Androgen suppresses proliferation of castration‐resistant LNCaP 104‐R2 prostate cancer cells through androgen receptor, Skp2, and c‐Myc

Abstract

Summary Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoring testosterone level may suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets implant. 24 out of 33 tumors adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen (PSA). We then study the molecular mechanism lying underneath the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G1 cell cycle arrest via reduction of Skp2 and c-Myc, and induction of p27Kip1. 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with slower growing rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27Kip1. Nuclear AR and PSA expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype, while knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells via AR, Skp2, and c-Myc.

Autophagic degradation of the androgen receptor mediated by increased phosphorylation of p62 suppresses apoptosis in hypoxia

“…Furthermore, the simultaneous knockdown of p62 and AR suppressed hypoxia-induced apoptosis. This pro-apoptotic action of higher AR levels is consistent with studies demonstrating that overexpression of AR suppresses cell proliferation in AR-negative PC3 and LNCaP 104-R2 [40,41]. Chronic hypoxia induces resistance to apoptosis in LNCaP cells [42,43], and hypoxia-induced autophagy promotes tumor cell survival and chemoresistance in hepatocellular carcinoma, glioblastoma, and prostate cancer [44][45][46].…”

Supraphysiological androgen levels induce cellular senescence in human prostate cancer cells through the Src-Akt pathway

“…Furthermore, it has been suggested that the androgen-activated AR acts also as a tumor suppressor for prostate cancer [9]. In line with this, treatment of mice with SAL inhibits the growth of human CRPCa cells in xenograft mouse model system in vivo [39, 42]. …”

Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

“…Deficiency of Skp2 in vivo triggers cellular senescence via up-regulation of p21 Cip1 , p27 Kip1 , and ATF4, therefore suppresses the development of PCa [60]. Skp2 was reported to cross-talk with PI3K/Akt [61], AR [62], PTEN [55], and BRCA2 [63] signaling pathways in PCa cells. As a result, Skp2 plays essential role in the development and progression of human PCa [49].…”

“…Relative cell number was analyzed by measuring the DNA content of cell lysates with the fluorescent dye Hoechst 33258 (Sigma, St. Louis, MO, USA) as described previously [18, 19, 26, 62]. All readouts were normalized to the average of the control condition in each individual experiment.…”

“…After 96 h of culture in the presence of different concentrations of CAPE, cells were processed as previously described [21, 26, 62]. Cell cycle profiles of LNCaP 104-R1, DU-145, 22Rv1, and LNCaP C4–2 cells were determined by flow cytometric analysis using a BD Facscan flow cytometer (BD Biosciences, San Jose, CA).…”