1) Inhibitors of BET domain-containing epigenetic factors inhibit growth of human lung adenocarcinoma cell lines. Bromodomain and extra terminal domain (BET) proteins function as epigenetic signaling factors that associate with acetylated histones and facilitate transcription of target genes. Inhibitors targeting the activity of BET proteins in hematological cancers have recently been shown to have potent anti-proliferative effects through the suppression of c-MYC and downstream target genes. However, as the epigenetic landscape of cells varies drastically depending on lineage, transcriptional co-activators such as BETs would be expected to have different targets in different cancer types. To test this hypothesis, Will Lockwood and Kreshnik Zejnullahu treated a panel of lung adenocarcinoma cell lines with the BET inhibitor JQ1 and found that a subset is acutely susceptible to BET inhibition Lockwood et al PNAS 2012 through a mechanism independent of c-MYC downregulation and dependent, at least in part, on the transcription factor FOSL1 and its targets. 2) Identification of targets for growth inhibitors of human lung adenocarcinoma cell lines. Romel Somwar, a former fellow, has used high throughput screening of large chemical libraries to identify small molecules that restrict the growth of human lung cancer cell lines (Somwar et al, J.Biomol Screen 14:1176, 2009). Four products of his Lung Cancer Screen (LCS 1-4) have been selected to identify the targets of these compounds. Using a combination of affinity chromatography and analysis of gene expression patterns, he has identified superoxide dismutase (SOD)-1 as the likely target for LCS-1(Somwar et al, PNAS, 2011). Using similar approaches, Will Lockwood has identified a potential molecular target for LCS-3 and has found that this compound inhibits the growth of diverse types of tumor cell lines. 3) Regulation and targeting of anti-apoptotic proteins. Three years ago, Levi Beverly, a former post-doctoral fellow, showed that six anti-apoptotic, BH3-containing proteins fell into two groups with respect to their ability to cooperate with c-Myc in a mouse leukemogenesis assay (Beverly and Varmus, Oncogene 28:1274, 2009). One of the weakly cooperative proteins, BCL-b, is unstable but can be converted into a strongly cooperative oncogenic protein by protecting it from ubiquitination, and we identified Ubiquitin (or Plic) as a BCL-b binding protein that can relocate BCL-b and retard degradation (Beverly et al PNAS 2012). More recently, Levi has extended chemical screens begun at the NIH to show that inhibition of ceramide metabolism sensitizes cells to inhibitors of these anti-apoptotic proteins (Beverly et al, submitted). 4) Further studies of a mouse model of lung adenocarcinoma induced by mutant EGFR. Our group makes extensive use of transgenic mice with inducible mutant EGFR genes engineered by Katie Politi, now at Yale Medical School (Politi et al, Genes Dev 20:1496, 2006). For example, we have shown that resistance to treatment with tyrosine-kinase inhibitors (TKIs) occurs by mechanisms observed in patients treated with the same drugs (Politi et al, DMM 2010);we are working with the laboratories of Tyler Jacks (MIT Cancer Center) and Greg Hannon (Cold Spring Harbor Laboratory) to sequence much or all of the exomes of drug-sensitive and drug-resistant tumors;and we are collaborating with Julian Downward (Cancer Research UK) to identify other mechanisms of drug resistance and with David Threadgill (UNC) on the role of ErbB3 in tumorigenesis and treatment with TKIs (manuscripts in preparation). 5) Mig6 as a tumor suppressor Previously published work by Udayan Guha catalogued many of the tyrosine phosphorylation sites that are differentially phosphorylated in human cancer cells and bronchial epithelial cells expressing mutant forms of the epidermal growth factor receptor (EGFR) (Guha et al, PNAS 105: 14112, 2008). Among those proteins was Mig-6, a possible tumor suppressor known to interact with and probably down-regulate EGFR. We have crossed mice carrying an engineered Mig-6 deficiency with our transgenic mice carrying inducible, gain-of-function oncogenic alleles of EGFR;the progeny show that loss of Mig-6 function accelerates EGFR-mediated tumorigenesis and death from lung cancer (Guha et al, in preparation). 6) Inflammation and its role in lung tumorigenesis The immune system can promote or retard cancer progression in different settings. Arun Unni has chosen to better understand this interplay by using our mutant EGFR-driven mouse models of lung cancer. He has observed early recruitment of macrophages to the lung parenchyma after induction of mutant EGFR. Experiments in various cell lines suggest that signaling by mutant EGFR is capable of driving an inflammatory response by induction of expression of cytokines. Experiments are underway to explore the exact mechanisms by which mutant EGFR induces expression of inflammatory genes. 7) Molecular events that cooperate with known lung cancer driver genes Pang Fan (a former post-doc) and Will Lockwood have adapted the Sleeping Beauty transposition system to generate mice that express both the Sleeping Beauty transposase and transposon along with mutant EGFR specifically in type 2 airway epithelial cells, and we are currently monitoring these mice for tumor development. Recently, Dennis Fei has started using the same Sleeping Beauty system to study small cell lung cancer. 8) Mutations in human lung adenocarcinomas Will Lockwood and Kreshnik Zejnullahu have been sequencing unusual lung adenocarcinomas, in a partnership with the NHGRI Sequencing Core and the University of British Columbia, to search for novel mutations that drive tumor development. Whole-exome sequences have been integrated with pre-existing data about single nucleotide polymorphisms, copy number variation, CGH, DNA methylation, and mRNA expression. Candidate driver genes are currently being interrogated in a larger cohort of 100 lung tumor/normal pairs and functional validation of candidates will be undertaken to complement our studies of secondary mutations in mouse models of lung adenocarcinoma. 9) Role of RNA processing in lung adenocarcinogenesis. New sequencing technology has revealed that cancer cells often harbor abnormal RNAs that indicate alterations in RNA processing events, including splicing. Dennis Fei is attempting to determine the functional significance of changes in RNA processing in lung carcinogenesis. In particular, he is characterizing the oncogenic properties of proteins that regulate RNA splicing are either highly expressed or mutated in human lung adenocarcinomas. He is also attempting to identify cancer-associated splicing isoforms that promote the pathogenesis of lung adenocarcinoma.
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