Novel chromatin-associated role of Protein Kinase C family members in regulating inducible genes and microRNAs during epithelial to mesenchymal transition

The epithelial to mesenchymal transition (EMT) is a key step in cancer progression and metastasis. A small subpopulation of tumor cells formed thereby, referred to as Cancer Stem Cell (CSC) cells, potentially have a significant role for metastatic tumor initiation and recurrence. The epigenetic regulation underpinning control of gene expression programs in EMT and CSC formation processes are not well understood, largely because an appropriate inducible in vitro human model is lacking. This thesis describes the establishment of a novel human in vitro rapidly inducible breast cancer EMT model amenable to detailed analysis of epigenetic processes. The human breast cancer epithelial cell line, MCF-7 was stimulated with the PKC pathway inducer, PMA. This results in the emergence of a significant cancer stem cell-like subpopulation amidst almost complete mesenchymal conversion. The epithelial cells, CSC-like and non-CSC (NCSC) mesenchymal subpopulations were shown to be distinct in their transcriptional programs and microRNA profiles. The novel role of PKC isoforms as chromatin-associated enzymes in EMT and CSC formation processes has been indentified for the first time in this thesis. Chromatin immuno-precipitation (ChIP) assays revealed that non-phosphorylated PKC-theta predominated in the epithelial state across the promoter of uPAR, whilst phosphorylated PKC-thata predominated in the mesenchymal state of MCF-7 cells. In contrast, PKC-beta recruitment on the uPAR promoter predominated in mesenchymal state with little enrichment detected in the epithelial state. Similarly, phosphorylated H3T6, which is a surrogate for active PKC-beta was highly recruited in the mesenchymal state in comparison to epithelial state. Interestingly, inactive forms of PKC-theta and PKC-beta form complexes with LSD1 and Zeb1 on the chromatin template in epithelial cells. However, in mesenchymal cells, active PKC-theta switches its association to the active mark Pol II instead of LSD1 and Zeb1. Importantly, the data suggest that although phosphorylated PKC-theta is required for PKC-beta recruitment in the mesenchymal state, the two PKC isozymes, PKC-theta and PKC-beta play opposing roles in regulating transcription of inducible EMT genes. For the first time it has been shown that PKC-theta and PKC-beta tether to chromatin on the promoters of microRNA 200 family members during the process of the epithelial to mesenchymal transition. Not only does Pol II co-exist with PKC-theta in the mesenchymal state on the miR 200c promoter but, also, epigenetic tags such as LSD1 and Zeb-1 co-exist with Pol II in mesenchymal state, suggesting that ""Pol II capturing"" occurs in a PKC-theta containing repressive complex. Additionally, it was shown that PKC activity is important in controlling EMT and CSC formation by regulating EMT associated inducible genes and microRNAs. Collectively, the data presented in this thesis suggest that PKC isozymes might have ""dual role"" consisting of signal transduction role and chromatin associated role in controlling EMT and CSC formation processes. Furthermore, a previously undescribed layer of chromatin-tethered enzymes with interconnected function regulates transcription of EMT-associated inducible genes and microRNAs, include PKC isozymes, histone demethylases and DNA methyltransferases. -- provided by Candidate