Executive Summary : | Cancer is the second leading cause of death worldwide. Majority of the cancer-related mortality is attributed to metastasis, a process by which cancer cells exit from the primary tumor site and colonize distant anatomical areas to form secondary tumours. Cancer cells initiate the metastatic process by re-engaging a developmental program called epithelial-to-mesenchymal transition (EMT). Activation of the EMT program enables the loss of epithelial characteristics and acquisition of mesenchymal properties such as invasion and migration. At the molecular level, execution of the EMT program is orchestrated by a core group of EMT-activating transcription factors, namely Twist1, Snail, Slug, Zeb1, and Zeb2. Twist1 is an evolutionarily conserved transcription factor that belongs to the basic helix-loop-helix family. Twist1 regulates EMT during normal development as well as cancer progression, i.e., metastasis. The activity of Twist1, in turn, is regulated by various post-translational modifications (PTMs) that affect its stability, nuclear translocation, and its interactions with other transcriptional co-activators. Various pathophysiological cues trigger the activation of EMT, including several stresses encountered during cancer progression, such as hypoxia, matrix deprivation, oxidative stress, and others. AMP-activated protein kinase (AMPK) is a bioenergetic stress sensor which coordinates metabolic pathways and thus balances nutrient supply with energy demand. Work done in our lab demonstrated that activation of AMPK led to a concomitant induction of EMT in multiple cancer cell types. Importantly, AMPK activity was found to be necessary for the induction of EMT by physiological cues such as hypoxia and TGFβ treatment. Furthermore, we showed that AMPK mediated its effects by enhancing the expression, stability, and nuclear translocation of the EMT-transcription factor Twist1. However, how AMPK mediates these effects on Twist1 remains largely unknown. More recently, a phospho-proteomic analysis done by our lab has identified a novel AMPK-responsive phosphorylation on Twist1 at serine 45 position. Since Twist1 function is critically regulated by PTMs, this project aims to investigate the role of the novel Ser 45 phosphorylation of Twist1 in mediating EMT and metastasis. We plan to generate phospho-dead and phospho-mimetic mutants of Twist1 at S45, and investigate their effects on the stability, nuclear localization, and transcriptional activity of Twist1, and on the biochemical and morphological changes associated with EMT. Identifying the PTMs that regulate EMT-transcription factor activity will expand our understanding of the mechanisms that regulate EMT and identify novel therapeutic vulnerabilities that can be targeted to curb the metastatic spread of cancer. |