The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

BACKGROUND: Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up‐regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re‐expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts.METHODS: Ovarian cancer cells (Hey and SKOv3) were treated with demethylating agents (5‐aza‐2′‐deoxycytidine [DAC] or 5‐azacitidine [AZA]) or with HDAC inhibitors (suberoylanilide hydroxamic acid [SAHA] or trichostatin A [TSA]) to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, autophagy, and re‐expression of 2 growth inhibitory imprinted tumor suppressor genes: guanosine triphosphate‐binding Di‐RAS‐like 3 (ARHI) and paternally expressed 3 (PEG3). The in vivo activities of DAC and SAHA were assessed in a Hey xenograft model.RESULTS: The combination of DAC and SAHA produced synergistic inhibition of Hey and SKOv3 cell growth by apoptosis and cell cycle arrest. DAC induced autophagy in Hey cells that was enhanced by SAHA. Treatment with both agents induced re‐expression of ARHI and PEG3 in cultured cells and in xenografts, correlating with growth inhibition. Knockdown of ARHI decreased DAC‐induced autophagy. DAC and SAHA inhibited the growth of Hey xenografts and induced autophagy in vivo.CONCLUSIONS: A combination of DAC and SAHA inhibited ovarian cancer growth while inducing apoptosis, G2/M arrest, autophagy, and re‐expression of imprinted tumor suppressor genes. Cancer 2011;. © 2011 American Cancer Society.