ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma

Pheochromocytoma (PCC) is related to germline mutations in 12 susceptibility genes. Although comparative genomic hybridization array has revealed some putative tumor suppressor genes on the short arm of chromosome 1 that are likely to be involved in PCC tumorigenesis, the molecules involved, except for those encoded by known susceptibility genes, have not been found in the generation of sporadic tumors. In the present work, we first identified that the unmethylated allele of Aplasia Ras homolog member I (ARHI) was deleted in most PCC tumors which retained a hypermethylated copy, while its mRNA level was significantly correlated with the unmethylated copy. De-methylation experiments confirmed that expression of ARHI was also regulated by the methylation level of the remaining allele. Furthermore, ARHI overexpression inhibited cell proliferation, with cell cycle arrest and induction of apoptosis, in ARHI-negative primary human PCC cells, whereas knockdown of ARHI demonstrated the opposite effect in ARHI-positive primary human PCC cells. Finally, we demonstrated that ARHI has the ability to suppress pAKT and pErK1/2, to promote the expression of p21^Waf1/Cip1 and p27^Kip1, and also to increase p27^Kip1 protein stability. In summary, ARHI was silenced or downregulated in PCC tissues harboring only one hypermethylated allele. ARHI contributes to tumor suppression through inhibition of PI3K/AKT and MAKP/ERK pathways, to upregulate cell cycle inhibitors such as p27^Kip1. We therefore reasoned that ARHI is a novel epigenetic silenced tumor suppressor gene on chromosome 1p that is involved in sporadic PCC tumorigenesis.