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Background:. Thyroid dysfunction is associated with cardiovascular diseases. However, the role of thyroid function in lipid metabolism remains partly unknown. The present study aimed to investigate the causal association between thyroid function and serum lipid metabolism via a genetic analysis termed Mendelian randomization (MR). Methods:. The MR approach uses a genetic variant as the instrumental variable in epidemiological studies to mimic a randomized controlled trial. A two-sample MR was performed to assess the causal association, using summary statistics from the Atrial Fibrillation Genetics Consortium (n = 537,409) and the Global Lipids Genetics Consortium (n = 188,577). The clinical measures of thyroid function include thyrotropin (TSH), free triiodothyronine (FT3) and free thyroxine (FT4) levels, FT3:FT4 ratio and concentration of thyroid peroxidase antibodies (TPOAb). The serum lipid metabolism traits include total cholesterol (TC) and triglycerides, high-density lipoprotein, and low-density lipoprotein (LDL) levels. The MR estimate and MR inverse variance-weighted method were used to assess the association between thyroid function and serum lipid metabolism. Results:. The results demonstrated that increased TSH levels were significantly associated with higher TC (β = 0.052, P = 0.002) and LDL (β = 0.041, P = 0.018) levels. In addition, the FT3:FT4 ratio was significantly associated with TC (β = 0.240, P = 0.033) and LDL (β = 0.025, P = 0.027) levels. However, no significant differences were observed between genetically predicted FT4 and TPOAb and serum lipids. Conclusion:. Taken together, the results of the present study suggest an association between thyroid function and serum lipid metabolism, highlighting the importance of the pituitary-thyroid-cardiac axis in dyslipidemia susceptibility.

BackgroundHigh lipoprotein(a) (Lp[a]) levels are associated with increased risks of cardiovascular events in Percutaneous Coronary Intervention (PCI) patients with diabetes mellitus (DM). Peri-procedural myocardial infarction (PMI) occurs commonly during the PCI, whereas the relationship between Lp(a) and PMI remains unclear. Our study aimed to evaluate the association between Lp(a) value and the incidence of PMI in a larger-scale diabetic cohort undergoing PCI throughout 2013.MethodsA total of 2,190 consecutive patients with DM were divided into two groups according to the median Lp(a) level of 175 mg/L: Low Lp(a) group (N = 1095) and high Lp(a) group (N = 1095). PMI was defined based on the 2018 universal definition of myocardial infarction.ResultsPatients with high Lp(a) levels exhibited higher rates of PMI compared to those with low Lp(a) levels (2.3% versus 0.8%, P = 0.006). The multivariable logistic analysis showed that PMI was independently predicted by Lp(a) as a dichotomous variable (OR 2.64, 95%CI 1.22–5.70) and as a continuous variable (OR 1.57, 95% CI 1.12–2.20). However, further investigation found that this association was only maintained in men, whose Lp(a) levels were significantly associated with the frequency of PMI, both as a dichotomous variable (OR 3.66, 95%CI 1.34–10.01) and as a continuous variable (OR 1.81, 95%CI 1.18–2.78). Lp(a) wasn’t a risk factor of PMI in women.ConclusionsHigh Lp(a) levels had forceful correlations with the increased frequency of PMI in male diabetic patients undergoing PCI. Lp(a) might act as a marker of risk stratification and a therapeutic target to reduce PCI-related ischemic events.

Cancer cells are characterized by the aberrant regulation of signaling pathways that govern response to external stimuli, resulting in uncontrolled cellular proliferation. The genomic alterations that accumulate to cause the malignant growth phenotype can also result in acquired vulnerabilities in the tumor. Loss-of-function screening using pooled short hairpin RNAs (shRNAs) is proving to be a powerful method by which new cancer therapeutic targets can be identified. A major limitation of these studies is that they are usually performed in vitro under low selection pressure, thus potentially missing key vulnerabilities that might be detected in a more clinically relevant setting. Here we tested the use of parallel in vitro and in vivo screens using a mini-library of shRNAs to identify previously unknown acquired vulnerabilities in lung cancer. Using a mini-library of 1062 lentiviral shRNAs targeting nuclear hormone receptors and their coregulators (120 genes total), we screened the lung adenocarcinoma cell line NCI-H1819 for dependency on these genes during in vitro and in vivo growth. Briefly, cells were transduced with the library, allowed to undergo a 4 day period of puromycin selection, and then were either injected subcutaneously into NOD-SCID mice or maintained in 2D tissue culture. Tumors were harvested after ~2 months when reaching a volume of 300 mm3. In a parallel in vitro screen, transduced cells were cultured for 20 population doublings. The relative abundance of individual shRNAs was quantified by next generation sequencing, and candidate essential genes were nominated by filtering for depleted shRNAs that scored in a combination of two statistical methods (t-test and ranked KS-test). We identified six genes required for survival of H1819 in vitro (BRCA1, CCND1, MED1, PHB, HNRNPU, and PELP1), and three genes that were required for tumor survival in vivo, but not in vitro: FOXA1, HDAC1, and NCOR2. None of these genes were mutated by full exome sequencing of H1819, however FOXA1 was found to be co-amplified with NKX2-1 on chromosome 14q. The NKX2-1 gene has previously been identified as one of the most frequent focally amplified genes in lung adenocarcinomas, and we found that FOXA1 is co-amplified with NKX2-1 in 80% of these cases. We also found that NSCLC cell lines and tumor samples have significantly higher FoxA1 expression than normal lung epithelial cells and tissues. Knockdown of Foxa1 in amplified NSCLC cell lines causes significant reduction of clonogenicity, anchorage-independent growth, and in vivo xenograft growth. FoxA1 is a pioneer transcription factor, able to interact with condensed chromatin and recruit other transcription factors to their target genes. Notably, both HDAC1 and NCOR2 are also known to play roles in epigenetic regulation of gene expression. The identification of FOXA1, HDAC1, and NCOR2 as genes that are specifically required for tumor growth in vivo suggests that there are transcriptional and epigenetic programs in lung cancer which depend on microenvironmental cues and/or regulation by paracrine signaling mechanisms. We conclude that our shRNA mini-library screen identifies unsuspected acquired vulnerabilities that are only detected in vivo, and thus new therapeutic targets in lung cancer. Citation Format: Suzie K. Hight, Elizabeth McMillan, Chunli Shao, Luc Girard, Jaime Rodriguez Canales, Michael A. White, Ignacio I. Wistuba, David Mangelsdorf, John D. Minna. An in vivo shRNA functional genomics screen identifies transcriptional and epigenetic regulators that are essential in lung tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-06.

Aggressive neuroendocrine lung cancers, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), represent an understudied tumor subset that accounts for approximately 40,000 new lung cancer cases per year in the United States. No targeted therapy exists for these tumors. We determined that achaete-scute homolog 1 (ASCL1), a transcription factor required for proper development of pulmonary neuroendocrine cells, is essential for the survival of a majority of lung cancers (both SCLC and NSCLC) with neuroendocrine features. By combining whole-genome microarray expression analysis performed on lung cancer cell lines with ChIP-Seq data designed to identify conserved transcriptional targets of ASCL1, we discovered an ASCL1 target 72-gene expression signature that (i) identifies neuroendocrine differentiation in NSCLC cell lines, (ii) is predictive of poor prognosis in resected NSCLC specimens from three datasets, and (iii) represents novel "druggable" targets. Among these druggable targets is B-cell CLL/lymphoma 2, which when pharmacologically inhibited stops ASCL1-dependent tumor growth in vitro and in vivo and represents a proof-of-principle ASCL1 downstream target gene. Analysis of downstream targets of ASCL1 represents an important advance in the development of targeted therapy for the neuroendocrine class of lung cancers, providing a significant step forward in the understanding and therapeutic targeting of the molecular vulnerabilities of neuroendocrine lung cancer.

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo. Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K–mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448–59. ©2017 AACR.

MafA is a transcriptional regulator expressed primarily in pancreatic beta cells. It binds to the RIPE3b/C1-binding site within the ins gene promoter, which plays a critical role in regulating ins gene expression in response to glucose. Here, we show that MafA is post-translationally modified by the small ubiquitin-related modifiers SUMO-1 and -2. Mutation of a single site in MafA, Lys(32), blocks its sumoylation in beta cells. Incubation of beta cells in low glucose (2 mm) or exposure to hydrogen peroxide increases sumoylation of endogenous MafA. Forced sumoylation of MafA results in reduced transcriptional activity toward the ins gene promoter and increased suppression of the CHOP-10 gene promoter. Sumoylation of MafA has no apparent effect on either its nuclear localization in beta cells or its ubiquitin-dependent degradation. This study suggests that modification of MafA by SUMO modulates gene transcription and thereby beta cell function.

MAPK pathways regulate transcription through phosphorylation of transcription factors and other DNA-binding proteins. In pancreatic β-cells, ERK1/2 are required for transcription of the insulin gene and several other genes in response to glucose. We show that binding of glucose-sensitive transcription activators and repressors to the insulin gene promoter depends on ERK1/2 activity. We also find that glucose and NGF stimulate the binding of ERK1/2 to the insulin gene and other promoters. An ERK1/2 cascade module, including MEK1/2 and Rsk, are found in complexes bound to these promoters. These findings imply that MAPK-containing signaling complexes are positioned on sensitive promoters with their protein substrates to modulate transcription in situ in response to incoming signals.

Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activities are modulated in a manner that reflects the secretory demand on beta cells to integrate long- and short-term nutrient sensing information. Our studies have focused on the mechanisms of ERK1/2 activation in beta cells and on the actions of ERK1/2 that regulate beta cell function. Insulin and growth factors regulate ERK1/2 in beta cells in a largely calcium-independent manner. Nutrients and anticipatory hormones, in contrast, activate ERK1/2 in a calcium-dependent manner in these cells. We are exploring the key intermediates in these distinct activation pathways and find that calcineurin is essential for the nutrient pathway but is not essential for the growth factor pathway. Using reporter assays, heterologous reconstitution, electrophoretic mobility shift assays, Northern analysis, Q-PCR and chromatin immunoprecipitation, we have examined several genes that are regulated by ERK1/2, primarily the insulin gene and the apoptotic factor C/EBP-homologous protein (CHOP)-10 (GADD153/DDIT-3), a bZIP protein. ERK1/2-sensitive transcriptional regulators common to these two genes are C/EBP-beta and MafA. The insulin promoter is both positively and negatively regulated by glucose and other nutrients. Exposure to glucose for minutes to hours causes an increase in the rate of insulin gene transcription. In contrast, exposure to elevated glucose for 48 h or more results in inhibition of the insulin gene promoter. Both of these processes depend on ERK1/2 activity. Expression of CHOP is induced by stresses including nutrient deprivation and endoplasmic reticulum stress. CHOP gene expression, especially that regulated by nutrients, is also ERK1/2-dependent in beta cells, These studies support the hypothesis that the genes regulated by ERK1/2 and the mechanisms employed are key to maintaining normal beta cell function.

Protein S-palmitoylation is a widespread and dynamic posttranslational modification that regulates protein–membrane interactions, protein–protein interactions, and protein stability. A large family of palmitoyl acyl transferases, termed the DHHC family due to the presence of a common catalytic motif, catalyzes S-palmitoylation; the role of these enzymes in cancer is largely unexplored. In this study, an RNAi-based screen targeting all 23 members of the DHHC family was conducted to examine the effects on the growth in non–small cell lung cancer (NSCLC). Interestingly, siRNAs directed against DHHC5 broadly inhibited the growth of multiple NSCLC lines but not normal human bronchial epithelial cell (HBEC) lines. Silencing of DHHC5 by lentivirus-mediated expression of DHHC5 shRNAs dramatically reduced in vitro cell proliferation, colony formation, and cell invasion in a subset of cell lines that were examined in further detail. The phenotypes were restored by transfection of a wild-type DHHC5 plasmid but not by a plasmid expressing a catalytically inactive DHHC5. Tumor xenograft formation was severely inhibited by DHHC5 knockdown and rescued by DHHC5 expression, using both a conventional and tetracycline-inducible shRNA. These data indicate that DHHC5 has oncogenic capacity and contributes to tumor formation in NSCLC, thus representing a potential novel therapeutic target. Implications: Inhibitors of DHHC5 enzyme activity may inhibit non–small cell lung cancer growth. Mol Cancer Res; 13(4); 784–94. ©2015 AACR.

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22+ Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non–small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins. Cancer Res; 74(1); 263–71. ©2014 AACR.

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non–small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH+ lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH− counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH+ cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH+ lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH+ component, implicating Notch signaling in lung cancer stem cell maintenance. Cancer Res; 70(23); 9937–48. ©2010 AACR.

Apoptosis contributes to immune-mediated pancreatic beta cell destruction in type I diabetes. Exposure of beta cells to interleukin-1beta (IL-1beta) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in beta cells treated with IL-1beta. In addition, prolonged exposure to high glucose further increases IL-1beta-triggered CHOP expression. IL-1beta also causes increased expression of C/EBP-beta and a reduction of MafA, NFATc2, and Pdx-1 expression in beta cells. Inhibition of the NF-kappaB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects beta cells from IL-1beta-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of beta cells.

During the onset of diabetes, pancreatic beta cells become unable to produce sufficient insulin to maintain blood glucose within the normal range. Proinflammatory cytokines have been implicated in impaired beta cell function. To understand more about the molecular events that reduce insulin gene transcription, we examined the effects of hyperglycemia alone and together with the proinflammatory cytokine interleukin-1beta (IL-1beta) on signal transduction pathways that regulate insulin gene transcription. Exposure to IL-1beta in fasting glucose activated multiple protein kinases that associate with the insulin gene promoter and transiently increased insulin gene transcription in beta cells. In contrast, cells exposed to hyperglycemic conditions were sensitized to the inhibitory actions of IL-1beta. Under these conditions, IL-1beta caused the association of the same protein kinases, but a different combination of transcription factors with the insulin gene promoter and began to reduce transcription within 2 h; stimulatory factors were lost, RNA polymerase II was lost, and inhibitory factors were bound to the promoter in a kinase-dependent manner.

MAP kinases transduce signals that are involved in a multitude of cellular pathways and functions in response to a variety of ligands and cell stimuli. Aberrant or inappropriate functions of MAPKs have now been identified in diseases ranging from cancer to inflammatory disease to obesity and diabetes. In many cell types, the MAPKs ERK1/2 are linked to cell proliferation. ERK1/2 are thought to play a role in some cancers, because mutations in Ras and B-Raf, which can activate the ERK1/2 cascade, are found in many human tumors. Abnormal ERK1/2 signaling has also been found in polycystic kidney disease, and serious developmental disorders such as cardio-facio-cutaneous syndrome arise from mutations in components of the ERK1/2 cascade. ERK1/2 are essential in well-differentiated cells and have been linked to long-term potentiation in neurons and in maintenance of epithelial polarity. Additionally, ERK1/2 are important for insulin gene transcription in pancreatic beta cells, which produce insulin in response to increases in circulating glucose to permit efficient glucose utilization and storage in the organism. Nutrients and hormones that induce or repress insulin secretion activate and/or inhibit ERK1/2 in a manner that reflects the secretory demand on beta cells. Disturbances in this and other regulatory pathways may result in the contribution of ERK1/2 to the etiology of certain human disorders.

MYC is one of the most commonly deregulated oncogenes in various cancers, including breast, colorectal and lung. While mutations are rare, we know that MYC is overexpressed and in some cases amplified in these (and other) cancers. Numerous reports have recently demonstrated the utility of various therapeutics 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. Our goal is to identify lung cancers that are addicted to MYC, determine their oncogenotype and molecular properties, and identify a biomarker to identify patients with MYC addicted tumors. We studied a large panel of clinically and molecularly annotated NSCLC lines for MYC mRNA, protein expression, and DNA copy number. Functional tests were performed on 17 NSCLC cell lines using three 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, effects were monitored by colony forming efficiency (CFE) assays and proliferation assays. OMOMYC results were confirmed via xenograft experiments. Each of the three MYC inhibitors tested elicited a viability response in a subset of the 17 NSCLC cell lines, though the sensitive subset was not significantly similar between any two drugs (highest correlation coefficient of 0.3). In order to determine which, if any, of the drugs truly targeted MYC-driven lung cancers, we stably expressed OMOMYC in all 17 parental NSCLC cells and performed functional assays. 6/17 cell lines were dramatically sensitive to OMOMYC (with up to 100 fold reduction in CFE), compared to 11/17 totally resistant. The viability in the presence of OMOMYC shows a statistically significant correlation with one of the three MYC inhibitors tested, which supports the notion 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 c-Myc target genes as measured by both qRT-PCR and microarray. These data suggest that the observed phenotype was the result of decreased MYC activity. 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 are classifying this subset of cancer cell lines, MYC “addicted.” Using the MYC “addicted” vs. non- addicted NSCLC panel, we are testing for gene expression, methylation and mutational differences in order to identify and eventually characterize a biomarker for MYC dependence in lung cancer. Citation Format: Patrick Dospoy, Chunli Shao, Elizabeth McMillan, Michael Peyton, Jill Larsen, Luc Girard, Ignacio Wistuba, Adi F. Gazdar, John D. Minna. Differential MYC dependence in NSCLC identified through pharmacological and genetic MYC inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A22.

Purpose: It is clear that Hedgehog (Hh) signaling pathway can promote small cell lung cancer development but is not sufficient to drive tumor formation. However, the functional role of Hh pathway in non-small cell lung cancer remains unclear. We found that Gli1, a transcription factor of Hh pathway, is highly expressed in lung squamous cell carcinoma (SSC). The purpose of our study is to identify the functional importance of Gli1 in lung SCC. Methods: We performed a genome-wide gene expression analysis to examine Hh pathway component expression in 275 lung cancer patient specimens. qRT-PCR and western blot were used to validate their expression in SCC lines. To further explore its biological function in lung SCC we manipulated Gli1 expression genetically or pharmacologically followed by colony formation and tumor formation assays. Results: Microarray analysis revealed that Gli1, Gli2, Gli3, and Smoothened are significantly upregulated in lung SCC compared to lung adenocarcinoma (AD). Consistently we found the increased expression of Glis in lung SCC lines relative to AD lines by real-time PCR and western blot. Knocking down Gli1 by siRNA decreased liquid colony formation ability and Gli1-luciferase reporter activity in SCC lines. A significant reduction of cell proliferation and liquid colony formation was observed in shGli1 expressing SCC cells compared to control cells. Gli1 specific inhibitor GANT61 strongly inhibited SCC cell proliferation through inducing cell apoptosis. However, Gli1 expression or transcriptional activity is not regulated by classical Hh pathway upstream regulators in lung SCC. We found that blocking PI3K or ERK pathways by pharmacological inhibitors significantly reduced Gli1 expression and cell survival. In addition, TGF-β stimulation promoted Gli1 expression and transcriptional activity in SCC cells. Conclusions: Our data suggest that Hh pathway downstream factor Gli1 expression is essential for cell proliferation and colony formation in lung SCC. Gli1 is not regulated by canonical Hh pathway regulators, whereas PI3K, ERK, and TGF pathways are involved in its regulation. Blocking Gli1 function is a novel therapeutic strategy for lung SCC treatment. Citation Format: Chunli Shao. Inhibition of Gli1 as a novel therapeutic target for lung squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2350. doi:10.1158/1538-7445.AM2014-2350

Post-translational palmitoylation of intracellular proteins is mediated by protein palmitoyltransferases (PAT) belonging to the DHHC family, which share a common catalytic Asp-His-His-Cys (DHHC) motif. According to a genome-wide siRNA screen scoring for growth suppression in over 50 non-small cell lung cancer (NSCLC) cell lines, DHHC family members are strikingly over-represented. Among the tested DHHC palmitoyltransferases, knockdown of DHHC5 showed cell growth inhibition in many cell lines in the initial screen. To further investigate the growth suppression in response to DHHC5 depletion, transient and stable knockdowns of DHHC5 were undertaken in a dozen of NSCLC cell lines identified in the genome-wide screen using siRNAs and lentiviral shRNAs. Such knockdown was also performed in three immortalized human lung bronchial epithelial cell lines (HBECs). The gene expression and protein levels of DHHC5 were analyzed by RT-PCR and immunoblotting, both of which showed efficient knockdown levels. Cell proliferation and anchorage-dependent/independent colony formation assays indicated that DHHC5 knockdown led to cell growth arrest and decreased cell clonogenicity in most of the tested NSCLC cell lines in vitro, but not in the HBECs. H1299, H358 and H2009 cell lines were particularly dependent on DHHC5 expression and the growth-suppression phenotype of DHHC5 stable knockdown H1299/H358/H2009 cells was rescued by overexpression of DHHC5. The catalytically inactive DHHC5 mutant was not able to provide rescue. In vivo tumor formation assays were then carried out by injecting H1299 and H358 control cells and DHHC5 stable knockdown cells subcutaneously into NOD/SCID mice. DHHC5 knockdown led to absence or dramatically decreased tumor xenograft formation. Furthermore, a Tet-On inducible system with TRIPZ shRNA lentiviral delivery was established in H1299. Mouse xenografts using this H1299 Tet-on inducible cell line showed similar results. As DHHC5 function is needed for optimal lung cancer cell growth, it will be important to identify physiologically relevant substrates in order to develop inhibitors of DHHC5 function. Future plans include microarray and protein kinase array analysis to distinguish possible signaling pathways in which DHHC5 may be involved, as well as palmitoyl-protein profiling of normal and knockdown DHHC5 cell lines by mass spectrometry. Citation Format: Hui Tian, Jui-Yun Lu, Chunli Shao, Kenneth Huffman, Ryan Carstens, John D. Minna, Sandra L. Hofmann. Protein palmitoylation in non-small cell lung cancer (NSCLC): DHHC5 palmitoyltransferase as a potential therapeutic target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2014-4619

We have been comprehensively screening for “vulnerabilities” that have been acquired during the multi-step pathogenesis of lung cancer cells but are not present in normal lung epithelial cells to identify genetic and chemical perturbations that will selectively kill lung cancer. We think many of these have occurred to allow the lung cancers to undergo/tolerate “oncogene addiction.” We tested a sub-panel of 12-15 non-small cell lung cancer (NSCLC) lines that covers the known molecular spectra of lung cancer with genome wide siRNA and large scale chemical library (~250,000 compounds) and natural products in vitro screens to identify “hits” that will kill (suppress the growth of) lung cancer cells but not normal human bronchial epithelial cells and that also only kill a subset of lung cancer cells providing two types of specificity. “Hits” from these broad screens are then tested (including detailed drug concentration curves) across a large panel of lung cancer lines (~100) representing a variety of lung cancer histologic and molecular oncogenotypes. Other versions of these screens include the intensive use of “mini-libraries” each containing 50 – 150 gene targets by siRNAs or shRNAs, or ~200 defined drugs to explore pathways in detail in tests of over 70 NSCLCs. Examples include: nuclear receptors and their co-regulators (120 genes); cancer stem cell pathways (50 genes); chromatin remodelers (75 genes) and identified lung cancer mutated driver oncogenes (175 genes). In addition to the in vitro tests, we have developed in vivo (xenograft) tests where shRNA mini-libraries are introduced into tumor cells at high representation which are grown as xenografts, analyzed by NexGen sequencing and shRNAs identified that drop out or are retained in xenografts compared to in vitro grown cells to identify vulnerabilities that are only detected in the in vivo situation. All of the data are then related to the large legacy molecular datasets associated with the lung cancer lines (including whole exome sequence analyses and genome wide mRNA, copy number variation, methylation, miR expression data and proteomics data). In addition, detailed chemical and pharmacokinetic analyses for favorable drug properties and subsequent chemical modifications also occur for the chemical compounds to progress those towards potential clinical studies. The results of all of these analyses have identified ~300 new chemical compounds and ~300 genetic hits all of which show selectivity for lung cancer over normal lung cells and selectivity for subtypes of lung cancer. The chemical and genes hits are being compared to the tumor molecular information and integrated in turn through a “connectivity map” type of approach – to identify drugs and gene hits involving the same pathways. The molecular correlates of the tumor lines are related to similar molecular changes in patient derived xenografts and patient tumor specimens to provide a connection of the molecular subtype-selective vulnerabilities (“enrollment biomarkers”) between the preclinical response phenotypes and patient tumor specimens. From these data we find lung cancers can be classified into groups (“clades”) that represent functional vulnerabilities to the gene and chemical compound hits and these in turn can be related to molecular abnormalities in tumors. One example of this is our detailed analyses a matched lung adenocarcinoma/normal lung epithelial cell model derived from the same patient which identified three distinct target/response-indicator pairings that are represented a significant frequencies (6-16%) in the lung adenocarcinoma population (Kim et al. Cell 155:552, 2013). These include three totally novel lung cancer selective targeted therapies: NLRP3 mutation/inflammasome activation-dependent FLIP addiction; co-occurring KRAS and LKB1 mutation-driven COPI addiction; and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Our panel of “hits” provide the opportunity to identify all potential therapeutic targets for lung cancer, while the molecular correlates will allow “personalization” of these new therapies going forward in preclinical and clinical translation. (Supported by NCI SPORE P50CA70907, NCI CTD2N, CPRIT, UTSW CCSG P30CA142543) Citation Format: John D. Minna, Adi Gazdar, Alexander Augustyn, Rebecca Britt, Ryan Carstens, Patrick Dospoy, Boning Gao, Luc Girard, Suzie Hight, Kenneth Huffman, Jill Larsen, Michael Peyton, Chunli Shao, David Mangelsdorf, Rolf Brekken, Ralph Deberardinis, Pei-Hsuan Chen, Carmen Behrens, Lauren Byers, John Heymach, Jack Roth, Ignacio Wistuba, Yang Xie, Caleb Davis, David Wheeler, Richard Gibbs, Edward Marcotte, Joseph Ready, Deepak Nijhawan, Noelle Williams, Steven McKnight, Bruce Posner, John MacMillan, Michael Roth, Michael White. Developing a new functional classification of lung cancer based on tumor acquired vulnerabilities. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr IA21.

Background: Accumulating evidence demonstrates that the malignant phenotype is driven and maintained by cancer stem cells (CSCs), which can self-renew, differentiate, and contribute to drug resistance and tumor metastasis. Our group has identified CSCs in both non-small cell lung cancer cell (NSCLC) lines and patient tumor samples by their enhanced ALDH activity and demonstrated that ALDH+ lung cancer cells are highly tumorigenic and clonogenic. We also found that ALDH1A3 is the major isozyme responsible for the ALDH activity and functionally important for lung CSCs. Aims and Methods: The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. We performed a siRNA screen of 40 stem cell pathway genes in a panel of NSCLC lines to identify potential targets related to stem cell self-renewal pathways and other regulators. qRT-PCR, western blot and flow cytometry based Aldefluor assay were used to validate the promising hits. To further explore their biological function in lung CSCs we manipulated their expression genetically or pharmacologically followed by colony formation and tumor formation assays. Results: siRNA screen revealed that knocking down ALDH1A3, Signal Transducers and Activators of Transcription 3 (STAT3), or certain components of WNT and Notch pathway caused a reduction of cell viability in many NSCLC lines. Western blot analysis confirmed that ALDH+ cells contained more activated STAT3 compared to ALDH− lung cancer cells. Inhibition of STAT3 activation by Stattic (a specific STAT3 inhibitor), GSK126 (an EZH2 inhibitor), or siRNA attenuated ALDH1A3 expression, ALDH+ lung cancer cells and tumor cell clonogenicity. In addition, FZD2, DVL3 and LBH were more commonly upregulated in ALDH+ cells across the penal of NSCLC lines. shRNA mediated knockdown of DVL3 in NSCLCs resulted in a significant reduction in ALDH activity, clonogenicity, and tumorigenicity. Conclusions: Our data suggest that ALDH1A3 expression, STAT3 pathway activation (via EZH2), and an active WNT pathway are all essential for maintenance of lung CSCs. Blocking ALDH1A3, WNT or STAT3 pathways appear to be rationale therapeutic strategies for eliminating NSCLC stem cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B50. Citation Format: Chunli Shao, Chris DeSevo, Luc Girard, Paul Yenerall, Carmen Behrens, Ignacio Wistuba, John D. Minna. Regulation of non-small cell lung cancer stem cells by STAT3 and WNT pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B50.

The cancer stem cell model hypothesizes that a small subpopulation of a given tumor is endowed with the ability to give rise to the bulk of the tumor mass. The ability to understand signaling pathways that maintain cancer stem cells would provide for novel therapeutic targets. Recent evidence has shown that in non-small cell lung cancer (NSCLC) the stem cell population can be defined by aldehyde dehydrogenase (ALDH) activity, with this fraction of cells demonstrating self-renewal, serial transplantation, resistance to therapy, and increased tumorigenicity. We are interested in studying the involvement of microRNAs in potentially regulating the ALDH+ cancer stem cell population in NSCLC. MicroRNAs have been shown to be critical for the differentiation of several cancer cell types and we hypothesize that they may be critical for the differentiation of lung cancer stem cells into terminally-differentiated, non-dividing cells. miR-10a is a microRNA located in the HOX cluster of genes, which is known to be important for the development of the body plane along the anterior-posterior axis. Additionally, miR-10a has been shown to be induced by all-trans-retinoic acid (ATRA), clinically used to induce differentiation in neuroblastoma and acute promyelocytic leukemia. We treated H2009 NSCLC cells with miR-10a mimic and observed a significant decrease in the ALDH+ cell population. This decrease was related to the loss of mRNA and protein levels of ALDH1A3, an isoform of the ALDH family of proteins. Increasing miR-10a levels inhibited the ability to form colonies and induced apoptosis. We then tested if depletion of ALDH+ cells using miR-10a mimic would affect the response to paclitaxel, which is a front line therapy for NSCLC. We observed a 10-fold decrease in cell viability in the presence of miR-10a mimic, suggesting that high levels of miR-10a may predict for sensitivity to taxane therapy. Assessing miR-10a levels in a clinically annotated database of NSCLC patients demonstrated that high miR-10a levels correlate with longer overall survival. A consensus target prediction search using multiple algorithms showed that ALDH1A3 is not a direct target of miR-10a. A targeted siRNA screen containing genes implicated in stem cell maintenance revealed that the WNT and Notch pathway are important for cell survival. Both pathways are down regulated when cells are treated with miR-10a mimic. TargetScan identify DVL3 as a miR-10a target gene. Manipulation of miR-10a levels resulted in significant changes in both mRNA and protein levels of DVL3. To determine if DVL3 was important in regulating the ALDH+ population, we used siRNAs targeting DVL3, which significantly decreased ALDH1A3 protein level and ALDH+ cell population. Several new small molecules have been shown to inhibit DVL3 docking to the frizzle receptor; we hypothesized that these molecules would reduce ALDH+ cell populations. After several days of treatment the molecule had no effect on colony formation but significantly reduced ALDH+ cells. The identification of miR-10a as a regulator of ALDH+ cancer stem cells in NSCLC presents a novel therapeutic target. In addition, miR-10a's role in modulating cellular response to taxane therapy may provide a biomarker for predicting paclitaxel response in NSCLC. This study will provide new insights into the molecular basis of lung cancer and deliver new targets for therapy. Citation Format: Chris G. Desevo, Chunli Shao, Jill Larsen, Carmen Behrens, Ignacio I. Wistuba, John D. Minna, Alex Pertsemlidis. miR-10a regulation of cancer stem cells in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B15.

Introduction: Gene expression signatures from large cohorts of lung tumors suggest that distinct neuroendocrine (NE) cancers with poor prognosis appear in ~10% of otherwise pathologically unremarkable NSCLCs. However, a complete molecular characterization is lacking because a similar subtype has yet to be identified in a significant number of lung cancer cell lines. Our goal is to develop a pre-clinical model for NSCLC-NE and use it to rationale targeted therapy for this important subtype of lung cancer. We are developing targeted siRNA-based synthetic lethal screens to identify acquired vulnerabilities in lung cancers by using “mini-libraries” of putative cancer stem cell genes (CSC, N = 40), nuclear receptors and their co-regulators (NR/CoReg, N = 120), chromatin remodelers (CR, N = 102), and ASCL1-regulated druggable genes (N = 40). The siRNA libraries were derived from multiple studies while the ASCL1-targeted library was developed from our own ChIP-Seq data. Identifying NSCLC-NE Cell Lines: Using mRNA expression signatures from 207 lung cell lines (NSCLC/SCLC/HBEC) we identified a cluster of NSCLC cell lines (9% of NSCLC) that express genes indicative of a neuroendocrine phenotype (NSCLC-NE). A highly expressed gene in this class of cell lines is the lineage-specific transcription factor ASCL1, which is required for the development of pulmonary neuroendocrine cells. ASCL1 loss-of-function studies demonstrate dramatic cell death compared to controls, suggesting these cells are addicted to ASCL1 for survival. ASCL1 ChIP-Seq Analysis: To better understand the molecular pathogenesis of ASCL1-dependent NSCLC-NEs and identify druggable therapeutic downstream targets, we performed ChIP-Seq analysis on six ASCL1+ and two ASCL1- cell lines. A 125-member “ASCL1-signature” was generated and tested on multiple resected NSCLC datasets (N > 800 patients) for prognostic utility and tumors that exhibited the “ASCL1 signature” had significantly worse prognosis. A target of ASCL1 as determined by ChIP-Seq analysis is the anti-apoptotic regulator BCL2. BCL2 knockdown using siRNA as well as inhibition of BCL2 using a small molecule (ABT-263) results in cell death that is specific to ASCL1+/BCL2+ cells. ASCL1 Upstream Regulation: A separate therapeutic avenue is to determine upstream regulators of ASCL1 and we have demonstrated that ASCL1 participates in a double-negative feedback loop with the MEK/ERK pathway. Pharmacological activation of the MEK/ERK pathway results in loss of ASCL1 mRNA and protein, and induces apoptosis in ASCL1-dependent NSCLC-NE lines. Additionally, siRNA-mediated knockdown of ASCL1 results in reciprocal activation of the MEK/ERK pathway even in the presence of an siRNA targeting MEK1. Combining MEK/ERK activators with ABT-263 results in dramatic induction of apoptosis in NSCLC-NE while sparing normal immortalized HBEC cells suggesting the possibility of a synthetic lethal combination therapy for this subset of tumors. Synthetic Lethal Screens: We have completed synthetic lethal siRNA screens for the CSC, NR/CoReg, and CR mini-libraries using a panel of lung cancer lines. Optimal transfection conditions were established for each line and siRNA pools were used with multiple (n = 9) replicates providing great reproducibility (r > 0.9). From these screens, preliminary data demonstrates that the NSCLC-NE tumor lines have response phenotypes distinct from all other classes of lung cancer. Conclusions: We show that NE gene expression in NSCLC is of clinical relevance, and ASCL1 appears to be a lineage-dependent oncogene for NSCLC-NE. ASCL1 ChIP-Seq and siRNA screen data provide a roadmap for systematically searching for therapeutic targets such as BCL2 while work focused on targeting pathways upstream of ASCL1 also demonstrates clinical potential for NSCLC with neuroendocrine features. Our results indicate that lung cancers driven by ASCL1 have distinct acquired vulnerabilities. (Supported by NCI SPORE P50CA70907, CPRIT) Citation Format: Alexander Augustyn, Mark Borromeo, Tao Wang, Chunli Shao, Patrick Dospoy, Kenneth Huffman, Ryan Carstens, Luc Girard, Carmen Behrens, Ignacio Wistuba, Yang Xie, Jane Johnson, Adi Gazdar, John Minna. Targeted synthetic lethal screens to identify acquired vulnerabilities in a novel class of ASCL1-dependent neuroendocrine non-small cell lung cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A24.

A considerable amount of evidence reveals the important roles of cancer stem cells (CSCs, also called “cancer initiating cells”) in tumor growth, metastasis, relapse and drug resistance. Our group has identified lung CSCs in both lung cancer cell lines and patient tumor samples by their elevated Aldehyde Dehydrogenase (ALDH) activity and demonstrated that NSCLC patients whose tumors are enriched in ALDH+ cells have inferior survival, and that the ALDH+ subpopulations (ranging from 0.5 to 30% of tumor cells) are self renewing, and significnatly more clonogenic and tumorigencic (in xenograft models) than the ALDH− population. To further define the molecular profile of lung CSCs and to identify key pathways involved in lung CSC function we performed genome wide microarray expression profiling of isolated ALDH+ and ALDH− cell populations from 8 non-small cell lung cancer (NSCLC) lines representing a variety of oncogenotypes to determine genes that are differentially expressed in these two populations. Strikingly we found that different lung cancers exhibited different ALDH+ cell population expression profiles but that the ALDH1A3 isozyme was one of the few commonly genes whose expression was elevated. (Previously our group and other investigators in lung and other cancers had found the ALDH1A1 and not ALDH1A3 isozyme to be a marker of the ALDH+ population). ALDH1A3 differential expression was confirmed by q RT PCR and elevated ALDH1A3 protein levels which in turn was correlated with the % of ALDH+ tumor cells in each tumor. ALDH1A3 mRNA levels in (N = 182) resected lung adenocarcinoma samples showe that the highest levels of ALDH1A3 were associated with inferior survival. Of great importance, shRNA mediated knockdown of ALDH1A3 dramatically reduced both NSCLC cell colony formation in vitro and tumor growth in vivo demonstrating the functional importance of ALDH1A3 expression in NSCLCs. However, ectopic expression of ALDH1A3 in NSCLC cells did not enhance lung cancer cell growth and tumor formation, suggesting that ALDH1A3 alone is not sufficient to promote lung cancer progression. We conclude that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity in NSCLCs of many oncogenotypes, is of potential prognostic importance, and a new therapeutic target for eliminating NSCLC stem cells.

Recent advances in detailed molecular understanding of lung cancer including complete genome sequences affords the possibility of developing molecularly targeted therapy for potentially all lung cancers. Currently the Lung Cancer Mutation Consortium (LCMC) has developed an integrated approach and network for identifying lung cancers with specific mutations and funneling these patients into clinical trials targeting the specific mutations identified. However, another approach is to use genome wide and chemical library screens to identify genetic and epigenetic changes that have been created in lung cancer cells during tumor pathogenesis that are absolutely required for the activated oncogenic pathways to function. These are often referred to as “synthetic lethal” changes and represent adaptations the cancer cell has to make to allow the “oncogene addictions” to drive tumor growth and survival. They are present in tumor but not normal cells and thus represent “acquired vulnerabilities” that can be therapeutically targeted. The NCI's “Cancer Target Discovery and Development Network” (CTD2N) exemplifies this combined genetic and pharmacologic approach. A most important subgroup of these vulnerabilities are changes that are required for the continued function and survival of a subpopulation of tumor cells that have acquired tumor initiating and often metastatic and drug resistant characteristics including many of the properties of stem cells that are referred to as cancer stem cells (CSCs) or “cancer initiating cells.” To achieve these goals we have developed a large integrated research platform. We have also: developed assays for identifying lung CSCs, “molecular portraits” (clades) that group lung cancers into subsets of clinical and molecular relevance; genome wide siRNA and chemical library functional screens to test for portrait/clade specificity. From these efforts we have learned that: There are a subset of cells within lung cancers (ranging from 0.1–30% of non small cell lung cancers, NSCLCs) and 50% + of small cell lung cancers, SCLCs) identified by elevated aldehyde dehydrogenase (ALDH) activity that have dramatically enhanced clonogenic, tumorigenic, and self renewal capacity; Patients whose tumors are enriched is such ALDH+ tumor cells have significantly impaired prognosis; The notch pathway (particularly Notch3) and ALDH1A3 are major vulnerabilities in lung CSCs; NSCLCs can be subdivided by mRNA expression profiles into “molecular portraits” (or “clades”) that have relevant clinical, oncogenotype, and drug response phenotypes; Genome wide siRNA and large chemical library screens identify targets that are specific for lung cancer over normal lung epithelial cells; The identified targets show dramatic clade and oncogenotype specificity as well as specificity for lung cancers with different responses to available chemotherapy and targeted therapy for lung cancer; The newly identified vulnerabilities provide coverage of essentially all lung cancers and as such, provide a new functional “vulnerability classification” of lung cancer. All of these findings set the stage for the development of a rational approach to developing therapy targeted at lung cancer acquired vulnerabilities including those in lung CSCs that will include personalizing the therapy for each patient. (Supported by Lung Cancer SPORE P50CA70907, DOD Prospect, CPRIT, and NCI CTD2N CA148225.)

Background: The Hedgehog (Hh) signaling pathway is critical for embryonic development and its deregulation is implicated in a number of tumor types. The role of the Hh signaling pathway, however, in the initiation and growth of non-small cell lung cancer is largely unknown. Here, we investigate the role of the Hh pathway transcription factor, GLI1, in lung squamous cell carcinoma (SCC) and as a potential therapeutic target for treatment of lung SCC. Methods: GLI1 expression in human SCC cell lines was evaluated by quantitative PCR and Western Blot. siRNA and shRNA of GLI1 in these cell lines were utilized in vitro and in vivo to test the requirement of GLI1 in tumor growth. Small molecule modulators of GLI1 were tested for their therapeutic potential. Results: GLI1 mRNA expression was significantly elevated in lung SCC compared to normal lung and lung adenocarcinoma patient specimens in several human genomic databases. Importantly, overexpression of GLI1 was correlated with poor overall survival in lung cancer patients. siRNA-mediated knock down of GLI1 in SCC cell lines decreased the expression of GLI1 target genes and caused a significant reduction in colony formation. Stable knock down of GLI1 in SCC cell lines caused a significant reduction in growth of xenograft tumors indicating the critical role of GLI1 in lung SCC progression. Inhibition or activation of SMO, an upstream component of Hh pathway, did not alter GLI1 expression level in lung SCC cell lines. However, inhibition of PI3K/AKT and MAPK signaling pathways down-regulated GLI1 expression, suggesting that GLI1 expression is dependent on PI3K/AKT and MAPK pathway activity rather than Hh ligand. Small molecule inhibition of PI3K/ mTOR pathway or GLI1 significantly reduced GLI1 expression, proliferation, and clonogenicity in SCC cell lines. Combinatorial inhibition of PI3K and GLI1 by BKM120 and arsenic trioxide (ATO), respectively, significantly abrogated the in vivo growth SCC tumors in mice and correlated with decreased tumor GLI1 expression. Conclusion: Our findings demonstrate that GLI1 is essential for lung SCC tumor progression. Furthermore, GLI1 expression in SCC is independent of Hh pathway ligand action and dependent on MAPK and PI3K pathway activity. Direct inhibition of GLI1 by repurposing ATO in combination with a PI3K inhibitor may represent a novel therapeutic strategy for lung SCC.