its precursor cholesterol and demonstrate that platelets of CRPC patients with ADT resistance are able to generate T. Overall, our findings reveal an unknown capacity of platelets to synthesize T at functionally relevant levels in patients with lethal prostate cancer. Importantly, it suggests a novel paracrine mechanism of T production that may act to sustain CRPC state and potentiate therapeutic resistance.

Figure 1

Platelet de novo synthesis of T. (A) Ratio of ¹³C-T to an internal standard (isotopically labeled estrone) in healthy donor platelets (n = 4) preincubated with cholesterol or pregnenolone. “Platelets”: untreated platelets; “Platelets + ¹³C-Cholesterol”: platelets incubated with ¹³C-cholesterol; “Platelets + ¹³C-Pregnenolone”: platelets incubated with ¹³C-pregnenolone. Significance: ***P < .001. (B) Western blot of platelet lysates demonstrating the presence of T biosynthetic enzymes (black arrows) in washed platelets of “patients” (men with prostate cancer) (from Table 1) and “controls” (healthy donors). Actin was used as loading control. (C) Ratio of ¹³C-T to an internal standard in healthy donor platelets (n = 4) preincubated with ¹³C-pregnenolone and abiraterone acetate at 10 μg/ml. “Platelets”: untreated platelets; “Platelets + ¹³C-Pregnenolone”: platelets incubated with ¹³C-pregnenolone. (D) Graph representing the ratio of ¹³C-T to an internal standard in platelets from abiraterone-resistant men with CRPC (n = 3). “Platelets”: untreated platelets; “Platelets + ¹³C-Cholesterol”: incubated with ¹³C-cholesterol. Significance: *P < .05. (E) Platelets from a man currently receiving abiraterone (castration-sensitive disease). “Platelets”: untreated platelets; “Platelets + ¹³C-Cholesterol”: incubated with ¹³C-cholesterol.

Figure 2

Platelet-derived androgens stimulate AR and promote tumor cell proliferation. (A) MS analysis of intraplatelet levels of androgens in platelets of men with prostate cancer. Concentrations of T and DHT in 1 × 10⁶ platelets from men with CRPC (n = 10) and from men with high-risk disease (n = 8). (B) MS analyses demonstrate ¹³C-T in LNCaP cells after addition of washed platelets preincubated with ¹³C-T for 24 hours in vitro. (C) Relative mRNA levels of TMPRSS2 (normalized to GAPDH) in LNCaP cells after the addition of pooled platelets from three healthy donors under androgen-deprived cell culture conditions for 6, 12, and 24 hours (significance: ***P < .001). (D) LNCaP cell proliferation over 72 hours in response to healthy donor platelets under androgen- and serum-deprived cell culture conditions. “PB treated”: cells incubated in platelet buffer only; “+ Healthy donor platelets in PB”: cells incubated with healthy donor’s platelets in platelet buffer (significance: ***P < .001). (E) LNCaP-AR (AR overexpressing) cell proliferation over 72 hours in response to prostate cancer patient platelets under androgen- and serum-deprived cell culture conditions. “PB treated”: cells incubated in platelet buffer only; “+ Patient # platelets in PB”: cells incubated with CRPC patient (Table 1) platelets in platelet buffer (significance: *P < .05; ***P < .001).

Figure 3

Platelet-derived T regulates AR target genes in LNCAP-AR cells. (A) Relative mRNA expression levels of PSA (left panel) and TMPRSS2 (right panel) normalized to GAPDH in LNCAP-AR cells after treatment with platelets for 24 hours under androgen- and serum-deprived cell culture conditions in the presence or absence of bicalutamide (10 μM). Control cells were treated with platelet buffer alone - “PB treated”; cells treated with platelets from individual healthy donors: “A”, “B” and “C” “+ healthy donor platelets in PB” - resuspended in platelet buffer; and cells treated with Bicalutamide and platelets (significance: *P < .05, ***P < .001). (B) Relative mRNA expression levels of PSA (left panel) and TMPRSS2 (right panel) normalized to GAPDH in LNCAP-AR cells after addition of washed platelets for 24 hours under androgen- and serum-deprived cell culture conditions. Control cells were treated with platelet buffer alone: “PB treated”; cells treated with platelets from individual healthy donors “D”, “E,” and “F” resuspended in platelet buffer: “+ healthy donors’ platelets in PB”; and cells treated with platelets from individual prostate cancer patients #3, #4, #5, and #6 (from Table 1) resuspended in platelet buffer: “+ Prostate cancer patients’ platelets in PB” (significance: *P < .05, ***P < .001).