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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.

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.

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

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

Background: MPM is caused by lethal neoplastic growth of the pleura surrounding lungs. It is resistance to most standard anti-cancer treatment regimens and needs discovery of newer therapeutic approaches. MPM is characterized by massive loco-regional invasion of the malignant pleural cells into the lung parenchyma. Twist1 is a transcription factor, which promotes invasion and metastasis of tumor cells, increases chemotherapeutic resistance and is involved in the pathobiology of many cancers. Also recent studies have highlighted the potential of twist1 as a therapeutic target in cancer. But there is no report investigating its function in mesothelioma. Methods: We extracted total RNA from 53 frozen resected tumor tissue specimens, comprised of 39 epitheloid, 7 sarcomatoid and 7 biphasic histotypes, along with paired normal tissue. The RNA was labeled and hybridized to Affymetrix U133 plus 2.0 microarray to obtain transcriptomic profiles. Bioinformatic analysis of the microarray data using a 2 sample t-test was applied, on a probe-by-probe basis followed by Beta-uniform Mixture for multiple comparisons, to determine the differences between tumor vs normal specimens. The microarray results were validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using Taqman assays on the ABI 7300 platform. For all qRT-PCR experiments the twist1 transcript levels were determined relative to endogenous GAPDH as control using ΔΔCT calculation. We performed Western blot analysis on a panel of 16 mesothelioma cell lines including Met-5A (SV-40 immortalized) and HCT-4012 (telomerase immortalized) pleural mesothelial control cell lines. Results: The bioinformatic analysis of microarray data showed that twist1 transcript level was 8.7 fold higher in tumors (p = 1.1E-16) compared to paired normal specimens. Using qRT-PCR, we compared twist1 transcript levels in 12 pairs of tumor vs paired normal tissue specimens and found that twist1 was upregulated to more than 10 fold in MPM tumors (p < E-4). Western blot showed that 10 MPM cancer cell lines had higher expression of twist1 protein compared to Met-5A and HCT-4012 cell lines. The highest expression was seen in 2 of the sarcomatoid cell lines - RS5 and DM3, suggesting a correlation with metastatic phenotype since sarcomatoid tumors are highly metastatic in nature. Conclusion: Our preliminary findings suggest that twist1 is upregulated in MPM tumors and cell lines and may play a role in the development of MPM. Further studies are needed to investigate its role in the process of tumorigenesis and metastasis. Supported by Grants: DoD W81XWH-07-1-0306 (I.I.W and AST), Fleming Foundation, IASLC Young Investigator Award 2011-2013 (MS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3404. doi:1538-7445.AM2012-3404

Purpose: While chemotherapy has improved lung cancer patient survival, relapse rates after neoadjuvant or adjuvant chemotherapy (minimal residual disease settings) are high. This could be due to cancer cells acquiring resistance to therapy or selective survival of pre-existing resistant cells. The molecular basis for primary or acquired resistance to standard platinum doublet chemotherapies are unknown and their elucidation is necessary for developing curative therapy that can be personalized for each patient. Methods: 3 complementary approaches were used to explore mechanisms underlying NSCLC resistance to platin-taxane chemotherapy: A) NSCLC lines were exposed in vitro to cycles of paclitaxel + carboplatin to mimic clinical treatment and resistance validated with cell viability and colony formation assays. Genome wide mRNA expression profiles of resistant and parental lines were compared using Illumina arrays and qRT-PCR. B) A large dataset of clinically and molecularly annotated NSCLC tumor specimens obtained after no chemotherapy or neoadjuvant platin-taxane chemotherapy were subjected to mRNA microarray analysis. C) Lung cancer xenografts were treated with chemotherapy in vivo in mice and profiled for mRNA expression. Biostatistical and bioinformatics approaches were used to identify genes that are differentially expressed in resistant cells in these three settings, and their function studied using over-expression or shRNA mediated knockdown. Results: NSCLC lines H1299 and H1355 were treated in vitro with paclitaxel + carboplatin and showed progressive increases in resistance to chemotherapy (after 5 to 15 cycles) ultimately achieving 30 (H1299) or 60 fold (H1355) differences compared to parental cells. In addition, multiple single cell clones also demonstrated chemotherapy resistance. Resistant NSCLC strains were also resistant to platin+docetaxel and platin+pemetrexed combinations. Expression profiles revealed upregulation of ABCB1/MDR1, down regulation of epithelial-specific genes suggesting EMT, differences in expression of epigenetic modulators as well as cancer testis family of GAGE genes and many of these genes had expression correlated with recurrence after neoadjuvant chemotherapy and with poor survival. Resistance was only partially reversed by verapamil indicating that it can be only partly explained by MDR mechanisms. Mechanistic studies of genes identified by these three approaches provide opportunities for overcoming chemotherapy resistance and developing novel therapies for NSCLC. Grant support: UT-Lung SPORE (5 P50 CA70907-11) and DoD PROSPECT (W81XWH-07-1-0306) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 821. doi:1538-7445.AM2012-821

The G protein-coupled receptor family C group 5 member A (hGPRC5A, mGprc5a) was previously shown to function as a lung-specific tumor suppressor gene evidenced by the development of lung adenomas and adenocarcinomas in mice with knockout of the gene. Moreover, we have demonstrated that the immunohistochemical expression of GPRC5A protein was significantly lower in non-small cell lung cancer (NSCLC) resected specimens compared to normal bronchial epithelia from healthy individuals. The latter findings prompted us to examine the expression patterns of GPRC5A in malignant pleural mesothelioma (MPM) because, like NSCLC, this malignancy develops in the thoracic cavity. Analysis of two publicly available MPM microarray datasets revealed that GPRC5A mRNA expression is significantly higher in MPMs compared to matched normal pleura (5.5 fold higher, p=0.002; 6.57 fold higher, p=0.04). GPRC5A immunohistochemical protein expression was semi-quantitatively assessed in formalin-fixed paraffin embedded (FFPE) MPM surgical specimens (n=73) comprised of 41 epitheloid, 14 biphasic and 18 sarcomatoid type MPMs. GPRC5A protein expression was also assessed in a subset of the MPM specimens (n=51) that were reconstructed as high-density spiral tissue microarrays (TMA) developed by J. Fukuoka and colleagues (Sakura-Finetek, Tokyo, Japan) comprised of 35 epitheloid, 9 biphasic and 7 sarcomatoid type MPMs. ANOVA analysis demonstrated significant differences in GPRC5A immunohistochemical expression among MPMs based on the histological subtype (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1711. doi:1538-7445.AM2012-1711

Background. The folate pathway is active in several cancers including malignant pleural mesothelioma (MPM). Membrane receptors folate receptor alpha (FRα), reduced folate carrier 1 (RFC1), proton coupled folate transporter (PCFT) and the enzyme thymidylate synthase (TS) regulate intra-cellular uptake of folate molecules and antifolate drugs. Due to the documented overexpression of folate markers in MPM they arise as potential targets for novel specific therapies with humanized monoclonal anti-FRα antibody and also to antifolate chemotherapy with pemetrexed. Material and Methods. We studied 79 surgically resected MPM (43 epithelioid, 28 biphasic and 13 sarcomatoid histology types). Protein expression of FRα, RFC1, PCFT and TS was examined by immunohistochemistry (IHC) in FFPE tissues placed in tissue microarray platform and results correlated to clinical-pathologic characteristics. Gene expression and DNA copy number changes of the FRα gene (FOLR1) were examined in 53 mesothelioma tumors using Affymetrix U133 Plus 2.0 microarray's and Illumina HumanOmni1-Quad v1.0 chips, respectively. The expression microarray data was analyzed in the GeneSpring GX 11 software (Agilent technologies Inc.) while DNA copy number changes were detected using the Nexus 5.0 software (BioDiscovery Inc.). Results. MPM frequently showed protein over-expression of FRα, RFC1 and PCFT. Nuclear TS expression positively correlated (P0) had a lower hazard ratio than those who did not (HR=0.309; 95%CI 0.159, 0.601; P=0.0005). Patients with any level (score >0) of cytoplasmic PCFT had lower hazard ratio than those with absence of the marker (HR=0.539; 95%CI 0.306, 0.952; P=0.0332). Our gene expression data analysis on 53 mesothelioma tumors showed greater than 2-fold increase in the FOLR1 and 3-fold increase in the TYMS messenger RNA levels compared to paired normal tissue. Additionally, at least 7 of these tumors show increased gene copy number at the FOLR1 locus (Ch11q 13.4). Conclusions. Folate membrane transporters (FRα, RFC1, PCFT) and the enzyme TS are frequently over expressed in MPM tumor tissues, and PCFT and TS are associated with tumors’ clinico-pathological features. The high level of expression of FRα in MPM supports the rational use of potential utilization of humanized monoclonal anti-FRα antibody and also standard antifolate chemotherapy in this disease. Supported by grant US DoD W81XWH-07-1-0306, ASCO CDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2011-4121

Background Malignant pleural mesothelioma (MPM) is a deadly disease with poor prognosis, few treatment options, and an increasing incidence worldwide. There is, therefore, a great need to identify new therapeutic targets and develop more effective therapies for patients with MPM. To better characterize the molecular changes occurring in MPM, we determined genetic and proteomic abnormalities in MPM cell lines and correlated them with those found in MPM tumor tissue specimens. Methods We performed SNP/copy number analysis using Affymetrix SNP 6.0 chips, messenger RNA (mRNA) analysis using Affymetrix U133 plus 2.0 chips, and Reverse Protein Phase Array (RPPA) analysis in 4 mesothelioma cell lines (H28, MSTO-211H, H2052 and H2452) along with the control normal cell line HCT-4012, for comparison. DNA copy number microarray data were analyzed using the Nexus 5.0 software (BioDiscovery Inc.), while mRNA expression array data were analyzed using the GeneSpring GX 11 software (Agilent technologies, Inc.). Additionally, we evaluated copy number gain (CNG) of the c-myc gene and expression of c-myc protein in the mesothelioma cell lines and control cell line by fluorescent in situ hybridization (FISH) and Western blot, respectively. Furthermore, we investigated CGN in MPM tumor tissue specimens by performing FISH using the c-myc probe on tissue microarrays (TMAs) containing 80 MPM samples from different histological subtypes (41 epithelioid, 27 biphasic, 12 sarcomatoid). Results DNA copy number analysis using the Affymetrix chip revealed CNG or amplification of the c-myc oncogenic locus in 3 out of 4 mesothelioma cell lines compared to the control HCT-4012 cell line. In concordance, the mRNA microarray data also showed increased number of transcripts from this locus; more importantly, the RPPA array data showed increased c-myc protein expression in these cell lines compared to control. These results were confirmed by FISH and Western blot analysis, which showed CNG or amplification at this locus and increased levels of the c-myc protein in the mesothelioma cell lines. FISH analysis of TMAs also revealed a relatively high frequency (21%) of CNG (≥4 copies in ≥70% of cells) for the c-myc locus in the MPM tumors. Interestingly, this amplification were seen in either epithelioid (10%) or biphasic (11%) histotype, and none were observed in the sarcomatoid cases. Similarly, within the biphasic cases, we observed that the amplification was present only in the epithelioid component, and not in the sarcomatoid component. Conclusion Our findings suggest that CNG of the c-myc locus is characteristic for MPM tumors of the epithelioid histotype or of the epitheloid component of biphasic histotype, and suggest that c-myc CNG could have a role in pathogenesis of this disease; however, further studies are needed to clarify the role of c-myc in MPM development and progression. Supported by grant US DoD W81XWH-07-1-0306. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4031. doi:10.1158/1538-7445.AM2011-4031

Background: Lung cancer is the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers. Brain metastases (BMs) are estimated to occur in up to 40% of patients with NSCLC. DNA copy number changes are common in cancer and lead to altered expression and function of genes residing within the affected region of the genome. The aim of this study was to compare DNA copy number abnormalities between primary tumors (PT) and their corresponding BM in NSCLC to identify genomic regions associated with BM development by using array comparative genomic hybridization (aCGH). Material and Method: From surgically resected NSCLC formalin-fixed and paraffin-embedded (FFPE) tissues, we selected 28 paired PTs and BMs (24 adenocarcinomas, and 4 squamous cell carcinomas, SCCs), and 55 additional BMs (43 adenocarcinomas, 2 SCCs, and 10 NSCLCs) specimens for analysis. Using DNA extracted from FFPE tumor tissues we performed oligo-based aCGH in 10 paired PT and BM adenocarcinomas. Statistical aberration detection algorithms of data were conducted by aCGH with Nexus Copy Number software v5.0. Immunohistochemical (IHC) analysis of two proteins (PSCA and LY6K) coded by genes located in 8q24.3 region was performed in both PT and BM specimens. Results: In the aCGH analysis, multiple chromosomal regions with significant (P≤0.05) copy number gain and loss were detected. Whereas PT demonstrated 32 regions (harboring 933 genes) of copy number gain and 16 regions (108 genes) of copy number loss, BM showed 31 regions (1274 genes) with gains and 16 regions (90 genes) with losses. BM demonstrated significantly (P≤0.01) higher frequency of copy number gains in 3 chromosomal regions: 8q24.3 (BM 100% vs. PT 40%), 19q13.33 (BM 90% vs. PT 20%), and 20q13.12 (BM 60% vs. PT 0%). The 8q24.3 region with copy number gain in tumors harbors 7 genes, including Prostate Stem Cell Antigen (PSCA) and Lymphocyte Antigen 6 Complex Locus K (LY6K). Interestingly, in our IHC analysis, BMs (n=84; mean score 141.8) showed a significantly (P=0.008) higher score of expression of PSCA than the corresponding PTs (n=27; mean score 97.8). Similar significant differences were detected when we analyzed only adenocarcinomas samples. No significant differences in the level of IHC expression was detected comparing BMs (n=83; mean score 64) and PTs (n=27; mean score 68.5) for LY6K. Conclusions: By aCGH and subsequent protein expression analysis we have identified that PSCA may play an important role in the development of NSCLC brain metastasis. Similarly to prostate cancer, PSCA represents a novel potential marker for NSCLC metastasis and therapeutic target for advanced lung tumors. Supported by grant US DoD W81XWH-07-1-0306 and Jimmy L Hewlett Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5166. doi:10.1158/1538-7445.AM2011-5166

MPM is a lethal neoplasm exhibiting low median survival of patients and lacks effective therapeutic options. There is an urgent need to understand the underlying pathobiology and discover novel therapeutic targets. We undertook a messenger RNA (mRNA) expression profiling strategy to determine the pathways and biomarkers significantly altered in MPM tumors. We isolated total RNA from 55 MPM tumors with 38 paired controls representing 39 epitheloid, 8 biphasic and 6 sarcomatoid cases. The paired controls were adjacent non-tumor tissue samples and histopathological analysis revealed a tumor content of greater than 70 % in most of these tumors cases. The RNA was labeled and hybridized onto Affymetrix U133 Plus 2.0 chips to ascertain the global expression profile in these tumors. Bioinformatic analysis of the microarray data using a two sample t-test was applied on a probe-by-probe basis followed by Beta-uniform Mixture for multiple comparisons. Finally paired t-test was applied to determine the differences between tumor vs normal samples. About ∼955 highly significant probesets representing ∼ 670 genes, at a FDR of e-09, were obtained and subjected to pathway analysis using MetaCore software suite (GeneGo, Inc.). The most significantly altered pathway in MPM tumors was the Mitotic Spindle Assembly Checkpoint (MSAC) pathway due to up-regulation of at least 15 genes including a ∼3.4 fold increase in Aurora kinase A, which is currently being explored in other cancers as a potential therapeutic target. The other genes belonging to this pathway, also up-regulated in tumors, include Mad2L1 and BUBR1 which together regulate cell division cycle 20 (Cdc20) protein, an essential cofactor needed by the Anaphase Promoting Complex to initiate the anaphase of cell cycle. Interestingly we also discovered that some of the MSAC pathway genes show a histotype-specific graded expression pattern with higher levels in sarcomatoid tumors compared to biphasic tumors and with lowest expression levels seen in epitheloid tumors. Additionally survivin, the product of which localizes to the mitotic spindle and negatively regulates apoptosis by inhibiting caspase activation, was expressed more than 2 fold in MPM tumors compared to normal samples. The microarray data also revealed other pathways significantly upregulated in MPM tumors including the Wnt and Cell-Adhesion signaling pathways. We are currently validating the expression microarray data using quantitative Polymerase Chain Reaction (PCR) platform with respect to key components of MSAC pathway. This will be followed by proteomic analysis of these components on tumor lysates to confirm the alterations in their expression profiles. Supported by Grants: DOD PROSPECT W81XWH-07-1-030602, Fleming Foundation for Mesothelioma Research, Aileen Dillon and Lee Bourg Mesothelioma Endowment and NIH K12 CA088084 08 award. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2172.