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Regulation of the pro-apoptotic BH3-only protein BIM
Authors
LEONA ROHRBECK
Publication date
1 January 2015
Publisher
Abstract
© 2015 Dr. Leona RohrbeckThis thesis is not authorised to be made available in the Baillieu Reading Room until July 2016The pro-apoptotic BH3-only protein BIM is a member of the BCL-2 family, which is critical for initiation of the intrinsic apoptotic pathway in response to several cytotoxic stimuli. Loss of BIM causes SLE-like autoimmune disease due to accumulation of self-reactive lymphocytes, and promotes tumour formation in humans and mice. In addition, BIM is essential for the killing of cancer cells by several conventional anti-cancer drugs and novel designer drugs that inhibit oncogenic kinases. These findings highlight the importance of BIM in maintaining homeostasis of haematopoietic cells and in functioning as a potent tumour suppressor. Because too much or too little BIM can cause severe abnormalities, expression of BIM needs to be tightly controlled. So far, the vast majority of studies on BIM regulation have been conducted in cell lines, using over-expression systems. Thus more work needs to be done to understand the transcriptional and post-transcriptional regulation of BIM expression. The transcription factor FOXO3a has been reported to be crucial for the initiation of BIM-dependent apoptosis of cytokine-starved haematopoietic cells. I analysed BimΔFoxo/ΔFoxo mice, in which the binding sites for FOXO proteins (including FOXO3a) in the Bim promoters were mutated to prevent FOXO proteins from binding to Bim. In these studies I could show that the development of the haematopoietic system was not impaired in the absence of FOXO-mediated regulation of Bim. In addition, I could demonstrate that cytokine-dependent cells, which I generated from the BimΔFoxo/ΔFoxo mice, were normally sensitive to cytokine withdrawal-induced apoptosis. These data reveal that binding of FOXO proteins to their recognition elements in the Bim promoters is dispensable for BIM expression and for BIM-dependent apoptosis of haematopoietic cells. I then turned my attention to post-transcriptional regulation of Bim because the Bim 3’UTR harbours multiple conserved AU-rich elements (ARE) to which RNA-binding proteins (RBPs) might bind. Several studies demonstrated that binding of RBPs, including members of the CCCH ZFP family, to AREs in the 3’UTR of genes regulate mRNA stability and thereby control gene expression. I generated GFP-Bim 3’UTR reporter constructs in which changes in GFP expression served as a read-out for Bim mRNA stability. In doing this, I identified several proteins of the CCCH ZFP family that act as potential post-transcriptional regulators of Bim mRNA. Additionally, I generated preliminary data that reveal a correlation between mRNA expression of identified ZFPs and Bim mRNA levels in different haematopoietic cell lines. As mentioned above, sensitivity of many cancers to anti-cancer therapies depends on the expression of BIM. Melanoma cells that harbour the BRAFV600E activating mutation initially respond well to treatment with the BRAF kinase specific inhibitor PLX4032, however tumours ultimately acquire resistance to the drug. When hepatocyte growth factor (HGF), which can be secreted from the tumour microenvironment or from the melanoma cells themselves binds to the receptor tyrosine kinase cMET, cMET-expressing melanoma cells acquire resistance to PLX4032-induced apoptosis. I studied the underlying molecular mechanisms of PLX4032-induced killing of BRAF mutant melanoma cells and found that the BH3-only proteins PUMA and to a lesser extent BIM are critical. Moreover, I showed that resistance of BRAF mutant melanoma cells to PLX4032, induced by HGF is accompanied by substantial downregulation of BIM and PUMA proteins. Understanding the mechanisms that lead to resistance of BRAF mutant melanoma cells to PLX4032 will help to develop novel strategies to resensitise these cancer cells to this drug
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Last time updated on 06/01/2019