Role of HSulf-1 Loss in Apoptosis and Drug Resistance

描述(申请人提供)：为了确定与卵巢癌发展有关的早期基因变化，我们构建了两个早期和两个晚期卵巢肿瘤与正常卵巢上皮细胞刷检的抑制消减杂交(SSH)cDNA文库，以确定这些组织中异常调控的基因。这些分析导致HSulf-1被鉴定为在75%的卵巢癌中下调的基因。HSulf-1编码一种新的871个氨基酸多肽，含有高度保守的硫酸盐酶结构域。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。更多的数据表明，HSulf-1编码的细胞表面多肽具有硫酸酶活性，并减少了硫酸肝素蛋白多糖(HSPGs)的硫酸盐化，特别是糖胺聚糖的N-乙酰氨基葡萄糖残基。新出现的数据表明，HSPGs的硫酸盐化状态可以通过肝素结合生长因子影响信号转导，这让我们假设HSulf-1下调调控卵巢癌生长因子信号转导。与这一假设一致，我们观察到HSulf-1缺陷卵巢癌细胞中HSulf-1的修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)的信号转导。因此，与HSulf-1缺陷克隆相比，HSulf-1转基因克隆的增殖速度更慢，对顺铂和星形孢子素诱导的细胞凋亡更敏感。这些观察表明，HSulf-1的下调与卵巢癌中观察到的增殖和凋亡的失调有关。尽管进行了广泛的手术和全身化疗，通常使用紫杉烷和铂剂，但绝大多数患有播散性(Ill期或更高)卵巢癌的患者死于他们的疾病。这一观察结果突显了提高对卵巢癌耐药的了解的必要性。为了进一步评估HSulf-1下调在卵巢癌中的生物学效应，我们现在提议：1)探讨HSulf-1调控细胞凋亡的机制；2)确定HSulf-1下调的遗传基础；3)评估HSulf-1下调是否导致对其他常用治疗卵巢癌药物的耐药性，包括紫杉醇、拓扑替康、吉西他滨和阿霉素，以及4)评估HSulf-1表达与临床药物敏感性的关系。

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.