Mechanism of PMT-Induced Anchorage-Independent Growth and mTOR Signaling

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We previously showed that 3T3 cell treatment with PMT leads to a Gaq-dependent activation of the mammalian target of rapamycin complex 1 (mTORC1), a key kinase involved in the regulation of protein synthesis, cell proliferation, and autophagy in a nutrient and energy-responsive manner. This mTOR activation is mediated by diffusible factors, generated by PMT treatment, which include connective tissue growth factor (CTGF). In addition, inhibition of mTOR with rapamycin causes a partial decrease in cell proliferation and protein synthesis induced by PMT, suggesting that PMT-mediated mTOR activation is involved in these events. However, the downstream signaling behind PMT-induced cell proliferation are not known. Microarray analysis showed that survivin is upregulated several fold in PMT-treated cells compared to that of control non-treated cells. Survivin is classified as a member of the inhibitor of apoptosis protein (IAP) family, due to its N terminal baculovirus IAP repeat (BIR). Expressed during fetal development, survivin is undetectable in terminally differentiated adult tissues. However, it becomes prominently expressed in transformed cell lines and in all the most common human cancers and correlates with reduced tumor cell apoptosis and resistance to cancer therapy. Besides its role as an antiapoptotic molecule, survivin acts as a subunit of the chromosomal passenger complex (CPC) composed of the mitotic kinase Aurora-B, Borealin and INCENP, and is essential for proper chromosome segregation and cytokinesis. As such, survivin has aroused keen interest in disparate areas of basic and translational research. The PMT-induced survivin mRNA was further confirmed by RT-PCR. Furthermore, using Western blot analysis, we showed a huge increase in survivin protein in PMT- treated cells relative to that in control non-treated cells. It has been shown that IGFR activation leads to the upregulation of survivin. Using mouse phosphor-receptor tyrosine kinase (phosphor-RTK) array, we did not observe IGF receptor activation in PMT-treated cells. However, inhibition of mTOR and Erk1/2 (p42/44) pathways by pharmacological inhibitors significantly reduced PMT-mediated survivin up-regulation at both mRNA and protein levels. These results indicate that the PMT-induced survivin expression may be regulated by the mTOR pathway. Since survivin expression is closely connected with cell cycle progression, it is possible that PMT induces cell proliferation through PMT-induced mTOR activation which in turn leading to survivin upregulation. However, it is not known whether the effect of rapamycin on PMT-mediated induction of survivin is a consequence or not of the inhibition of cell cycle progression by rapamycin. It is worth mentioning that, following PMT treatment, we did not observe any increase in XIAP, another IAP family member. Interestingly, PMT treatment also upregulates Aurora kinase B in a time-and concentration-dependent manner. Taken together, these results may suggest that PMT induces cell proliferation through survivin/aurora upregulation mediated by the mTOR pathway.