Regulation of Serine Protease HtrA1 and Chemoresponse

描述(由申请人提供)：大多数被诊断为卵巢癌的女性最终死于这种疾病，原因是耐药细胞的生长[4]。提高对化疗耐药的细胞机制的理解对于开发新的有效的抗癌治疗以克服化疗耐药问题是至关重要的。我们已经证明，HtrA1是一种功能未知的丝氨酸蛋白酶，在大多数卵巢癌细胞系和原发肿瘤中表达下调。更多研究表明，HtrA1可能调节卵巢癌细胞的细胞死亡和锚定独立生长[2]。我们最近的数据表明HtrA1被顺铂和紫杉醇上调，提示它可能与化疗敏感性有关。HtrA1表达后细胞死亡和化疗敏感性的增加依赖于HtrA1的蛋白酶活性，这表明丝氨酸蛋白酶活性在细胞程序性死亡(PCD)中发挥作用。此外，在HtrA1高表达的肿瘤患者中，90%(27/30)的患者对化疗有完全或部分临床缓解，而HtrA1低水平和中等水平的肿瘤患者的有效率分别为62%(8/13)和65%(11/17)。低染色与高染色(P=0.0276)、中染色与高染色(P=0.0342)对化疗的反应差异有统计学意义。这些发现确认HtrA1是顺铂诱导的细胞毒性的一种新的调节剂，并提示HtrA1在卵巢癌中的缺失可能与化疗耐药有关[5]。本应用的目的是确定HtrA1在癌症中表达的调控机制，并确定HtrA1在细胞程序性死亡和化疗耐药中的作用。基于我们的初步研究，我们提出了以下三个假设：第一，HtrA1在癌症中的表达在表观遗传中受到调控，当其表达没有受到表观遗传机制的沉默时，它在癌细胞中的转录水平上调；第二，HtrA1参与了“丝氨酸蛋白酶体”介导的PCD，该过程与caspase介导的细胞死亡途径相互作用；第三，化疗后HtrA1对特定底物的靶向降解导致了化疗诱导的细胞毒性。与公共卫生相关：新出现的证据表明，导致肿瘤转化的一些相同的变化也会导致耐药性。特别是，通过使细胞抵抗不利生长条件的压力和失去重要的动态平衡过程而导致表型转化的相同的抗凋亡变化，似乎也使细胞对癌症化疗的抵抗力更强。基于这一观点，提高我们对肿瘤发生过程的理解的研究也有可能为耐药问题提供新的见解。我们的研究表明，丝氨酸蛋白酶HtrA1的表达变化调节了化疗诱导的细胞毒性。在这些研究的结论中，我们希望阐明HtrA1表达下调在卵巢癌中的意义，以及这种HtrA1表达降低如何促进化疗耐药卵巢癌的发展。更好地了解HtrA1在鲜为人知的丝氨酸蛋白酶介导的程序性细胞死亡中的作用，可能有助于发现克服耐药性的新治疗方法。耐药卵巢癌是一种致命的疾病，该项目将更好地确定卵巢癌耐药和程序性细胞死亡的新机制。

DESCRIPTION (provided by applicant): The majority of women diagnosed with ovarian cancer ultimately succumb to the disease due to the outgrowth of cells resistant to chemotherapy [4]. An improved understanding of cellular mechanisms of chemoresistance is essential in developing new effective anticancer therapy to overcome the problem of chemoresistance. We have shown that HtrA1, a serine protease with previously unknown function, is down-regulated in a majority of ovarian cancer cell lines and primary tumors. Additional studies indicated that HtrA1 may modulate cell death and anchorage independent growth of ovarian cancer cells [2]. Our more recent data indicate that HtrA1 is upregulated by cisplatin and paclitaxel and suggests that it may contribute to sensitivity to chemotherapy. Increased cell death and chemosensitivity following HtrA1 expression depends on the protease activity of HtrA1, suggesting a role of serine protease activity in programmed cell death (PCD). Moreover, 90% of patients (27/30) with tumors expressing high levels of HtrA1 responded to chemotherapy with complete or partial clinical remissions, compared to 62% (8/13) and 65% (11/17) response rates in tumors with low and moderate levels of HtrA1, respectively. Response to chemotherapy was significantly different between low and high (P = 0.0276) or moderate and high (P = 0.0342) staining groups. These findings identify HtrA1 as a novel modulator of cisplatin-induced cytotoxicity and suggest that loss of HtrA1 in ovarian cancer may contribute to chemoresistance [5]. The objective of this application is to determine the mechanisms by which HtrA1 expression is regulated in cancer and define the role of HtrA1 in programmed cell death and chemoresistance. Based on our preliminary studies, we are proposing the following three hypotheses: first, HtrA1 expression is epigenetically regulated in cancer and is transcriptionally upregulated by chemotherapy in cancer cells when its expression is not silenced by epigenetic mechanisms; second, HtrA1 participates in a "serine proteasome"-mediated PCD that cross-talks with caspase-mediated cell death pathways; and third, targeted degradation of specific substrates by HtrA1 following chemotherapy treatment contributes to chemotherapy-induced cytotoxicity. PUBLIC HEALTH RELEVANCE: Emerging evidence suggests that some of the same changes that contribute to neoplastic transformation also contribute to drug resistance. In particular, the same anti-apoptotic changes that contribute to the transformed phenotype by making cells resistant to the stresses of unfavorable growth conditions and loss of important homeostatic processes also appear to make cells more resistant than they would otherwise be to cancer chemotherapy. Based on this view, studies that improve our understanding of the process of tumorigenesis have the potential to also provide new insight into the problem of drug resistance. Our studies have shown that altered expression of the serine protease HtrA1 modulates chemotherapy induced cytotoxicity. At the conclusion of the proposed studies, we hope to elucidate the significance of HtrA1 down-regulation in ovarian cancer and how this reduced HtrA1 expression contributes to the development of chemoresistant ovarian cancer. A better understanding of the role of HtrA1 in a poorly understood serine protease mediated programmed cell death may contribute to discoveries of new therapeutic approaches to overcome drug resistance. Drug resistant ovarian cancer is a lethal disease, and this project will better define novel mechanisms of drug resistance and programmed cell death in ovarian cancer.