Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer.
We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5'aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.
Department of Surgery, VAGLAHS/David Geffen School of Medicine at UCLA, Los Angeles 90073, CA.
Mysore S
MS
Veena
Grant
G
Lee
Daniel
D
Keppler
Marc S
MS
Mendonca
J Leslie
JL
Redpath
Eric J
EJ
Stanbridge
Sharon P
SP
Wilczynski
Eri S
ES
Srivatsan
D001483:Base Sequence; D045744:Cell Line, Tumor; D055332:Cystatin M; D015891:Cystatins / Q000235:genetics*; D019175:DNA Methylation; D005091:Exons; D005260:Female; D005455:Fluorescent Antibody Technique; D016147:Genes, Tumor Suppressor; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000378:metabolism
C525104:CST6 protein, human; D055332:Cystatin M; D015891:Cystatins