雄激素受体在前列腺癌细胞中作用及其靶向治疗的研究进展

雄激素受体(androgen receptor, AR)在前列腺癌的发生发展中扮演重要角色。雄激素剥夺疗法(androgen deprivationtherapy, ADT)早期可成功抑制肿瘤的生长, 但最终导致肿瘤复发并进入激素抵抗阶段。AR对前列腺癌基质细胞起促进肿瘤增殖和转移作用, 是上皮腔样细胞的存活因子, 而对肿瘤干细胞样细胞及上皮基底细胞的增殖起抑制作用, AR在不同类型细胞中的不同作用向当前ADT传统的疗法提出挑战, 为发展新的治疗策略提供理论依据。目前以AR为靶点的靶向治疗药物研发也取得一些进展。本文就AR在前列腺癌不同类型细胞中的作用及靶向治疗方面的进展加以综述。      

Adaptive responses of androgen receptor signaling in castration-resistant prostate cancer

Prostate Cancer (PCa) is an important age-related disease being the most common cancer malignancy and the second leading cause of cancer mortality in men in Western countries. Initially, PCa progression is androgen receptor (AR)- and androgen-dependent. Eventually advanced PCa reaches the stage of Castration-Resistant Prostate Cancer (CRPC), but remains dependent on AR, which indicates the importance of AR activity also for CRPC. Here, we discuss various pathways that influence the AR activity in CRPC, which indicates an adaptation of the AR signaling in PCa to overcome the treatment of PCa. The adaptation pathways include interferences of the normal regulation of the AR protein level, the expression of AR variants, the crosstalk of the AR with cytokine tyrosine kinases, the Src-Akt-, the MAPK-signaling pathways and AR corepressors. Furthermore, we summarize the current treatment options with regard to the underlying molecular basis of the common adaptation processes of AR signaling that may arise after the treatment with AR antagonists, androgen deprivation therapy (ADT) as well as for CRPC, and point towards novel therapeutic strategies. The understanding of individualized adaptation processes in PCa will lead to individualized treatment options in the future.     

Isosilybin A induces apoptosis in human prostate cancer cells via targeting Akt,NF‐κB,and androgen receptor signaling

Prostate cancer (PCA) is the second most malignancy in American men. Advanced stage PCA cells possess unlimited replication potential as well as resistance to apoptosis. Therefore, targeting survival mechanisms and activating apoptotic machinery in PCA cells using nontoxic phytochemicals is suggested as an attractive strategy against this deadly malignancy. In the present study, we assessed the effect of one such botanical agent, namely isosilybin A, on apoptotic machinery and key members of cell survival signaling [Akt, NF‐κB, and androgen receptor (AR)] in different PCA cells. Results showed that isosilybin A (90–180 µM) treatment significantly induces apoptotic death by activating both extrinsic (increased level of DR5 and cleaved caspase 8) and intrinsic pathways (caspase 9 and 3 activation) of apoptosis in three different human PCA cell lines namely 22Rv1, LAPC4, and LNCaP. Further, isosilybin A treatment decreased the levels of phospho‐Akt (serine‐473), total Akt, and the nuclear levels of NF‐κB constituents (p50 and p65). Isosilybin A treatment also decreased the AR and PSA level in 22Rv1, LAPC4, and LNCaP cells. Employing pan‐caspase inhibitor (Z‐VAD.fmk), we confirmed that isosilybin A‐mediated decreased AR is independent of caspases activation. Temporal kinetics analysis showed that the primary effect of isosilybin A is on AR, as decrease in AR was evident much earlier (4 h) relative to caspase activation and apoptosis induction (12 h). Overall, our results demonstrated that isosilybin A activates apoptotic machinery in PCA cells via targeting Akt–NF‐κB–AR axis; thereby, indicating a promising role for this phytochemical in the management of clinical PCA. © 2010 Wiley‐Liss, Inc.     

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.     

大黄素通过抑制雄激素受体表达抑制前列腺癌细胞生长的体内实验研究

目的进一步证实大黄素在体内对前列腺癌的作用和作用机制.方法选用PC3-AR荷瘤裸鼠和C3(1)/SV40转基因小鼠作为动物模型,分别设对照组和治疗组,对照组腹腔注射DMSO,治疗组腹腔注射大黄素40mg/(kg·d).每周测动物体重及肿瘤大小.结果在PC3-AR荷瘤裸鼠中,治疗组小鼠肿瘤体积明显小于对照组(P＜0.001).在C3(1)/SV40转基因小鼠中,治疗组小鼠的生存时间明显长于对照组(P＜0.001).与治疗组相比,对照组小鼠的体重明显降低,皮毛光滑度差,活动能力差.对C3(1)/SV40转基因小鼠肿瘤进行组织化学染色和Western bloting分析表明:治疗组小鼠肿瘤表达雄激素受体阳性细胞明显减少(P＜O.05);雄激素受体的表达与对照组相比也有明显的降低.此外,对照组小组(1/7)与治疗组(7/7)相比更具有向周围组织转移和浸润的倾向.以上结果证明了大黄素在体内对前列腺癌的作用机制与体外相同.结论大黄素不但对前列腺癌的生长有明显的抑制作用,还可抑制前列腺癌的发生.提示大黄素不仅可用于前列腺癌的临床治疗,也可用于前列腺癌的预防.     

Equol inhibits prostate cancer growth through degradation of androgen receptor by S‐phase kinase‐associated protein 2

Chemopreventive and potential therapeutic effects of soy isoflavones have been shown to be effective in numerous preclinical studies as well as clinical studies in prostate cancer. Although the inhibition of androgen receptor signaling has been supposed as one mechanism underlying their effects, the precise mechanism of androgen receptor inhibition remains unclear. Thus, this study aimed to clarify their mechanism. Among soy isoflavones, equol suppressed androgen receptor as well as prostate‐specific antigen expression most potently in androgen‐dependent LNCaP cells. However, the inhibitory effect on androgen receptor expression and activity was less prominent in castration‐resistant CxR and 22Rv1 cells. Consistently, cell proliferation was suppressed and cellular apoptosis was induced by equol in LNCaP cells, but less so in CxR and 22Rv1 cells. We revealed that the proteasome pathway through S‐phase kinase‐associated protein 2 (Skp2) was responsible for androgen receptor suppression. Taken together, soy isoflavones, especially equol, appear to be promising as chemopreventive and therapeutic agents for prostate cancer based on the fact that equol augments Skp2‐mediated androgen receptor degradation. Moreover, because Skp2 expression was indicated to be crucial for the effect of soy isoflavones, soy isoflavones may be applicable for precancerous and cancerous prostates.     

雄激素非依赖性前列腺癌的研究现状与进展

前列腺癌是泌尿、男生殖系常见的恶性肿瘤之一,在我国发病率虽较低,但近年来呈显著增长的趋势.在我国,前列腺癌被临床上发现时大多已有转移,经雄激素祛除治疗大部分患者病情好转,但最终仍发展为雄激素非依赖性前列腺癌(AIPC).目前临床上将雄激素非依赖性前列腺癌分为三类,其临床表现和预后不一.AIPC的发生、发展机制主要有两方面:①雄激素受体途径;②雄激素受体外的旁路途径,即不依赖于雄激素受体.事实上,大部分AIPC患者两条途径是共同存在、密不可分的.在治疗方面,最近两项大规模的Ⅲ期临床随机对照研究显示,以泰索帝为基础的化疗方案可明显延长AIPC的生存期.其预后与AIPC患者的生存状态、血红蛋白、乳酸脱氢酶等水平有关.     

Collateral resistance to taxanes in enzalutamide‐resistant prostate cancer through aberrant androgen receptor and its variants

Currently, the optimal sequential use of androgen receptor (AR) axis‐targeted agents and taxane chemotherapies remains undetermined. We aimed to elucidate the resistance status between taxanes and enzalutamide, and the functional role of the AR axis. Enzalutamide‐resistant 22Rv1 cells showed collateral resistance to taxanes, including docetaxel and cabazitaxel. However, taxane‐resistant cells showed no collateral resistance to enzalutamide; taxane‐resistant cells expressed comparable protein levels of full‐length AR and AR variants. Knockdown of both full‐length AR and AR variants rendered cells sensitive to taxanes, whereas knockdown of AR variants sensitized cells to enzalutamide, but not to taxanes. In contrast, overexpression of full‐length AR rendered cells resistant to taxanes. Consistently, the prostate‐specific antigen response and progression‐free survival in docetaxel chemotherapy were worse in cases with prior use of ARAT agents compared with cases without. Collateral resistance to taxanes was evident after obtaining enzalutamide resistance, and aberrant AR signaling might be involved in taxane resistance.     

Darolutamide is a potent androgen receptor antagonist with strong efficacy in prostate cancer models

Darolutamide is a novel androgen receptor (AR) antagonist with a distinct chemical structure compared to other AR antagonists and currently in clinical Phase 3 trials for prostate cancer. Using cell-based transactivation assays, we demonstrate that darolutamide, its diastereomers and its main metabolite keto-darolutamide are strong, competitive antagonists for AR wild type, and also for several mutants identified in prostate cancer patients for which other AR antagonists show reduced antagonism or even agonism. Darolutamide, its two diastereomers and main metabolite are also strong antagonists in assays measuring AR N/C interaction and homodimerization. Molecular modeling suggests that the flexibility of darolutamide allows accommodation in the W742C/L mutated AR ligand-binding pocket while for enzalutamide the loss of the important hydrophobic interaction with W742 leads to reduced AR interaction. This correlates with an antagonistic pattern profile of coregulator recruitment for darolutamide. In vitro efficacy studies performed with androgen-dependent prostate cancer cell lines show that darolutamide strongly reduces cell viability and potently inhibits spheroid formation. Also, a marked down-regulation of androgen target genes paralleled by decreased AR binding to gene regulatory regions is seen. In vivo studies reveal that oral dosing of darolutamide markedly reduces growth of the LAPC-4 cell line-derived xenograft and of the KuCaP-1 patient-derived xenograft. Altogether, these results substantiate a unique antagonistic profile of darolutamide and support further development as a prostate cancer drug.     

Receptor tyrosine kinase recepteur d'origine nantais as predictive marker for aggressive prostate cancer in African Americans

Published evidence shows a correlation between several molecular markers and prostate cancer (PCa) progression including in African Americans (AAs) who are disproportionately affected. Our early detection efforts led to the identification of elevated levels of antiapoptotic protein, c‐FLIP and its upstream regulatory factors such as androgen receptor (AR), recepteur d'origine nantais (RON), a receptor tyrosine kinase in human prostate tumors. The primary objective of this study was to explore whether these markers play a role in racial disparities using immunohistochemistry in prostatectomy samples from a cohort of AA, Hispanic Whites (HWs), and non‐Hispanic Whites (NHWs) . Bivariable and multivariable logistic regression analyses were used to identify a statistical association between molecular markers, possible correlation with risk factors including race, obesity, prostate‐specific antigen (PSA) and disease aggressiveness. Further, changes in the levels and expression of these molecular markers were also evaluated using human PCa cell lines. We found significantly elevated levels of RON ( P = 0.0082), AR ( P = 0.0001), c‐FLIP ( P = 0.0071) in AAs compared with HWs or NHWs. Furthermore, a higher proportion of HW and NHWs had a high Gleason score (>6) but not PSA as compared to AAs ( P = 0.032). In summary, our findings suggest that PSA was important in predicting aggressive disease for the cohort overall; however, high levels of RON may play a role in predisposing AA men to develop aggressive disease. Future research is needed using large datasets to confirm these findings and to explore whether all or any of these markers could aid in race‐specific stratification of patients for treatment.     

Synergistic antiproliferative and apoptotic effects induced by epidermal growth factor receptor and protein kinase a inhibitors in human prostatic cancer cell lines

Our results revealed that the blockade of epidermal growth factor receptor (EGFR) tyrosine kinase and protein kinase A (PKA) signaling pathways by specific inhibitors (PD153035 and Rp-cAMPs) leads to a synergistic inhibition of EGF- and serum-stimulated growth of human prostatic cancer cells (LNCaP, DU145 and PC3) concomitant with an arrest in the G1 phase of cellular cycle. Of particular interest, the combination of PD153035 and Rp-cAMPs also caused a more substantial apoptotic/necrotic death of these prostatic cancer cells as compared to drugs alone. Moreover, we observed that the inhibition of acidic sphingomyelinase and caspase cascades results in a marked reduction of DNA fragmentation and apoptotic death induced by PD153035, alone or in combination with Rp-cAMPs, in EGF stimulated PC3 cells. This suggests that these agents might mediate their cytotoxic effects at least in part via the ceramide generation and activation of caspase signaling pathways. N-oleoylethanolamine (OE), an inhibitor of acidic ceramidase, consistently potentiated the apoptotic effects of PD153035 in all the prostatic cancer cell lines tested. Additionally, the cellular ceramide content estimated for PC3 cells was increased after treatment with PD153035, alone or in combination, at a lower dose with OE and Rp-cAMPs.The synergistic apoptotic effect of PD153035 plus Rp-cAMPs induced in PC3 was also accompanied by a significant rate of mitochondrial membrane depolarization and release of cytochrome c into cytosol as compared to drugs alone. Combined, the results indicated that the simultaneous inhibition of EGFR and PKA signaling cascades might lead to a more massive apoptotic death of metastatic prostatic cancer cells by increasing ceramide accumulation and activating of caspase cascade of a mitochondrial dependent manner.     

Phosphorylation of the androgen receptor is associated with reduced survival in hormone-refractory prostate cancer patients

Cell line studies demonstrate that the PI3K/Akt pathway is upregulated in hormone-refractory prostate cancer (HRPC) and can result in phosphorylation of the androgen receptor (AR). The current study therefore aims to establish if this has relevance to the development of clinical HRPC. Immunohistochemistry was employed to investigate the expression and phosphorylation status of Akt and AR in matched hormone-sensitive and -refractory prostate cancer tumours from 68 patients. In the hormone-refractory tissue, only phosphorylated AR (pAR) was associated with shorter time to death from relapse (P=0.003). However, when an increase in expression in the transition from hormone-sensitive to -refractory prostate cancer was investigated, an increase in expression of PI3K was associated with decreased time to biochemical relapse (P=0.014), and an increase in expression of pAkt(473) and pAR(210) were associated with decreased disease-specific survival (P=0.0019 and 0.0015, respectively). Protein expression of pAkt(473) and pAR(210) also strongly correlated (P<0.001, c.c.=0.711) in the hormone-refractory prostate tumours. These results provide evidence using clinical specimens, that upregulation of the PI3K/Akt pathway is associated with phosphorylation of the AR during development of HRPC, suggesting that this pathway could be a potential therapeutic target.     

前列腺癌组织雄激素受体N端区基因突变的研究

目的 :研究雄激素受体 (AR)基因突变与前列腺癌 (PC)发生、发展的关系。方法 :采用自行设计的 3对引物 ,检测了 2 5例PC石蜡切片组织 ,对其AR的N端转录激活区进行了银染聚合酶链式反应 单链构象多态性分析 (PCR SSCP)和异常SSCP片段的DNA测序。结果 :发现一患者的A片段有SSCP异常 ,经测序证实A外显子存在一错义突变 (C96 6 →A ,Ser2 96Arg)。结论 :这是一新发现的突变 ,这一突变位于转录激活区内 ,推测可改变AR的转导活性 ,从而影响AR的功能。