Regulation of SPDEF expression by DNA methylation in advanced prostate cancer.
Regulation of SPDEF expression by DNA methylation in advanced prostate cancer.
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DOI：
10.3389/fendo.2023.1156120
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发表时间：
2023
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
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作者：
前列腺癌(PCA)是对男性健康的重大挑战，相当多的死亡归因于转移性去势抵抗前列腺癌(MCRPC)。此外，由于前列腺癌，非裔美国人男性的死亡率高得不成比例。本研究探讨了前列腺特异性ETS转录因子SPDEF的关键作用，以及DNA甲基化对其在前列腺癌进展中的调节作用。我们进行了表观遗传学重编程，每天使用无毒剂量的5-氮-2-脱氧胞苷(5-氮-2-脱氧胞苷)治疗两周，以评估其对前列腺癌细胞PDEF表达的影响。接下来，我们对重新编程的细胞进行了功能研究，包括细胞迁移(创伤愈合试验)、侵袭(Boyden-Chamber试验)和增殖(四甲基偶氮唑盐试验)，以全面评估PDEF表达改变的后果。我们使用亚硫酸氢盐测序(BSP)来检测SPDEF启动子上的DNA甲基化。同时，我们利用siRNA介导的关键DNMT(DNMT1、DNMT3a和Dnmt3b)的靶向来阐明它们在调节PDEF中的特定作用。我们分别用定量逆转录聚合酶链式反应(qRT-PCR)和免疫印迹技术检测基因和蛋白的表达。在这篇报道中，我们观察到：a)在前列腺癌从低到高的Gleason分级过程中，SPDEF的表达逐渐降低，同时伴随着SPDEF基因内甲基化CpG位点的增加；b)DNMT(DNMT1、3a和3b)的表达在前列腺癌的进展过程中增加，并且SPDEF和DNMT的表达呈负相关；c)在起源于非裔美国人的PCa细胞系RC77/T中，与雄激素依赖细胞系LNCaP细胞相比，SPDEF水平降低；(D)在SPDEF阴性的CRPC(PC3、DU145和RC77/T)细胞系中，SPDEF基因的5‘CpG岛发生高甲基化，而在SPDEF阳性的去势敏感(LNCaP)细胞系中，SPDEF基因的5’CpG岛发生低甲基化；(E)在缺乏SPDEF的PCa细胞中，SPDEF的表达减少了细胞的迁移和侵袭，但对细胞的增殖没有显著影响；(F)去甲基化试剂5-aza-2‘-脱氧胞苷处理，或通过siRNA沉默DNMT，部分恢复了SPDEF在SPDEF阴性的细胞系中的表达，并减少了细胞的迁移和侵袭。这些结果表明，高甲基化是前列腺癌进展过程中降低SPDEF表达的一种普遍机制。数据表明，前列腺癌细胞中SPDEF表达的丧失是细胞可塑性的关键步骤，是潜在的可逆的DNA异常甲基化过程的结果。这些研究表明，DMNT活性是一种潜在的治疗脆弱性，可用于限制前列腺癌的细胞可塑性、肿瘤进展和治疗耐药性。前列腺癌的进展与DNMT表达增加和SPDEF基因甲基化增加以及随后SPDEF表达降低有关。抑制DNMTs可恢复SPDEF的表达，并导致表型逆转。
Prostate cancer (PCa) presents a significant health challenge in men, with a substantial number of deaths attributed to metastatic castration resistant PCa (mCRPC). Moreover, African American men experience disproportionately high mortality rates due to PCa. This study delves into the pivotal role of SPDEF, a prostate specific Ets transcription factor, and its regulation by DNA methylation in the context of PCa progression. We performed Epigenetic reprogramming using daily treatment with non-toxic dose of 5Aza-2-deoxycytidine (5Aza-dC) for two weeks to assess its impact on PDEF expression in prostate cancer cells. Next, we conducted functional studies on reprogrammed cells, including cell migration (wound-healing assay), invasion (Boyden-Chamber test), and proliferation (MTT assay) to comprehensively evaluate the consequences of altered PDEF expression. We used bisulfite sequencing (BSP) to examine DNA methylation at SPDEF promoter. Simultaneously, we utilized siRNA-mediated targeting of key DNMTs (DNMT1, DNMT3A, and DNMT3B) to elucidate their specific role in regulating PDEF. We measured mRNA and protein expressions using qRT-PCR and immune-blotting techniques, respectively. In this report, we observed that: a) there is a gradual decrease in SPDEF expression with a concomitant increase in methylated CpG sites within the SPDEF gene during prostate cancer progression from lower to higher Gleason grade; b) Expression of DNMT’s (DNMT1, 3a and 3b) is increased during prostate cancer progression, and there is an inverse correlation between SPDEF and DNMT expression; c) SPDEF levels are decreased in RC77/T, a line of PCa cells from African American origin similar to PC3 and DU145 cells (CRPC cells), as compared to LNCaP cells , a line of androgen dependent cells,; d) the 5′ CpG island of SPDEF gene are hypermethylated in SPDEF-negative CRPC ( PC3, DU145 and RC77/T) cell lines but the same regions are hypomethylated in SPDEF-positive castrate sensitive (LNCaP) cell line ; (e) expression of SPDEF in PCa cells lacking SPDEF decreases cell migration and invasion, but has no significant effect on cell proliferation, and; (f) treatment with the demethylating agent, 5-aza-2′-deoxycytidine, or silencing of the DNMT’s by siRNA, partially restores SPDEF expression in SPDEF-negative PCa cell lines, and decreases cell migration and invasion. These results indicate hypermethylation is a prevalent mechanism for decreasing SPDEF expression during prostate cancer progression. The data demonstrate that loss of SPDEF expression in prostate cancer cells, a critical step in cellular plasticity, results from a potentially reversible process of aberrant DNA methylation. These studies suggest DMNT activity as a potential therapeutic vulnerability that can be exploited for limiting cellular plasticity, tumor progression, and therapy resistance in prostate cancer. Prostate cancer progression is associated with increased expression of DNMTs and a consequent increased methylation on SPDEF gene and subsequent decrease in SPDEF expression. Inhibition of DNMTs restores SPDEF expression and results in phenotype reversal.
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影响因子：
--
作者：
Findlay, Victoria J.;LaRue, Amanda C.;Turner, David P.;Watson, Patricia M.;Watson, Dennis K.
通讯作者：
Watson, Dennis K.
影响因子：
82.9
作者：
Chen, CD;Welsbie, DS;Sawyers, CL
通讯作者：
Sawyers, CL
影响因子：
82.9
作者：
Beltran H;Prandi D;Mosquera JM;Benelli M;Puca L;Cyrta J;Marotz C;Giannopoulou E;Chakravarthi BV;Varambally S;Tomlins SA;Nanus DM;Tagawa ST;Van Allen EM;Elemento O;Sboner A;Garraway LA;Rubin MA;Demichelis F
通讯作者：
Demichelis F
影响因子：
64.5
作者：
Hnisz D;Abraham BJ;Lee TI;Lau A;Saint-André V;Sigova AA;Hoke HA;Young RA
通讯作者：
Young RA
DOI：
10.1016/j.urolonc.2007.05.010
发表时间：
2007-09-01
影响因子：
2.7
作者：
Hadaschik, Boris A.;Gleave, Martin E.
通讯作者：
Gleave, Martin E.