Browsing by Subject "Proto-Oncogene Proteins c-myc"
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Item Characterizing c-Myc Dependent Lung Cancers(2015-07-27) Dospoy, Patrick; Scaglioni, Pier Paolo; Shay, Jerry W.; White, Michael A.; Minna, John D.MYC is one of the most commonly deregulated oncogenes in human cancer, including breast, colorectal and lung. While mutations in myc are rare, MYC is overexpressed and in some cases amplified in these (and other) cancers. Recent reports demonstrate the utility of various drugs in selectively targeting MYC-driven cancers. However, given the lack of consistency across tissue types, particularly lung cancer, a multimodal approach to delineate MYC-dependent lung cancers is required. My goal is to characterize MYC deregulation in lung cancer, investigate the degree of differential dependence on MYC in lung cancer, and to elucidate the mechanism for resistance to MYC inhibition. A large panel of clinically and molecularly annotated NSCLC lines was investigated for MYC mRNA, protein expression, and DNA copy number. In addition, publically available databases were interrogated to characterize the degree of MYC deregulation in lung cancer. Functional tests were performed on a large panel of NSCLC cell lines (n = 83) using four drugs that were recently shown to selectively target MYC-driven cancers. Further, we utilized the dominant negative mini-protein OMOMYC for functional classification. In all cases, drug effects were monitored by colony forming efficiency (CFE) assays. OMOMYC results were confirmed via xenograft experiments. Each of the four MYC inhibitors tested elicited a variable response in a subset of the 83 NSCLC cell lines, though the sensitive subset was not similar between any two drugs (highest correlation coefficient of 0.24). In order to determine which, if any, of the drugs targeted MYC-driven lung cancers, we stably expressed OMOMYC in a subset of the NSCLC cell line panel and performed functional assays. Most of the cell lines were sensitive to OMOMYC (with up to 100 fold reduction in CFE), compared to 3/8 that were totally resistant. The variability in the presence of OMOMYC showed a significant correlation with one of the four MYC inhibitors tested. These results support the idea that this sensitive subset represents a truly MYC-dependent class of lung cancers. Surprisingly, there was no correlation between MYC dependence and either MYC mRNA, protein expression or DNA copy number. OMOMYC levels were normalized in all cell lines tested and quantified using qRT-PCR. Additionally, in all cases, exogenous OMOMYC expression led to down regulation of MYC target genes as measured by both qRT-PCR and microarray. These data could be interpreted to suggest that the observed phenotype was the result of decreased MYC activity. Last, the NSCLC probed with OMOMYC showed a variable response in the Wnt pathway, with some cell lines showing a dramatic activation of the Wnt pathway upon OMOMYC induction. This activation proved to be functionally important, as dual inhibition of β-catenin and MYC proved more effective than either approach alone. To investigate the clinical significance of this approach, a subset of the original panel of NSCLC (n = 15) was screened with the MYC inhibitor 10058-F4, Wnt inhibitor Wnt-C59, or a combination of both drugs. Here, 8/15 cell lines displayed a statistically significant increase in sensitivity to MYC inhibition when Wnt pathway inhibition was added. We conclude: there is a subset of NSCLCs that demonstrates dramatic growth inhibition by a single MYC-inhibitor, and these data are phenocopied by the more specific MYC-dominant negative protein, OMOMYC. We further conclude that activation of the Wnt pathway serves as a compensatory response in some cell lines that confers resistance to MYC inhibition. In conclusion, simultaneously targeting MYC and the Wnt pathway elicits superior sensitivity in a subset of NSCLCs, and thus provides rationale for a combinatorial approach in a subset of lung cancer patients.Item Oncogene-Induced Signaling Heterogeneity in Lung Cancer(2015-07-22) Deb, Dhruba; Garcia, Christine K.; White, Michael A.; Cobb, Melanie H.; Altschuler, Steven J.; Wu, Lani; Minna, John D.Lung cancer causes the maximum number of cancer related deaths worldwide. In recent years, the cancer genome atlas (TCGA) initiative has identified 138 frequently occurring driver oncogenes and tumor suppressor genes in lung cancer. Currently, only 15 of these genes can be targeted therapeutically. Study of downstream signaling alterations of these oncogenes and tumor suppressor genes may identify novel therapeutic targets. Although studies on genetic heterogeneity in subclonal populations within one tumor using deep sequencing and multiple sectioning have gained popularity recently, the signaling heterogeneity within tumor cells with identical genetic changes remain poorly understood. Hence, I focus on TP53, KRas and C-Myc as they are among the most frequently occurring oncogenic alterations in lung adenocarcinoma. The downstream signaling changes of these genes may be different from one cell to another. Here, I develop high throughput approaches to study alterations of 6 major signaling readouts - phospho-Erk1/2, phospho-Stat3, Smad2/3, β-catenin, P65, and Foxo1 and quantitatively analyze thousands of cells with defined set of genetic changes. I ask - Can I utilize oncogene-induced signaling alterations in single cells to identify novel targetable vulnerabilities? Using single-cell image analysis I show that the genetically transformed HBECs with all 3 oncogenic changes (TP53, KRas and C-Myc) show significant signaling heterogeneity. They exhibit downregulated Smad2/3 signaling in single cells. Next, using a dominant negative construct, I confirm that this phenotype is partially reversible by the removal of C-Myc oncogenic stress. I further observe that the transformed HBECs exhibit upregulated Stat3 signaling in single cells. In addition, the Stat3 inhibitor Stattic causes more cell death in transformed HBECs. Interestingly, our single-cell image analysis suggests that Stat3 upregulation and Smad2/3 downregulation are mutually exclusive. Hence, Stattic will not be able to target the Smad2/3 downregulated cells. To target Smad2/3 downregulated cells, I identify Bcl6, a downstream target of Smad2/3, and I show that Bcl6 is a novel targetable vulnerability in transformed HBECs. I observe that C-Myc and Bcl6 gene expressions are strongly correlated in cell populations as well as in single-cell level. I further show that Bcl6 can be a targetable vulnerability in a subset of c-Myc addicted non-small cell lung cancers. I conclude that single-cell analysis of driver oncogenes and their downstream signaling can identify novel targetable vulnerabilities.Item Regulation of Pyruvate Kinase M2 (PKM2) Expression and Activity in Cardiac Hypertrophy(2013-01-22) Hogen, Victor; Wang, Zhao V.; Wang, Bo; Hill, Joseph A.BACKGROUND: Cardiac hypertrophy is characterized by robust structural, metabolic, and signaling events, which include increased myocyte size and width, increased glycolytic flux, aerobic glycolysis, and induction of transcriptional programs governed by such factors as c-Myc, Fos, and Jun. We have noted that this phenotypic profile exhibits similarities to cancer, where c-Myc, HIF-1α and PKM2 contribute to tumorigenesis and enhanced cancer cell survival in the setting of oxidative stress. PKM2, highly expressed in heart, is the sole pyruvate kinase M isoform expressed in a variety of tumors and is thought to participate in shifts between anabolic and catabolic flux in glycolysis. METHODS AND RESULTS: First, we set out to determine mechanisms underlying aerobic glycolysis in cardiac hypertrophy. We hypothesized that hypertrophic growth cues, including hypoxia, mediate increases in PKM2 protein levels and oxidation at Cys-358. To test this, we first measured protein levels and activity of glycolytic PKM2 in neonatal rat ventricular myocytes maintained in culture. We evaluated four pro-growth stimuli: phenylephrine, endothelin-1, angiotensin II, and hypoxia. We observed that phenylephrine and angiotensin II did not increase normalized PKM2 protein levels, whereas hypoxia and endothelin-1 did. None of these growth stimuli increased PKM2 fractional oxidation. Further, no change in fractional oxidation of PKM2 was observed in mouse hearts subjected to one week of TAC (thoracic aortic constriction). However, an increase in total normalized PKM2 oxidation was readily detected. CONCLUSIONS: Together, these data suggest that hypoxia increases PKM2 protein levels via mechanisms mediated in part by localized ET-1 signaling. Additionally, these data suggest that TAC triggers an increase in the abundance of oxidized PKM2, mediated in part by increased PKM2 protein production. Finally, as phenylephrine did not increase PKM2 oxidation, this suggests that a non-NOX2-dependent mechanism is involved.