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. 2008 Dec;118(12):3917-29.
doi: 10.1172/JCI35512. Epub 2008 Nov 20.

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

Zhen Lu  1 , 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
Affiliations

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

Zhen Lu et al. J Clin Invest. 2008 Dec.

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.

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Figures

Figure 1
Figure 1. Expression of ARHI induces autophagy in ovarian cancer cells.
(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.
Figure 2
Figure 2. ARHI is required for autophagy.
(A–J) ARHI and rapamycin (RM) induce autophagy in ovarian cancer cells. (A–D) SKOv3-ARHI cells were transfected with GFP-LC3 and treated with or without DOX to induce ARHI expression or with or without rapamycin to inhibit mTOR activity. Scale bar: 1 μm. (E–J) ES2 and OC316 cells were not transfected or were transfected with ARHI expression vector and ARHI siRNA or control siRNA for 24 hours before they were transfected with GFP-LC3. Cells were treated with 50 nM rapamycin at the time of siRNA transfection and examined for autophagy by fluorescence microscopy 48 hours later. Scale bar: 1 μm. (K) ARHI expression is necessary for rapamycin-induced autophagy in ovarian cancer cells. ES2 and OC316 ovarian cancer cells were not transfected or were transfected with ARHI or control siRNA for 48 hours. Expression of ARHI and the control GAPDH were examined by RT-PCR. (L–U) NOSE cells undergo spontaneous autophagy. (L–O) Two NOSE cell lines were transfected with GFP-LC3 and treated with or without rapamycin. Scale bar: 1 μm. (P–U) GFP-LC3 plasmid was transfected into OSE106 cells alone or was cotransfected with ARHI siRNA or control siRNA. Transfected cells were treated with or without rapamycin. Scale bar: 1 μm.
Figure 3
Figure 3. ARHI colocalizes with LC3 in the autophagosome.
(A) ARHI enhances ATG4 expression. SKOv3-ARHI or -NTD cells were treated with DOX and harvested at the indicated times. ARHI or control siRNA was transfected to knockdown ARHI expression. Western blots of ATG4 and ARHI were examined. (B) ARHI is essential for conversion of LC3-I to LC3-II. SKOv3-ARHI and -NTD cells were treated with DOX and transfected with ARHI or control siRNA. Cell lysates were blotted for LC3. (C–F) Colocalization of ARHI and LC3 in autophagosomes. DOX-treated SKOv3-ARHI cells were transfected with pGFP-LC3, stained for ARHI, and examined by fluorescence microscopy. Punctate spots mark LC3 or ARHI localization. Arrowheads indicate a ring-like shape and a colocalized ARHI-LC3 signal. Scale bars: 1 μm.
Figure 4
Figure 4. Expression of ARHI inhibits PI3K pathway and upregulates TSC1/2 expression.
(A–F) SKOv3-ARHI cells were transfected with GFP-PHAKT reporter and cultured with or without DOX before they were treated with EGF alone (10 ng/ml for 8 min) or with EGF and wortmannin (EGF+ Wm+; 100 nM). Arrows indicate membrane accumulation of GFP-PHAKT. Scale bar: 1 μm. (G) Phosphorylation levels of AKT were measured before (blue arrows) and after (red arrows) stimulation with 10 nM of lysophosphatidic acid (LPA). Fold changes compared with the basal levels in DOX-treated cells (assigned as 1.0) are indicated. (H) Decreased MAPK and PI3K pathway activity in ARHI-expressing cells. SKOv3-ARHI and -NTD cells were treated with DOX, and the activities of signaling molecules were determined. (I–T) AKT-CA prevents ARHI-induced autophagy. DOX-induced (I–K and O–T) or non-induced (L–N) SKOv3-ARHI cells were cotransfected with GFP-LC3 and AKT-CA or with GFP-LC3 and AKT-DN plasmids. pGFP served as a control. Cells were stained with anti-AKT and examined with fluorescence microscopy. Green and red arrowheads indicate GFP- and AKT-expressing cells, respectively. Orange arrowheads indicate cells expressing both GFP and AKT. Scale bar: 1 μm. (U) Expression of ARHI enhances TSC1/2 expression. Blot shows TSC1/2 expression following the induction of ARHI in SKOv3-ARHI cells. (V) ARHI expression inhibits Rheb activity. Top: Thin-layer chromatography of [32P]-labeled guanine nucleotides from cell lysates of SKOv3-ARHI cells cotransfected with or without Rheb, dominant-positive Rheb-R15V, and/or TSC1/2 in the presence or absence of DOX. Bottom: Calculated GTP/GDP ratios of the TLC density scanning as an indication of GTPase activity.
Figure 5
Figure 5. ARHI-induced autophagy results in cell death in cultured cells.
(A) Induction of ARHI expression results in cell death of cultured cells. SKOv3 or SKOv3-ARHI cells were cultured with or without DOX for 14 days and stained with Coomassie blue, and colonies were counted with light microscopy. **P < 0.01 compared with SKOv3 cells. Scale bar: 2 mm. (B) Re-expression of ARHI induces cell death within a week. DOX was added to induce ARHI expression and then withdrawn at indicated times. Cell colonies were counted with light microscopy. *P < 0.05, **P < 0.01 compared with no DOX.
Figure 6
Figure 6. ARHI-induced autophagy results in tumor dormancy in xenografts.
(A–D) BALB/c nu/nu mice were injected with SKOv3 (A), SKOv3-ARHI (B), or SKOv3-NTD (C) cells and provided with or without DOX in their drinking water. (D) In 2 subgroups, DOX was withdrawn after 32 (green triangles) or 42 (blue diamonds) days of treatment. Arrowheads indicate day of DOX withdrawal. Results shown are from 3 independent experiments. *P < 0.01 compared with absence or withdrawal of DOX. (E–I) TEM images of tumor xenografts showing tumor cells with autophagosomes (arrows) in groups with DOX (F) but not in groups without DOX (G) groups. (I) Withdrawal of DOX resulted in disappearance of autophagosomes and replacement with large number of mitochondria (M), an indication of high metabolism and rapid growth. N, nucleus. Scale bar: 10 μm (E and H), 1 μm (F, G, and I). (J) Inhibition of autophagy blocks tumor dormancy. Nu/nu mice were injected with SKOv3-ARHI cells, and drinking water was supplemented or not with DOX for 5 weeks. One group of DOX-treated mice was injected with CQ for 5 weeks, and a second group was injected with CQ for the final 2 weeks before DOX withdrawal. Top: Growth curve of xenografts with indicated treatments. Bottom: Tumor sizes from day 76 were used for statistical analysis. All pairs comparisons between individual groups were done using the Tukey-Kramer test (P < 0.05). The dashed line represents the mean of all groups; solid lines represent the mean of individual groups. The diameter of the circles reflects the within-group variance, and overlapping of circles indicates a lack of statistical significance.
Figure 7
Figure 7. Growth and angiogenic factors and cell matrix proteins can rescue cultured cells from ARHI-induced autophagic cell death.
(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.
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