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.
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.