Novel Mechanisms of ROS/RNS Signaling Underlying Castration-Resistant Prostate Cancer Emergence and Progression

摘要 对去势抵抗性前列腺癌（CRPC）的分子机制的了解有限， 这是对这种致命疾病进行有效治疗的障碍。我们鉴定了一氧化氮受体 复合物，可溶性鸟苷酸环化酶（sGC），作为一种新的CRPC抑制靶点，通过无偏转录组学 筛选我们实验室开发的紧急CRPC模型。分析人类PC数据集和我们的 初步结果支持在CRPC从雄激素依赖性PC进展期间sGC活性被抑制， 并且sGC复合物被氧化灭活。然而，我们发现氧化还原保护机制诱导 通过雄激素剥夺（AD）保护CRPC细胞免于凋亡提供了治疗窗口，在此期间， sGC可被刺激至最大生物活性。因此，我们假设sGC活性抑制CRPC生长， 通过临床批准的激动剂的刺激将是治疗上有益的， 标准护理AD。我们的假设得到了我们强有力的临床前数据的支持，这些数据表明FDA批准的 血管扩张剂和sGC激动剂利奥西呱可减少去势抵抗性异种移植肿瘤的体内生长， 降低PSA并增加肿瘤内环cGMP，sGC信号传导的产物和一种对 目标利奥西呱疗效。与其生物学功能一致，sGC刺激诱导稳健的肿瘤 氧合以及CD 44 PC干细胞标志物的丢失，表明它破坏了缺氧干细胞， 壁龛去势抵抗与肿瘤缺氧和随后的放射抵抗有关。我们发现 利奥西呱增加了放射治疗在CRPC异种移植肿瘤中的肿瘤抑制疗效。我们的目标是 全面建立刺激sGC通路限制的分子机制 CRPC生长和进展，并确定预测sGC激动剂在CRPC中抗CRPC疗效的因素。 临床前模型。我们将评估1）控制sGC水平和分子还原的机制 再生氧化失活sGC的伴侣在去势敏感细胞与去势抗性细胞中发生改变， 2)sGC生物活性的生理效应如何产生抗CRPC结局，并考虑缺氧- 相关的PC相关代谢和氧化还原应激机制，和3）测试sGC激动剂在 CRPC疾病谱。我们的体外研究将利用强大的临床前模型的出现，生长， 骨环境的进展和转移性定植。我们将利用遗传学和药理学手段， 调节金标准培养模型中sGC的表达和活性， CRPC的特征，我们将利用稳健的皮下、原位和转移性临床前小鼠模型 以及患者来源的异种移植物（PDX）。我们将验证去识别PC中的关键分子发现 患者来源的标本，包括固定和冷冻组织、血清和血浆。我们的研究将揭示新的 CRPC生长和进展的生物学基础，并可能为重新利用提供临床前依据 sGC激动剂与标准护理AD的组合治疗。

Abstract Limited understanding of molecular mechanisms underlying castration-resistant prostate cancer (CRPC)is a barrier to effective therapeutic development for this fatal disease. We identified the nitric oxide receptor complex, soluble guanylyl cyclase (sGC), as a novel CRPC-inhibitory target via unbiased transcriptomics screening of an emergent CRPC model developed in our lab. Analyses of human PC datasets and our preliminary results support that sGC activity is inhibited during CRPC progression from androgen-dependent PC, and that the sGC complex is oxidatively inactivated. However we find the redox-protective mechanisms induced by androgen deprivation (AD) to protect CRPC cells from apoptosis provides a therapeutic window during which sGC can be stimulated to maximal bioactivity. Thus, we hypothesize sGC activity inhibits CRPC growth and that its stimulation by clinically-approved agonists will be therapeutically beneficial in combination with standard-of-care AD. Our hypothesis is supported by our strong preclinical data showing that the FDA-approved vasodilator and sGC agonist, riociguat, reduces in vivo growth of castration-resistant xenograft tumors, decreases PSA and increases intratumoral cyclic cGMP, the product of sGC signaling and a measure of on- target riociguat efficacy. Consistent with its biological function, sGC stimulation induces robust tumor oxygenation as well as loss of the CD44 PC stem cell marker, suggesting that it destroys hypoxic stem cell niches. Castration resistance is associated with tumor hypoxia and consequent radioresistance. We find that riociguat increases the tumor-suppressive efficacy of radiation in CRPC xenograft tumors. Our objective is to comprehensively establish molecular mechanisms underlying how and why stimulating the sGC pathway limits CRPC growth and progression and to identify factors that predict anti-CRPC efficacy of sGC agonists in preclinical models. We will assess 1) how mechanisms that control sGC levels and molecular reducing partners that regenerate oxidized inactive sGC are altered in hormone-sensitive vs. castration-resistant cells, 2) how the physiologic effects of sGC bioactivity enact anti-CRPC outcomes, with consideration of hypoxia- associated PC- relevant metabolic and redox stress mechanisms, and 3) test the efficacy of sGC agonists in the spectrum of CRPC disease. Our in vitro studies will utilize robust preclinical models of emergence, growth, progression and metastatic colonization of the bone milieu. We will utilize genetic and pharmacologic means to modulate sGC expression and activity in gold standard culture models that recapitulate the relevant clinical features of CRPC and we will utilize robust subcutaneous, orthotopic and metastatic preclinical mouse models as well as patient-derived xenografts (PDXs). We will validate key molecular findings in de-identified PC patient-derived specimens including fixed and frozen tissue, serum, and plasma. Our studies will uncover novel biology underlying CRPC growth and progression, and potentially provide preclinical rationale for re-purposing sGC agonists in combinatorial treatments with standard-of-care AD.