ABSTRACT
Autophagy can sustain or kill tumor cells depending upon the context. The mechanism of autophagy-associated cell death has not been well elucidated and autophagy has enhanced or inhibited sensitivity of cancer cells to cytotoxic chemotherapy in different models. ARHI (DIRAS3), an imprinted tumor suppressor gene, is downregulated in 60% of ovarian cancers. In cell culture, re-expression of ARHI induces autophagy and ovarian cancer cell death within 72 h. In xenografts, re-expression of ARHI arrests cell growth and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks, dormancy is broken and xenografts grow promptly. In this study, ARHI-induced ovarian cancer cell death in culture has been found to depend upon autophagy and has been linked to G1 cell-cycle arrest, enhanced reactive oxygen species (ROS) activity, RIP1/RIP3 activation and necrosis. Re-expression of ARHI enhanced the cytotoxic effect of cisplatin in cell culture, increasing caspase-3 activation and PARP cleavage by inhibiting ERK and HER2 activity and downregulating XIAP and Bcl-2. In xenografts, treatment with cisplatin significantly slowed the outgrowth of dormant autophagic cells after reduction of ARHI, but the addition of chloroquine did not further inhibit xenograft outgrowth. Taken together, we have found that autophagy-associated cancer cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells are still vulnerable to cisplatin-based chemotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Ovarian Neoplasms/drug therapy , rho GTP-Binding Proteins/genetics , Autophagy/genetics , Caspase 3/genetics , Caspase 3/metabolism , Cell Line, Tumor , Chloroquine/pharmacology , Drug Resistance, Neoplasm/genetics , Female , G1 Phase Cell Cycle Checkpoints/drug effects , Humans , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Proteolysis , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Reactive Oxygen Species/metabolism , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Signal Transduction , X-Linked Inhibitor of Apoptosis Protein/genetics , X-Linked Inhibitor of Apoptosis Protein/metabolism , Xenograft Model Antitumor Assays , rho GTP-Binding Proteins/metabolismABSTRACT
The process of autophagy has been described in detail at the molecular level in normal cells, but less is known of its regulation in cancer cells. Aplasia Ras homolog member I (ARHI; DIRAS3) is an imprinted tumor suppressor gene that is downregulated in multiple malignancies including ovarian cancer. Re-expression of ARHI slows proliferation, inhibits motility, induces autophagy and produces tumor dormancy. Our previous studies have implicated autophagy in the survival of dormant ovarian cancer cells and have shown that ARHI is required for autophagy induced by starvation or rapamycin treatment. Re-expression of ARHI in ovarian cancer cells blocks signaling through the PI3K and Ras/MAP pathways, which, in turn, downregulates mTOR and initiates autophagy. Here we show that ARHI is required for autophagy-meditated cancer cell arrest and ARHI inhibits signaling through PI3K/AKT and Ras/MAP by enhancing internalization and degradation of the epidermal growth factor receptor. ARHI-mediated downregulation of PI3K/AKT and Ras/ERK signaling also decreases phosphorylation of FOXo3a, which sequesters this transcription factor in the nucleus. Nuclear retention of FOXo3a induces ATG4 and MAP-LC3-I, required for maturation of autophagosomes, and also increases the expression of Rab7, required for fusion of autophagosomes with lysosomes. Following the knockdown of FOXo3a or Rab7, autophagolysosome formation was observed but was markedly inhibited, resulting in numerous enlarged autophagosomes. ARHI expression correlates with LC3 expression and FOXo3a nuclear localization in surgical specimens of ovarian cancer. Thus, ARHI contributes to the induction of autophagy through multiple mechanisms in ovarian cancer cells.
