BACKGROUND Androgen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance. METHODS Gene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium. RESULTS Gene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect. CONCLUSIONS From our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa. Video abstract.

中文翻译：

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癌症相关的成纤维细胞通过上调胆固醇和类固醇的生物合成来促进前列腺肿瘤的生长和进展。

背景技术雄激素受体靶向疗法已经成为治疗晚期前列腺癌（PCa）的有效工具。然而，治疗抗性的频繁发生代表患者临床管理中的主要挑战，这也是因为尚未完全理解治疗抗性背后的分子机制。因此，在本研究中，我们旨在使用PCa共培养球体的基因表达分析来鉴定干预治疗耐药性的新靶点，其中PCa细胞在存在癌症相关的成纤维细胞（CAF）的情况下生长，并且先前已证明能够是抗雄激素耐药性的可靠模型。方法通过Illumina微阵列分析技术鉴定共培养球体（LNCaP和DuCaP与CAF一起接种）的基因表达变化。实时PCR，蛋白质印迹，进行了2D和3D培养物中的免疫组织化学和细胞生存力测定，以验证体外和体内所选靶标的表达。进行细胞因子谱分析以分析CAF条件培养基。结果共培养球体的基因表达分析表明，CAFs在PCa细胞中诱导胆固醇和类固醇生物合成途径的显着上调。细胞因子分析显示CAF上清液中存在大量促炎，促迁移和促血管生成因子。特别是，在与CAF共同培养后，PCa细胞中的3个基因，即3-羟基-3-甲基戊二酰辅酶A合酶2（HMGCS2）和醛基酮还原酶家族1成员C3（AKR1C3）被显着上调。与具有AKR1C3表达的良性组织相比，这两种酶在人PCa中也显着增加，甚至与格里森评分和转移状态有关。抑制HMGCS2和AKR1C3导致2D和3D培养中的共培养球体以及各种去势和enzalutamide耐药细胞系的生长显着延迟，从而强调了它们在PCa中的假定作用。重要的是，用辛伐他汀（一种常用的胆固醇合成抑制剂）和一种针对AKR1C3的抑制剂双重靶向胆固醇和类固醇的生物合成具有最强的生长抑制作用。结论从我们的结果可以得出结论，CAFs诱导PCa细胞中胆固醇和类固醇生物合成的上调，从而使它们成为AR靶向治疗耐药性。因此，用辛伐他汀和AKR1C3抑制剂阻断这两种途径可能是克服PCa对AR靶向疗法耐药的有前途的方法。录像摘要。