The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells
Zhen Lu, Robert Z. Luo, Yiling Lu, Xuhui Zhang, Qinghua Yu, Shilpi Khare, Seiji Kondo, Yasuko Kondo, Yinhua Yu, Gordon B. Mills, Warren S.-L. Liao, Robert C. Bast Jr.
Zhen Lu, Robert Z. Luo, Yiling Lu, Xuhui Zhang, Qinghua Yu, Shilpi Khare, Seiji Kondo, Yasuko Kondo, Yinhua Yu, Gordon B. Mills, Warren S.-L. Liao, Robert C. Bast Jr.
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Research Article

The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells

Abstract

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.

Authors

Zhen Lu, Robert Z. Luo, Yiling Lu, Xuhui Zhang, Qinghua Yu, Shilpi Khare, Seiji Kondo, Yasuko Kondo, Yinhua Yu, Gordon B. Mills, Warren S.-L. Liao, Robert C. Bast Jr.

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Figure 1

Expression of ARHI induces autophagy in ovarian cancer cells.

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Expression of ARHI induces autophagy in ovarian cancer cells. (A) Kineti...
(A) Kinetics of induction of ARHI. Lysates from SKOv3-ARHI or SKOv3-NTD cells cultured with DOX for the indicated time periods were probed for ARHI or NTD on western blots. (B) Cell proliferation of inducible ovarian cancer cells in the presence of DOX (DOX+) or absence of DOX (DOX–). *P < 0.05, **P < 0.01 compared with no DOX. (C–H) DOX (1 μg/ml) or cisplatin (5 μg/ml) was added to cultured SKOv3-ARHI cells for 72 hours. Propidium iodide (PI) staining and terminal dUTP transferase (TdT) assay were used to detect apoptosis. Scale bar: 10 μm. (I–N) Inducible SKOv3 cells were stained with acridine orange and examined by fluorescence microscopy. Large orange punctate spots were considered to be AVOs, markers for autophagosomes. (K and N) Hey and SKOv3 cells, transfected with pcDNA3-ARHI and incubated for 48 hours, were stained with acridine orange to visualize AVOs (orange arrowheads). (I, J, L, and M) No AVOs were seen in nontransfected cells (data not shown). Scale bar: 1 μm. (O–T) TEM images of induced and uninduced SKOv3-ARHI cells. White arrows indicate autophagosome vesicles, and black arrows indicate typical double-membrane of autophagosome. Scale bars: 5 μm (O and R); 1 μm (P and S); 500 nm (Q and T). (U) Expression of ARHI increases degradation of long-lived proteins. SKOv3-ARHI or -NTD cells were labeled with [35S]-methionine/cysteine for 6 hours after incubation with or without DOX for 48 hours. Data are representative of the mean ± SEM from at least 3 experiments. *P < 0.05.