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PDF file - 129KB, Supplemental Figure 3. ACBP downregulation decreases proliferation and induces apoptosis in A549 NSCLC cells.

We identified acyl-coenzyme A–binding protein (ACBP) as part of a proteomic signature predicting the risk of having lung cancer. Because ACBP is known to regulate β-oxidation, which in turn controls cellular proliferation, we hypothesized that ACBP contributes to regulation of cellular proliferation and survival of non–small cell lung cancer (NSCLC) by modulating β-oxidation. We used matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) and immunohistochemistry (IHC) to confirm the tissue localization of ABCP in pre-invasive and invasive NSCLCs. We correlated ACBP gene expression levels in NSCLCs with clinical outcomes. In loss-of-function studies, we tested the effect of the downregulation of ACBP on cellular proliferation and apoptosis in normal bronchial and NSCLC cell lines. Using tritiated-palmitate (3H-palmitate), we measured β-oxidation levels and tested the effect of etomoxir, a β-oxidation inhibitor, on proliferation and apoptosis. MALDI-IMS and IHC analysis confirmed that ACBP is overexpressed in pre-invasive and invasive lung cancers. High ACBP gene expression levels in NSCLCs correlated with worse survival (HR = 1.73). We observed a 40% decrease in β-oxidation and concordant decreases in proliferation and increases in apoptosis in ACBP-depleted NSCLC cells as compared with bronchial airway epithelial cells. Inhibition of β-oxidation by etomoxir in ACBP-overexpressing cells produced dose-dependent decrease in proliferation and increase in apoptosis (P = 0.01 and P < 0.001, respectively). These data suggest a role for ACBP in controlling lung cancer progression by regulating β-oxidation. Cancer Prev Res; 7(7); 748–57. ©2014 AACR.

PDF file - 103KB, Supplemental Figure 1. ACBP protein expression in lung cancer cell lines.

PDF file - 90KB, Supplemental Figure 2. ACBP downregulation has no effect on proliferation or apoptosis in 16-HBE cells.

PDF file - 68KB, Supplemental Table 1. Clinical correlates of microarray data sets.

PDF file - 83KB, Supplemental Figure 4. Downregulation of ACBP reduces ATP levels in H520 cells.

Supplementary Figures 1-5 - PDF file 342K, Figure S1. The IGV plot of copy number variation for 152 TCGA samples in 3q regions. The samples are in descending order according to the average expression scores. Figure S2. The effect of knockdown SENP2 expression on NSCLC cells. Figure S3. Three-gene expression-based 3q26-29 stratification informs response to adjuvant chemotherapy on different stage patients. Figure S4 Relationship between patient stratification based on expression value of each of three genes and response to adjuvant chemotherapy. Figure S5. Copy number alteration of SENP family members in TCGA lung SCC dataset (n=177)

PDF file - 16K, SLC1A5 protein is overexpressed in NSCLC tissues

Purpose: Chromosome 3q26-29 is a critical region of genomic amplification in lung squamous cell carcinomas (SCC). Identification of candidate drivers in this region could help uncover new mechanisms in the pathogenesis and potentially new targets in SCC of the lung.Experimental Design: We conducted a meta-analysis of seven independent datasets containing a total of 593 human primary SCC samples to identify consensus candidate drivers in 3q26-29 amplicon. Through integrating protein–protein interaction network information, we further filtered for candidates that may function together in a network. Computationally predicted candidates were validated using RNA interference (RNAi) knockdown and cell viability assays. Clinical relevance of the experimentally supported drivers was evaluated in an independent cohort of 52 lung SCC patients using survival analysis.Results: The meta-analysis identified 20 consensus candidates, among which four (SENP2, DCUN1D1, DVL3, and UBXN7) are involved in a small protein–protein interaction network. Knocking down any of the four proteins led to cell growth inhibition of the 3q26-29–amplified SCC. Moreover, knocking down of SENP2 resulted in the most significant cell growth inhibition and downregulation of DCUN1D1 and DVL3. Importantly, a gene expression signature composed of SENP2, DCUN1D1, and DVL3 stratified patients into subgroups with different response to adjuvant chemotherapy.Conclusion: Together, our findings show that SENP2, DCUN1D1, and DVL3 are candidate driver genes in the 3q26-29 amplicon of SCC, providing novel insights into the molecular mechanisms of disease progression and may have significant implication in the management of SCC of the lung. Clin Cancer Res; 19(20); 5580–90. ©2013 AACR.

PDF file - 28K, SLC1A5 mediates response to Gln-dependent growth

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PDF file - 20K, Inhibition of SLC1A5 activity reduces Gln uptake and cell growth of NSCLC cells

PDF file - 15K, Silencing SLC1A5 in SCC cells by siRNA attenuates cell growth and reduces glutamine uptake

Purpose: We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an overexpressed protein in a shotgun proteomic analysis of stage I non–small cell lung cancer (NSCLC) when compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the metabolic demand for lung cancer cell growth and survival.Experimental Design: To test our hypothesis, we first analyzed the protein expression of SLC1A5 in archival lung cancer tissues by immunohistochemistry and immunoblotting (N = 98) and in cell lines (N = 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of l-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor of SLC1A5, gamma-l-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung cancer cell lines.Results: Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the cytoplasmic membrane and is significantly associated with SCC histology and male gender; (iii) 68% of Gln is transported in a Na+-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv) pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an effect mediated in part by mTOR signaling.Conclusions: These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer cells' metabolism, growth, and survival. Clin Cancer Res; 19(3); 560–70. ©2012 AACR.

Supplementary Tables 1-13 - PDF file 135K, Supplementary Table S1: Amplification scores and average expression scores for all paired samples based on the TCGA copy number data and gene expression data Table S2: The corr_exp_amp and corr_exp_ave for 164 genes in the 3q26-39 regions Table S3 Description of six datasets using to identify candidate driver genes Table S4 Predicted information based on GSE2109 Table S5 Predicted information based on GSE3141 Table S6 Predicted information based on GSE4573 Table S7 Predicted information based on GSE8894 Table S8 Predicted information based on GSE17710 Table S9 Predicted information based on Roepman et al. dataset Table S10 Order statistic result for 181 genes in 3q26�29 Table S11 Correlation between gene expression and 3q26-29 amplification level for identified 20 genes in head and neck SCC Table S12 Correlation between gene expression and 3q26-29 amplification level for identified 20 genes in cervical SCC Table S13 Copy number alteration and mRNA expression of four genes in a panel of NSCLC cell lines (data from Cancer Cell Line Encyclopedia database)

PDF file - 39K, Expression of SLC1A5 in lung cancer cell lines

Diagnosis of lung cancer patients with indeterminate pulmonary nodules (IPNs) presents a significant clinical challenge, with morbidity and management costs of $28 billion/year. We show that a quantitative free-solution assay (FSA), coupled with a compensated interferometric reader (CIR), improves the diagnostic performance of CYFRA 21-1 as a lung cancer biomarker. FSA-CIR is a rapid, mix-and-read, isothermal, label- and enzyme-free, matrix-insensitive, and target and probe-agnostic assay. Operating FSA-CIR at ∼40, 0.75 μL samples/day delivered a serum CYFRA 21-1 limit of quantification (LOQ) of 81 pg/mL with intra-assay and interassay CVs of 4.9% and 9.6% for four-day replicate determinations. Blinded analysis of a 225 patient cohort, consisting of 75 nonmalignant nodules, 45 adenocarcinomas, 44 squamous cell carcinomas, and 61 small cell lung cancers, gave a clear separation of cases and controls, not observed in the Cobas ECL analysis. The area under the curve (AUC) for the Mayo model increased from 0.595 to 0.923 when combined with the FSA-CIR CYFRA 21-1 measurements. In a population with nodules between 6 and 30 mm, the AUC increased from 0.567 to 0.943. In this subgroup, the positive predictive value (PPV) for all tumors by the CYFRA 21-1 assay was 98.7%. Our results demonstrate increased performance of the CYFRA 21-1 assay using FSA-CIR and represents a proof of concept for redefining the performance characteristics of this important candidate biomarker.

Somatostatin receptor 2 (SSTR2) is overexpressed in a majority of neuroendocrine neoplasms, including small-cell lung carcinomas (SCLCs). SSTR2 was previously considered an inhibitory receptor on cell growth, but its agonists had poor clinical responses in multiple clinical trials. The role of this receptor as a potential therapeutic target in lung cancer merits further investigation. We evaluated the expression of SSTR2 in a cohort of 96 primary tumors from patients with SCLC and found 48% expressed SSTR2. Correlation analysis in both CCLE and an SCLC RNAseq cohort confirmed high-level expression and identified an association between NEUROD1 and SSTR2. There was a significant association with SSTR2 expression profile and poor clinical outcome. We tested whether SSTR2 expression might contribute to tumor progression through activation of downstream signaling pathways, using in vitro and in vivo systems and downregulated SSTR2 expression in lung cancer cells by shRNA. SSTR2 downregulation led to increased apoptosis and dramatically decreased tumor growth in vitro and in vivo in multiple cell lines with decreased AMPKα phosphorylation and increased oxidative metabolism. These results demonstrate a role for SSTR2 signaling in SCLC and suggest that SSTR2 is a poor prognostic biomarker in SCLC and potential future therapeutic signaling target.

We hypothesized that distinct protein expression features of benign and malignant pulmonary nodules may reveal novel candidate biomarkers for the early detection of lung cancer. We performed proteome profiling by liquid chromatography— tandem mass spectrometry to characterize 34 resected benign lung nodules, 24 untreated lung adenocarcinomas (ADCs), and biopsies of bronchial epithelium. Group comparisons identified 65 proteins that differentiate nodules from ADCs and normal bronchial epithelium and 66 proteins that differentiate ADCs from nodules and normal bronchial epithelium. We developed a multiplexed parallel reaction monitoring (PRM) assay to quantify a subset of 43 of these candidate biomarkers in an independent cohort of 20 benign nodules, 21 ADCs, and 20 normal bronchial biopsies. PRM analyses confirmed significant nodule-specific abundance of 10 proteins including ALOX5, ALOX5AP, CCL19, CILP1, COL5A2, ITGB2, ITGAX, PTPRE, S100A12, and SLC2A3 and significant ADC-specific abundance of CEACAM6, CRABP2, LAD1, PLOD2, and TMEM110-MUSTN1. Immunohistochemistry analyses for seven selected proteins performed on an independent set of tissue microarrays confirmed nodule-specific expression of ALOX5, ALOX5AP, ITGAX, and SLC2A3 and cancer-specific expression of CEACAM6. These studies illustrate the value of global and targeted proteomics in a systematic process to identify and qualify candidate biomarkers for noninvasive molecular diagnosis of lung cancer.

T and B lymphocytes undergo metabolic re-programming upon activation that is essential to allow bioenergetics, cell survival, and intermediates for cell proliferation and function. To support changes in the activity of signaling pathways and to provide sufficient and necessary intracellular metabolites, uptake of extracellular nutrients increases sharply with metabolic re-programming. One result of increased metabolic activity can be reactive oxygen species (ROS), which can be toxic when accumulated in excess. Uptake of cystine allows accumulation of cysteine that is necessary for glutathione synthesis and ROS detoxification. Cystine uptake is required for T cell activation and function but measurements based on radioactive labeling do not allow analysis on single cell level. Here we show the critical role for cystine uptake in T cells using a method for measurement of cystine uptake using a novel CystineFITC probe. T cell receptor stimulation lead to upregulation of the cystine transporter xCT (SLC7a11) and increased cystine uptake in CD4+ and CD8+ human T cells. Similarly, lipopolysaccharide stimulation increased cystine uptake in human B cells. The CystineFITC probe was not toxic and could be metabolized to prevent cystine starvation induced cell death. Furthermore, blockade of xCT or competition with natural cystine decreased uptake of CystineFITC. CystineFITC is thus a versatile tool that allows measurement of cystine uptake on single cell level and shows the critical role for cystine uptake for T cell ROS regulation and activation.

Meta-analyses have demonstrated that low-dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the antiplatelet action of low-dose aspirin likely contributes to its antimetastatic effect. Cycloxooxygenase-2 (COX-2)–derived prostaglandin E2 (PGE2) also contributes to metastasis, and we addressed the hypothesis that low-dose aspirin also inhibits PGE2 biosynthesis. We show that low-dose aspirin inhibits systemic PGE2 biosynthesis by 45% in healthy volunteers (P < 0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells, its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells upregulates COX-2 expression and PGE2 biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE2 production by the platelet–tumor cell aggregates. In conclusion, low-dose aspirin has a significant effect on extraplatelet cyclooxygenase and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low-dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE2 biosynthesis in metastasizing tumor cells. Cancer Prev Res; 9(11); 855–65. ©2016 AACR.

New therapeutic strategies are desperately needed in lung cancer. Targeting cancer-specific biochemical phenotypes, including those determined metabolically, represents an alternative approach to treating patients with lung cancer, the leading cause of cancer deaths in the USA and worldwide.1 L-Glutamine (Gln) is an essential amino acid for non-small cell lung cancer (NSCLC) growth in vitro and in vivo.2–4 Gln has been shown to sustain tumor growth under hypoxia,5 to mediate K-RAS-driven lung cancer growth6 and to support the autophagy-mediated prosurvival pathway in B-RAFV600-driven lung tumors.7 Nonetheless, the specific contribution of Gln transporters to lung cancer remains largely unknown. What’s new? New strategies to overcome lung cancer mortality depend heavily on the discovery of novel therapeutic targets. In non-small cell lung cancer (NSCLC), a possible target is solute linked carrier family 1A, member 5 (SLC1A5), a major glutamine transporter in NSCLC. This study furthers the promise of SLC1A5 by showing that its expression levels in lung cancer cells can predict cell sensitivity to the inhibitor gamma-L-glutamyl-p-nitroanilide (GPNA). In NSCLC cell lines, SLC1A5 inactivation led to glutamine starvation and oxidative stress-mediated autophagy and apoptosis. In NSCLC patients, SLC1A5 expression was associated with poor overall survival. We recently identified by in-depth shotgun proteomic analysis of stage I NSCLCs several therapeutic targets including the solute linked carrier family 1A, member 5 (SLC1A5).8 We were the first to report that SLC1A5 functions as the primary transporter of Gln in a sodium-dependent manner in NSCLC.9 Inhibition of SLC1A5 attenuates cell growth and mTOR signaling.9,10 Although pharmacological strategies to inhibit Gln metabolism using amino acid analogs such as acivicin and 6-diazo-5-oxo-L-norleucine (DON) have been investigated,3,11 the lack of selectivity of these agents has shifted the efforts to developing agents directed at specific nodes of glutamine metabolism instead.12 Given the emerging role of Gln metabolism in cancer13 and the differential expression of SLC1A5 in NSCLC,9 we tested the hypothesis that elevated SLC1A5 expression is a key prosurvival mechanism that promotes NSCLC progression by increasing tumor cells to transport and utilize Gln available in the microenvironment. We investigated the prognostic value of SLC1A5 expression in NSCLC and examined the therapeutic potential of targeting its Gln transport activity in cell and xenografts by inhibiting SLC1A5-dependent Gln transport.

Somatostatin receptor 2 (SSTR2) is overexpressed in a majority of neuroendocrine neoplasms, including small cell lung carcinomas (SCLCs). SSTR2 was previously considered an inhibitory receptor on cell growth, but its agonists had poor clinical responses in multiple clinical trials. The role of this receptor as a potential therapeutic target in lung cancer merits further investigation. We evaluated the expression of SSTR2 in a cohort of 96 primary tumors from patients with SCLC and found 48% expressed SSTR2. Correlation analysis in both CCLE and an SCLC RNAseq cohort confirmed high level expression and identified an association between NEUROD 1 and SSTR2. There was a significant association with SSTR2 expression profile and poor clinical outcome. We tested whether SSTR2 expression might contribute to tumor progression through activation of downstream signaling pathways, using in vitro and in vivo systems and down regulated SSTR2 expression in lung cancer cells by shRNA. SSTR2 downregulation led to increased apoptosis and dramatically decreased tumor growth in vitro and in vivo in multiple cell lines with decreased AMPKα phosphorylation and increased oxidative metabolism. These results demonstrate a role for SSTR2 signaling in small cell lung carcinoma and suggest that SSTR2 is a poor prognostic biomarker in SCLC and potential future therapeutic signaling target.

Realizing personalized medicine, which promises to enable early disease detection, efficient diagnostic staging, and therapeutic efficacy monitoring, hinges on biomarker quantification in patient samples. Yet, the lack of a sensitive technology and assay methodology to rapidly validate biomarker candidates continues to be a bottleneck for clinical translation. In our first direct and quantitative comparison of backscattering interferometry (BSI) to fluorescence sensing by ELISA, we show that BSI could aid in overcoming this limitation. The analytical validation study was performed against ELISA for two biomarkers for lung cancer detection: Cyfra 21-1 and Galectin-7. Spiked serum was used for calibration and comparison of analytical figures of merit, followed by analysis of blinded patient samples. Using the ELISA antibody as the probe chemistry in a mix-and-read assay, BSI provided significantly lower detection limits for spiked serum samples with each of the biomarkers. The limit of quantification (LOQ) for Cyrfa-21-1 was measured to be 230 pg/mL for BSI versus 4000 pg/mL for ELISA, and for Galectin-7, it was 13 pg/mL versus 500 pg/mL. The coefficient of variation for 5 day, triplicate determinations was

Pierian Biosciences has developed the ChemoINTEL platform for high-throughput, semi-automated quantification of cellular responses to chemotherapeutics. ChemoINTEL relies on a microscopy-based process that captures and analyzes single cell behavior to reflect responses within a population. The platform is customizable to a variety of small molecule panels, either specific to certain tumor types or for titration screening of small molecules in development. Automated imaging of each well of a 384-well plate over time provides real-time kinetic response data under a variety of treatment conditions. Brightfield and fluorescent images are used to determine quantitative changes in morphology and molecular metrics reflecting induced apoptosis and cell death. Unique to ChemoINTEL as a drug screening platform is the quantification of intensity values, at the single cell level versus whole well fluorescence, for each fluorescent probe at each timepoint through a high-throughput analysis routine. These data are further processed through an internally developed algorithm to compare a treated population’s response relative to an untreated control and reports a sensitivity score. By combining different treatment conditions, the platform provides intelligent design of single agent or combination treatment approaches. By analyzing individual cells in the population, ChemoINTEL aids in better understanding response of tumor cell sub-populations to different chemotherapeutic agents. As a compound library screening tool or potential clinical diagnostic, Pierian Biosciences’ quality-controlled processes ensure all its equipment, reagents and processes follow ISO17025 guidelines to ensure the quality of all data generated. Each assay is internally controlled to include a standard cell line, whose response is monitored for accuracy of the reported results. In collaboration with several biorepositories and through the development of a standardized dissociation approach resulting in purification of viable primary tumor cells, the ChemoINTEL platform is also under development to assess chemotherapeutic response within patient populations. In conjunction with the ChemoINTEL platform, Pierian Biosciences has also developed a sophisticated multi-color flow cytometry platform (ImmunoINTEL) that when used in conjunction with ChemoINTEL provides information on the purity of isolated primary tumor cells prior to plating, as well as information on the cell populations present within the tumor microenvironment. These two platforms can be further complexed to evaluate immunotherapies in combination with front-line therapies. In summary, Pierian Biosciences has developed a high-throughput, multi-parametric platform for both drug development and diagnostic applications. Note: This abstract was not presented at the meeting. Citation Format: Kellye C. Kirkbride, Kevin J. Polach, Samantha J. Braxton, Megan D. Hoeksema, Dustin C. Rogers, Patricia Ladd-Ward, Durdica Vojnic Zelic, Santosh Putta, Matt Westfall, Norman Purvis. ChemoINTEL: A high-throughput, multi-parametric compound screening platform for intelligent lead compound and therapeutic combination identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 294.

Small cell lung cancer (SCLC) is a devastating disease due to its propensity for early invasion and refractory relapse after initial treatment response. Although these aggressive traits have been associated with phenotypic heterogeneity, our understanding of this association remains incomplete. To fill this knowledge gap, we inferred a set of 33 transcription factors (TF) associated with gene signatures of the known neuroendocrine/epithelial (NE) and non-neuroendocrine/mesenchymal-like (ML) SCLC phenotypes. The topology of this SCLC TF network was derived from prior knowledge and was simulated using Boolean modeling. These simulations predicted that the network settles into attractors, or TF expression patterns, that correlate with NE or ML phenotypes, suggesting that TF network dynamics underlie the emergence of heterogeneous SCLC phenotypes. However, several cell lines and patient tumor specimens failed to correlate with either the NE or ML attractors. By flow cytometry, single cells within these cell lines simultaneously expressed surface markers of both NE and ML differentiation, confirming the existence of a “hybrid” phenotype. Upon exposure to standard-of-care cytotoxic drugs or epigenetic modifiers, NE and ML cell populations converged toward the hybrid state, suggesting possible escape from treatment. Our findings indicate that SCLC phenotypic heterogeneity can be specified dynamically by attractor states of a master regulatory TF network. Thus, SCLC heterogeneity may be best understood as states within an epigenetic landscape. Understanding phenotypic transitions within this landscape may provide insights to clinical applications. Cancer Res; 77(5); 1063–74. ©2016 AACR.

The molecular determinants of lung cancer risk remain largely unknown. Airway epithelial cells are prone to assault by risk factors and are considered to be the primary cell type involved in the field of cancerization. To investigate risk-associated changes in the bronchial epithelium proteome that may offer new insights into the molecular pathogenesis of lung cancer, proteins were identified in the airway epithelial cells of bronchial brushing specimens from risk-stratified individuals by shotgun proteomics. Differential expression of selected proteins was validated by parallel reaction monitoring mass spectrometry in an independent set of individual bronchial brushings. We identified 2,869 proteins, of which 312 proteins demonstrated a trend in expression. Pathway analysis revealed enrichment of carbohydrate metabolic enzymes in high-risk individuals. Glucose consumption and lactate production were increased in human bronchial epithelial BEAS2B cells treated with cigarette smoke condensate for 7 months. Increased lipid biosynthetic capacity and net reductive carboxylation were revealed by metabolic flux analyses of [U-13C5] glutamine in this in vitro model, suggesting profound metabolic reprogramming in the airway epithelium of high-risk individuals. These results provide a rationale for the development of potentially new chemopreventive strategies and selection of patients for surveillance programs.

Aberrant expression of RNA-binding proteins has profound implications for cellular physiology and the pathogenesis of human diseases such as cancer. We previously identified the Fragile X-Related 1 gene (FXR1) as one amplified candidate driver gene at 3q26-29 in lung squamous cell carcinoma (SCC). FXR1 is an autosomal paralog of Fragile X mental retardation 1 and has not been directly linked to human cancers. Here we demonstrate that FXR1 is a key regulator of tumor progression and its overexpression is critical for nonsmall cell lung cancer (NSCLC) cell growth in vitro and in vivo. We identified the mechanisms by which FXR1 executes its regulatory function by forming a novel complex with two other oncogenes, protein kinase C, iota and epithelial cell transforming 2, located in the same amplicon via distinct binding mechanisms. FXR1 expression is a candidate biomarker predictive of poor survival in multiple solid tumors including NSCLCs. Because FXR1 is overexpressed and associated with poor clinical outcomes in multiple cancers, these results have implications for other solid malignancies.

Introduction: Small cell lung cancer (SCLC) is a high grade neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths. Treatment in SCLC has not changed significantly in the last 20 years. SCLC initially responds well to chemotherapy, but inevitably recurs. Characterization of tumor heterogeneity and changes in SCLC cell signaling and phenotypes after chemotherapy could yield new insights and therapeutic options. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of more than 40 proteins in single cells and offers the opportunity to identify new subpopulations including potential cancer stem cell populations as well as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with one cycle of carboplatin/etoposide or saline injection. Tumors were harvested at ~2000mm3, disaggregated, and cryopreserved. PDX samples were stained with a 22 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. ViSNE analysis and biaxial gating were used to identify major subpopulations of interest. Results: PDX tumors released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. ACK buffer and enzymatic dissociation yielded the best quality cells by depleting red blood cells. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and iridium intercalator was used to identify nucleated cells. Single cell protein expression and phosphorylation was analyzed using viSNE and yielded at least 9 distinct subpopulations based on density islands with neuroendocrine (CD56+) and non-neuroendocrine (CD56-) populations. Chemotherapy treated cells had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included 2-3 fold expansion of SOX2+, CD117+, and pSTAT3+ populations with chemotherapy treatment. A small CD44+ tumor subpopulation identified in the chemotherapy treated cells was not present in the matched mock treated tumor suggesting a potential chemotherapy resistant/ stem- like subpopulation. Kinase activity showed stable p-AKT overall, but increased p-S6 in the chemotherapy treated cells. Conclusions: Mass cytometry was able to identify multiple neuroendocrine and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of multiple stem-like signaling factors. This work demonstrates the utility of mass cytometry and viSNE as novel techniques to identify subpopulations associated with chemotherapy resistance for future targeting and demonstrates the feasibility of this technique for characterizing signaling heterogeneity in human SCLC tumors. Citation Format: Jonathan M. Lehman, Nalin Leelatian, Bradford Harris, Megan Hoeksema, Zou Yong, Deon B. Doxie, Jonathan M. Irish, Pierre P. Massion. Dissecting small cell lung carcinoma heterogeneity and chemotherapy resistance with mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3935. doi:10.1158/1538-7445.AM2017-3935

We identified acyl-coenzyme A–binding protein (ACBP) as part of a proteomic signature predicting the risk of having lung cancer. Because ACBP is known to regulate β-oxidation, which in turn controls cellular proliferation, we hypothesized that ACBP contributes to regulation of cellular proliferation and survival of non–small cell lung cancer (NSCLC) by modulating β-oxidation. We used matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) and immunohistochemistry (IHC) to confirm the tissue localization of ABCP in pre-invasive and invasive NSCLCs. We correlated ACBP gene expression levels in NSCLCs with clinical outcomes. In loss-of-function studies, we tested the effect of the downregulation of ACBP on cellular proliferation and apoptosis in normal bronchial and NSCLC cell lines. Using tritiated-palmitate (3H-palmitate), we measured β-oxidation levels and tested the effect of etomoxir, a β-oxidation inhibitor, on proliferation and apoptosis. MALDI-IMS and IHC analysis confirmed that ACBP is overexpressed in pre-invasive and invasive lung cancers. High ACBP gene expression levels in NSCLCs correlated with worse survival (HR = 1.73). We observed a 40% decrease in β-oxidation and concordant decreases in proliferation and increases in apoptosis in ACBP-depleted NSCLC cells as compared with bronchial airway epithelial cells. Inhibition of β-oxidation by etomoxir in ACBP-overexpressing cells produced dose-dependent decrease in proliferation and increase in apoptosis (P = 0.01 and P < 0.001, respectively). These data suggest a role for ACBP in controlling lung cancer progression by regulating β-oxidation. Cancer Prev Res; 7(7); 748–57. ©2014 AACR.

Purpose: Chromosome 3q26-29 is a critical region of genomic amplification in lung squamous cell carcinomas (SCC). Identification of candidate drivers in this region could help uncover new mechanisms in the pathogenesis and potentially new targets in SCC of the lung. Experimental Design: We conducted a meta-analysis of seven independent datasets containing a total of 593 human primary SCC samples to identify consensus candidate drivers in 3q26-29 amplicon. Through integrating protein–protein interaction network information, we further filtered for candidates that may function together in a network. Computationally predicted candidates were validated using RNA interference (RNAi) knockdown and cell viability assays. Clinical relevance of the experimentally supported drivers was evaluated in an independent cohort of 52 lung SCC patients using survival analysis. Results: The meta-analysis identified 20 consensus candidates, among which four (SENP2, DCUN1D1, DVL3, and UBXN7) are involved in a small protein–protein interaction network. Knocking down any of the four proteins led to cell growth inhibition of the 3q26-29–amplified SCC. Moreover, knocking down of SENP2 resulted in the most significant cell growth inhibition and downregulation of DCUN1D1 and DVL3. Importantly, a gene expression signature composed of SENP2, DCUN1D1, and DVL3 stratified patients into subgroups with different response to adjuvant chemotherapy. Conclusion: Together, our findings show that SENP2, DCUN1D1, and DVL3 are candidate driver genes in the 3q26-29 amplicon of SCC, providing novel insights into the molecular mechanisms of disease progression and may have significant implication in the management of SCC of the lung. Clin Cancer Res; 19(20); 5580–90. ©2013 AACR.

RNA-binding proteins (RBPs) are the master regulators of mRNA processing and translation and are often aberrantly expressed in cancer. We have recently identified Fragile X Mental Retardation-Related 1 (FXR1) as a novel cancer gene in non-small cell lung cancer (NSCLC) and its expression is correlated with poor prognosis in multiple human cancers. FXR1 encodes an RNA binding protein (RBP) that belongs to the family of fragile X-related proteins including the fragile X mental retardation protein (FMR1) and FXR2. Inactivation of FMR1 expression is the cause of the Fragile X syndrome in humans. Little is known for the function of FXR1 in human cancers. In this study, endogenous FXR1 or a Flag-tagged FXR1 was immunoprecipitated from H520, a lung squamous carcinoma cell line, and analyzed by shotgun proteomics. The Flag-tagged FXR1 was also transfected into human HEK293 cells and followed by co-immunoprecipitation and shotgun proteomic analysis. In total we found 206 proteins enriched in H520 with more than two-fold change spectral counts over the IgG control. Of the 206 proteins, 49 were detected in HEK293 cells as well and 157 were only detected in H520. KEGG pathway analysis indicated enrichment of proteins involved in ribosomal function, RNA transport and proteasome. To identify lung cancer related proteins, we interrogated the mRNA expression of 206 proteins in The Cancer Genome Atlas (TCGA) lung cancer dataset (1013 tumors,109 normal), in a combined NSCLC dataset (1392 tumors, 240 normal) from the Gene Expression Omnibus (GEO) and in a previously reported dataset of putative FXR1 target mRNAs and found 75 altered genes in common. Among these, we found that 24 genes were not only unregulated in all NSCLC samples (n = 2405, FDR Citation Format: Jun Qian, Yong Zou, Megan Hoeksema, Bradford Harris, Heidi Chen, Pierre Massion. Identification of FXR1-associated protein complexes in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2873.

e20090Background: Small cell lung cancer (SCLC) is a high grade poorly differentiated neuroendocrine carcinoma responsible for ~15% of diagnosed lung cancers and up to 25% of lung cancer deaths. Tr...

Purpose: We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an overexpressed protein in a shotgun proteomic analysis of stage I non–small cell lung cancer (NSCLC) when compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the metabolic demand for lung cancer cell growth and survival. Experimental Design: To test our hypothesis, we first analyzed the protein expression of SLC1A5 in archival lung cancer tissues by immunohistochemistry and immunoblotting (N = 98) and in cell lines (N = 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of l-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor of SLC1A5, gamma-l-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung cancer cell lines. Results: Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the cytoplasmic membrane and is significantly associated with SCC histology and male gender; (iii) 68% of Gln is transported in a Na+-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv) pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an effect mediated in part by mTOR signaling. Conclusions: These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer cells' metabolism, growth, and survival. Clin Cancer Res; 19(3); 560–70. ©2012 AACR.

Advances in proteomic analysis of human samples are driving critical aspects of biomarker discovery and the identification of molecular pathways involved in disease etiology. Toward that end, in this report we are the first to use a standardized shotgun proteomic analysis method for in-depth tissue protein profiling of the two major subtypes of nonsmall cell lung cancer and normal lung tissues. We identified 3621 proteins from the analysis of pooled human samples of squamous cell carcinoma, adenocarcinoma, and control specimens. In addition to proteins previously shown to be implicated in lung cancer, we have identified new pathways and multiple new differentially expressed proteins of potential interest as therapeutic targets or diagnostic biomarkers, including some that were not identified by transcriptome profiling. Up-regulation of these proteins was confirmed by multiple reaction monitoring mass spectrometry. A subset of these proteins was found to be detectable and differentially present in the peripheral blood of cases and matched controls. Label-free shotgun proteomic analysis allows definition of lung tumor proteomes, identification of biomarker candidates, and potential targets for therapy.

Introduction: Many tumors increase uptake and dependence on nutrients such as glucose, cysteine or glutamine. These basic observations on cancer cell metabolism have opened multiple new diagnostic and therapeutic avenues in cancer. Here, we investigated the function of SLC7A11 (xCT), a cystine/glutamate anti-porter, in the pathogenesis of lung cancer. Recently, we found xCT to be strongly overexpressed at the cell surface of lung cancers and began to investigate the mechanisms by which xCT may contribute to lung cancer progression. Methods: The expression of xCT and its related genes in 411 patients with non small cell lung cancer (NSCLC) were evaluated in publically available lung cancer gene expression datasets. The association between xCT expression and overall survival, stage of diseases were tested in tissue microarray datasets. Gain of function studies for xCT were undertaken in the normal airway epithelium cell lines (BEAS2B and 16HBE) to investigate the role of xCT in regulating cell metabolism, growth and survival in normal and malignant lung epithelial cells. Results: Our results shows that xCT proein is overexpressed in 38% of NSCLC patients. We also found that expression of xCT mRNA is strongly associated with disease stage (p Conclusion: Our results demonstrate that xCT is highly expressed in a subset of NSCLCs. The induction of xCT in transformed aiway epithelial cells causes tumor formation in nude mice. The mechanism of malignant transformation involves the Warburg effect. Further investigation into the mechanisms of transformation may help in answering fundamental questions pertaining to how glutamate/cystine exchange contributes to lung cancer development and progression and also in establishing xCT as a potential novel therapeutic target in lung cancer. Citation Format: Xiangming Ji Ji, Jun Qian, Jamshedur Rahman, Brad Harris, Megan Hoeksema, Heidi Chen, Rosana Eisenberg, Jamey Young. SLC7A11 contributes to the pathogenesis of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A10.

Background: Meta-analysis of the trials evaluating the effect of aspirin on cardiovascular outcomes demonstrated that aspirin prevented mortality from adenocarcinomas by 47% and reduced metastasis by 46%. Equivalent benefit was seen with both high and low doses of aspirin. We hypothesize that low dose aspirin blocks metastasis by inhibiting platelet COX-1 and also by inhibiting the biosynthesis of the pro-metastatic PGE2. Methods and Results: The effect of a range of doses of aspirin on PGE2 biosynthesis in three lung adenocarcinoma cell lines was compared with that in washed platelets. PGE2 was measured by GC/MS. In the cancer cell lines, aspirin inhibits COX-2-dependent PGE2 production with IC50s equivalent to or less than that for platelet COX-1. The IC50s were: Platelet = 19.8 ± 1.5 μM; H2122 = 19.5 ± 5 μM; HCC827 = 3.9 ± 2 μM; A549 = 1.6 ± 0.4 μM. To explore the extra-platelet effects of low dose aspirin in vivo, we examined the effect of aspirin 81 mg daily for 2 weeks on the biosynthesis of PGE2 and prostacyclin as reflected by their respective urinary metabolites in 54 healthy humans. The PGE2 metabolite was measured by LC/MS/MS and the prostacyclin metabolite by GC/MS. This dose of aspirin inhibited PGE2 production by 45% (p < 0.0001) and reduced prostacyclin by 37% (p < 0.0001). Adherence of platelets to tumor cells facilitates metastasis. We determined the effect of adherence of ADP-activated washed platelets to lung adenocarcinoma cells (A549) on the PGE2 biosynthetic pathway. COX-2 expression was determined by Western blot, and cytosolic calcium by Fura-2 AM. Activated platelets increased cytosolic calcium in A549 cells by 22% (p < 0.001), increased expression of COX-2 by 8.6 fold (p Conclusion: These findings form the basis for a hypothesis that the remarkable effect of low dose aspirin on adenocarcinoma prevention and reduction in metastatic behavior could result from inhibition of platelet activation in concert with inhibition of platelet-induced PGE2 biosynthesis and direct inhibition of the catalytic activity COX-2 in circulating tumor cells. Work funded by the Thoracic Program of the Vanderbilt Ingram Cancer Center Citation Format: John Oates, Pierre Massion, Bjorn Knollmann, James Smith, Elias Haddad, Philip Lammers, Denise Oram, Taneem Amin, Bradford Harris, Megan Hoeksema, Hyun Hwang. Low dose aspirin that reduces mortality from lung adenocarcinoma inhibits both platelet COX-1 and the biosynthesis of PGE2. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR05.

INTRODUCTION: In a recent study, we showed that SLC1A5, a key glutamine (Gln) transporter, regulates tumor growth and survival in NSCLC. Here we sought to characterize the expression of other Gln transporters in lung cancer and to test whether the inhibition of SLC1A5 activity induced a change in expression of other amino acid transporters in NSCLC. METHODS: Neutral amino acid transporters reported in the cancer literature were evaluated in publically available lung cancer gene expression datasets. Among those, SLC38A1, SLC1A4, SLC1A5, SLC7A5, SLC7A11, SLC7A8 were selected for the in vitro validation. The expression of these genes was evaluated in two lung squamous carcinoma cell lines (HCC15, H226) and one adenocarcinoma cell line (A549) by RT-PCR and then quantified by qPCR, using SYBR® Green Supermix protocol. The expression of the same genes was assessed in response to transient SLC1A5 knockdown in HCC15 and A549 cell lines by qPCR. RESULTS: Amino acid transporters SLC38A1, SLC1A5, SLC7A5 and SLC7A11 are expressed in NSCLCs, a finding that was confirmed in the three cell lines studied. SLC1A4 was found to have the lowest expression and SLC7A8 no expression in any of cell lines. In response to SLC1A5 transient knockdown in A549, SLC38A1, SLC1A4 and SLC7A5 were significantly up-regulated (p CONCLUSION: Our results show that SLC38A1, SLC1A5, SLC7A5 and SLC7A11 have the highest level of gene expression in NSCLC. We identified a possible compensatory mechanism to satisfy Gln transport and cellular function upon SLC1A5 silencing involving different transporters in a cell-specific manner. Compensatory mechanisms in response to SLC1A5 knockdown deserve further investigation as it may have implications in strategies aiming at inhibiting its activity. Further expression, localization and functional studies will follow to better understand changes occurring in lung cancer progression. Key words: Non-small cell lung cancer, SLC1A5, expression, amino acid, transporters Citation Format: Pierre P. Massion, Maria Senosain, Mohamed Hassanein, Xiangming Ji, Jun Qian, Megan hoeksema. Regulation of neutral amino acid transporters gene expression profile in non-small cell lung cancer. [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 1185. doi:10.1158/1538-7445.AM2015-1185

Small-cell lung cancer (SCLC), a lethal neuroendocrine cancer, lacks targetable oncogenes and is monolithically treated with standard combination chemotherapy. Interestingly, distinct subpopulations of neuroendocrine and non-neuroendocrine cells have been identified in mouse models of SCLC, but their existence in human SCLC remains unknown. Phenotypic heterogeneity, an important phenomenon in cellular reprogramming and therapeutic resistance, can arise from cell state transitions (i.e. network-level changes dynamically controlled by transcriptional regulators). The motivation for this study is to determine whether phenotypically distinct cell states exist in human SCLC, and investigate transcription factor (TF) dynamics that maintain these states. We implemented a mixed bioinformatics and experimental approach that defines inter-tumor heterogeneity in SCLC patients and cell lines as a spectrum of neuroendocrine (NE) and mesenchymal (MC) differentiation delineated by two anti-correlated gene co-expression networks. Features such as adhesion, surface markers and kinases effectively summarize heterogeneity in SCLC cell lines and patients as three distinct phenotypic states: NE, MC and intermediate. To characterize the transcriptional influence that governs these distinct phenotypic states, we constructed a TF regulatory network using the mutual information based method ARACNE. Boolean network model dynamics of the top 19 predicted TFs of the NE and MC networks gives rise to 3 distinct clusters of stable phenotypic states or ‘attractors’, each given by a unique TF network configuration. These TF network states were validated at both gene and protein level in SCLC cell lines and patients, identifying specific network states governing NE, MC and intermediate attractors. In silico TF perturbation experiments (single or combination TF activation/knockdown) performed to explore the possibility of state transitions, indicated that the NE state is more easily reprogrammable via single TF manipulations than the MC state, which required a combination of 3-4 simultaneous TF manipulations. SOX2, FOXA2 and OVOL2 were identified as master regulators of MC → NE state transitions while NOTCH1, MYC, SMAD3, and NFKB1 were master regulators of the NE → MC state transitions. Experimentally, phenotypic state transitions such as NE → Intermediate and MC → Intermediate were successfully induced using HDAC inhibitors, but not with demethylating agents or cisplatin. Etoposide treatment could also induce NE → Intermediate state transitions in NE cell lines but MC cell lines were resistant. Drug rebound experiments reveal that the transitioned cells fall back into NE/MC differentiated states upon removal of the drug. Thus, classification of human SCLC into 3 distinct phenotypic states - NE, MC and intermediate serves as a useful mapping tool for defining heterogeneity that could lead into personalized treatment strategies. Citation Format: Akshata R. Udyavar, Megan Hoeksema, David J. Wooten, Mukesh Bansal, Andrea Califano, Lourdes Estrada, Jonathan Irish, Pierre Massion, Vito Quaranta. Distinct transcriptional programs drive phenotypic heterogeneity in small cell lung cancer. [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 3761. doi:10.1158/1538-7445.AM2015-3761

7569 Background: Small cell lung cancer (SCLC) is a neuroendocrine cancer of the lung responsible for up to 25% of lung cancer deaths. Targeted therapies for SCLC are sorely needed. Somatostatin re...

The amplification of the distal portion of chromosome 3q in lung cancer is a major signature of neoplastic transformation, particularly in the squamous cell carcinoma (SCC) of the lung. A number of potential drivers in the 3q amplicon have been identified and are proposed to contribute to the development of lung SCC. miRNAs have emerged as a new class of small, non-coding RNAs that regulate gene expression and might act as integral parts of the molecular architecture of oncogene and tumor suppressor networks in human cancer, including non-small cell lung cancer (NSCLC). miR 1224-5p, 1248 and 944 are located at 3q amplicon and overexpressed in 50%, 26% and 53% of 30 primary SCCs when compared to normal tissues. The overexpression of these miRNAs was further validated in 237 TCGA lung SCC samples compared with 35 normal tissues (p=0.007, 1.18E-16 and 7.71E-20, respectively). The expression of the three miRNAs was higher in lung SCC compared to lung adenocarcinomas (p=8.19E-05, 1.04E-33 and 3.37E-95, respectively), suggesting unique roles of these miRNAs in the lung SCC development. Using miRNA mRNA target prediction tools (miRANDA and TargetScan) and in the combination with a literature search, we found RUNX1T1 and SMAD4 were candidate tumor suppressor gene target for miRNA-1224-5p and miRNA-1248. Downregulation of RUNX1T1 and SMAD4 were confirmed in TCGA lung SCC dataset. Using real-time RT-PCR, we found that the same three miRNAs 1224-5p, 1248 and 944 are overexpressed in 3q amplified lung SCC cell lines H520 and HCC95. When miRNA inhibitors were used to repress the expression of miR-1224-5p or miR-1248 in H520, we found that RUNX1T1 and SMAD4 protein expression levels were increased, suggesting that these tumor suppressor genes are regulated by miR-1224-5p or miR-1248. Lastly, knockdown of miR-1224-5p or miR-1248 using miRNA inhibitors led to 35-60% growth inhibition in H520 and HCC95 cells. Together, these results indicate potential roles for miR-1245-5p and 1248 in regulating cell proliferation by directly targeting tumor suppressor genes RUNX1T1 or SMAD4 in lung SCC. This work provides, for the first time, evidence for overexpression of miRNAs at the 3q26-29 amplicon that might be implicated in the oncogenesis of lung SCC. The work is supported by the NIH Grant R01 CA102353 to PPM and Lung Cancer Research Foundation grant (2011) to JQ. Note: This abstract was not presented at the meeting. Citation Format: Jun Qian, Xiangming Ji, Yong Zou, Megan Hoeksema, Heidi Chen, Pierre P. Massion. Overexpression of chromosome 3q26-29 miRNAs in squamous cell carcinoma of the lung. [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 526. doi:10.1158/1538-7445.AM2014-526

e13563 Background: Squamous cell carcinoma of the lung (SCC) is a subset of lung cancer responsible for ~ 25% of non small cell lung carcinoma. Unfortunately, targeted therapies in SCC have not to ...

Background: SCLC (15% of lung cancers) exhibits: 1) rapid growth and early fatal metastasis; 2) neuroendocrine features; 3) high initial responsiveness to chemotherapy and radiation; 4) aggressive recurrence with 5% 5-year patient survival. Gene expression and mutation profiling efforts to identify oncogenic mutations, gene amplifications or signatures with clinical utility in SCLC have thus far been unfruitful. In addition, prognostic or diagnostic markers for SCLC are scarce. Hence, there is a dire need for investigating molecular subtypes and oncogenic drivers in SCLC. We hypothesize that deregulated networks, rather than single genes, drive SCLC phenotype. Results: We previously identified a SCLC-specific gene co-expression network (Blue module, by Weighted Gene Co-expression Network Analysis - WGCNA) from a lung cancer patient dataset, and derived a SCLC-specific hub network (SSHN) signature that: 1) separated SCLC from other lung cancer types and normal lung in both genomic and proteomic independent datasets; 2) identified 2 SCLC subtypes with high and low SSHN expression in both patient specimens and cultured cell lines. Spleen tyrosine kinase (SYK) was validated as a candidate oncogenic driver of one subtype, as SYK targeted small-interfering RNA significantly decreased viability via increased death in high SYK-expressing SCLC cell lines. Due to the lack of larger SCLC patient datasets, we have now applied the SSHN classifier to the 53 SCLC cell lines from the Cancer Cell Line Encyclopedia (CCLE) and validated the SSHN-high and low subtypes. From this larger dataset, it is evident that the SSHN-defined subtypes are not totally separate. Rather, they are connected by gradual intermediate shades. This gradation became clearer by applying WGCNA to SCLC cell lines from CCLE, which identified 2 gene co-expression modules – Blue and Turquoise, that overlap with modules from patient datasets described above. The Blue module is enriched in neuroendocrine signaling, the Turquoise in mesenchymal adhesion-related pathways. Eigengene expression of the 2 modules (MEblue, MEturquoise) is anti-correlated, and all 53 SCLC cell lines are distributed along this anti-correlation diagonal. Expression of the neuroendocrine marker CD56 is highest in cells at one end of this diagonal (MEblue-high cell lines), and decreases towards the other end (MEturquoise-high cell lines), whereas the mesenchymal marker CD44 has an opposite trend. Multi-dimensional flow cytometry data, visualized with viSNE, indicated that SCLC cell lines are heterogeneous with respect to several additional cell surface and cytoplasmic markers and that, in general, there is a gradient of expression of these markers that tends to correlate with the neuroendocrine (e.g., SYK) to mesenchymal (e.g., TGFbeta receptor II) phenotype gradient. Finally, at the neuroendocrine end of the phenotypic spectrum (MEblue-high) cells grow in suspension, whereas they become increasingly adherent towards the mesenchymal end (MEturquoise-high). Conclusion: Our data provide strong evidence for a heterogeneous phenotypic space in SCLC that may define distinct subtypes. This heterogeneity was previously unsuspected in human SCLC, although evidence for it was reported in genetic mouse models of SCLC {Calbo J, et.al, Cancer Cell, 2011}. Classification of human SCLC cell lines along a neuroendocrine to mesenchymal differentiation gradient should apply to human tumors as well, since the WGCNA network classifiers overlap. However, further studies in patients are warranted to prove the existence of distinct SCLC subtypes, as well as to probe their translational value for biomarkers and targeted treatment. Citation Format: Akshata Ramrao Udyavar, Megan Hoeksema, Kirsten Diggins, Jonathan Irish, Pierre P. Massion, Vito Quaranta. Phenotypic plasticity and heterogeneity in small cell lung cancer (SCLC): Novel molecular subtypes and potential for targeted therapy. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B27.

Introduction: We recently reported that solute carrier family A1 member 5 (SLC1A5) controls glutamine (GLN) uptake and modulates cell growth, oxidative stress and mTOR signalling pathway. SLC1A5 is a transmembrane, high affinity glutamine transporter that is overexpressed and located at the plasma membrane in non-small cell lung cancer (NSCLC). GLN deprivation has been shown to induce cell death in several types of cancers, however the exact mechanism by which SLC1A5 targeting affects NSCLC survival remains unknown. We hypothesize that inhibition of SLC1A5 deprives cells from GLN which induces autophagy which eventually leads to cell starvation and ultimately apoptotic cell death. Methods: To test our hypothesis we targeted SLC1A5 by siRNA or by its specific inhibitor, GPNA, in a panel of 6 NSCLC lung cancer cell lines (express high level of SLC1A5). For comparison 2 human bronchial epithelial cell lines (HBEs) (express low level of SLC1A5) were used. The effects of targeting SLC1A5 on growth, GLN uptake, ATP level, autophagy, and cell death were examined. Markers of autophagy and cell death were analysed using western blot, nuclear staining and cell cycle analysis. Results: SLC1A5 inactivation by GPNA or by siRNA resulted in a significant decrease in cellular GLN uptake in NSCLC cell lines, while HBE cell lines were unaffected. Decrease in GLN uptake was accompanied by an increase in autophagy as evident by decrease in cell size, increase of LC3-II and decrease if LC3-I markers of autophagy. In addition, we observed a significant drop in ATP levels by 2 folds with increasing doses of GPNA. Markers of internal apoptotic pathway that include decrease of mitochondrial potential and cleaved caspases-3 and 9 were detected in response to increasing doses of GPNA. No change was observed in external apoptotic markers, caspases 8 or 1. In addition a 2-10 fold increase (based on the cell line) of sub-G1 was observed in NSCLC cell lines but not in HBE cell lines. Conclusion: Our results show that targeting SLC1A5 in NSCLC cells induces apoptotic cell death. Cells starve due to GLN deprivation and undergo autophagy which leads to a decrease in ATP level resulting in apoptotic cell death. These findings suggest that targeting SLC1A5 may have therapeutic implications in NSCLC. Grant funding: Lung Cancer Research Foundation Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B161. Citation Format: Mohamed Hassanein, Jun Qian, Megan Hoeksema, Marie Jacobovitz, Fredrick T. Harris, Bradford Harris, Pierre P. Massion. SLC1A5 inactivation induces apoptosis-mediated cells death in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B161.

Chromosome 3q26-29 amplification is a critical genomic alteration in squamous cell carcinomas (SCC) of the lung. There remains no consensus on what driver genes may carry this amplicon throughout tumor progression and their functional implications. We hypothesized that the amplification level of a predefined amplicon in a tumor sample can be inferred based on gene expression data. The hypothesis was tested using the TCGA dataset with paired copy number and gene expression data and achieved an accuracy of 90%. Based on the TCGA dataset and six other independent gene expression datasets (GSE4573, GSE8894,GSE2109,GSE3141 and GSE17710), we inferred candidate 3q26-29 drivers by searching for genes that were consistently over-expressed in SCCs with a high-level of 3q26-29 amplification in all seven datasets. The top 20 candidate driver genes includes PSMD2, DCUN1D1,SENP2, ZNF639, FXR1, SENP5,PRKCI, PIK3CA and UBXN7, et al. Using NetWalker we further identified a potential driver network consisting of SENP2, DCUN1D1, UBXN7 and DVL3, which are involved in SUMOlyation and Neddylation pathway. The amplification and overexpression of four genes were confirmed by performing genome-wide Agilent 244k CGH profiling and 44k gene expression profiling on twenty-four SCC tumors. Knocking down using siRNA against SENP2 resulted in greatest cell growth inhibition on 3q26-28 amplified SCC cell line H520. Immunoprecipitation assay showed that SENP2 binds to endogenous MDM2 in H520 cells. Loss of SENP2 leads to increased SUMO1 expression and elevated p53 expression associated with a decrease in MDM2 expression. Furthermore, knocking down SENP2 reduced the expression of DCUN1D1 and DVL3 but not UBXN7 in H520 cells. Together these data suggest that SENP2-mediated desumoylation and crosstalk with the Neddylation pathway contribute to lung SCC progression. Citation Format: Jun Qian, Jing Wang, Megan D. Hoeksema, Yong Zou, Allan V. Espinosa, Jamshedur Rahman, Bing Zhang, Pierre P. Massion. Integrative genomics and expression analysis reveals enhanced SENP2-mediated desumoylation and a crosstalk with Neddylation pathway in squamous cell carcinoma of the lung. [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 1804. doi:10.1158/1538-7445.AM2013-1804

Oncogenic mechanisms in SCLC remain poorly understood leaving this tumor with the worst prognosis among all lung cancers. Unlike other cancer types, traditional genomic approaches have been of limited success in SCLC. For instance, no patterns of prevalent mutations associated with SCLC progression or targeted therapeutics have emerged. Gene expression profiling based on differential expression have yielded potentially useful signatures in SCLC, but they have proven to be independent of each other and with minimal overlap, reducing translational potential. Nonetheless, since integrative systems biology studies of SCLC are warranted, we hypothesized that gene co-expression analyses can unveil key signaling hubs of specific networks that regulate SCLC proliferation and survival. We applied weighted gene co-expression network analysis (WGCNA) to a lung cancer patient gene expression dataset comprising of normal lung (NL), SCLC and Non-SCLC (NSCLC) such as squamous cell carcinoma and adenocarcinoma, and identified hierarchical gene networks highly specific to SCLC. The hubs of these SCLC-specific networks formed a SCLC-specific hub network (SSHN) signature that: 1) classified SCLC from NSCLC and NL on 3 independent patient and 1 cell line datasets; 2) was validated in independent RNAseq and shotgun proteomic patient datasets; 3) identified 2 SCLC subtypes with high and low SSHN expression in patients and cell lines. Three top SSHN hubs, Fyn, Syk and BRCA1, are involved in oxidative stress (OSR) and DNA damage response (DDR), respectively. At the protein level, Fyn, Syk and BRCA1 were significantly co-expressed in tissue microarrays and classified SCLC patients into 2 subtypes: High and Low Fyn/Syk/BRCA1 (F/S/B). High F/S/B staining significantly correlated with longer recurrence-free survival of patients treated with standard chemotherapy, suggesting that efficient OSR and DDR may enhance chemo response. This possibility was tested in established SCLC cell lines, which also could be separated into high vs low F/S/B groups corresponding to the clinical subtypes. High F/S/B cell lines exhibited lower proliferative rates, suggesting that OSR and DDR may still exert influence over cell cycle. Syk knockdown in these cell lines caused significant reduction in cellular viability and proliferation, as well as increased levels of Reactive Oxygen Species (ROS). Induction of oxidative stress in High F/S/B cell lines resulted in early activation of Fyn, Syk, p38, Erk and Akt. Thus, OSR may be overactive in SCLC and play a role in its natural history and response to treatment. In summary, our WGCNA combinatorial approach yielded novel insights into SCLC pathogenesis, separated two novel SCLC molecular subtypes with distinct functional OSR and DDR properties, and pointed to unsuspected potential therapeutic targets. Citation Format: Akshata R. Udyavar, Megan D. Hoeksema, Jonathan Clark, Yong Zou, Ming Li, Chiu-Lan (Heidi) Chen, Rosana Eisenberg, Alexander Statnikov, Daniel C. Liebler, John Field, Yu Shyr, Lourdes Estrada, Vito Quaranta, Pierre P. Massion. Gene co-expression network analysis of small cell lung cancer (SCLC) identifies new subclasses and novel therapeutic targets including Src family kinase Fyn and Spleen Tyrosine Kinase Syk. [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 5216. doi:10.1158/1538-7445.AM2013-5216

15 Background: More than 60% of lung cancer patients are diagnosed with advanced disease due to a lack of early diagnosis tools. These patients are ineligible for surgical resection and have a poor prognosis. This situation could be avoided if we could quantify lung cancer specific biomarkers at sufficiently low concentrations, providing an indication of disease threat. Backscattering Inerferometry (BSI) is a label-free sensor with a simple optical train that is used to quantify biomarkers in complex matrices at picomolar to femtomolar levels. Here we demonstrate that BSI can enable lung cancer biomarker validation in complex, volume constrained samples and at detection limits significantly better than ELISA. Methods: A BSI dose response curve for Galectin-7 was constructed by incubating increasing concentrations (0-20 ng/mL) of recombinant galectin-7 with 100 ng/mL of polyclonal antibody. The samples were mixed at 300 RPM for 2 before being injected into the BSI instrument and measured in triplicate each day. All reagents used were obtained from a commercial ELISA kit. An ELISA dose response curve was similarly constructed using spiked serum and spiked plasma according to the manufacturer’s recommended procedure. Finally, 9 patient serum samples were quantified using BSI and ELISA for direct comparison of the two technologies. Results: BSI was used to quantify galectin-7 in spiked serum and patients samples. The lower limit of detection with standards was determined to be 0.5 ng/mL for ELISA in serum, 10 ng/mL for ELISA in plasma, and 0.04 ng/mL for BSI in serum. In the analysis of 9 patient serum samples, all were quantifiable using BSI, which enjoys low pg/mL detection limits, yet with ELISA only 5 samples contained a galectin-7 concentration high enough to be measured. In this small sample set there was a good correlation between disease state and galectin-7 concentration as measured by BSI. Conclusions: This study demonstrates that BSI is well-suited for biomarker detection and validation, having a greater dynamic operating range and much lower limits of detection than standard commercially available ELISA kits. Future work will focus on detecting other NSCLC biomarkers that are undetectable with currently available technology.