ARHI (ras homolog member I) encodes a 26 kD GTPase with 50-60% amino acid homology to Ras and Rap. ARHI and Ras share similar GTP/GDP-binding domains, but exert opposite functions. ARHI is the first reported putative tumor suppressor in the Ras superfamily. Structural analysis has demonstrated that ARHI has a distinctive N-terminal extension that is critical for the suppression of tumor growth.
ARHI is expressed consistently in normal breast and ovarian epithelial cells, but not in breast or ovarian cancers. The loss of ARHI can be related to tumor progression. In breast cancer samples, ARHI is down-regulated in ductal carcinoma in situ and further down-regulated in invasive carcinoma. Re-expression of ARHI inhibits growth of cancers that have lost their expression, induces p21, down-regulates cyclin Dl, blocks signaling through RAS/MAP, activates JNK, and induces apoptosis through a calpain-dependent pathway. Transgenic expression of ARHI in mice produces small stature and inhibits lactation.
ARHI is maternally imprinted with methylation of the three CpG islands in the maternal allele of normal cells. ARHI is expressed only from the paternal allele whose three CpG islands are not methylated. Down-regulation of the functioning allele can occur through multiple mechanisms, including loss of heterozygosity, methylation, and transcriptional regulation. ARHI maps to chromosome 1p31 at the center of a common allelic deletion site observed in 40% of breast, ovarian, and pancreatic cancers. Analysis of the genomic structure of ARHI has demonstrated two exons and three CpG islands. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region of exon II. CpG island II is particularly important, since it is near the TATA box and extends into the untranslated exon I. The ARHI promoter has been linked to a luciferase reporter. Treatment of the entire construct with SsI methylase destroyed promoter activity when transfected into SKBr3 breast cancer cells. Selective methylation of CpG island II also eliminated promoter activity, but methylation of CpG island I only minimally decreased promoter activity.
Down-regulation of ARHI in breast cancer cell lines can be associated with different patterns of methylation. Three types of methylation have been observed. In type 1 methylation, CpG island II is hypermethylated. In type 2 methylation, CpG island II is hypomethylated, and CpG islands I and III are either partially methylated or hypermethylated. In type 3 methylation, all three CpG islands are partially methylated. Using microdissected specimens, we studied methylation of ARHI in 20 human breast cancers. Seven of 20 had type 2 methylation, whereas 3 of 20 had type 3 methylation. In 12 cases, hypermethylation was found in CpG island III in the coding region of exon 2.
Treatment with the demethylating agent 5-deoxy-aza-cytidine (5-AZA) partially activated ARHI expression in four of six cell lines associated with decreased methylation of CpG islands I, II, and III. Treatment with 5-AZA did not affect ARHI expression in cancer cells with only methylation of the imprinted maternal allele, suggesting that this inhibitor could not reverse imprinting. Treatment with the histone deacetylase inhibitor trichostatin (TSA) increased ARHI expression in four cell lines which did not have hypermethylation in CpG island II. Increased ARHI expression was also observed in breast cancer cell lines that exhibited methylation of only the imprinted maternal allele, raising the possibility that TSA could reverse imprinting. Robust expression of ARM was observed with a combination of 5-AZA and TSA in all six cell lines. Taking advantage of a G/A polymorphism at +231 in the coding region of ARHI, we demonstrated that the paternal allele of ARHI could be reactivated by TSA. The imprinted maternal allele could also be reactivated with a combination of 5-AZA and TSA.
A chromatin immunoprecipitation assay was used to study acetylation of the chromatin-DNA complex in normal breast epithelial cells and breast cancer cells with known methylation status. In normal breast epithelial cells, we found ARHI DNA associated with acetylated histone H3 in all three CpG islands. In breast cancer cells, much lower levels of acetylated histone H3 were observed. After treatment with 5-AZA and TSA, association of ARHI with acetylated histone H3 was increased.
Our data suggest that ARHI can be silenced through methylation of particular CpG islands of the non-imprinted allele with a concomitant deacetylation of associated histones. Reactivation of both the silenced paternal and imprinted maternal alleles can be achieved by demethylation and inhibition of histone deacetylation.