The role of viruses in carcinogenesis has long been accepted because of the direct mechanistic effects of often a single viral gene in cell transformation. The involvement of bacteria in carcinogenesis, however, remains controversial partly because there is no clear agreement on the molecular mechanism(s) by which they might promote the development of cancer. Nonetheless, epidemiological evidence has linked prior and chronic bacterial infection to tumor formation. Although most attention has been focused on H. pylori, other bacterial infections have been shown to cause cancer in animal models. Bacterial toxins are known to modulate intracellular signaling pathways directly in a way that could promote tumor development. However their carcinogenic role is not fully studied. Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage-independent growth for certain type of cells. These properties suggest that PMT might have the potential to act as a tumor promoter especially in the case of chronic infections.The detailed mechanism behind mitogenic properties of PMT is unknown. We have shown that rPMT induces protein and ATP synthesis, cell migration and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces a sustained activation of mTOR signaling pathway. The PMT-mediated mTOR activation was observed in MEF WT but not in MEF Gαq/11 knockout cells, consistent with our results indicating that PMT-induced mTOR activation proceeds via the deamidation of Gαq/11 and leads to a constituvely active phenotype of G proteins which activate PLCβto generate diacylglycerol (DAG) and inositol trisphosphate (IP3), two known activators of PKC. In essence, PMT hijacks cellular signal transduction pathways via deamidation of heterotrimeric G-proteins and leads to a sustained activation of mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, which in part, leads to cell proliferation and migration. Cell division involves coordinated chromosomal and cytoskeletal rearrangements to ensure the faithful segregation of genetic materials into the daughter cells. To this end, a class of proteins known as chromosomal passengers (CPC) has been reported to regulate several steps of this process. In most organisms, the core CPC comprises of Aurora-B kinase and three regulatory non-enzymatic subunits, identified as inner centromer protein (INCENP), survivin and borealin. In addition to its role as a CPC component, survivin is classified as a member of the inhibitor of apoptosis protein (IAP) family, due to its N‑terminal baculovirus IAP repeat (BIR). While, it is largely undetectable in differentiated tissues, survivin is highly expressed in most human tumors. Under normal conditions, the expression of survivin peaks at mitosis and is tightly regulated through various posttranscriptional and translational mechanisms. However, regulatory mechanisms of survivin expression remain not well understood. We tested whether PMT can induce survivin gene expression in fibroblast cells. Swiss 3T3 cells were serum-starved overnight then treated with PMT (0 to 50 ng/ml) for 24h. A significant increase in survivin protein levels was observed in all PMT-treated cells. The survivin level was undetected in control non-treated cells. Interestingly, under the same conditions, PMT treatment did not alter the protein level of XIAP, an X-chromosome-linked inhibitor of apoptosis proteins, relative to that found in the control non-treated cells. Time course analysis of PMT-induced survivin monitored by Western blot revealed that the level of surviving protein increased with a lag phase. After it reached its peak, the surviving level decreased after two days incubation, but the level was still significantly higher than that detected with the controlled cells. Contrary to survivin, XIAP levels remained unchanged in cells treated with PMT for up to 48h. To investigate the biochemical mechanism responsible for the observed increase in survivin protein levels induced by PMT, we first evaluated potential changes in survivin mRNA monitored by semiquantitative RT-PCR. After the cells were incubated with PMT (0 to 50 ng/ml) for 24 hours, we found that PMT treatment induced a substantial increase of surviving mRNA relative to that observed in the control cells. Under the same conditions, PMT exerted no effect on XIAP mRNA expression. This result suggests that PMT treatment upregulates surviving gene transcription in 3T3 cells. To investigate whether PMT treatment could affect survivin protein turnover, we treated serum- starved cells with PMT for 24h, followed by cycloheximide treatment to inhibit new protein synthesis, for different period of times ranging from 30 to 120 min. PMT treated cell showed high levels of survivin in comparison the control cells. However, PMT-induced surviving protein expression quickly dropped after cycloheximide treatment for only 30 min. This observation indicates that PMT treatment did not affect the stability of survivin protein. To investigate the role of PMT-induced ERK signaling in the regulation of survivin gene expression, we treated serum-starved cells with PMT in the presence or absence of U0126, a MAPK inhibitor. We observed that PMT-mediated survivin expression decreases at both mRNA and protein when the cells were treated with PMT in the presence of 10 uM of MEK inhibitor, U0126. This result suggests that PMT-mediated ERK activation plays an importent role in surviving upregulation, at least at the transcriptional levels. To investigate the role of PMT-induced mTOR activation in the expression of survivin protein and mRNA, we made use of rapamycin, a specific inhibitor of mTORC1. To this end, we observed that PMT-induced survivin protein expression is hampered by the presence of rapamycin. Furthermore, additional study showed that the PMT-induced upregulation of surviving mRNA expression was also abrogated by rapamycin. In control experiments, PMT exerted no effect on XIAP expression in the presence or absence of rapamycin. Together these results indicate clearly that mTOR signaling pathway is involved in the regulation of the survivin gene expression at least at the transcriptional level in PMT treated cells.
| Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells. Cell Signal 25:1136-48 |
| Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Mammalian target of rapamycin complex 1 (mTORC1) plays a role in Pasteurella multocida toxin (PMT)-induced protein synthesis and proliferation in Swiss 3T3 cells. J Biol Chem 288:2805-15 |
