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

我们之前的研究表明，用PMT处理3T3细胞会导致哺乳动物雷帕霉素靶蛋白1 （mTORC1）的gaq依赖性激活，这是一种以营养和能量响应方式参与蛋白质合成、细胞增殖和自噬调节的关键激酶。这种mTOR激活是由PMT治疗产生的扩散因子介导的，其中包括结缔组织生长因子（CTGF）。此外，用雷帕霉素抑制mTOR会导致PMT诱导的细胞增殖和蛋白质合成的部分减少，这表明PMT介导的mTOR激活参与了这些事件。然而，pmt诱导的细胞增殖背后的下游信号尚不清楚。微阵列分析显示，与未处理的对照细胞相比，pmt处理细胞中的survivin上调了数倍。Survivin因其N端杆状病毒IAP重复序列（BIR）而被归类为凋亡抑制蛋白（IAP）家族的成员。survivin在胎儿发育期间表达，在终末分化的成人组织中检测不到。然而，它在转化细胞系和所有最常见的人类癌症中显著表达，并与肿瘤细胞凋亡减少和对癌症治疗的耐药性相关。除作为抗凋亡分子外，survivin还作为由有丝分裂激酶Aurora-B、Borealin和INCENP组成的染色体客运复合体（CPC）的一个亚基，对染色体分离和细胞分裂至关重要。因此，survivin在基础和转化研究的不同领域引起了浓厚的兴趣。RT-PCR进一步证实pmt诱导的survivin mRNA表达。此外，通过Western blot分析，我们发现PMT处理的细胞中survivin蛋白的表达明显高于未处理的细胞。研究表明，IGFR激活可导致survivin的上调。使用小鼠磷酸化受体酪氨酸激酶（磷酸化- rtk）阵列，我们未观察到pmt处理细胞中IGF受体的激活。然而，通过药物抑制剂抑制mTOR和Erk1/2 （p42/44）通路可显著降低pmt介导的survivin mRNA和蛋白水平上调。这些结果表明pmt诱导的survivin表达可能受到mTOR通路的调控。由于survivin的表达与细胞周期进展密切相关，PMT可能通过诱导mTOR激活而诱导细胞增殖，进而导致survivin上调。然而，目前尚不清楚雷帕霉素对pmt介导的survivin诱导的作用是否是雷帕霉素抑制细胞周期进程的结果。值得一提的是，在PMT治疗后，我们没有观察到IAP家族成员XIAP的任何增加。有趣的是，PMT治疗也以时间和浓度依赖的方式上调极光激酶B。综上所述，这些结果可能表明PMT通过mTOR途径介导的survivin/aurora上调诱导细胞增殖。

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.