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Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Mutations in the Epidermal Growth Factor Receptor (EGFR) and Kirsten Rat Sarcoma (KRAS) genes occur in a mutually exclusive manner in ~15% and ~30% of all lung adenocarcinomas (LACs), respectively. Using Doxycycline (Dox)-regulated gene expression vectors, we have previously demonstrated that the forced co-expression of EGFR and KRAS mutants in LAC cells induces lethality through the hyperactivation of the RAS-mitogen-activated protein kinase (MAPK) pathway. A subsequent phosphoproteomic assay using Tet-O-KRASG12V-PC9 cells, which carry an endogenous EGFR mutation and was engineered to express KRASG12V upon Dox treatment, revealed that phosphorylation of extracellular signal-regulated kinases (ERKs) increased acutely and dramatically compared to the Tet-O-GFP-PC9 control. This suggested that early activation of ERK1/2 is a crucial event in mediating the observed lethality. Additionally, genetic and pharmacological inhibition of ERK1/2 rescued multiple co-expression LAC cells, confirming that ERK is the main mediator of this phenomena. Here, I aim to investigate whether KRAS- or EGFR-driven LAC cells exploit any existing negative regulatory mechanisms of the ERK to maintain its levels below its upper signalling threshold. Because MAPK signalling is typically regulated by phosphatases, our group performed an analysis of the MAPK phosphatase expression data comparing two LAC TCGA tumor subsets – tumors with (n=107) and without (n=123) either EGFR or KRAS mutation. This analysis revealed that Dual-specificity phosphatase 6 (DUSP6) is the only phosphatase that is up-regulated in tumors with a mutation in either two genes in comparison to their wildtype counterparts, suggesting that these tumors may be dependent on a robust DUSP6 activity to moderate the P-ERK1/2 levels and prevent ERK hyperactivation. Furthermore, when DUSP6 was inhibited in mutant KRAS or mutant EGFR bearing LAC cells using DUSP6 small-interfering RNAs (siRNAs) or a DUSP6 inhibitor called (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), we observed that only the mutant bearing LAC cells were more sensitive to DUSP6 inhibition than the KRAS and EGFR wildtype cells. Such findings suggest a potential therapeutic scenario in EGFR or KRAS mutant LACs can be targeting through inhibiting DUSP6, a key negative feedback regulator that prevents the hyperactivation of ERK.
Lung cancer is the leading cause of cancer related death in both men and women worldwide, mainly due to the lack of effective therapies. The development of specific chemical compounds that target epigenetic post-translational modifications has recently emerged as an excellent approach for validating new treatment strategies for diseases that have complex underlying mechanisms. JQ1 is a small-molecule inhibitor of the bromodomain and extraterminal (BET) family proteins, which function as important reader molecules of acetylated histones and recruit transcriptional activators to specific promoter sites. In many cancer lines the down-regulation of MYC, a known oncogenic transcription factor and contributor to the pathogenesis in certain cancer types, has been linked to BET inhibitor (BETi) treatment. In addition, resistance to BETis has only been examined in MYC-dependent cancers, with all forms of resistance involving re-expression of MYC, through several mechanisms. Previously, our lab has shown that lung adenocarcinoma (LAC) cells are inhibited by JQ1 through a mechanism independent of MYC down-regulation, identifying FOSL1 as a mediator of response. This suggests that the epigenetic landscape of cells from different origins and differentiation states influences response to JQ1. Therefore, I aim to investigate how LAC cells, independent of MYC down-regulation, acquire resistance to BET inhibition, to elucidate mechanisms of primary resistance and potential treatment strategies for LAC.Here, I establish resistance in two JQ1 sensitive LAC cell lines and demonstrate that MYC levels were not significantly altered, nor was FOSL1 expression reactivated in resistant lines, indicating a novel mechanism of resistance. Interestingly, resistant lines were still dependent on the BET protein BRD4, as demonstrated by siRNA knockdown, suggesting that BRD4 may drive resistance through regulating gene transcription independent of its acetyl-binding domain. Both resistant lines showed increased levels of phosphorylated BRD4, and also up-regulation of casein kinase 2 (CK2), a kinase previously shown to phosphorylate BRD4. Furthermore, combining JQ1 with a CK2 inhibitor showed synergistic effects in both resistant lines, with treatment leading to decreased levels of pBRD4. Overall, we have determined that LAC cells develop JQ1 resistance through mechanisms independent of MYC, identifying CK2 phosphorylation of BRD4 as a likely mechanism of resistance.