| Summary: | 碩士 === 國立成功大學 === 微生物及免疫學研究所 === 96 === The WWOX gene encodes a candidate tumor suppressor WW domain–containing oxidoreductase (designated as WWOX, FOR or WOX1). Overexpression of WOX1 induces apoptosis in several types of cancer cells. Previous study in our laboratory has demonstrated that methotrexate (MTX), an anticancer drug, increases WOX1 expression in squamous cell carcinoma (SCC)-15 cells and sensitizes these cells to apoptosis. Interestingly, MTX treatment inhibits autophagy, a bulk degradation process for survival during starvation, in SCC-15 cells. Phosphorylation of autophagy-inhibitory mammalian target of rapamycin (mTOR) is upregulated in MTX-treated SCC-15 cells. In light of these findings, we hypothesize that WOX1 regulates mTOR signalling. Our results showed that ectopic overexpression of WOX1 increased phosphorylation of mTOR downstream substrates p70 S6 Kinase (p70 S6K) and eIF4E binding protein 1 (4E-BP1) in SCC-15 cells. WOX1-induced phosphorylation of p70 S6K was downregulated by the treatment of rapamycin, an mTOR inhibitor. To further explore the regulatory mechanism of WOX1, the upstream kinase Akt was examined. Our data showed that WOX1 induced phosphorylation of Akt at Ser473 in SCC-15 cells. WOX1-induced phosphorylation of Akt and p70 S6K was suppressed by starvation or a PI3 kinase inhibitor wortmannin. Most strikingly, we determined that WOX1 downregulated the protein expression and methylation of protein phosphatase 2A catalytic subunit (PP2Ac). Glutathione S-transferase pull-down assay and coimmunoprecipitation demonstrated the interaction of WOX1 with PP2Ac in SCC-15 cells. Together, our results suggest that WOX1 modulates Akt/mTOR signaling pathway by downregulating the protein expression and methylation of PP2Ac in SCC-15 cells.
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