Loss of Androgen Receptor-Dependent Growth Suppression by Prostate Cancer Cells Can Occur Independently from Acquiring Oncogenic Addiction to Androgen Receptor Signaling

The conversion of androgen receptor (AR) signaling as a mechanism of growth suppression of normal prostate epithelial cells to that of growth stimulation in prostate cancer cells is often associated with AR mutation, amplification and over-expression. Thus, down-regulation of AR signaling is commonly therapeutic for prostate cancer. The E006AA cell line was established from a hormone naïve, localized prostate cancer. E006AA cells are genetically aneuploid and grow equally well when xenografted into either intact or castrated male NOG but not nude mice. These cells exhibit: 1) X chromosome duplication and AR gene amplification, although paradoxically not coupled with increased AR expression, and 2) somatic, dominant-negative Serine-599-Glycine loss-of-function mutation within the dimerization surface of the DNA binding domain of the AR gene. No effect on the growth of E006AA cells is observed using targeted knockdown of endogenous mutant AR, ectopic expression of wild-type AR, or treatment with androgens or anti-androgens. E006AA cells represent a prototype for a newly identified subtype of prostate cancer cells that exhibit a dominant-negative AR loss-of-function in a hormonally naïve patient. Such loss-of-function eliminates AR-mediated growth suppression normally induced by normal physiological levels of androgens, thus producing a selective growth advantage for these malignant cells in hormonally naïve patients. These data highlight that loss of AR-mediated growth suppression is an independent process, and that, without additional changes, is insufficient for acquiring oncogene addiction to AR signaling. Thus, patients with prostate cancer cells harboring such AR loss-of-function mutations will not benefit from aggressive hormone or anti-AR therapies even though they express AR protein.     

Inactivation of ATM/ATR DNA Damage Checkpoint Promotes Androgen Induced Chromosomal Instability in Prostate Epithelial Cells

The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene have been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR) to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen-induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced autophosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis.     

Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.     

Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells

Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.     

p21-activated Kinase 6 (PAK6) Inhibits Prostate Cancer Growth via Phosphorylation of Androgen Receptor and Tumorigenic E3 Ligase Murine Double Minute-2 (Mdm2)

The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.     

Reevesioside A,a Cardenolide Glycoside,Induces Anticancer Activity against Human Hormone-Refractory Prostate Cancers through Suppression of c-myc Expression and Induction of G1 Arrest of the Cell Cycle

In the past decade, there has been a profound increase in the number of studies revealing that cardenolide glycosides display inhibitory activity on the growth of human cancer cells. The use of potential cardenolide glycosides may be a worthwhile approach in anticancer research. Reevesioside A, a cardenolide glycoside isolated from the root of Reevesia formosana, displayed potent anti-proliferative activity against human hormone-refractory prostate cancers. A good correlation (r² = 0.98) between the expression of Na⁺/K⁺-ATPase α₃ subunit and anti-proliferative activity suggested the critical role of the α₃ subunit. Reevesioside A induced G1 arrest of the cell cycle and subsequent apoptosis in a thymidine block-mediated synchronization model. The data were supported by the down-regulation of several related cell cycle regulators, including cyclin D1, cyclin E and CDC25A. Reevesioside A also caused a profound decrease of RB phosphorylation, leading to an increased association between RB and E2F1 and the subsequent suppression of E2F1 activity. The protein and mRNA levels of c-myc, which can activate expression of many downstream cell cycle regulators, were dramatically inhibited by reevesioside A. Transient transfection of c-myc inhibited the down-regulation of both cyclin D1 and cyclin E protein expression to reevesioside A action, suggesting that c-myc functioned as an upstream regulator. Flow cytometric analysis of JC-1 staining demonstrated that reevesioside A also induced the significant loss of mitochondrial membrane potential. In summary, the data suggest that reevesioside A inhibits c-myc expression and down-regulates the expression of CDC25A, cyclin D1 and cyclin E, leading to a profound decrease of RB phosphorylation. G1 arrest is, therefore, induced through E2F1 suppression. Consequently, reevesioside A causes mitochondrial damage and an ultimate apoptosis in human hormone-refractory prostate cancer cells.     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.     

Androgen Receptor as a Driver of Therapeutic Resistance in Advanced Prostate Cancer

The role of the androgen receptor (AR) signaling axis in the progression of prostate cancer is a cornerstone to our understanding of the molecular mechanisms causing castration-resistant prostate cancer (CRPC). Resistance of advanced prostate cancer to available treatment options makes it a clinical challenge that results in approximately 30,000 deaths of American men every year. Since the historic discovery by Dr. Huggins more than 70 years ago, androgen deprivation therapy (ADT) has been the principal treatment for advanced prostate cancer. Initially, ADT induces apoptosis of androgen-dependent prostate cancer epithelial cells and regression of androgen-dependent tumors. However, the majority of patients with advanced prostate cancer progress and become refractory to ADT due to emergence of androgen-independent prostate cancer cells driven by aberrant AR activation. Microtubule-targeting agents such as taxanes, docetaxel and paclitaxel, have enjoyed success in the treatment of metastatic prostate cancer; although new, recently designed mitosis-specific agents, such as the polo-kinase and kinesin-inhibitors, have yielded clinically disappointing results. Docetaxel, as a first-line chemotherapy, improves prostate cancer patient survival by months, but tumor resistance to these therapeutic agents inevitably develops. On a molecular level, progression to CRPC is characterized by aberrant AR expression, de novo intraprostatic androgen production, and cross talk with other oncogenic pathways. Emerging evidence suggests that reactivation of epithelial-mesenchymal-transition (EMT) processes may facilitate the development of not only prostate cancer but also prostate cancer metastases. EMT is characterized by gain of mesenchymal characteristics and invasiveness accompanied by loss of cell polarity, with an increasing number of studies focusing on the direct involvement of androgen-AR signaling axis in EMT, tumor progression, and therapeutic resistance. In this article, we discuss the current knowledge of mechanisms via which the AR signaling drives therapeutic resistance in prostate cancer metastatic progression and the novel therapeutic interventions targeting AR in CRPC.     

Down-regulation of Id-1 expression is associated with TGF beta 1-induced growth arrest in prostate epithelial cells

Transforming growth factor beta1 (TGF beta 1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGF beta 1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGF beta 1 was able to suppress the expression of Id-1, a helix-loop-helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGF beta 1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGF beta 1 signaling pathway. The fact that up-regulation of p21(WAF1), one of the downstream effectors of Id-1, was observed after exposure to TGF beta 1 further indicates the involvement of Id-1 in the TGF beta 1-induced growth arrest in HPr-1 cells. However, increased expression of p27(KIP1) was also observed in the TGF beta 1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGF beta 1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGF beta 1 may be one of the upstream regulators of Id-1.     

雄激素受体共调节因子与雄激素非依赖性前列腺癌

雄激素介导的雄激素受体（AR）信号途径对雄性胚胎的发育及雄激素依赖性靶组织的分化发育是必需的。异常的AR活性与前列腺癌由雄激素依赖转变为雄激素非依赖性密切相关。已证实AR共调节因子参与前列腺癌的发生和发展,并在雄激素非依赖性前列腺癌细胞的增殖中扮演着重要角色。它们的表达失衡,可导致AR转录活性的改变,促进晚期前列腺癌的进展。简要综述了AR共调节因子的类型和功能,及其与雄激素非依赖性前列腺癌的关系。     

Down-regulation of Id-1 expression is associated with TGFβ1-induced growth arrest in prostate epithelial cells

Transforming growth factor β1 (TGFβ1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGFβ1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGFβ1 was able to suppress the expression of Id-1, a helix–loop–helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGFβ1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGFβ1 signaling pathway. The fact that up-regulation of p21^WAF1, one of the downstream effectors of Id-1, was observed after exposure to TGFβ1 further indicates the involvement of Id-1 in the TGFβ1-induced growth arrest in HPr-1 cells. However, increased expression of p27^KIP1 was also observed in the TGFβ1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGFβ1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGFβ1 may be one of the upstream regulators of Id-1.     

Divergent Androgen Receptor and Beta-Catenin Signaling in Prostate Cancer Cells

Despite decades of effort to develop effective therapy and to identify promising new drugs, prostate cancer is lethal once it progresses to castration-resistant disease. Studies show mis-regulation of multiple pathways in castration-resistant prostate cancer (CRPC), reflecting the heterogeneity of the tumors and also hinting that targeting androgen receptor (AR) pathway alone might not be sufficient to treat CRPC. In this study, we present evidence that the Wnt/β-catenin pathway might be activated in prostate cancer cells after androgen-deprivation to promote androgen-independent growth, partly through enhanced interaction of β-catenin with TCF4. Androgen-independent prostate cancer cells were more prone to activate a Wnt-reporter, and inhibition of the Wnt/β-catenin pathway increased sensitivity of these cells to the second-generation antiandrogen, enzalutamide. Combined treatment of enzalutamide and Wnt/β-catenin inhibitor showed increased growth repression in both androgen-dependent and -independent prostate cancer cells, suggesting therapeutic potential for this approach.     

Tumor-suppressive functions of 15-Lipoxygenase-2 and RB1CC1 in prostate cancer

15-Lipoxygenase-2 (15-LOX2) is a human-specific lipid-peroxidizing enzyme most prominently expressed in epithelial cells of normal human prostate but downregulated or completely lost in > 70% of prostate cancer (PCa) cases. Transgenic expression of 15-LOX2 in the mouse prostate surprisingly causes hyperplasia. Here we first provide evidence that 15-LOX2-induced prostatic hyperplasia does not progress to PCa even in p53^+/− or p53^−/− background. More important, by generating 15-LOX2; Hi-Myc double transgenic (dTg) mice, we show that 15-LOX2 expression inhibits Myc-induced PCa development, such that in the 3-month- and 6-month-old dTg mice, there is a significant reduction in prostate intraneoplasia (PIN) and PCa prevalent in age-matched Hi-Myc prostates. The dTg prostates show increased cell senescence and expression of several senescence-associated molecules, including p27, phosphorylated Rb, and Rb1cc1. We further show that in HPCa, 15-LOX2 and c-Myc manifest reciprocal protein expression patterns. Moreover, RB1CC1 accumulates in senescing normal human prostate (NHP) cells, and in both NHP and RWPE-1 cells, the 15-LOX2 metabolic products 15(S)-HPETE and 15(S)-HETE induce RB1CC1. We finally show that unlike 15-LOX2, RB1CC1 is not lost but rather frequently overexpressed in PCa samples. RB1CC1 knockdown in PC3 cells enhances clonal growth in vitro and tumor growth in vivo. Together, our present studies provide evidence for tumor-suppressive functions for both 15-LOX2 and RB1CC1.