Research Projects

Radioresistance in cancer cells is a serious concern in managing radiotherapy of cancer patients. Various molecular and cellular studies have shown that these cells are robust in their tolerance to oxidative stresses and DNA damage as compared to the closest control. Since, the role of RqkA, a serine/threonine protein kinase in radioresistance of D. radiodurans (thermophilic bacterium) has been demonstrated through phosphorylation of DNA repair and cell division proteins, a possibility of RqkA expression affecting DSB repair leading to radioresistance in cancer cells is hypothesized. The rqkA Deinococcus showed nearly 4 log cycle drop in the radioresistance of wild type bacterium. Further, studies have established RqkA role in DNA damage response and cell cycle regulation in this bacterium, as an alternative to canonical SOS response. Unlike most of the eSTPKs that are promiscuous in nature, RqkA is regulated by an antioxidant and radioprotector named pyrroloquinoline quinone (PQQ). Preliminary studies in cancer cells, has demonstrated that RqkA-GFP when expressed in A549 and Hela cells and exposed to 5Gy of dose gamma radiation, exhibited significant resistance when compared with cells harboring vector as control. Interestingly, when these cells were observed under microscope, they showed a typical morphological transition from epithelial to mesenchymal phenotype (EMT). EMT is an hallmark of metastasizing cancer cells The molecular basis of RqkA action in both radioresistance and EMT phenotype in cancer cells are not known and would be worth studying. Here, we propose to express bacterial as well as codon optimized coding sequences of RqkA in mammalian cancer and non-cancer cell lines and monitor the cellular and molecular changes associated with cell morphology (EMT phenotypes), cell growth under normal and DNA damaging stress conditions and DSB repair. An in depth transcriptomic and proteomic analyses of the cancer cells expressing the ectopic RqkA would aid in identification of novel mammalian substrates for the bacterial kinase whose role in radioresistance and DNA damage response shall be validated using loss-of-function studies. These results would throw light on novel molecules involved in EMT that would act as potential therapeutic targets. OBJECTIVES 1. Construction of mammalian expression plasmids for targeted expression of RqkA into nucleus and mitochondria and confirmation of RqkA expression in organelles of mammalian cells. 2. Study the effects of cytosolic and organelle expressing RqkA on resistance to DNA damage and acquisition of EMT phenotypes using DNA damage response and EMT markers. 3. Phosphoproteome and transcriptome profiling of transgenic stable cancer cell lines expressing RqkA in cytosol, mitochondria and nucleus. 4. Data analysis, identification of potential candidate proteins and validation of their role in resistance to DNA damage and EMT phenotype by reverse genetics and knockdown approaches.