Isolation of a cervical cancer tumor suppressor gene

描述（由申请人提供）： 摘要。我们以前已经表明，宫颈癌抑制基因定位于染色体11 q13的300 kb间隔。我们现在已经确定了半胱氨酸蛋白酶抑制剂E/M映射到这个区间作为宫颈癌抑制基因。RT-PCR和Western blotting研究显示，6个宫颈癌细胞系和21个原发性肿瘤中的17个中没有cystatin E/M表达。20例肿瘤标本的免疫组化显示15例肿瘤表达缺失或减少。一些样品的正常区域中的基因表达作为阳性对照。在肿瘤前宫颈肿瘤内（CINs）病变中也观察到表达，表明表达丧失是肿瘤特异性事件。此外，正常和含有半胱氨酸蛋白酶抑制剂E/M的CIN显示溶酶体蛋白酶组织蛋白酶L的表达减少。然而，在肿瘤中观察到组织蛋白酶L的过表达，表明胱抑素E/M和组织蛋白酶L的表达之间呈反比关系。 对19例原发性肿瘤和21例正常组织中CystatinE/M基因的3个外显子进行检测，发现1例肿瘤中存在外显子1的纯合性缺失。在其他六个肿瘤中观察到点突变。两个肿瘤在组织蛋白酶L的共有结合位点含有突变。使用定点诱变引入结合位点突变导致半胱氨酸蛋白酶抑制剂E/M与组织蛋白酶L的结合减少。虽然在细胞系中没有观察到突变，但4个细胞系和18个肿瘤中的12个含有启动子超甲基化。在5 '氮杂2-脱氧胞苷和/或曲古抑菌素A处理宫颈癌细胞系后观察到胱抑素E/M的再表达，证实了启动子高甲基化的存在。此外，对正常人表皮角质形成细胞（宫颈癌的细胞类型来源）和HPV 16 E6、E7或E6和E7转化的角质形成细胞的分析显示，含E7的角质形成细胞中半胱氨酸蛋白酶抑制剂E/M的表达降低。混合培养（HeLa与E6或E7角质形成细胞）研究显示E6细胞对HeLa细胞生长的抑制，表明E7角质形成细胞中半胱氨酸蛋白酶抑制剂E/M的降解。免疫沉淀研究表明HPV 16 E7与胱抑素E/M蛋白结合。cystatin E/M在宫颈癌细胞系中的异位表达导致生长抑制，伴随着细胞内和细胞外组织蛋白酶L水平的降低。最后，组织蛋白酶L的过表达导致细胞生长增加，并且半胱氨酸蛋白酶抑制剂E/M的引入导致生长抑制。 我们现在提出使用以下目标来确定半胱氨酸蛋白酶抑制剂E/M介导的肿瘤抑制的机制：1）鉴定与原发性宫颈肿瘤中半胱氨酸蛋白酶抑制剂E/M表达丧失相关的肿瘤特异性突变，2）证实半胱氨酸蛋白酶抑制剂E/M通过组织蛋白酶L失活抑制细胞生长，3）确定HPV 16 E7蛋白降解半胱氨酸蛋白酶抑制剂E/M的机制，4）利用四环素诱导型载体系统研究cystatin E/M的抑癌作用; 5）探讨cystatin E/M和cathepsin L的表达与宫颈癌表型的关系。 公共卫生相关性： 对退伍军人保健的潜在影响：虽然妇女在退伍军人中占少数，但近年来有更多的妇女在武装部队服役。宫颈癌是世界上第二大常见的癌症，是导致女性癌症相关死亡的原因。癌症影响到处于特权和服务不足的妇女。它也是发展中世界的一大癌症。这种疾病常见于有多个性伴侣、吸烟习惯和免疫系统功能障碍的女性。

DESCRIPTION (provided by applicant): ABSTRACT. We have previously shown that a cervical cancer suppressor gene is localized to a 300 kb interval of chromosome 11q13. We have now identified cystatin E/M mapped to this interval as a cervical cancer suppressor gene. RT-PCR (reverse transcriptase PCR), and western blotting studies revealed absence of cystatin E/M expression in 6 cervical cancer cell lines and 17 of 21 primary tumors. Immunohistochemistry on 20 tumor samples revealed loss or reduced expression in 15 tumors. Expression of the gene in normal areas of some samples served as positive controls. Expression was also seen in pre-neoplastic cervical intraneoplasisa (CINs) lesions, indicating loss of expression as a tumor specific event. Further, normal and CINs containing cystatin E/M showed reduced expression of the lysosomal protease cathepsin L. However, overexpression of cathepsin L was observed in tumors pointing to an inverse relationship between the expression of cystatin E/M and cathepsin L. Examination of the three exons of cystatin E/M in 19 primary tumors and 21 normal tissues revealed homozygous deletion of exon 1 in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L. Introduction of the binding site mutations using site directed mutagenesis resulted in reduced binding of cystatin E/M to cathepsin L. Although mutations were not observed in the cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5'aza 2-deoxycytidine and/or Trichostatin A treatment of cervical cancer cell lines confirming the presence of promoter hypermethylation. Further, analysis of normal human epidermal keratinocytes (cell type origin of cervical cancer), and HPV16 E6, E7 or E6 & E7 transformed keratinocytes showed decreased expression of cystatin E/M in E7 containing keratinocytes. Mixed culture (HeLa with E6 or E7 keratinocytes) studies showed HeLa cell growth inhibition by E6 cells indicating degradation of cystatin E/M in E7 keratinocytes. Immunoprecipitation studies indicated binding of HPV 16 E7 to the cystatin E/M protein. Ectopic expression of the cystatin E/M in cervical cancer cell lines resulted in growth inhibition accompanied by decreased intracellular and extracellular level of cathepsin L. Finally, overexpression of cathepsin L led to increased cell growth and introduction of cystatin E/M resulted in growth inhibition. We now propose to determine the mechanism of cystatin E/M mediated tumor suppression using the following objectives: 1) Identification of tumor specific mutations correlating to loss of cystatin E/M expression in primary cervical tumors, 2) confirmation of cell growth inhibition by cystatin E/M through the inactivation of cathepsin L, 3) determine the mechanism of cystatin E/M degradation by the HPV 16 E7 protein, 4) use of tetracycline inducible vector system to study the tumor suppressor function of cystatin E/M, and 5) determine the relationship between the expression of cystatin E/M and cathepsin L to cervical cancer phenotypes. PUBLIC HEALTH RELEVANCE: Potential Impact on General VA Health care: Although women comprise minority of the veteran population, more women are serving in the armed services in recent years. Cervical cancer is the second most common cancer responsible for cancer related death in women around the world. The cancer affects women who are under privileged and under served. It is also a major cancer of the developing world. The disease is frequently found in women having multiple sex partners, smoking habits, and immune system dysfunctions.