Elevated AKR1C3 expression promotes prostate cancer cell survival and prostate cell-mediated endothelial cell tube formation: Implications for prostate cancer progression

Background: Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we provide initial evidence for potential roles of AKR1C3 in PCa progression. Methods: Spatial distribution of AKR1C3 was analyzed using immunohistochemical staining in prostate adenocarcinoma tissue array. Human PCa PC-3 cells were stably transfected with AKR1C3 cDNA to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analyses were performed to identify pathways that are activated by elevated AKR1C3 expression in PCa cells. Functional confirmation of microarray and bioinformatics results was performed by immunoblot analysis and an in vitro Matrigel angiogenesis assay. Results: Elevated AKR1C3 expression was specifically limited to human prostate adenocarcinoma. Microarray and bioinformatics analysis suggested that elevated AKR1C3 expression in PC-3 cells modulates estradiol and androgen metabolism and activates insulin growth factor (IGF)-1 and Akt signaling pathways. Immunoblots confirmed that phosphorylated levels of IGF-1 receptor (IGF-1R) and Akt are significantly up-regulated in PC3-AKR1C3 as compared to mock transfectants. PC3-AKR1C3 transfectants promoted endothelial cell tube formation in Matrigel as compared to parental PC-3 cells and mock transfectants. Conclusion: Microarray and bioinformatics data followed by biological analyses suggest that elevated AKR1C3 expression in PC-3 cells promotes PCa angiogenesis and aggressiveness. These results suggest AKR1C3 can promote the aggressiveness of PCa through modulating estrogen and androgen metabolism with subsequent activation of growth factor IGF-1 and cytoplasmic Akt signaling pathways. Total RNA from mock- and ACR1C3 transfected PC-3 cells was isolated, with 2 or 3 biological replicates each. Gene expression data from AKR1C3 transfected PC-3 cells were compared with mock-transfected data.

研究背景：醛酮还原酶1C家族成员3（Aldo-keto reductase 1C family member 3, AKR1C3）是已鉴定的四种人类AKR酶之一，可催化类固醇、前列腺素及外源性物质的代谢。在前列腺组织中，AKR1C3在局限性及进展期前列腺腺癌中表达上调，并与前列腺癌（prostate cancer, PCa）的侵袭性密切相关。本研究首次提供了AKR1C3在前列腺癌进展中潜在作用的初步实验证据。
实验方法：采用免疫组化染色分析AKR1C3在前列腺腺癌组织芯片中的空间分布特征。将AKR1C3互补脱氧核糖核酸（complementary DNA, cDNA）稳定转染至人类前列腺癌细胞PC-3中，成功构建PC3-AKR1C3转染细胞株。通过微阵列分析与生物信息学分析，鉴定前列腺癌细胞中AKR1C3高表达所激活的信号通路。采用免疫印迹分析及体外Matrigel血管生成实验，对微阵列与生物信息学分析的结果进行功能验证。
实验结果：AKR1C3高表达特异性局限于人类前列腺腺癌组织。微阵列及生物信息学分析结果显示，PC-3细胞中AKR1C3高表达可调节雌二醇与雄激素的代谢，并激活胰岛素样生长因子（insulin growth factor, IGF）-1及Akt信号通路。免疫印迹实验证实，与空白转染组相比，PC3-AKR1C3细胞中胰岛素样生长因子-1受体（IGF-1 receptor, IGF-1R）及Akt的磷酸化水平显著上调。与亲本PC-3细胞及空白转染组相比，PC3-AKR1C3转染细胞可显著促进Matrigel基质中的内皮细胞管形成。
研究结论：经微阵列、生物信息学分析及后续生物学实验验证，PC-3细胞中AKR1C3高表达可促进前列腺癌的血管生成与侵袭性。上述结果表明，AKR1C3可通过调节雌、雄激素代谢，后续激活生长因子IGF-1及胞质Akt信号通路，进而促进前列腺癌的侵袭性。本研究提取了空白转染及AKR1C3转染的PC-3细胞的总RNA，每组各设置2或3次生物学重复。将AKR1C3转染的PC-3细胞的基因表达数据与空白转染组的数据进行对比分析。