ARHI: AN IMPRINTED TUMOR SUPPRESSOR GENE

University Of California San Francisco, San Francisco CA

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Abstract

Differential display PCR ha been used to search for genes that are expressed in normal ovarian surface epithelial cells, but not in ovarian cancer cells. NO3EY2 (designated ARHI, by the Human Gene Nomenclature Committee) is expressed consistently by normal ovarian and breast epithelial cells, but not be ovarian or breast cancers. This gene encodes a small G protein of 26 Kd that is homologous to ras and rap. Loss of ARHI expression results from multiple mechanisms. The gene is expressed monoallelically and is maternally imprinted. Loss of heterozygosity of the gene was detected in 41% of ovarian and breast cancers on chromosome 1p31. In tumo5rs with LOH, the non-imprinted functional allele was deleted in 7 of 9 instances. Expression of ARHI can also be transcriptionally regulated with promoter activity in normally ovarian epithelial cells, but not in most cancers. The activities of promoter segments of different lengths have been evaluated in normal and malignant ovarian epithelial cells. A truncated construct of 180 bp was significantly less active in ovarian cancers that in normal cells. A 26 bp DNA element in this region binds to a putative regulatory protein in ovarian cancer cells that fail to express ARHI, but does not bind to nuclear proteins in normal ovarian surface epithelial cells that express ARHI. Reexpression of the ARHI gene by transfection inhibits the clonogenic growth of ovarian and breast cancer cell lines that have lost expression of ARHI. Growth inhibition is associated with down- regulation of cyclin D1 promoter activity and induction of P21/WAF1/CIP1. EGF-induced signaling through Fas/map is reduced and truncated by expression of ARHI whereas the JNK kinase pathway is activated. Mice bearing the human ARHI transgene are small in size, resembling cyclin D1 knockout mice. In addition, strong expression of the ARHI transgene is associated with a defect in mammary gland development, sterility, atrophy of multiple organs and degeneration of the hippocampus and cerebellar cortex. Thus, ARHI is a putative growth inhibitory tumor suppressor gene whose loss may be important for the development of a significant fraction of ovarian cancers. The overall goal of this proposals is to explore the role of ARHI as a growth regulatory gene. Our specific aims are three fold: 1) to evaluate (a) the clinical significance and (b) the mechanism of loss of ARHI expression in malignant ovarian epithelial cells; 2) to determine the effect ARHI expression on proliferation, invasion, metastasis and survival of ovarian cells; 3) to determine mechanisms by which ARHI interferes with signal transduction, inhibits cell proliferation or induces apoptosis