前列腺癌雄激素受体和PI3K/Akt信号通路相互作用研究进展

前列腺癌的发生、进展依赖于雄激素,因此去势手术成为治疗晚期前列腺癌的标准疗法。但是去势后大多前列腺癌最终将转化为雄激素非依赖性前列腺癌,甚至进展为激素难治性前列腺癌,使得肿瘤的进展不受低水平雄激素的影响。即使如此,大多数激素非依赖性前列腺癌,依然阳性表达雄激素受体。因而雄激素受体在前列腺癌发生发展中起着重要作用。而PI3K/Akt信号通路能够通过维持细胞生存、抑制细胞凋亡、促进细胞代谢及血管生成等促进前列腺癌进展。本综述旨在总结前人研究,阐述雄激素受体和PI3K/Akt信号通路之间相互作用关系。研究表明Akt信号通路能够正性或者负性调控AR蛋白表达、蛋白的稳定性及其转录活性,从而维持细胞的生存、代谢。而AR即可以通过基因转录途径抑制Akt活化又能通过非转录基因途径激活Akt及其下游蛋白。因此,AR和Akt信号通路相互协同促进前列腺癌的发生及其向雄激素非依赖性前列腺癌进展。     

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。     

雄激素和雄激素受体对骨代谢的调控及机制研究进展

骨代谢由成骨细胞介导的骨形成和破骨细胞介导的骨吸收构成。雄激素能调控骨代谢,即促进骨形成、抑制骨吸收,在骨骼生长、骨峰值的获得和骨量维持中起重要作用;且该作用主要通过雄激素受体(androgen receptor,AR)介导。AR调控骨代谢的作用,一方面是通过直接调控骨代谢相关的AR靶基因(如与成骨相关的I型胶原蛋白α1、骨钙素、组织非特异性碱性磷酸酶、小整合素结合配体N-端连接糖蛋白和与破骨相关的核因子κB受体活化因子配体(RANKL)、组织蛋白酶K的表达;另一方面是通过间接调控骨代谢的多个信号通路[如Wnt/β-catenin、骨形态发生蛋白(BMP)/Smads-Runt相关转录因子2(Runx2)、RANKL/骨保护蛋白(OPG)、PI3K/Akt和MAPK信号通路]实现的。该文主要就雄激素/AR在骨代谢调控中的作用及机制作一综述,对丰富AR调控骨代谢的理论认识和骨代谢性疾病的药物研发具有重要意义。     

雄激素非依赖性前列腺癌细胞雄激素受体靶基因的筛选与初步鉴定

采用染色质免疫共沉淀技术在全基因组水平筛选雄激素非依赖性前列腺癌细胞LNCaP-AI的雄激素受体结合位点,行高通量测序及生物信息学分析共得到2 876个peak（pvalue〈1×10–5）,peak平均长度为673 bp;将peak序列定位到Hg19基因组,共有1 865个靶基因,其中fold enrichment≥10的基因有425个。对peak相关基因进行GO分析发现,与细胞、细胞组分、细胞过程、结合、细胞器相关的基因位列前五位;对peak相关基因进行通路分析发现,与黏着斑、代谢通路、癌症中的转录错误调控、嘌呤代谢等信号通路相关的基因占大多数。筛选出7个候选AR靶基因,采用Real-time qPCR技术分析它们在LNCaP-AI细胞和雄激素依赖性前列腺癌细胞LNCaP中对DTH刺激的反应性,发现DHT刺激可改变7个候选AR靶基因在LNCaP-AI细胞中的表达,为进一步研究雄激素依赖性前列腺癌向非依赖性前列腺癌发展的过程中雄激素受体及其调控的下游靶基因功能起着至关重要的作用。     

雄激素受体共调节因子及其在前列腺癌进展中的作用

雄激素受体（AR）信号通路在前列腺癌的发生、进展和转移中发挥着重要作用,但AR介导组织对雄激素的特异应答是通过与其相互作用的AR共调节因子共同完成的,许多AR共调节因子的功能已被广泛研究。简要综述了目前发现的部分AR共调节因子在调节AR转录活性及前列腺癌发生、进展中的生物学作用。     

谷氨酰胺激活PI3K/Akt信号通路促进痘苗病毒复制

谷氨酰胺(glutamine,Gln)在细胞生长和代谢过程中起重要作用,能够激活磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素蛋白(mammalian target of rapamycin,m TOR)信号通路。多种病毒能通过PI3K/Akt信号通路维持细胞生存和抑制细胞凋亡,维持病毒的感染。为了探讨Gln在痘苗病毒(vaccinia virus strain western reserve,WR)感染人肺癌细胞A549过程中的调控作用及其潜在机制,该文通过蛋白免疫印迹(Western blot)检测PI3K/Akt信号通路蛋白质磷酸化水平,流式细胞术检测细胞阳性率,结晶紫染色检测WR滴度,实时定量PCR(quantitative Real-time PCR,q PCR)检测WR基因E3L和A46R m RNA水平。结果显示,Gln处理后PI3K/Akt信号通路蛋白质磷酸化水平显著升高。抑制PI3K/Akt信号通路后,细胞阳性率显著降低(P0.05),WR滴度显著降低(P0.05),WR基因E3L和A46R m RNA水平显著降低(P0.05)。该研究结果表明,Gln通过激活PI3K/Akt信号通路从而促进A549细胞中WR的复制。     

雄激素受体信号转导途径及其在前列腺癌发展中的作用

前列腺癌是西方男性发病率最高的癌症之一,在采用雄激素阻断疗法后,大部分患者的病情可得到控制,但经过一段时间又会转变为雄激素非依赖型前列腺癌。雄激素受体(AR)在前列腺细胞中扮演重要的角色,它可调节大量基因的表达。在前列腺癌由雄激素依赖型向雄激素非依赖型的转变过程中,AR及其信号途径通过多种方式发挥作用,AR基因的扩增、AR的突变,以及与共激活子之间作用的改变都可能使细胞获得雄激素非依赖型的生长能力。此外,AR还受到多肽生长因子和细胞因子等的调节,表现激素非依赖型的转录激活活性。AR在前列腺癌中作用的阐明对前列腺癌的诊断与治疗有着重大的意义。     

Prosaposin对细胞增殖和凋亡的调控及其分子机制

为研究鞘脂激活蛋白原(Prosaposin)对细胞增殖、细胞凋亡的调控及其可能的分子机制, 以pcDNA3.1 in NIH3T3阴性对照细胞株和过表达prosaposin的Psap-Myc in NIH3T3细胞株为模型, 噻唑蓝(MTT)比色法检测prosaposin对细胞增殖的影响; Annexin V联合碘化丙啶(Propidium iodide, PI)法检测血清饥饿状态下prosaposin对细胞凋亡的影响; Western blotting检测PI3K/Akt信号通路中蛋白磷酸化水平的变化; Real-time PCR检测PI3K/Akt信号通路下游靶分子表达水平的改变。结果表明prosaposin可活化PI3K/Akt信号通路, 提高Akt^Ser473的磷酸化水平, 抑制细胞周期抑制基因P27^KIP1的表达, 上调细胞周期蛋白Cyclin D1的表达, 促进细胞周期从G1→S期进展; 诱导survival基因cIAP1、cIAP2的表达, 促进细胞存活。这些结果提示, prosaposin对细胞增殖和凋亡的调控可能是通过PI3K/Akt信号通路及其下游靶分子进行的。     

固醇激素代谢及其在前列腺癌治疗中的应用

固醇激素参与调控一系列的生理病理过程,其中,雄激素代谢紊乱导致前列腺癌发生发展。雄激素代谢途径及其相关的雄激素受体信号通路是前列腺癌治疗的主要靶点。通过对雄激素代谢途径中小分子生理功能和代谢酶活性调节机制进行研究,有助于理解癌症治疗耐受的原因及治疗个体化差异的来源,有助于寻找新的靶点进行药物筛选和探索新的生物标志物进行临床诊治。该文主要介绍前列腺癌病情发展过程中雄激素代谢的途径以及与雄激素代谢相关的治疗手段、耐药机制和生物标志物。     

阻断MAPK通路促进雄激素受体招募NCoR并抑制前列腺癌细胞生长

雄激素受体(androgen receptor,AR)是配体调节的转录因子,与前列腺癌的生长和内分泌治疗密切相关.醋酸环丙孕酮(cyproterone acetate,CPA)作为雄激素的拮抗剂已用于前列腺癌的治疗.结合了CPA的AR可与核受体协同抑制因子作用.已证实丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)可介导生长因子和雄激素受体的信号转导通路的交互作用.我们报道,激活的MAPK抑制结合了CPA的AR招募核受体协同抑制因子(nuclear receptor corepressor,NCoR)至雄激素反应元件上.应用MEK的抑制剂U0126阻断MAPK通路可促进结合了CPA的AR和NCoR相互作用并通过对NCoR的招募增加抑制AR的功能从而阻遏AR靶基因的表达.此外,联合使用CPA和U0126处理稳定表达NCoR的LNCaP细胞可显著抑制前列腺癌细胞的生长.本研究表明,联合应用AR的拮抗剂和MAPK抑制剂有助于前列腺癌的治疗.     

Metabolomic profiling reveals a role for androgen in activating amino acid metabolism and methylation in prostate cancer cells

Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer.     

PC-１基因表达增强C4-2B前列腺癌细胞生存

建立稳定表达外源PC-１基因的人前列腺癌骨转移C4-2B细胞模型,初步探讨PC- １基因表达对前列腺癌发展的影响.通过脂质体介导的方法,将融合PC-１基因的真核表达载体pcDNA3.1PC-１稳定转染C4-2B细胞,Western 印迹和RT-PCR技术,分别从蛋白水平和RNA水平确定外源PC-１基因表达. MTT和软琼脂集落形成能力等一系列方法,研究PC-１基因的功能,RT-PCR和实时定量PCR检测前列腺癌发生发展相关基因表达的变化. 结果表明,PC-１基因的高表达能够诱导雄激素受体（AR）调控基因和一系列重要的信号通路成员基因PSA、PSMA、NKX31、Jagged1、EphA3、SGEF和 NOTCH3等表达发生变化. 实验结果初步证明,PC-１基因表达在晚期前列腺癌中,以及在雄激素非依赖的转变中可以发挥作用,PC-１基因表达可调控一些重要信号通路.对PC-１基因功能深入研究将有可能为发现新的前列腺癌的诊断治疗分子靶标提供线索.     

Androgen dependent regulation of protein kinase A subunits in prostate cancer cells

Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cbeta isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cbeta splice variants are differentially regulated during this process. Whereas the Cbeta2 splice variant is down-regulated in growth arrested LNCaP cells, the Cbeta1, Cbeta3 and Cbeta4 variants, as well as the RIIbeta subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cbeta expression could be mimicked by androgen stimulation, implying the Cbeta gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cbeta subunits were also observed in human prostate specimens, and the Cbeta2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cbeta isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.     

Differential requirements for ras and the retinoblastoma tumor suppressor protein in the androgen dependence of prostatic adenocarcinoma cells.

Prostate cells are dependent on androgen for proliferation, but during tumor progression prostate cancer cells achieve independence from the androgen requirement. We report that androgen withdrawal fails to inhibit cell cycle progression or influence the expression of cyclin-dependent kinase (CDK)/cyclins in androgen-independent prostate cancer cells, indicating that these cells signal for cell cycle progression in the absence of androgen. However, phosphorylation of the retinoblastoma tumor suppressor protein (RB) is still required for G1-S progression in androgen-independent cells, since the expression of constitutively active RB (PSM-RB) or p16ink4a caused cell cycle arrest and mimicked the effects of androgen withdrawal on downstream targets in androgen-dependent LNCaP cells. Since Ras is known to mediate mitogenic signaling to RB, we hypothesized that active V12Ras would induce androgen-independent cell cycle progression in LNCaP cells. Although V12Ras was able to stimulate ERK phosphorylation and induce cyclin D1 expression in the absence of androgen, it was not sufficient to promote androgen-independent cell cycle progression. Similarly, ectopic expression of CDK4/cyclin D1, which stimulated RB phosphorylation in the presence of androgen, was incapable of inactivating RB or driving cell cycle progression in the absence of androgen. We show that androgen regulates both CDK4/cyclin D1 and CDK2 complexes to inactivate RB and initiate cell cycle progression. Together, these data show that androgen independence is achieved via deregulation of the androgen to RB signal, and that this signal can only be partially initiated by the Ras pathway in androgen-dependent cells.