Modulation of cancer cell progression by HBP1 through epigenetic mechanisms

Cancer is fundamentally a genetic and epigenetic disease. Mortality from most solid cancers is invariably due to metastasis to distant tissue and organs rather than the primary tumor. Cancer cell invasion out of its local contained environment represents an early step in the metastatic process. Cancer cells co-opt an embryonic process, termed EMT, that enables these cells to invade and metastasize. At the molecular level, loss of E-cadherin is the defining characteristic of EMT and is associated with a poor clinical course. Epigenetic silencing of TSGs, such as E-cadherin, is a common occurrence in breast cancers and is closely associated with EMT. This thesis is concerned with exploring the molecular mechanisms whereby the green tea polyphenol, EGCG, can mediate the prevention and/or treatment for breast cancer progression.;Our lab has previously demonstrated that the tumor suppressive function of EGCG is in part mediated by HBP1. HBP1 is a transcriptional repressor that complexes with Rb to repress gene expression and have a role in cell cycle arrest, differentiation and senescence. HBP1 has been shown to inhibit the canonical Wnt signaling pathway that is frequently dysregulated in many cancers. Recent data has identified HBP1 variants/mutants that have been isolated from individuals with invasive disease. Reduced HBP1 and another important secreted Wnt inhibitor antagonist, sFRP1, are associated with reduced survival.;In Chapter 1, we identify a novel HBP1 target gene that may shed light on the mechanics of HBP1. We show that HBP1 represses DNMT1 by binding to a consensus HBP1 binding motif located within the DNMT1 promoter. Further, we characterize a common variant/mutant of HBP1 that behaves as a dominant negative with regards to DNMT1. We asked what are the consequences of dysregulated DNMT1? We show data that loss of HBP1 results in repression of E-cadherin and coincident increase in mesenchymal markers. While DNMT inhibitors increase E-cadherin levels, we provide evidence that the CDH1 locus is unmethylated. To confirm that DNMT1 is responsible for E-cadherin repression, ectopic expression of DNMT1 nicely recapitulates E-cadherin repression as seen with HBP1 knockdown or dominant negative expression.;In Chapter 2, we ask whether EGCG can act in concert with the clinically relevant DNMT inhibitors to de-repress the epigenetically silenced sFRP1 locus. We reason that EGCG, by increasing HBP1, will work to repress DNMT1 and de-repress silenced genes. We present data showing that EGCG and a DNMT inhibitor synergize to de-repress sFRP1. We convincingly show that the EGCG component of this combo treatment is dependent on HBP1. Finally, the data we show rules out our prediction that EGCG works through HBP1 to repress DNMT1. Instead, the mechanism by which EGCG shuts down DNMT1 remains unknown.;In Chapter 3, we explore an alternate mechanism for EGCG increasing HBP1 levels. In silico analysis of the HBP1 protein sequence predicts that HBP1 is a target of the PI3Kinase/Akt pathway. We present evidence to confirm that HBP1 is negatively regulated by the PI3K pathway and is targeted for proteasomal degradation. It appears unlikely that HBP1 is phosphorylated on the predicted Akt phosphorylation motifs. Instead, it appears that the PDK1 kinase may directly or indirectly phosphorylate HBP1 and target it for degradation. Post-translational degradation of HBP1 may provide an alternate mechanism for its loss in cell growth and cancer.