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 7

Growth and angiogenic factors and cell matrix proteins can rescue cultured cells from ARHI-induced autophagic cell death.

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Growth and angiogenic factors and cell matrix proteins can rescue cultur...
(A and B) Growth and angiogenic factors and cell matrix proteins rescue ARHI-induced autophagic death. Clonogenic assays were carried out with SKOv3-ARHI cells cultured in medium with added cytokines and growth factors (A) on plastic with or without different cell matrix proteins (B). #P < 0.05, compared with control without DOX. *P < 0.05, compared with control with DOX. (C) Most of the detected growth factors were of host origin. Antibody array analyses of cytokines, growth factors, and inflammatory factors from cultured cells or xenograft tissues were performed with antibodies specific for human or mouse antigens. Each antibody was spotted in duplicate and 2 sets of positive and negative controls were spotted at the top left corner of each membrane (see Supplemental Figure 7). *P < 0.05, compared with cultured cell lysates. (D) Detection of angiogenic factors in cultured cells and xenografts. Antibodies specific for human or mouse angiogenic factors or inflammatory proteins were used. Densitometry scanning of antibody spots is represented as arbitrary density units. *P < 0.05, compared with in vitro samples. (E) Hypoxia promotes VEGF expression. VEGF levels were determined in culture medium and in cell lysates under hypoxic conditions. *P < 0.05, compared with normoxic conditions. (F) ARHI downregulates HIF-1α in SKOv3-ARHI cells cultured in hypoxic conditions. Western blots of HIF-1α were carried out with lysates from cells cultured under normoxic or hypoxic conditions and from xenograft tissues. Numbers under protein bands are densitometry units.