Role of HSulf-1 Loss in Apoptosis and Drug Resistance

描述（申请人提供）：为了鉴定卵巢癌发展中涉及的早期遗传变化，我们构建了来自两个早期和两个晚期卵巢肿瘤的抑制性消减杂交（SSH）cDNA文库，这些cDNA文库减去正常卵巢上皮细胞刷拭，以鉴定这些组织中异常调节的基因。这些分析导致HSulf-1的鉴定，其编码含有高度保守的硫酸酯酶结构域的新的871个氨基酸的多肽，作为在75%的卵巢癌中下调的基因。在乳腺、胰腺、肾细胞和肝细胞癌细胞系中也观察到类似的下调。其他数据表明，HSulf-1编码一种细胞表面多肽，其表现出硫酸酯酶活性并减少硫酸肝素蛋白聚糖（HSPG）的硫酸化，特别是糖胺聚糖的N-乙酰葡糖胺残基。新出现的数据表明HSPG的硫酸化状态可以影响肝素结合生长因子的信号传导，这使我们假设HSulf-1下调调节卵巢癌中的生长因子信号传导。与这一假设相一致，我们观察到HSulf-1缺陷卵巢癌细胞中HSulf-1的恢复减弱了成纤维细胞生长因子（FGF）和肝素结合表皮生长因子（HB-EGF）的信号传导。结果，HSulf-1转染的克隆增殖更慢，更敏感的诱导细胞凋亡的顺铂和星形孢菌素比他们的HSulf-1缺陷的同行。这些观察结果表明，HSulf-1下调有助于卵巢癌中观察到的增殖和凋亡的失调。尽管广泛的手术和全身化疗，通常与紫杉烷和铂剂，绝大多数患者谁目前与播散（III期或更高）卵巢癌死于他们的疾病。这一观察结果凸显了需要更好地了解卵巢癌的耐药性。为了进一步评价HSulf-1下调在卵巢癌中的生物学效应，我们现在提出：1）探索HSulf-1调节细胞凋亡能力的机制基础; 2）确定HSulf-1下调的遗传基础;和3）评估HSulf-1下调是否导致对常用于治疗卵巢癌的其它药剂的抗性，所述其它药剂包括紫杉醇，托泊替康，吉西他滨和多柔比星，在体外和体内，和4）评估HSulf-1表达和药物敏感性之间的关系，在临床设置。

DESCRIPTION (provided by applicant): In an effort to identify early genetic changes involved in the development of ovarian cancer, we constructed suppression subtraction hybridization (SSH) cDNA libraries from two early and two late stage ovarian tumors subtracted against normal ovarian epithelial cell brushings to identify aberrantly regulated genes in these tissues. These analyses led to the identification of HSulf-1, which encodes a novel 871 amino acid polypeptide containing a highly conserved sulfatase domain, as a gene that is downregulated in (75% of ovarian cancers. Similar downregulation is also observed in breast, pancreatic, renal cells and hepatocellular carcinoma lines. Additional data indicated that HSulf-1 encodes a cell surface polypeptide that exhibits sulfatase activity and diminishes the sulfation of heparin sulfate proteoglycans (HSPGs), specifically the N-acetylglucosamine residue of glycosaminoglycans. Emerging data suggesting that the sulfation state of HSPGs can influence signaling by heparin binding growth factors led us to hypothesize that HSulf-1 down-regulation modulates growth factor signaling in ovarian cancer. Consistent with this hypothesis, we observed that HSulf-1 restoration in HSulf-1-deficient ovarian cancer cells blunted signaling by fibroblast growth factor (FGF) and heparin-binding epidermal growth factor (HB-EGF). As a result, HSulf-1-transfected clones proliferated more slowly and were more sensitive to the induction of apoptosis by cisplatin and staurosporine than their HSulf-1-deficient counterparts. These observations suggest that HSulf-1 down-regulation contributes to the dysregulation of proliferation and apoptosis observed in ovarian cancer. Despite extensive surgery and systemic chemotherapy, usually with a taxane and a platinating agent, the vast majority of patients who present with disseminated (stage Ill or greater) ovarian cancer die of their disease. This observation highlights the need for improved understanding of drug resistance in ovarian cancer. To further evaluate the biological effects of HSulf-1 downregulation in ovarian cancer, we now propose to: 1) explore the mechanistic basis for the ability of HSulf-1 to modulate apoptosis; 2) determine the genetic basis of HSulf-1 downregulation; and 3) assess whether HSulf-1 downregulation results in resistance to other agents commonly used to treat ovarian cancer, including paclitaxel, topotecan, gemcitabine and doxorubicin, in vitro and in vivo and 4) evaluate the relationship between HSulf-1 expression and drug sensitivity in the clinical setting.