The role for alcohol-induced Golgi disorganization in the progression of prostate cancer

摘要 长期酗酒和酗酒被认为是前列腺癌(PCA)进展的危险因素，但 其机制尚不清楚。目前的项目将解决临床医生提出的一个重要问题： 戒酒是前列腺癌重要的治疗干预措施。此前，我们发现：(1)碎片化 高尔基复合体与前列腺癌的进展有关；(2)乙醇(Etoh)可诱导高尔基体的解体，即 由最大的高尔基体基质蛋白Giantin的二聚化受损引起的，这反过来又改变了高尔基体内的 一些高尔基体蛋白的定位。事实上，我们最近观察到，在雄激素反应性前列腺癌细胞中，乙醇- 诱导的高尔基体碎裂导致糖原合成酶β(β)从高尔基体转位到 胞质，随后激活HDAC6-HSP90-AR通路。此外，我们还报告了非肌肉组织 肌球蛋白IIA(NMIIA)马达蛋白迫使乙醇诱导高尔基体破坏。此外，酒精还能诱导内质 网状(ER)应激和未折叠蛋白反应(UPR)是Pca进展的已知驱动因素； 然而，乙醇在肿瘤中诱导UPR的机制在很大程度上仍不清楚。在这里，我们发现在Low 传代的LNCaP细胞是UPR感受器之一ATF6，在高尔基体中被S1P和S2P蛋白酶切割；然而， 乙醇处理改变S1P和S2P的高尔基体定位，使它们被困在内质网并促进ATF6 激活。此外，乙醇诱导糖基转移酶MGAT3从高尔基体转移到 细胞质紧随其降解。然而，MGAT5，与MGAT3竞争N-糖链的酶 分枝，仍保留在高尔基山脉。这导致了MGAT5介导的前转移蛋白的糖基化 (包括Mattritase和整合素)及其在细胞表面的过表达。最后，我们检测到失去了 NMIIA功能可防止酒精诱导的前列腺癌细胞高尔基体碎裂。这反过来又恢复了MGAT3的S内部 高尔基体定位，减少整合素的表达。鉴于这些事实，我们建议检验这一假设 酒精通过高尔基体碎裂加速前列腺癌进展：(A)通过以下途径增强内质网应激反应 诱导ATF6介导的UPR，以及(B)在其异常情况下刺激促转移蛋白的表达 MGAT5介导的糖基化。因此，靶向酒精诱导的高尔基体碎裂在治疗上具有重要意义。 这项研究的三个具体目的是：1)阐明酒精诱导的神经元激活的机制 通过确定酒精代谢产物如何诱导高尔基体定位的S1P和S2P蛋白酶的易位来实现内质网应激 2)检测乙醇诱导的MGAT5介导的糖基化在前列腺癌进展中的作用；3) 研究抑制或敲除NMIIA是否可以防止乙醇诱导的高尔基体碎裂，并减弱 PCa细胞的侵袭性表型。该方法将包括各种体外和体内实验，使用 乙醇处理的前列腺癌细胞系、动物模型和酒精消耗性前列腺癌患者的临床样本。这个 完成拟议研究的目标将扩大我们对前列腺癌基础生物学的了解 并阐明了酒精促癌作用的机制。

ABSTRACT Chronic alcohol abuse and alcoholism are considered risk factors for prostate cancer (PCa) progression, but the mechanism is unknown. The current project will address an important question raised by clinicians: whether alcohol abstinence is an important therapeutic intervention in PCa. Previously, we found that: (1) fragmentation of the Golgi complex correlates with the progression of PCa; (2) ethanol (EtOH) induces Golgi disorganization that is caused by the impaired dimerization of the largest Golgi matrix protein giantin, which, in turn, alters intra-Golgi localization of some Golgi proteins. Indeed, we recently observed that in androgen-responsive PCa cells, EtOH- induced Golgi fragmentation leads to translocation of glycogen synthase kinase β (GSK3β) from the Golgi to the cytoplasm, followed by the activation of HDAC6-HSP90-AR pathway. Additionally, we reported that non-muscle myosin IIA (NMIIA) motor protein forces EtOH-induced Golgi disruption. Also, alcohol induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR), which are the known drivers of PCa advancement; however, the mechanism of EtOH-induced UPR in cancer remains largely uncovered. Here, we found that in low passage LNCaP cells, one of the UPR sensor, ATF6, is cleaved in Golgi by S1P and S2P proteases; however, EtOH treatment alters the Golgi localization of S1P and S2P, trapping them in the ER and facilitating ATF6 transactivation. Further, EtOH induces translocation of the glycosyltransferase MGAT3 from the Golgi to the cytoplasm followed by its degradation. However, MGAT5, the enzyme that competes with MGAT3 for N-glycan branching, is still retained in the Golgi. This results in MGAT5-mediated glycosylation of pro-metastatic proteins (including matriptase and integrins) and their overexpression at the cell surface. Finally, we detected that loss of NMIIA function prevents alcohol-induced Golgi fragmentation in PCa cells. This, in turn, recovers MGAT3’s intra- Golgi localization and reduces the expression of integrins. In light of these facts, we propose to test the hypothesis that alcohol accelerates PCa progression through Golgi fragmentation, which: (a) enhances ER stress response via induction of ATF6-mediated UPR, and (b) stimulates expression of pro-metastatic proteins over their abnormal MGAT5-mediated glycosylation. Hence, targeting alcohol-induced Golgi fragmentation is therapeutically important. The three specific aims of the proposed study are to: 1) elucidate the mechanism of alcohol-induced activation of ER stress by determining how alcohol metabolites induce translocation of Golgi localized S1P and S2P proteases to the ER; 2) examine the role of EtOH-induced MGAT5-mediated glycosylation in the progression of PCa; and 3) examine whether inhibition or knockdown of NMIIA prevents EtOH-induced Golgi fragmentation and attenuates the aggressive phenotype of PCa cells. The approach will include a variety of in vitro and in vivo experiments using EtOH-treated PCa cell lines, animal models and clinical samples from alcohol consuming PCa patients. The accomplishment of the goal of the proposed study would expand our understanding of the basic biology of PCa carcinogenesis, and elucidate the mechanisms of alcohol's tumor promotion action.