Role of HSulf-1 Loss in Apoptosis and Drug Resistance

描述（由申请人提供）：为了确定卵巢癌发展过程中涉及的早期遗传变化，我们从两个早期和两个晚期卵巢肿瘤中构建了抑制减法杂交（SSH） cDNA文库，以确定这些组织中异常调节的基因。这些分析鉴定出HSulf-1基因在75%的卵巢癌中下调，该基因编码一种含有高度保守的磺化酶结构域的新型871个氨基酸多肽。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编码一种具有磺化酶活性的细胞表面多肽，并减少硫酸肝素蛋白聚糖（HSPGs）的磺化，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下调可调节卵巢癌中生长因子的信号传导。与这一假设一致，我们观察到HSulf-1缺失卵巢癌细胞中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，转染hsulf -1的克隆比缺乏hsulf -1的克隆增殖更慢，对顺铂和斯桃孢素诱导的细胞凋亡更敏感。这些观察结果表明，HSulf-1的下调参与了卵巢癌细胞增殖和凋亡的失调。尽管进行了广泛的手术和全身化疗（通常使用紫杉烷和铂化剂），但绝大多数患有弥散性（ii期或以上）卵巢癌的患者仍死于疾病。这一观察结果突出了提高对卵巢癌耐药认识的必要性。为了进一步评估HSulf-1下调在卵巢癌中的生物学作用，我们建议：1)探索HSulf-1调节细胞凋亡的机制基础；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卵巢癌治疗药物（包括紫杉醇、拓扑替康、吉西他滨和阿霉素）的体外和体内耐药；4)评估临床环境中HSulf-1表达与药物敏感性的关系。

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.