Lung Cancer Tissue Microarray Research Articles

Cytokeratin 18 knockdown decreases cell migration and increases chemosensitivity in non-small cell lung cancer.

Cytokeratin 18 (CK18) is a structural protein that is normally expressed in many single-layer epithelia. Previous studies have indicated that aberrant CK18 expression is associated with cancer progression. However, the functions of CK18 in lung cancer have not been fully elucidated. Here, we investigate the roles of CK18 in non-small cell lung cancer (NSCLC). CK18 protein expression was evaluated by immunohistochemistry in a lung cancer tissue microarray containing 129 cancer samples, and correlations between CK18 expression and clinicopathological characteristics and prognosis were analyzed. We then studied the effects of CK18 knockdown on cell motility and chemosensitivity in lung cancer cells. High CK18 expression was detected in 101/129 (78.3%) lung cancers. CK18 expression was significantly correlated to clinical stage, lymph node metastasis, the number of pathologically positive lymph nodes and recurrence and metastasis. Kaplan-Meier survival analysis showed that CK18 was a prognostic factor for overall survival (P=0.016) and disease-free survival (P=0.014). In addition, CK18 knockdown decreased cell migration and enhanced the sensitivity of lung cancer cells to paclitaxel. These findings indicate that CK18 plays an important role in lung cancer progression and may be a therapeutic target for NSCLC.

Elevated integrin α6β4 expression is associated with venous invasion and decreased overall survival in non–small cell lung cancer

Lung cancer carries a poor prognosis and is the most common cause of cancer-related death worldwide. The integrin α6β4, a laminin receptor, promotes carcinoma progression in part by cooperating with various growth factor receptors to facilitate invasion and metastasis. In carcinoma cells with mutant TP53, the integrin α6β4 promotes cell survival. TP53 mutations and integrin α6β4 overexpression co-occur in many aggressive malignancies. Because of the high frequency of TP53 mutations in lung squamous cell carcinoma (SCC), we sought to investigate the association of integrin β4 expression with clinicopathologic features and survival in non-small cell lung cancer (NSCLC). We constructed a lung cancer tissue microarray and stained sections for integrin β4 subunit expression using immunohistochemistry. We found that integrin β4 expression is elevated in SCC compared with adenocarcinoma (P<.0001), which was confirmed in external gene expression data sets (P<.0001). We also determined that integrin β4 overexpression associates with the presence of venous invasion (P=.0048) and with reduced overall patient survival (hazard ratio, 1.46; 95% confidence interval, 1.01-2.09; P=.0422). Elevated integrin β4 expression was also shown to associate with reduced overall survival in lung cancer gene expression data sets (hazard ratio, 1.49; 95% confidence interval, 1.31-1.69; P<.0001). Using cBioPortal, we generated a network map demonstrating the 50 most highly altered genes neighboring ITGB4 in SCC, which included laminins, collagens, CD151, genes in the EGFR and PI3K pathways, and other known signaling partners. In conclusion, we demonstrate that integrin β4 is overexpressed in NSCLC where it is an adverse prognostic marker.

MiR-1207-5p suppresses lung cancer growth and metastasis by targeting CSF1.

We previously reported that miR-1207-5p can inhibit epithelial-mesenchymal transition (EMT) induced by growth factors such as EGF and TGF-β, but the exact mechanism is unclear. Here we identified that Colony stimulating factor 1 (CSF1) is a target gene of miR-1207-5p. CSF1 controls the production, differentiation and function of macrophage and promotes the release of proinflammatory chemokines. We showed that miR-1207-5p inhibited lung cancer cell A549 proliferation, migration and invasion in vitro, and suppressed the STAT3 and AKT signalings. miR-1207-5p overexpression can increase HUVEC angiogenesis, and can modulate the M2 phenotype of macrophage. miR-1207-5p also significantly inhibited A549 cells metastasis in a nude mouse xenograft model. miR-1207-5p and CSF1 expression levels and their relationship with lung cancer survival and metastasis status were assayed by means of a lung cancer tissue microarray. Macrophage is an essential part of the tumor microenvironment, thus the miR-1207-5p-CSF1 axis maybe a new regulator of lung cancer development through modulating the tumor microenvironment.

High expression of AGR2 in lung cancer is predictive of poor survival.

BackgroundAnterior gradient 2 (AGR2) is a protein disulfide isomerase-like protein widely expressed in many normal tissues as well as cancers. In our study, non-neoplastic bronchial epithelial cells as well as non-small cell lung cancer (NSCLC) cells express AGR2 protein.MethodsAGR2 expression was analyzed on lung tissue microarrays. Tumor staining was correlated with clinical outcomes.ResultsOn a lung cancer tissue microarray using immunohistochemistry, expression levels in cancer showed generally decreasing intensities in order from adenocarcinomas with mucinous components, other adenocarcinomas, squamous carcinomas, to large cell carcinomas. The study cohort was comprised of 400 cases. As a group, there was a slight trend of lower expression with increasing tumor grade. AGR2 expression level was a significant predictor of overall survival in younger patients only. Patients under 65 with lower levels showed a significantly better survival for both men and women. Patients over 65, in contrast, showed no such trend.ConclusionsNearly all NSCLC tumors show AGR2 expression. Lung cancer expression of AGR2 has prognostic value for younger patients.

Abstract 2275: S100A4/metastasin-1 promotes lung cancer cell invasion and associates with decreased overall survival among patients with adenocarcinoma

Abstract The metastatic nature of advanced non-small cell lung cancer (NSCLC) is associated with therapeutic resistance, quick time to progression, and poor prognosis. This dismal outcome remains until we gain a better understanding of the crucial drivers of the metastatic process and the power to effectively target them. S100A4 (metastasin-1) is a tumor metastasis associated protein and an epithelial to mesenchymal transition (EMT) marker. S100A4 contributes to several hallmarks of cancer such as anti-apoptosis, proliferation, and therapeutic resistance. This protein has been implicated in the progression of different types of cancer, including breast, colon and pancreas. However, the impacts of S100A4 signaling in lung cancer progression and its potential use as a target for therapy have not been properly explored. Here, using established lung cancer cell lines, we demonstrate that S100A4 is overexpressed in about 45% of lung cancer cell lines tested, both at the mRNA and protein levels. To address the biological action of S100A4 overexpression in lung cancer cells, loss-of function and gain-of function experiments were performed. We found that inhibition of S100A4 by shRNA in A549 and H460 lung cancer cell lines reduced cell proliferation and invasion and decreased 3D invasive growth, while exogenous overexpression of S100A4 in H1299 cells increased invasive potential. In order to examine S100A4 expression in patient-derived tissues, we constructed a lung cancer tissue microarray (TMAs, N = 212) and analyzed it using immunohistochemistry. We found that S100A4 is preferentially overexpressed in lung adenocarcinoma (P = 0.0007) when compared to squamous cell carcinoma, which was confirmed using publicly available gene expression datasets (TCGA). Furthermore, we found that S100A4 overexpression is associated with the presence of lymphovascular invasion (P = 0.0189) and with decreased overall survival (Hazard ratio 2.14, 95% confidence interval 1.08-4.22, P = 0.0243) among patients with lung adenocarcinoma. To elucidate the mechanisms by which S100A4 promotes lung cancer invasive capacity, we focused on the NF-κB pathway. We observed that depletion of S100A4 by shRNA inhibited NF-κB activity and decreased TNFα-induced MMP9 expression. Furthermore, inhibition of the NF-κB/MMP9 axis decreased lung cancer cell invasive potential. Collectively, this study highlights the importance of the S100A4/NF-κB/MMP9 axis in lung cancer invasion and metastasis and provides rationale for targeting S100A4 to combat lung cancer. Citation Format: Rachel L. Stewart, Brittany L. Carpenter, Dava S. West, Teresa Knifley, Chi Wang, Heidi L. Weiss, Tamas S. Gal, Kathleen L. O'Connor, Min Chen. S100A4/metastasin-1 promotes lung cancer cell invasion and associates with decreased overall survival among patients with adenocarcinoma. [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 2275. doi:10.1158/1538-7445.AM2015-2275

An aberrant nuclear localization of E-cadherin is a potent inhibitor of Wnt/β-catenin-elicited promotion of the cancer stem cell phenotype.

Several studies suggest that Wnt signaling contributes to reprogramming and maintenance of cancer stem cell (CSC) states activated by loss of membranous E-cadherin expression. However, E-cadherin's exact role in Wnt/β-catenin-mediated promotion of the CSC phenotype remains unclear. Recently, a significant positive correlation has been observed between the expression of nuclear (an aberrant nuclear localization) E-cadherin and β-catenin in gastric and colorectal carcinomas. Here we conducted a series of in-vitro and in-vivo studies to show that the β-catenin/TCF4 interaction was abolished by E-cadherin and was correlated with its nuclear localization, and consequently decreased β-catenin/TCF4 transcriptional activity. Nuclear E-cadherin was a negative regulator of Wnt/β-Catenin-elicited promotion of the CSC phenotype. Using immunohistochemistry on lung cancer tissue microarrays, we found that changes in subcellular location of E-cadherin may be described by tumor grade and stage, suggesting cellular redistribution during lung tumorigenesis. Furthermore, nuclear E-cadherin expression was more significantly inversely correlated with CD133 (a lung CSC marker) expression (P<0.005) than total E-cadherin expression (P<0.05), suggesting that lung cancer as defined by nuclear E-cadherinLow/nuclear β-cateninHigh/CD133High biomarkers has superior prognostic value over total E-cadherinLow/nuclear β-cateninHigh/CD133High.

Ribonucleotide reductase subunit M2 predicts survival in subgroups of patients with non-small cell lung carcinoma: effects of gender and smoking status.

BackgroundRibonucleotide reductase catalyzes the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. The functional enzyme consists of two subunits - one large (RRM1) and one small (RRM2 or RRM2b) subunit. Expression levels of each subunit have been implicated in prognostic outcomes in several different types of cancers.Experimental DesignImmunohistochemistry for RRM1 and RRM2 was performed on a lung cancer tissue microarray (TMA) and analyzed. 326 patients from the microarray were included in this study.ResultsIn non-small cell lung cancer (NSCLC), RRM2 expression was strongly predictive of disease-specific survival in women, non-smokers and former smokers who had quit at least 10 years prior to being diagnosed with lung cancer. Higher expression was associated with worse survival. This was not the case for men, current smokers and those who had stopped smoking for shorter periods of time. RRM1 was not predictive of survival outcomes in any subset of the patient group.ConclusionRRM2, but not RRM1, is a useful predictor of survival outcome in certain subsets of NSCLC patients.

Targeting Nucleophosmin 1 Represents a Rational Strategy for Radiation Sensitization

To test the hypothesis that small molecule targeting of nucleophosmin 1 (NPM1) represents a rational approach for radiosensitization. Wilde-type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine whether radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived non-small-cell lung cancer (NSCLC) tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double- strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci that colocalized with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is overexpressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity.

JNK Signaling Mediates EPHA2-Dependent Tumor Cell Proliferation, Motility, and Cancer Stem Cell–like Properties in Non–Small Cell Lung Cancer

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here, we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a c-JUN-NH2-kinase (JNK) inhibitor, recapitulated defects in EPHA2-deficient tumor cells, whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells (CSC). RNA interference-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH(+) cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH(+) populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung CSCs. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in CSC function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. Cancer Res; 74(9); 2444-54. ©2014 AACR.

The utility of a novel triple marker (combination of TTF1, napsin A, and p40) in the subclassification of non-small cell lung cancer.

In lung cancer, targeted therapies depend on accurate histological subclassification of the tumor. The majority of lung cancers can be subclassified based on hematoxylin and eosin staining; however, classification may be difficult in small biopsies. In this study, we investigated the utility of a newly developed triple marker (combination of TTF1/Napsin A/p40) and compared the sensitivity and specificity of this novel marker with individual markers in the subclassification of non-small cell lung carcinomas. Lung cancer tissue microarrays were constructed using surgical resection material from the Johns Hopkins Hospital. They included 77 adenocarcinomas (ADCs), 77 squamous cell carcinomas (SqCCs), and 46 cases of metastatic lung ADCs. Immunostaining patterns of all markers were scored semi-quantitatively and compared. In ADCs, the sensitivity and specificity of the triple marker were 93.5% and 77.5%, respectively. The sensitivity and specificity of TTF1 and Napsin A were 85.7% and 75.0%, and 89.6% and 90.0%. In SqCCs, the sensitivity and specificity of the triple marker were 88.3% and 92.5%, while the p40, p63 and CK5/6 showed 80.5% and 90.0%; 93.5% and 80.0%; and 89.6% and 80.0%. In addition, the sensitivity and specificity of the triple marker in metastatic ADCs showed 71.7% and 73.5%, respectively. Our triple marker (combination of TTF1/Napsin A/p40) showed a similar sensitivity and specificity for the subclassification of NSCLC when compared to individual markers. Our study not only demonstrates a useful combination of immunomarkers but also optimally conserves tissue for molecular marker testing.

Abstract B17: Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma.

Abstract Background: Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify the upstream pathways that regulate Sox9 expression in lung cancer as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling pathways. SOX9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse SOX9 promoter are not conserved in humans. Results: We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. We validated the data by identifying significant overlap in Sox9 and Hes1 protein expression levels in a human lung cancer tissue microarray. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is immediately downstream of Notch1, but not Notch3, in lung adenocarcinoma cell lines. By ChIP and luciferase reporter assays, we located the previously unidentified human RBPjk binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the SOX9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling, that TGF-β upregulates both Sox9 and Notch1 expression, and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Sox9 overexpression led to an induced EMT phenotype, characterized by increased cell invasion/migration, and EMT-related gene expression changes. Sox9 mRNA expression correlated with EMT-like gene expression signatures in several gene expression microarray datasets, and Sox9 protein levels were negatively correlated with expression of the epithelial marker, E-cadherin, in a lung cancer tissue microarray. Our data also demonstrate that Notch1-induced EMT in lung adenocarcinoma is mediated, at least partly, through Sox9. Conclusion: These results establish Sox9 as a key Notch1 target gene mediating Notch1- and TGF-β- induced EMT, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen Capaccione, Xuehui Hong, Katherine M. Morgan, Wenyu Liu, Thaddeus Allen, J. Michael Bishop, Sharon R. Pine. Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. [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 B17.

TUSC1, a putative tumor suppressor gene, reduces tumor cell growth in vitro and tumor growth in vivo.

We previously reported the identification of TUSC1 (Tumor Suppressor Candidate 1), as a novel intronless gene isolated from a region of homozygous deletion at D9S126 on chromosome 9p in human lung cancer. In this study, we examine the differential expression of TUSC1 in human lung cancer cell lines by western blot and in a primary human lung cancer tissue microarray by immunohistochemical analysis. We also tested the functional activities and mechanisms of TUSC1 as a tumor suppressor gene through growth suppression in vitro and in vivo. The results showed no expression of TUSC1 in TUSC1 homozygously deleted cells and diminished expression in some tumor cell lines without TUSC1 deletion. Interestingly, the results from a primary human lung cancer tissue microarray suggested that higher expression of TUSC1 was correlated with increased survival times for lung cancer patients. Our data demonstrated that growth curves of tumor cell lines transfected with TUSC1 grew slower in vitro than those transfected with the empty vector. More importantly, xenograph tumors in nude mice grew significantly slower in vivo in cells stably transfected with TUSC1 than those transfected with empty vector. In addition, results from confocal microscopy and immunohistochemical analyses show distribution of TUSC1 in the cytoplasm and nucleus in tumor cell lines and in normal and tumor cells in the lung cancer tissue microarray. Taken together, our results support TUSC1 has tumor suppressor activity as a candidate tumor suppressor gene located on chromosome 9p.

Polyisoprenylated methylated protein methyl esterase as a potential novel target in lung cancer therapy

Polyisoprenylated monomeric G‐proteins such as Ras, Rho and Rac play important roles in lung carcinogenesis. Targeting the polyisoprenylation pathway enzymes presents therapeutic opportunities but polyisoprenylated methylated protein methyl esterase (PMPMEase) has not been explored. PMPMEase expression and activity were investigated in lung cancer cells, tissues and tissue microarrays (TMAs). PMPMEase activity was significantly elevated in lung cancer cases relative to normal adjacent tissues (p &lt; 0.001), correlating with PMPMEase protein levels. Intense PMPMEase immunoreactivity was observed in 88.3% of over 400 lung cancer cases on TMAs (p &lt; 0.0001). Specific PMPMEase activities in A549 and H460 cells were 1.6‐ and 1.5‐fold higher than in WI‐38 cells, respectively. Exposure of the cells to specific PMPMEase inhibitor altered F‐actin organization and induced apoptosis with EC50 of 3.0 μg/ml for A549 and 9.6 μg/ml for H460 cells. The PMPMEase overexpression and hyperactivity in lung cancer and the apoptosis resulting from its inhibition is a strong indication of its role in lung cancer progression. This implies that PMPMEase would be a strong candidate biomarker for diagnosis and target for therapy in lung cancer.

Reproducibility of Immunohistochemical Scoring for Epidermal Growth Factor Receptor Expression in Non–Small Cell Lung Cancer: Round Robin Test

The addition of cetuximab to first-line chemotherapy substantially prolonged survival in patients with advanced non-small cell lung cancer whose tumors expressed high levels of epidermal growth factor receptor (EGFR; immunohistochemistry score of ≥200 on a scale of 0-300). To evaluate the interobserver reproducibility of this EGFR immunohistochemistry scoring system, based on both the tumor cell membrane staining intensity (graded 0-3+) and the percentage of cells staining at each intensity. In parts 1 (initial feasibility study) and 2 of this 2-part round robin test, sections of different non-small cell lung cancer tissue microarrays were stained in a central reference laboratory. Following reference evaluation, EGFR expression in 30 selected tumor cores was characterized in serial sections by lung cancer pathology specialists. The reproducibility of scoring by different raters was assessed. Analysis of between-rater agreement was based on the allocation of EGFR immunohistochemistry scores into low- (<200) and high- (≥200) EGFR expression groups. After discussion with raters of the issues impacting reproducibility identified in part 1 and following adjustment of processes, part 2 of the round robin test showed a high interobserver agreement in EGFR immunohistochemistry scoring, with an overall concordance rate of 90.9% and a mean κ coefficient of 0.812. Specimens with a reference EGFR immunohistochemistry score of lower than 200 and of 200 or higher showed mean concordance rates of 94.7% and 85.6%, respectively. After appropriate training, assessing EGFR expression by this immunohistochemistry-based method allowed a highly reproducible allocation of non-small cell lung cancers into clinically relevant high- or low-EGFR expression groups.

Effects of Cancer-Associated EPHA3 Mutations on Lung Cancer

Cancer genome sequencing efforts recently identified EPHA3, which encodes the EPHA3 receptor tyrosine kinase, as one of the most frequently mutated genes in lung cancer. Although receptor tyrosine kinase mutations often drive oncogenic conversion and tumorigenesis, the oncogenic potential of the EPHA3 mutations in lung cancer remains unknown. We used immunoprecipitation, western blotting, and kinase assays to determine the activity and signaling of mutant EPHA3 receptors. A mutation-associated gene signature was generated from one large dataset, mapped to another training dataset with survival information, and tested in a third independent dataset. EPHA3 expression levels were determined by quantitative reverse transcription-polymerase chain reaction in paired normal-tumor clinical specimens and by immunohistochemistry in human lung cancer tissue microarrays. We assessed tumor growth in vivo using A549 and H1299 human lung carcinoma cell xenografts in mice (n = 7-8 mice per group). Tumor cell proliferation was measured by bromodeoxyuridine incorporation and apoptosis by multiple assays. All P values are from two-sided tests. At least two cancer-associated EPHA3 somatic mutations functioned as dominant inhibitors of the normal (wild type) EPHA3 protein. An EPHA3 mutation-associated gene signature that was associated with poor patient survival was identified. Moreover, EPHA3 gene copy numbers and/or expression levels were decreased in tumors from large cohorts of patients with lung cancer (eg, the gene was deleted in 157 of 371 [42%] primary lung adenocarcinomas). Reexpression of wild-type EPHA3 in human lung cancer lines increased apoptosis by suppression of AKT activation in vitro and inhibited the growth of tumor xenografts (eg, for H1299 cells, mean tumor volume with wild-type EPHA3 = 437.4 mm(3) vs control = 774.7 mm(3), P < .001). Tumor-suppressive effects of wild-type EPHA3 could be overridden in trans by dominant negative EPHA3 somatic mutations discovered in patients with lung cancer. Cancer-associated EPHA3 mutations attenuate the tumor-suppressive effects of normal EPHA3 in lung cancer.

RUNX3 Downregulation in Human Lung Adenocarcinoma is Independent of p53, EGFR or KRAS Status

RUNX3 aberrations play a pivotal role in the oncogenesis of breast, gastric, colon, skin and lung tissues. The aim of this study was to characterize further the expression of RUNX3 in lung cancers. To achieve this, a lung cancer tissue microarray (TMA), frozen lung cancer tissues and lung cell lines were examined for RUNX3 expression by immunohistochemistry, while the TMA was also examined for EGFR and p53 expression. RUNX3 promoter methylation status, and EGFR and KRAS mutation status were also investigated. Inactivation of RUNX3 was observed in 70% of the adenocarcinoma samples, and this was associated with promoter hypermethylation but not biased to EGFR/KRAS mutations. Our results suggest a central role of RUNX3 downregulation in pulmonary adenocarcinoma, which may not be dependent of other established cancer-causing pathways and may have important diagnostic and screening implications.

Abstract 2416: Neuropilin-2 is upregulated during EMT in lung cancer

Abstract Neuropilins (NRP1 and NRP2) are high affinity receptors for the class-3 semaphorins, cell guidance molecules involved in tissue development, immune responses, angiogenesis and cancer. Semaphorins affect cell protrusion, spreading and adhesion, and can trigger migratory responses often in a repulsive manner depending on the cellular context. Interestingly, neuropilins are also receptors for galectin-1 and growth factors including VEGF, PlGF, EGF and TGFβ. We previously cloned the SEMA3F gene from chromosome 3p21.3, which undergoes homozygous deletion and frequent loss of heterozygosity in lung cancer. We found that SEMA3F levels were inversely correlated with tumor aggressiveness in human lung cancer and confirmed its tumor suppressor activity in experimental xenograft models. We subsequently discovered that SEMA3F is directly downregulated by ZEB1, a transcription factor involved in the epithelial to mesenchymal transition (EMT). In the present study, we asked whether SEMA3F specific receptor, NRP2, was also regulated during the EMT process. TGFβ, a physiologic EMT inducer, stimulated NRP2 expression in two NSCLC cell lines. Forced expression of ZEB1, but not Snail, also induced NRP2. Conversely, ZEB1 and Snail inhibition blocked NRP2 upregulation by TGFβ. Importantly, inhibiting NRP2 or ZEB1 expression reduced TGFβ-induced migration in an equivalent manner. In lung cancer tissue microarrays, NRP1 and NRP2 were preferentially expressed in the tumor compartment, whereas the EMT-related transcription factors, ZEB1 and Snail, were predominantly expressed in the stroma. Of note, NRP2 was also expressed in stromal cells and was significantly associated with both ZEB1 and a higher (worse) tumor grade. Since NRP2 is the highest affinity receptor for SEMA3F, our results suggest that loss of SEMA3F coupled with increased NRP2 would facilitate the binding of growth factors to NRP2 to further promote EMT and metastasis. Therefore, targeting NRP2 could be an important therapeutic approach against EMT in lung cancer. 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 2416. doi:1538-7445.AM2012-2416

Abstract 1042: Down-regulation of UTX contributes to lung cancer progression

Abstract Smoking, the main risk factor leading to lung cancer, causes both genetic and epigenetic changes resulting in aberrant gene expression. UTX is a histone demethylase that has been implicated in many cancers and has been shown to regulate both cell cycle and differentiation genes. However, the role of UTX has not been elucidated in lung cancer. We hypothesized that smoking injury leads to a down regulation in UTX expression. This results in an inability of repairing airway epithelial cells to differentiate, resulting in premalignant lesions and ultimately invasive lung cancer. Examination of published lung cancer data sets revealed a down-regulation of Utx gene expression in patients with poorer prognosis, tumor grade, tumor recurrence and in smokers. To assess the involvement of UTX protein expression in lung cancer we stained a lung cancer tissue microarray and found a significant correlation between down-regulation of UTX protein expression and poorer prognosis in metastasis (p=0.0005) and smokers (p= 0.02). UTX expression was found to be significantly lower in squamous cell carcinoma compared to adenocarcinoma, linking down-regulation of UTX in a diseased caused solely by smoking. To test if smoking directly affects Utx expression we treated mouse airway epithelial cells at air-liquid interface with media infused with cigarette smoke for 9 days. After this short time point of acute smoke exposure there was a significant down regulation of Utx gene expression (p=0.03). We carried out UTX overexpression studies to assess the role of UTX in lung cancer cells lines and found up-regulation of UTX results in up-regulation of genes involved in lung development and differentiation, including Nrf2, FoxJ2 and FZD4. Our finding support the hypothesis that smoking causes a reduction in UTX that affects the proliferation and differentiation of airway epithelial cells contributing to lung cancer progression. Re-activation of UTX therefore may be a potential therapeutic target for the prevention of lung cancer progression. 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 1042. doi:1538-7445.AM2012-1042

Highly expressed SLC35F2 in non-small cell lung cancer is associated with pathological staging

Homo sapiens solute carrier family 35 member F2 (SLC35F2) is highly homologous to the lung squamous cell cancer-related gene, LSCC3, which is highly expressed in lung squamous cell tumour tissues. However, the clinical implication of the SLC35F2 gene in tumour development and progression remains unclear. An affinity-purified polyclonal antibody raised against the human SLC35F2 peptide was used in the immunohistochemical analysis of a non-small cell lung cancer (NSCLC) tissue microarray of human NSCLC (n=129). SLC35F2 protein was also analysed with the same antibody using Western blotting. Total RNAs were extracted from tumour tissues (n=43) and from laser-dissected tumour cells (n=9). SLC35F2 gene expression was detected by fluorescent real-time quantitative PCR and compared to the expression in the corresponding adjacent normal lung tissues. It was found that both the SLC35F2 protein (by IHC analysis) and the SLS35F2 gene transcripts (by Q-PCR analysis) were expressed at significantly higher levels in the NSCLC tumour tissues than in the corresponding adjacent normal lung tissues (p<0.001 and =0.015, respectively). There was a significant correlation between the SLC35F2 transcript and pathological staging (r=0.219, p=0.029), although the correlation between SLC35F2 protein and the staging was not significant (r=0.175). SLC35F2 was highly expressed in NSCLC tissues and the levels of expression, in particular the levels of the SLC35F2 transcript, were associated with NSCLC pathological staging. SLC35F2 appears to have a significant prognostic value in NSCLC.

Loss of Cytoplasmic CDK1 Predicts Poor Survival in Human Lung Cancer and Confers Chemotherapeutic Resistance

The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery.