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PDF file - 521K, Different expression profiles of hub genes between the lung adenocarcinoma (ADC) group and lung squamous cell carcinoma (SCC) group.

Purpose: To understand the role of nuclear factor erythroid-2–related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in non–small cell lung cancer (NSCLC), we studied their expression in a large series of tumors with annotated clinicopathologic data, including response to platinum-based adjuvant chemotherapy.Experimental Design: We determined the immunohistochemical expression of nuclear Nrf2 and cytoplasmic Keap1 in 304 NSCLCs and its association with patients' clinicopathologic characteristics, and in 89 tumors from patients who received neoadjuvant (n = 26) or adjuvant platinum-based chemotherapy (n = 63). We evaluated NFE2L2 and KEAP1 mutations in 31 tumor specimens.Results: We detected nuclear Nrf2 expression in 26% of NSCLCs; it was significantly more common in squamous cell carcinomas (38%) than in adenocarcinomas (18%; P < 0.0001). Low or absent Keap1 expression was detected in 56% of NSCLCs; it was significantly more common in adenocarcinomas (62%) than in squamous cell carcinomas (46%; P = 0.0057). In NSCLC, mutations of NFE2L2 and KEAP1 were very uncommon (2 of 29 and 1 of 31 cases, respectively). In multivariate analysis, Nrf2 expression was associated with worse overall survival [P = 0.0139; hazard ratio (HR), 1.75] in NSCLC patients, and low or absent Keap1 expression was associated with worse overall survival (P = 0.0181; HR, 2.09) in squamous cell carcinoma. In univariate analysis, nuclear Nrf2 expression was associated with worse recurrence-free survival in squamous cell carcinoma patients who received adjuvant treatment (P = 0.0410; HR, 3.37).Conclusions: Increased expression of Nrf2 and decreased expression of Keap1 are common abnormalities in NSCLC and are associated with a poor outcome. Nuclear expression of Nrf2 in malignant lung cancer cells may play a role in resistance to platinum-based treatment in squamous cell carcinoma. Clin Cancer Res; 16(14); 3743–53. ©2010 AACR.

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PDF file - 124K, The SWEAVE report contains all the details about the model, parameters and procedures used to generate the results in the paper.

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PDF file - 49K, Multivariate Cox regression analysis of the 18-hub-gene prognostic signature and clinical variables in the UT Lung SPORE.

PDF file - 108K, Comparison with the gene signatures with other signatures.

PDF file - 64K, Meta-analysis of 18-hub-gene prognostic signature on ADC patients and SCC patients.

Cardiac glycosides such as oleandrin are known to inhibit the Na,K-ATPase pump, resulting in a consequent increase in calcium influx in heart muscle. Here, we investigated the effect of oleandrin on the growth of human and mouse cancer cells in relation to Na,K-ATPase subunits. Oleandrin treatment resulted in selective inhibition of human cancer cell growth but not rodent cell proliferation, which corresponded to the relative level of Na,K-ATPase α3 subunit protein expression. Human pancreatic cancer cell lines were found to differentially express varying levels of α3 protein, but rodent cancer cells lacked discernable expression of this Na,K-ATPase isoform. A correlation was observed between the ratio of α3 to α1 isoforms and the level of oleandrin uptake during inhibition of cell growth and initiation of cell death; the higher the α3 expression relative to α1 expression, the more sensitive the cell was to treatment with oleandrin. Inhibition of proliferation of Panc-1 cells by oleandrin was significantly reduced when the relative expression of α3 was decreased by knocking down the expression of α3 isoform with α3 siRNA or increasing expression of the α1 isoform through transient transfection of α1 cDNA to the cells. Our data suggest that the relative lack of α3 (relative to α1) in rodent and some human tumor cells may explain their unresponsiveness to cardiac glycosides. In conclusion, the relatively higher expression of α3 with the limited expression of α1 may help predict which human tumors are likely to be responsive to treatment with potent lipid-soluble cardiac glycosides such as oleandrin. [Mol Cancer Ther 2009;8(8):2319–28]

Purpose: Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment, including the modification of inflammatory responses from antitumorigenic to protumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis.Experimental Design: K-rasLA1 mice were irradiated with various doses and dose regimens and then monitored until death. Microarray analyses were performed using Illumina BeadChips on whole lung tissue 70 days after irradiation with a fractionated or acute dose of radiation and compared with age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets.Results: Radiation exposure accelerates lung cancer progression in the K-rasLA1 lung cancer mouse model with dose fractionation being more permissive for cancer progression. A nonrandom inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature.Conclusions: These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by affecting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis. Clin Cancer Res; 20(6); 1610–22. ©2014 AACR.

Purpose: To investigate the mechanisms of regulation and role associated with enhancer of zeste homolog 2 (EZH2) expression in lung cancer cells.Experimental Design: We investigated the mechanisms of EZH2 expression associated with the VEGF/VEGFR-2 pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. In addition, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients' clinical characteristics.Results: In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 and hypoxia-inducible factor-1α (HIF1α), and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. In addition, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patients who did not receive this therapy. Furthermore, we demonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti–VEGFR-2 drug AZD2171.Conclusion: Our results suggest that the VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF1α, and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy. Clin Cancer Res; 20(14); 3849–61. ©2014 AACR.

PDF file - 136KB, Supplementary Table 1. Sumary of the clinicopathological characteristics of the patients with lung adecarcinomas examined for EZH2 and miR-101 expressions. Supplementary Table 2. Association between markers' expression and lung adenocarcinoma patient's clinical and pathological characteristics. Supplementary Table 3. Summary of Multivariate Analysis of Outcome in Lung Adenocarcinoma Patients by Adjuvant Chemoterapy and EZH2, miR-101 and Combination of EZH2/miR-101 Expression.

PDF file - 1617KB, Figure S1. Malignant Characteristics of Invasive Adenocarcinoma in K-rasLA1 Mice; Figure S2. Radiation Effects on the Incidence of Various Endpoints in K-rasLA1 Mice; Figure S3. Comparative Genomic Analyses and Classifier Isolation from Irradiated Versus Unirradiated Control K-rasLA1 Mice; Figure S4. Comparative Genomic Analysis of Whole Lungs Reveals Unique Gene Classifiers Capable of Specifying Individual Experimental Cohorts; Figure S5. Only "Fractionated" Classifier Demonstrates Clinical Relevance for Lung Cancer Patient Survival; Figure S6. "Fractionated" Classifier Capable of Predicting Overall Survival in Patients with Breast, but not Lung Squamous Cell Cancer; Supplementary Figure S7. Cox Regression Analysis Exposes 6 Genes Within "Fractionated" Classifier Which Retain Predictive Capacity; Table S1. Logistic Regression Analysis of Unirradiated K-rasLA1 Mice for Gender and Strain Effects on Various Endpoints; Table S2. Logistic Regression Analysis for Gender and Strain Effects on Various Endpoints Controlling for Experiment; Table S3. Logistic Regression Analysis of Radiation Effects on the Incidence of Invasive Adenocarcinoma Controlling for Gender and Strain; Table S4. Multivariate Cox Analysis for Gender and Strain Effects on Overall Survival Controlling for Experiment; Table S5. Multivariate Cox Analysis of Radiation Effects on Overall Survival Controlling for Gender and Strain Effects; Table S6. IPA Network Annotations Associated with Corresponding Gene Lists.

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Supplementary Data from Nrf2 and Keap1 Abnormalities in Non–Small Cell Lung Carcinoma and Association with Clinicopathologic Features

Purpose: Prospectively identifying who will benefit from adjuvant chemotherapy (ACT) would improve clinical decisions for non–small cell lung cancer (NSCLC) patients. In this study, we aim to develop and validate a functional gene set that predicts the clinical benefits of ACT in NSCLC.Experimental Design: An 18-hub-gene prognosis signature was developed through a systems biology approach, and its prognostic value was evaluated in six independent cohorts. The 18-hub-gene set was then integrated with genome-wide functional (RNAi) data and genetic aberration data to derive a 12-gene predictive signature for ACT benefits in NSCLC.Results: Using a cohort of 442 stage I to III NSCLC patients who underwent surgical resection, we identified an 18-hub-gene set that robustly predicted the prognosis of patients with adenocarcinoma in all validation datasets across four microarray platforms. The hub genes, identified through a purely data-driven approach, have significant biological implications in tumor pathogenesis, including NKX2-1, Aurora Kinase A, PRC1, CDKN3, MBIP, and RRM2. The 12-gene predictive signature was successfully validated in two independent datasets (n = 90 and 176). The predicted benefit group showed significant improvement in survival after ACT (UT Lung SPORE data: HR = 0.34, P = 0.017; JBR.10 clinical trial data: HR = 0.36, P = 0.038), whereas the predicted nonbenefit group showed no survival benefit for 2 datasets (HR = 0.80, P = 0.70; HR = 0.91, P = 0.82).Conclusions: This is the first study to integrate genetic aberration, genome-wide RNAi data, and mRNA expression data to identify a functional gene set that predicts which resectable patients with non–small cell lung cancer will have a survival benefit with ACT. Clin Cancer Res; 19(6); 1577–86. ©2013 AACR.

PDF file - 200K, Summary of the prognostic performance for the 12 hub-gene set.

Supplementary Figures 1-3 from Oleandrin-mediated inhibition of human tumor cell proliferation: Importance of Na,K-ATPase α subunits as drug targets

PDF file - 104K, Members of the 18-hub gene signature have been included in MSigDB database C2 signature catalogues.

PDF file - 453KB, Figure 1. EZH2, VEGFR-2 and miR-101 expression in in additional cell lines. Figure 2. Effect of VEGF stimulation in the E2F expression. Figure 3. Effect of Hypoxia in the expression of EZH2,HIF-1alpha and miR-101. Figure 4. Effect of miR-101 overexpression in the expression of EZH2. Figure 5. Effect of treatment with DZNep on lung adenocarcinoma cell viability. Figure 6. Effect of VEGFR-2 overexpression in sensitivity to cisplatin.

Supplementary Figure 2. Expression and response in cells derived from a mouse model of adenocarcinoma.

Supplementary Figure 3. Transcriptional profiling of HBEC30KT and HBEC30KT-shTP53 identifies context-dependent de-repression of the miR-17~92 targetome after p53 loss driven by target genes of the miR-17-92 polycistron.

Supplementary Figure 1. Activity of miR-92a related inhibitors.

While several molecular targets have been identified for adenocarcinoma (ACA) of the lung, similar drivers with squamous cell carcinoma (SCC) are sparse. We compared signaling pathways and potential therapeutic targets in lung SCC and ACA tumors using reverse phase proteomic arrays (RPPA) from two independent cohorts of resected early stage NSCLC patients: a testing set using an MDACC cohort (N=140) and a validation set using the Cancer Genome Atlas (TCGA) cohorts. We identified multiple potentially targetable proteins upregulated in SCC, including NRF2, Keap1, PARP, TrkB, and Chk2. Of these potential targets, we found that TrkB also had significant increases in gene expression in SCC as compared to adenocarcinoma. Thus, we next validated the upregulation of TrkB both in vitro and in vivo and found that it was constitutively expressed at high levels in a subset of SCC cell lines. Furthermore, we found that TrkB inhibition suppressed tumor growth, invasiveness and sensitized SCC cells to tyrosine kinase EGFR inhibition in a cell-specific manner.

Lung cancer is the leading cause of cancer-related fatalities. Recent success developing genotypically targeted therapies, with potency only in well-defined subpopulations of tumors, suggests a path to improving patient survival. We used a library of oligonucleotide inhibitors of microRNAs, a class of posttranscriptional gene regulators, to identify novel synthetic lethal interactions between miRNA inhibition and molecular mechanisms in non–small cell lung cancer (NSCLC). Two inhibitors, those for miR-92a and miR-1226*, produced a toxicity distribution across a panel of 27 cell lines that correlated with loss of p53 protein expression. Notably, depletion of p53 was sufficient to confer sensitivity to otherwise resistant telomerase-immortalized bronchial epithelial cells. We found that both miR inhibitors cause sequence-specific downregulation of the miR-17∼92 polycistron, and this downregulation was toxic only in the context of p53 loss. Mechanistic studies indicated that the selective toxicity of miR-17∼92 polycistron inactivation was the consequence of derepression of vitamin D signaling via suppression of CYP24A1, a rate-limiting enzyme in the 1α,25-dihydroxyvitamin D3 metabolic pathway. Of note, high CYP24A1 expression significantly correlated with poor patient outcome in multiple lung cancer cohorts. Our results indicate that the screening approach used in this study can identify clinically relevant synthetic lethal interactions and that vitamin D receptor agonists may show enhanced efficacy in p53-negative lung cancer patients. Cancer Res; 75(4); 666–75. ©2014 AACR.

Purpose: To investigate the mechanisms of regulation and role associated with enhancer of zeste homolog 2 (EZH2) expression in lung cancer cells. Experimental Design: We investigated the mechanisms of EZH2 expression associated with the VEGF/VEGFR-2 pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. In addition, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients' clinical characteristics. Results: In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 and hypoxia-inducible factor-1α (HIF1α), and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. In addition, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patients who did not receive this therapy. Furthermore, we demonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti–VEGFR-2 drug AZD2171. Conclusion: Our results suggest that the VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF1α, and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy. Clin Cancer Res; 20(14); 3849–61. ©2014 AACR.

Introduction: Malignant pleural mesothelioma (MPM) is a deadly disease with poor prognosis and few treatment options. We characterized and elucidated the roles of C-MYC and PVT1 involved in the pathogenesis of MPM. Methods: We used small interfering RNA (siRNA)-mediated knockdown in MPM cell lines to determine the effect of C - MYC and PVT1 abrogation on MPM cells undergoing apoptosis, proliferation, and cisplatin sensitivity. We also characterized the expression of microRNAs spanning the PVT1 region in MPM cell lines. Copy number analysis was measured by quantitative polymerase chain reaction and fluorescence in situ hybridization. Results: Copy number analysis revealed copy number gains (CNGs) in chromosomal region 8q24 in six of 12 MPM cell lines. MicroRNA analysis showed high miR-1204 expression in MSTO-211H cell lines with four copies or more of PVT1. Knockdown by siRNA showed increased PARP-C levels in MSTO-211H transfected with siPVT1 but not in cells transfected with si C-MYC . C-MYC and PVT1 knockdown reduced cell proliferation and increased sensitivity to cisplatin. Analysis of the expression of apoptosis-related genes in the MSTO-211H cell line suggested that C-MYC maintains a balance between proapoptotic and antiapoptotic gene expression, whereas PVT1 and, to a lesser extent, miR-1204 up-regulate proapoptotic genes and down-regulate antiapoptotic genes. Fluorescence in situ hybridization analysis of MPM tumor specimens showed a high frequency of both CNGs (11 of 75) and trisomy (three copies; 11 of 75) for the C-MYC locus. Conclusion: Our results suggest that C-MYC and PVT1 CNG promotes a malignant phenotype of MPM, with C-MYC CNG stimulating cell proliferation and PVT1 both stimulating proliferation and inhibiting apoptosis.

1. International Agency for Research on Cancer, World Health Organization. Estimatedcancer Incidence, Mortality, Prevalence and Disability-adjusted life years (DALYs)Worldwide in2008. http://globocan.iarc.fr/(17August2013,datelastaccessed).2. Vital Statistics Japan (Ministry of Health, Labour and Welfare). http://ganjoho.jp/professional/statistics/statistics.html (17 August 2013, date last accessed).3. Son T, Hyung WJ, Lee JH et al. Clinical implication of an insufficient number ofexamined lymph nodes after curative resection for gastric cancer. Cancer 2012;118: 4687–4693.4. Edge SB BD, Compton CC, Fritz AG et al. AJCC Cancer Staging Manual, 7th ed.NewYork: Springer2010.5. Zu H, Wang F, Ma Y et al. Stage-stratified analysis of prognostic significance oftumor size inpatients withgastric cancer.PLoS One2013; 8(1):e545026. Kunisaki C, Makino H, Kimura J et al. Impact of lymphovascular invasion inpatients withstage I gastric cancer.Surgery 2010; 147:204–211.7. Li C, Oh SJ, Kim S et al. Macroscopic Borrmann type as a simple prognostic indicatorinpatientswithadvancedgastriccancer.Oncology2009; 77: 197 –204.8. Kunisaki C, Akiyama H, Nomura M et al. Clinicopathologic characteristics and surgicaloutcomesof mucinousgastriccarcinoma. Ann SurgOncol 2006; 13: 836 –842.9. Talamonti MS, Kim SP, Yao KA et al. Surgical outcomes of patients with gastriccarcinoma: the importance of primary tumor location and microvessel invasion.Surgery 2003; 134:720–727;discussion 727–9.10. Iasonos A, Schrag D, Raj GV et al. How to build and interpret a nomogram forcancer prognosis. JClin Oncol2008; 26: 1364–1370.11. Han DS, Suh YS, Kong SH et al. Nomogram predicting long-term survival after d2gastrectomyfor gastric cancer. JClin Oncol2012; 30: 3834–3840.12. Kattan MW, Karpeh MS, Mazumdar M et al. Postoperative nomogram for disease-specific survival after an R0 resection for gastric carcinoma. J Clin Oncol 2003;21:3647–3650.13. Kim JH, Kim HS, Seo WY et al. External validation of nomogram for the predictionof recurrence after curative resection in early gastric cancer. Ann Oncol 2012; 23:361–367.14. Kim DH, Kim SM, Hyun JK et al. Changes in postoperative recurrence and prognosticrisk factors for patients with gastric cancer who underwent curative gastric resectionduring different time periods. AnnSurgOncol 2013; 20:2317 –2327.15. Japanese Gastric Cancer Association. Japanese Classification of GastricCarcinoma,13 ed. Tokyo:Kandera 1999.16. Akazawa K, Nakamura T, Palesch Y. Power of logrank test and Cox regression modelinclinical trialswith heterogeneoussamples.StatMed1997; 16: 583 –597.17. Harrell FE, Jr, Califf RM, Pryor DB et al. Evaluating theyield of medical tests. JAMA1982;247: 2543–2546.18. Akaike H. Information Theory and an Extension of the Maximum LikelihoodPrinciple.Budapest: Hungary AkademiaiKiado 1973.19. NakajimaT, Kinoshita T, Nashimoto A et al. Randomized controlled trial of adjuvanturacil-tegafur versus surgery alone for serosa-negative, locally advanced gastriccancer.Br JSurg 2007; 94: 1468–1476.20. Sakuramoto S, Sasako M, Yamaguchi T et al. Adjuvant chemotherapy forgastric cancer with S-1, an oral fluoropyrimidine. N Engl J Med 2007; 357:1810–1820.21. Bang YJ, Kim YW, Yang HK et al. Adjuvant capecitabine and oxaliplatin for gastriccancer after D2 gastrectomy (CLASSIC): a phase 3 open-label, randomisedcontrolled trial. Lancet 2012; 379:315–321.

PDGF/PDGFR pathway has been implicated in malignant pleural mesothelioma (MPM) carcinogenesis, and evidence suggests autocrine mechanisms of proliferation. We sought to evaluate the incidence of PDGFRB gene copy number gain (CNG) by fluorescence in situ hybridization and PDGFR pathway protein expression by immunohistochemistry (IHC) and correlate it to patient clinical outcome. Eighty-eight archived tumor blocks from resected MPM with full clinical information were used to perform IHC biomarkers (PDGFRα, PDGFRβ, p-PDGFRβ) and fluorescence in situ hybridization analysis of PDGFRB gene CNG. Spearman rank correlation, Wilcoxon rank-sum test, Kruskal-Wallis test, BLiP plots, and Kaplan-Meier method were used to analyze the biomarkers and correlation to clinical outcome. Several correlations between the IHC biomarkers were seen; however, none correlated to clinically relevant patient demographics or histology. In the CNG analysis, PDGFRB gene CNG in >10% of tumor cells had lower cytoplasmic p-PDGFRβ ( P =.029), while PDGFRB gene CNG in >40% of tumor cells had a higher cytoplasmic PDGFRβ ( P =.04). PDGFRB gene CNG status did not associate with patient demographics or tumor characteristics. PDGFR pathway IHC biomarkers did not associate with survival outcomes. However, patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P =.0096) and improved overall survival (HR 0.32 [95% CI 0.11-0.89], P =.029). PDGFRB CNG >40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients. Future validation of this biomarker in prospective trials is needed. From a retrospective review of archived tissue specimens from patients with resected malignant pleural mesothelioma tumors, we show that patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P =.0096) and improved overall survival (HR 0.32 [95% CI 0.11-0.89], P =.029). PDGFRB CNG >40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients.

Aberrant expression of microRNAs (miRNAs) and the enzymes that control their processing have been reported in multiple biological processes including primary and metastatic tumours1–6, but the mechanisms governing this are not clearly understood. Here we show that TAp63, a p53 family member, suppresses tumorigenesis and metastasis, and coordinately regulates Dicer and miR-130b to suppress metastasis. Metastatic mouse and human tumours deficient in TAp63 express Dicer at very low levels, and we found that modulation of expression of Dicer and miR-130b markedly affected the metastatic potential of cells lacking TAp63. TAp63 binds to and transactivates the Dicer promoter, demonstrating direct transcriptional regulation of Dicer by TAp63. These data provide a novel understanding of the roles of TAp63 in tumour and metastasis suppression through the coordinate transcriptional regulation of Dicer and miR-130b and may have implications for the many processes regulated by miRNAs.

Purpose: To understand the role of nuclear factor erythroid-2–related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in non–small cell lung cancer (NSCLC), we studied their expression in a large series of tumors with annotated clinicopathologic data, including response to platinum-based adjuvant chemotherapy. Experimental Design: We determined the immunohistochemical expression of nuclear Nrf2 and cytoplasmic Keap1 in 304 NSCLCs and its association with patients' clinicopathologic characteristics, and in 89 tumors from patients who received neoadjuvant (n = 26) or adjuvant platinum-based chemotherapy (n = 63). We evaluated NFE2L2 and KEAP1 mutations in 31 tumor specimens. Results: We detected nuclear Nrf2 expression in 26% of NSCLCs; it was significantly more common in squamous cell carcinomas (38%) than in adenocarcinomas (18%; P < 0.0001). Low or absent Keap1 expression was detected in 56% of NSCLCs; it was significantly more common in adenocarcinomas (62%) than in squamous cell carcinomas (46%; P = 0.0057). In NSCLC, mutations of NFE2L2 and KEAP1 were very uncommon (2 of 29 and 1 of 31 cases, respectively). In multivariate analysis, Nrf2 expression was associated with worse overall survival [P = 0.0139; hazard ratio (HR), 1.75] in NSCLC patients, and low or absent Keap1 expression was associated with worse overall survival (P = 0.0181; HR, 2.09) in squamous cell carcinoma. In univariate analysis, nuclear Nrf2 expression was associated with worse recurrence-free survival in squamous cell carcinoma patients who received adjuvant treatment (P = 0.0410; HR, 3.37). Conclusions: Increased expression of Nrf2 and decreased expression of Keap1 are common abnormalities in NSCLC and are associated with a poor outcome. Nuclear expression of Nrf2 in malignant lung cancer cells may play a role in resistance to platinum-based treatment in squamous cell carcinoma. Clin Cancer Res; 16(14); 3743–53. ©2010 AACR.

Cardiac glycosides such as oleandrin are known to inhibit the Na,K-ATPase pump, resulting in a consequent increase in calcium influx in heart muscle. Here, we investigated the effect of oleandrin on the growth of human and mouse cancer cells in relation to Na,K-ATPase subunits. Oleandrin treatment resulted in selective inhibition of human cancer cell growth but not rodent cell proliferation, which corresponded to the relative level of Na,K-ATPase α3 subunit protein expression. Human pancreatic cancer cell lines were found to differentially express varying levels of α3 protein, but rodent cancer cells lacked discernable expression of this Na,K-ATPase isoform. A correlation was observed between the ratio of α3 to α1 isoforms and the level of oleandrin uptake during inhibition of cell growth and initiation of cell death; the higher the α3 expression relative to α1 expression, the more sensitive the cell was to treatment with oleandrin. Inhibition of proliferation of Panc-1 cells by oleandrin was significantly reduced when the relative expression of α3 was decreased by knocking down the expression of α3 isoform with α3 siRNA or increasing expression of the α1 isoform through transient transfection of α1 cDNA to the cells. Our data suggest that the relative lack of α3 (relative to α1) in rodent and some human tumor cells may explain their unresponsiveness to cardiac glycosides. In conclusion, the relatively higher expression of α3 with the limited expression of α1 may help predict which human tumors are likely to be responsive to treatment with potent lipid-soluble cardiac glycosides such as oleandrin. [Mol Cancer Ther 2009;8(8):2319–28]

Background The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies. Results A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC). Conclusion The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1935-0) contains supplementary material, which is available to authorized users.

Lung adenocarcinoma is characterized by complex biology involving alterations at the genomic and protein expression levels. FGFR2 mutation and/or amplification are key drivers of disease progression and drug resistance in lung adenocarcinoma patients. These genetic alterations drive oncogenic downstream signalling due to the deregulated activity of the receptor. We have previously reported that wild type FGFR2 provides a binding site for which two proteins, Grb2 and Plcγ1, compete in a concentration-dependent manner. Metastasis and invasion ensue when Plcγ1 prevails on the receptor giving rise to oncogenic outcome in the absence of gene mutation/deletion. The effect of this signalling mechanism on FGFR2-driven lung adenocarcinoma has not previously been considered. In this study we show that fluctuation in the combinatorial expression levels of FGFR2, Grb2 and Plcγ1 modulates cell invasive properties, tumor formation and is linked to recurrence-free survival in 150 lung adenocarcinoma patients. High levels of expression of FGFR2 and Plcγ1 in a low background of Grb2 significantly correlates with poor prognosis. On the other hand, low levels of expression of FGFR2 and Plcγ1 in a high background of Grb2 correlates with favourable prognosis. This study defines the expression pattern of FGFR2, Plcγ1 and Grb2 as a novel prognostic marker in human lung adenocarcinoma. Thus, consideration of the Grb2 and Plcγ1-mediated mechanism of FGFR2 regulation will enhance the therapeutic targeting of aberrant FGFR2 activity to provide the much-needed improvement to the treatment regimen of this high mortality disease.

Stromal-epithelial interactions and the bioactive molecules produced by these interactions maintain tissue homeostasis and influence carcinogenesis. Bioactive prostaglandins produced by prostaglandin synthases and secreted by the prostate into seminal plasma are thought to support reproduction, but their endogenous effects on cancer formation remain unresolved. No studies to date have examined prostaglandin enzyme production or prostaglandin metabolism in normal prostate stromal cells. Our results show that lipocalin-type prostaglandin D synthase (L-PGDS) and prostaglandin D2 (PGD2) metabolites produced by normal prostate stromal cells inhibited tumor cell growth through a peroxisome proliferator–activated receptor γ (PPARγ)–dependent mechanism. Enzymatic products of stromal cell L-PGDS included high levels of PGD2 and 15-deoxy-Δ12,14-PGD2 but low levels of 15-deoxy-Δ12,14-prostaglandin J2. These PGD2 metabolites activated the PPARγ ligand-binding domain and the peroxisome proliferator response element reporter systems. Thus, growth suppression of PPARγ-expressing tumor cells by PGD2 metabolites in the prostate microenvironment is likely to be an endogenous mechanism involved in tumor suppression that potentially contributes to the indolence and long latency period of this disease.

Kinases can phosphorylate and regulate androgen receptor activity during prostate cancer progression. In particular, we showed that glycogen synthase kinase-3 beta phosphorylates the androgen receptor, thereby inhibiting androgen receptor-driven transcription. Conversely, the glycogen synthase kinase-3 beta inhibitor lithium chloride suppressed the glycogen synthase kinase-3 beta-mediated phosphorylation of the androgen receptor, thereby enabling androgen receptor-driven transcription to occur. The androgen receptor hinge and ligand-binding domains were important for both the phosphorylation and the inhibition of transcriptional activity of the receptor by glycogen synthase kinase-3 beta. Furthermore, androgen receptor phosphorylation was augmented by LY294002, an indirect inhibitor of protein kinase B/Akt that inhibits glycogen synthase kinase-3 beta. We also showed that the mutation of various phosphorylation sites on glycogen synthase kinase-3 beta affected the ability of these mutants to co-distribute with the androgen receptor in the cell nucleus, also that both glycogen synthase kinase-3beta and androgen receptor proteins can be found in cell nuclei of prostate cancer tissue samples. Because glycogen synthase kinase-3 beta activity is suppressed after the enzyme is phosphorylated by protein kinase B/Akt and Akt activity frequently increases during the progression of prostate cancer, nullification of the glycogen synthase kinase-3 beta-mediated suppression of androgen receptor activity by Akt likely contributes to prostate cancer progression.

Kaplan Meier curves for the validation set of ADC (Figure S9) and SCC (Figure S10) using the genomic model (A) and the clinical-genomic model (B) highlighted in red. Patients were divided into two risk groups according to the predicted risk. Survival curves were compared using the log-rank test p-values. (PPTX 6554 kb)

Immunosuppression of tumour-infiltrating lymphocytes (TIL) is a common feature of advanced cancer, but its biological basis has remained obscure. We demonstrate here a molecular link between epithelial-to-mesenchymal transition (EMT) and CD8(+) TIL immunosuppression, two key drivers of cancer progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumour cells, leading to CD8(+) T-cell immunosuppression and metastasis. These findings are supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets. In addition to revealing a link between EMT and T-cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists.

The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 overexpression has an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by downregulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression levels with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.

Phosphorylation and activation of Akt1 is a crucial signaling event that promotes adipogenesis. However, neither the complex multistep process that leads to activation of Akt1 through phosphorylation at Thr³⁰⁸ and Ser⁴⁷³ nor the mechanism by which Akt1 stimulates adipogenesis is fully understood. We found that the BSD domain-containing signal transducer and Akt interactor (BSTA) promoted phosphorylation of Akt1 at Ser⁴⁷³ in various human and murine cells, and we uncovered a function for the BSD domain in BSTA-Akt1 complex formation. The mammalian target of rapamycin complex 2 (mTORC2) facilitated the phosphorylation of BSTA and its association with Akt1, and the BSTA-Akt1 interaction promoted the association of mTORC2 with Akt1 and phosphorylation of Akt1 at Ser⁴⁷³ in response to growth factor stimulation. Furthermore, analyses of bsta gene-trap murine embryonic stem cells revealed an essential function for BSTA and phosphorylation of Akt1 at Ser⁴⁷³ in promoting adipocyte differentiation, which required suppression of the expression of the gene encoding the transcription factor FoxC2. These findings indicate that BSTA is a molecular switch that promotes phosphorylation of Akt1 at Ser⁴⁷³ and reveal an mTORC2-BSTA-Akt1-FoxC2-mediated signaling mechanism that is critical for adipocyte differentiation.

Folate receptor alpha (FRα) and reduced folate carrier-1 (RFC1) regulate uptake of folate molecules inside the cell. FRα is a potential biomarker of tumors response to antifolate chemotherapy, and a target for therapies using humanized monocloncal antibody. Information on the protein expression of these receptors in non-small-cell lung carcinoma (NSCLC) is limited.Expressions of FRα and RFC1 were examined by immunohistochemistry (IHC) in 320 surgically resected NSCLC (202 adenocarcinomas and 118 squamous cell carcinomas) tissue specimens and correlated with patients' clinico-pathologic characteristics. Folate receptor α gene (FOLR1) mRNA expression was examined using publicly available microarray datasets. FRα expression was correlated with thymidylate synthase and p53 expression in NSCLCs, and with epidermal growth factor receptor (EGFR) and V-Ki-ras2 Kirsten rat sarcoma viral (KRAS) gene mutations in adenocarcinomas.NSCLC overexpressed FRα and RFC1. In a multivariate analysis, lung adenocarcinomas were more likely to express FRα in the cytoplasm (OR = 4.39; p0.0001) and membrane (OR = 5.34; p0.0001) of malignant cells than squamous cell carcinomas. Tumors from never-smokers were more likely to express cytoplasmic (OR = 3.35; p0.03) and membrane (OR = 3.60; p=0.0005) FRα than those from smokers. In adenocarcinoma, EGFR mutations correlated with higher expression of membrane FRα and FOLR1 gene expressions. High levels of FRα expression was detected in 42 NSCLC advanced metastatic tumor tissues.FRα and RFC1 proteins are overexpressed in NSCLC tumor tissues. The high levels of FRα in lung adenocarcinomas may be associated to these tumors' better responses to antifolate chemotherapy and represents a potential novel target for this tumor type.

Sarcomatoid non-small cell lung cancer (NSCLC) is an uncommon histologic variant that has not been molecularly well-characterized. We conducted immunohistochemical and fluorescence in situ hybridization studies of PDGF-B/PDGFR-b on archived surgically resected specimens and showed high PDGFR-b IHC expression and gene copy number gain. Further studies are warranted to determine whether PDGFR-b is a feasible therapeutic target in this population.Sarcomatoid non-small cell lung cancer (NSCLC) is an uncommon histologic variant that has not been molecularly well-characterized. We hypothesized that the PDGF-B/PDGF-Rβ pathway may be dysregulated in sarcomatoid lung cancer.We conducted immunohistochemical (IHC) and gene copy number gain studies of PDGF-B/PDGFR-β on archived surgically resected specimens, 43 sarcomatoid NSCLCs and 42 control NSCLCs that were age, gender and stage-matched. Biomarkers were correlated to patient demographics, tumor characteristics, and survival.Sarcomatoid tumors had higher PDGFR-β IHC expression than control NSCLC (median score 2.69 vs. 1.93; P0.0001). No difference was seen between the two groups of PDGF-B IHC expression; and neither PDGF-B nor PDGFR-β IHC levels correlated with gender, age, clinical or pathologic TNM status, or overall survival. PDGFRB gene copy number was evaluated by FISH using three ways: presence of amplification, gene copy number gain, and gene copy ratio between tumor and normal tissue. PDGFRB gene copy number gain was associated with sarcomatoid histology (P = 0.006), lower clinical and pathologic T-stage (P = 0.07, P = 0.048), and higher pathologic N-stage (P = 0.013). Sarcomatoid NSCLC patients (P = 0.006) and female patients (P = 0.03) had higher gene copy ratios above 1.83. Higher PDGFR-β IHC expression in tumor cells was associated with gene copy number gain (P = 0.021) and higher gene copy ratio status (P = 0.005).This is the first study to demonstrate high PDGFR-β IHC expression and gene copy number gain in sarcomatoid NSCLC tumors and suggests that further studies are warranted to determine whether PDGFR-β is a feasible therapeutic target in this population.

Background: Proteomic profiling has elucidated several dysregulated pathways in NSCLC. We sought to identify patterns of protein expression that are enriched following neoadjuvant chemotherapy in patients with resected lung cancers. Methods: Tissue samples were selected from the PROSPECT trial at MD Anderson Cancer Center, the goal of which was to correlate molecular profiles with treatment response. Samples from 189 patients were analyzed, which included 26% squamous tumors; 27% with neoadjuvant chemotherapy (n = 49); and predominantly localized disease (distribution: I = 91, II = 35, III = 58, IV = 5). Reverse phase protein array (RPPA) analysis was utilized to quantify 127 total or phosphorylated proteins. Interactions between protein expression and the receipt of neoadjuvant chemotherapy were assessed by analysis of variance (ANOVA). Cox regression was performed to determine the relationship between protein expression and recurrence-free survival (RFS). Results: Twenty one of the 127 proteins (16%) were expressed at significantly different levels in patients receiving neoadjuvant chemotherapy. Specifically, patients receiving neoadjuvant chemotherapy had higher expression of multiple DNA repair proteins, including MSH2 (p Conclusions: The receipt of neoadjuvant chemotherapy was associated with higher expression of DNA repair proteins, suppression of the PI3K pathway, and an EMT shift. Increased expression of DNA repair proteins was also associated with reduced RFS. These findings suggest that higher expression of DNA repair proteins may contribute to treatment resistance, and support the combination of standard chemotherapy with targeted agents such as Chk inhibitors or immunotherapy (to address EMT-mediated immune escape). Citation Format: Daniel R. Gomez, Lixia Diao, Jing Wang, Ignacio I. Wistuba, Cesar Moran, Neda Kalhor, Milind B. Suraokar, Stephen G. Swisher, Carmen Behrens, Youhong Fan, John V. Heymach, Lauren A. Byers. Neoadjuvant chemotherapy is associated with increased expression of DNA repair proteins and epithelial to mesenchymal transition (EMT) in patients with non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3623. doi:10.1158/1538-7445.AM2015-3623

Purpose To develop a molecular based classification of non-small cell lung carcinomas based on genome wide copy number variations (CNVs). Background For a number of clinical and biologic reasons, the accurate classification of non small cell lung carcinoma (NSCLC) into adenocarcinoma (ADC) and squamous cell carcinoma (SCC) is essential. DNA based tests, which are not currently used, are more robust when applied to formalin fixed paraffin embedded tissues. Materials and Methods TCGA Dataset: The Cancer Genome Atlas project (TCGA) level 3 CNVs data (Affymetrix Genome-Wide Human SNP Array 6.0) of resected ADC (n = 241) and SCC (n = 210) patients were utilized as a Training set along with 1091 non malignant lung samples. SPORE Patient Dataset: The UT Lung SPORE cohort consists of 248 resected lung cancers (168 ADC and 74 SCC samples) run with the Agilent 244K Human Genome CGH Microarray. Molecular signature identification based on the morphologic subclassification of NSCLC: We used statistical algorithms to identify potential CNV biomarkers and combined them with known amplified oncogenes to identify 28 CNV signature genes that were highly correlated with histological classification. Identification of CNV classifier genes. The CNV signature genes were identified through the following four sequential steps: 1) Paired t-test. 2) Elastic Net. 3) Partial lease squares algorithm. 4) Naive Bayes classifier. Results: The 28 gene CNV signature accurately separated squamous cell carcinomas from adenocarcinomas in the Training and Validation sets as well as distinguished tumors from non-malignant tissues (Table 1). Table 1. The classification results between ADC and SCC DataSensitivitySpecificityAccuracyADC vs SCCTraining set (TCGA)0.940.870.91Validation set (SPORE)0.870.840.86Tumor vs Non-malignantADC0.910.960.94SCC0.970.980.98 Conclusions: A 28 gene CNV signature distinguished lung adenocarcinomas from squamous cell carcinomas with great accuracy (86-91%) and the lung tumor samples can be distinguished from the non-malignant lung samples with an accuracy of 94-98%. Citation Format: Kai Song, Guangqiang Zheng, Luc Girard, Ignacio I. Wistuba, Jack A. Roth, Carmen Behrens, Milind B. Suraokar, John D. Minna, Adi F. Gazdar. Copy number variations distinguish lung adenocarcinomas from squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 613. doi:10.1158/1538-7445.AM2015-613

Purpose: Non-small cell lung cancer (NSCLC) patients show benefit from neoadjuvant chemotherapy given before surgical resection or adjuvant chemotherapy administered after tumor resection. However, several of these patients still relapse and die with metastatic NSCLC. These relapses represent resistance of the tumors to chemotherapy. NSCLC resistance to standard taxane and platin doublet chemotherapy is a major problem. Identifying and overcoming resistance mechanisms are thus urgently needed and the focus of this research. Methods: (A) NSCLC cell lines were exposed long term to cycles of paclitaxel + carboplatin in clinically relevant dosage ratios, to select and subsequently characterize resistant cells. (B) Subcutaneous xenografts of parental and resistant cell lines were established in NOD/SCID mice to include expression changes that are retained in vivo. (C) Molecularly and clinically annotated dataset of NSCLC patient tumors obtained after neoadjuvant taxane + platin chemotherapy (N = 66; some had long term disease free survival and some had tumor relapses) were subjected to gene expression and survival analyses. Results and Significance: NSCLC cell lines NCI-H1299 and NCI-H1355 treated for 16-18 cycles with paclitaxel + carboplatin developed >50 fold resistance. Cells also developed cross-resistance to doxorubicin and vinorelbine. In addition, these resistant cell lines showed decreased response to docetaxel + cisplatin doublet in vivo. Consistent with the literature, cells showed increased expression of multi-drug transporter ABCB1/MDR1. This corresponded to decreased intracellular docetaxel accumulation in resistant cells. Cell line variants showed reversal in resistance when exposed to MDR1 inhibitors or with ABCB1 knockdown. Reversal in both cases was however partial, suggesting additional resistance mechanisms. This was also indicated by repeating long term drug selection in presence of MDR inhibitor, wherein cells still developed resistance. Microarray profiles of resistant cell lines suggested mRNA expression changes in stem cell pathways, epithelial-to-mesenchymal transition (EMT) genes and certain epigenetic modulators. Cross-comparison of gene hits from cell lines and xenografts yielded 14 commonly up-regulated and 21 down-regulated genes that formed a resistance signature from preclinical models. This 35-gene resistance signature could successfully classify neoadjuvant treated patient tumors into two main clusters with significant differences in tumor recurrence and cancer-free survival. Studies are underway to determine whether this signature is purely prognostic or also has a functional role in causing resistance. Such information will provide both clinically useful biomarkers to type tumor response in patients and also potentially new therapeutic targets to overcome standard taxane-platin drug resistance in NSCLC. Citation Format: Maithili Prafulla Dalvi, Carmen Behrens, Milind Suraokar, Rui Zhong, Brenda Timmons, Luc Girard, Yang Xie, Ignacio Wistuba, John D. Minna. Developing a molecular understanding of non-small cell lung cancer resistance to standard taxane-platin chemotherapy. [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 3772. doi:10.1158/1538-7445.AM2014-3772

Introduction. The aim of the study is to characterize the role of LMO1 in tumorigenesis of neuroendocrine lung cancer. LMO1 is a transcription factor that belongs to the LMO protein family containing the cysteine-rich LIM domains. LMO1 has been demonstrated to function as an important oncogene in the development of T-lineage leukemia and lymphoma. A recent study in neuroblastoma suggests that aberrant overexpression of LMO1 may play a critical role in the development of neuroendocrine cancers. The role of LMO1 in neuroendocrine lung cancer, however, was not investigated. Experimental Approaches. By analyzing a large panel of cell lines including lung cancer cell lines and normal lung epithelial cells, we compared the relative expression of LMO1 in different subtypes of lung cancers, and examined the correlation of LMO1 expression levels with neuroendocrine differentiation markers. Using in vitro approaches, we investigated the function of LMO1 in regulating lung cancer cell growth. To investigate the clinical significance of LMO1 dysregulation in lung cancer patients, we analyzed the correlation of LMO1 tumor levels with lung cancer patient survival. Results. We show that LMO1 is significantly overexpressed in small cell lung cancer (SCLC), an aggressive and major subtype of neuroendocrine lung cancers, relative to non-small cell lung cancer (NSCLC) and normal lung cells. In addition, in NSCLC cells, LMO1 mRNA levels are significantly correlated with expression of neuroendocrine differentiation markers. Functional in vitro investigations indicate that LMO1 overexpression significantly promotes growth of cultured lung cancer cells, suggesting its oncogenic function in lung cancer. More strikingly, our investigations of two independent lung cancer patient populations indicate that high tumor LMO1 mRNA level is an independent predictor of poor patient survival. Conclusions. Altogether, our findings strongly suggest that LMO1 is a neuroendocrine-specific oncogene in lung cancer that plays an important role in determining the cancer aggressiveness. Further studies are certainly warranted to define the mechanisms underlying the oncogenic function of LMO1 in neuroendocrine lung cancer and to further evaluate the clinical significance of LMO1 as a diagnostic and prognostic marker for neuroendocrine lung cancers in prospective studies. Citation Format: Zhenze Zhao, Xiuye Ma, Xiaojie Yu, Tzu-Hung Hsiao, Yidong Chen, Milind Suraokar, Ignacio Wistuba, John D. Minna, Alexander Pertsemlidis, Liqin Du. LMO1 is a novel oncogene in neuroendocrine lung cancer. [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 466. doi:10.1158/1538-7445.AM2014-466

8063 Background: Tumor cell regulation of the activity of diverse inflammatory cells creates an immunosuppressive microenvironment that favors tumor growth and metastasis. However, the mechanistic ...

Background: Approximately 85-90% of lung cancers are non-small cell lung cancers (NSCLCs) consisting of adenocarcinoma (ADC) and squamous cell carcinoma (SCC) and several rare types including undifferentiated large cell carcinomas. Most NSCLCs are diagnosed from small biopsies or cytology materials and, even with the use of immunostains, some cannot be classified further and are referred to as NSCLC - not otherwise specified (NSCLC-NOS). For several clinically relevant reasons and for selection of therapy the correct identification of NSCLC typing is important. Specific Aim: To develop a highly specific and sensitive RNA expression signature as an adjunct test for routine pathological classification. Methods: A microarray dataset of 263 resected NSCLC cases (183 ADC, 80 SCC) obtained from MD Anderson Cancer Center and profiled on the Illumina WG-6 V3 platform was used as the learning set. The Cancer Genome Atlas (TCGA; http://cancergenome.nih.gov/) microarray dataset (105 ADC, 205 SCC), which were profiled on an RNAseq platform, was used as the validation set. Histologic typing on the TCGA specimens was performed by light microscopy according to the WHO classification. Materials for immunostaining were not always available. Results: We developed a 44-gene signature which contained many of the genes encoding proteins routinely used in immunostains for NSCLC typing, including TP63, TTF1, SOX2, and several high molecular weight keratins. For testing, a nearest neighbor approach with Pearson metric was used, which yielded two scores (Pearson correlations, ranging from -1 to +1) for ADC and SCC histologies. Scores above 0.4 were considered positive for the respective tumor type, while values below 0.4 were considered to be null (equivalent to poorly/undifferentiated histology). The learning set had 7% discrepancies for the major tumor type and 1% was scored as null. Initial validation of the TCGA data indicated a relatively high percentage of discrepancies (10% of ADC, 12% of SCC). After discussions with TCGA reference pathologists Drs. Travis and Rekhtman and further evaluation by them a revised pathological classification was generated, which included 25 NSCLC-NOS diagnoses. The discrepancies were reduced to 9 % and 4 % for ADC and SCC respectively. The NSCLC-NOS cases were classified as ADC, SCC or null types. Future plans: We are utilizing the expression signature to develop and validate a quantitative nuclease protection assay (qNPA by High Throughput Genomics) that can be applied to small biopsies and 5-micron sections of archival paraffin embedded formalin fixed material in a 96 well format. Summary and significance: The development and application of a sensitive and specific molecular signature for the classification of NSCLC provides an important adjunct test for routine pathological diagnosis, especially for application to small specimens (which constitute about 70% of current diagnostic lung cancer samples). Citation Format: Luc Girard, Ignacio Wistuba, William D. Travis, Natasha Rekhtman, Milind Suraokar, Carmen Behrens, John D. Minna, Adi F. Gazdar. Expression signature for classification of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1202. doi:10.1158/1538-7445.AM2013-1202

Background. EZH2 overexpression has been described in a wide variety of tumors, including lung cancer. EZH2 has been implicated in neoplastic transformation, tumor progression and activation of angiogenesis. The mechanisms associated with the regulation of EZH2 expression in lung cancer cells are mostly unknown. In this study, we investigated the role of EZH2 in cell proliferation/migration and response to chemotherapy in lung adenocarcinoma (LADCa) cell lines. Moreover, we studied the mechanisms of EZH2 regulation associated to the VEGF/VEGFR2 pathway expression. Methods. LADCa cell lines were inhibited pharmacologically with different doses of DZNep (0, 2.5 and 5μM) and by knockdown of EZH2 by siRNA. EZH2, H3K27me3 and PARP-C expressions were determined by Western-blots (WB). Cisplatin and carboplatin sensitivity (IC50) and proliferation were determined by MTS assay. Cell migration was measured using Boyden chamber assay. After stimulation of cell lines with VEGF-A, EZH2, VEGFR-2, E2F3 and HIF-1α expression were determined using WBs, and EZH2 and miR-101 expressions were determined by quantitative PCR. To study the role of VEGFR-2 and HIF-1α in the regulation of EZH2, we examined the effect of knockdown VEGFR-2 and HIF-1α by siRNA. Results. We found that inhibition with DZNep and knockdown by siRNA decreased the expression of EZH2, reduced H3K27me3 level, and increased PARP-C levels in LADCa cell lines. Treatment with DZNep and knockdown of EZH2 significantly increased sensitivity to cisplatin and carboplatin (p Conclusion. Our in vitro findings suggest that EZH2 overexpression may promote a more malignant phenotype of lung adenocarcinoma. The pharmacologic inhibition with DZNep and knockdown of EZH2 by siRNA increased sensitivity of LADCa cell lines to cisplatin and carboplatin, and reduced cell migration capabilities. Our results suggest that VEGF/VEGFR-2 axis plays a role in the regulation of EZH2 through E2F3/HIF-1α and miR-101 regulations. EZH2 is a potential therapeutic target in lung cancer that regulates the epigenome to overcome drug resistance. (Supported in part by grants DoD PROSPECT W81XWH-07-1-0306, NCI/UT Lung SPORE 5P50CA70907-11, and Becas Chile) Citation Format: Erick M. Riquelme, Carmen Berhens, Milind Suraokar, Ignacio I. Wistuba. EZH2 promote a malignant phenotype in lung adenocarcinoma and VEGF/VEGFR2 pathways mediates regulation of EZH2 through E2F3/HIF-1α and downregulation of miR-101. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4643. doi:10.1158/1538-7445.AM2013-4643

We are developing in vitro drug response signatures based on profiling of mRNA (Illumina WG6-V3 arrays), DNA mutation (COSMIC and deep sequencing), DNA copy number (Illumina Human1M-Duov3 SNP array) and DNA methylation (Illumina HumanMethylation450) from lung cancer cell lines to predict which drugs a patient's tumor is most likely to respond to. We have generated drug response phenotypes (MTS colorimetric assays) for ∼25 standard, targeted, and new chemotherapy agents and combinations for ∼ 100 non-small cell lung cancer (NSCLC) lines. All assays were done in triplicates or more and were very reproducible over time (r > 0.8). More than 10,000 MTS assays were generated and we designed a high-throughput database software named DIVISA (Database of In VItro Sensitivity Assays) for the purpose of storing and analyzing these assays. Some drugs showed a wide range of sensitivities (> 10,000-fold in IC50 values) and IC50 clustering indicates that drug response phenotypes can be grouped according to drug types. As part of a joint NCI SPORE, NCI SPECS, and DOD PROSPECT effort we have collected 275 clinically annotated frozen tumors with drug response information including 94 that represent lung cancer resection followed by adjuvant treatment. These specimens have also been profiled on Illumina expression arrays to formally test the clinical relevance of the tumor cell line signatures, and to verify that the signatures predict for response only in the presence of treatment and thus are not prognostic of survival in the absence of treatment. In addition, we have 3 primary tumor datasets totaling 96 specimens with EGFR mutation information, thus providing a validation set for EGFR tyrosine kinase inhibitor signatures. Using a weighted voting classification, cell line signatures predicted drug response in primary tumors with accuracies of ∼65% for targeted therapy (EGFR) but with somewhat lower accuracies for platin/taxane therapies suggesting that cell line predictive signatures may be better suited for targeted drugs. To facilitate translation to clinical trials we are working with High Throughput Genomics (HTG) to develop quantitative mRNA profiles that are performed on formalin fixed paraffin embedded (FFPE) material on a platform that can be transferred to a CLIA certified environment. These studies thus provide a preclinical human tumor model platform for systematically testing new drugs and for developing signatures to guide their most efficient use in early clinical tests. Funded by University of Texas SPORE in Lung Cancer (P50CA70907) and NCI SPECS Lung Cancer (CA114771). Citation Format: Luc Girard, Michael Peyton, Ignacio Wistuba, Yang Xie, Rachel Greer, Milind B. Suraokar, Carmen Behrens, Guanghua Xiao, John Heymach, David A. Wheeler, Caleb F. Davis, Kenneth Huffman, David S. Shames, Kevin R. Coombes, Adi F. Gazdar, David CL Lam, David G. Beer, John D. Minna. Molecular signatures of in vitro drug response in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5589. doi:10.1158/1538-7445.AM2013-5589