ARH1/NOEY2, An Imprinted Tumor Suppressor Gene, is Downregulated in Cancers at Multiple Sites

Yinhua Yu
M.D. Anderson Cancer Center; University of Texas

ARHI (NOEY2) is an imprinted putative tumor suppressor gene in ovarian and breast cancers reported in 1999. This gene encodes a small GTP-binding protein that belongs to the RaslRap superfamily. Although little is known about its function and signaling pathway(s), ARHI has the characteristics of a tumor suppressor gene despite its homology to the protooncogene Ras.

Our previous work confirmed ARHI is expressed monoallelically and is imprinted maternally. Loss of heterozygosity of the gene was detected in 41% of ovarian and breast cancers. In most of cancer samples with loss of heterozygosity, the nonimprinted functional allele was deleted. ARHI is expressed in normal ovarian and breast epithelial cells but dramatically downregulated in ovarian and breast cancers. Reexpression of ARHI through transfection or gene delivery mediated by adenovirus suppresses clonogenic growth of breast and ovarian cancer cells.

To study the biological function of ARHI in vivo, we have developed a transgenic mouse model that overexpressed ARHI. Our data suggest that ARHI can inhibit prolactin secretion and act as a negative regulator in murine growth and development. This model will be useful to continually study the biological function of this novel gene.

Genomic structure analysis has found two potential CpG islands within the promoter and adjacent exon 1 of the ARHI gene. CpG island I is located about 1 kb upstream of the transcription initiation site and CpG island II is near the transcription initiation region. To explore the underlying mechanisms by which ARHI expression is regulated, we have studied the methylation status of CpG islands I and II. Consistent with imprinting, island I and II were semi-methylated in normal breast epithelial cells. Hypermethylation of CpG island I was found in 37.5% of 8 breast cancer cell lines. Hypermethylation of CpG island II was found in 25% of 8 breast cancer cell lines and hypomethylation was observed in 37.5% of 8 breast cancer cell lines. Treatment with the demethylating agent, 5'-aza-2'-deoxycytidine, partially restored ARHI expression in the cells that exhibited hypermethylation of CpG island I or II. Our study suggests that hypermethylation of the promoter region of ARHI may be one mechanism by which ARHI gene expression is downregulated.

ARHI has been introduced into Ad/GT with five GAL4 binding sites and a TATA box (Ad/GT-ARHI). A second adenoviral construct, Ad/PGK-GV16, expresses GAL4 fused to a highly acidic portion of the herpes simplex virus VP16 protein. Co-infection of Ad/GT-ARHI and Ad/PGK-GV16 induced ARHI p26 protein after 2-4 days, whereas infection with Ad/GT-ARHI alone or co-infection with Ad/GT-LacZ and Ad/PGK-GV16 did not induce ARHI p26. When human ovarian and breast cancer lines were co-infected with Ad/PGK-GV 16 and Ad/GT-ARHI, clonogenic growth was inhibited by 90-99%. Co-infection with Ad/PGK-GV16 and Ad/GT-LacZ or infection with Ad/GT-ARHI alone exerted significantly less inhibition. To study the mechanism of growth inhibition, FACS analysis with a TUNEL assay demonstrated a significant induction of apoptosis (20-40%) 2-3 days after co-infection with Ad/PGK-GV16 and Ad/GT-ARHI, whereas controls co-infected with AdPGK-GV16 and Ad/GT-LacZ or with Ad/GT-ARHI alone exhibited

Recent studies from our group and other groups have shown that ARHI is downregulated not only in ovarian and breast cancers, but also in pancreas, liver, bladder and prostate cancers. LOH was detected at the ARHI locus in 45% of pancreas cancer specimens. Using histochemical staining and in situ hybridization, only 22% of pancreatic cancer cells expressed ARHI, compared to ARHI expression in 64% of normal pancreatic duct epithelial cells. DNA array analysis from 4 pairs of liver hepatomas and surrounding normal tissues also showed ARHI was lost or downregulated in all liver cancer samples but not in their adjacent normal liver tissues, Consequently, lost or reduced ARHI expression could play an important role in the oncogenesis of cancers at multiple sites.