Non-tumor Lung Tissues Research Articles

Expression of RNA-binding motif 10 is associated with advanced tumor stage and malignant behaviors of lung adenocarcinoma cancer cells.

This study assessed RNA-binding motif 10 expression in lung adenocarcinoma tissues and examined the role and mechanism of RNA-binding motif 10 in the regulation of lung adenocarcinoma malignancy. Lung adenocarcinoma and corresponding adjacent non-tumor lung tissues from 41 patients were subjected to reverse transcription-polymerase chain reaction and Western blot assessment to detect RNA-binding motif 10 expression. Recombinant lentivirus carrying RNA-binding motif 10 complementary DNA was used to infect lung adenocarcinoma cell lines, A549 and H1299 cells. Complementary DNA microarray was used to profile RNA-binding motif 10-regulated genes. Levels of RNA-binding motif 10 messenger RNA and protein were significantly lower in lung adenocarcinoma tissues than those in paired non-tumor tissues (p < 0.001). Reduced RNA-binding motif 10 expression was found to be associated with an advanced tumor stage. RNA-binding motif 10 overexpression inhibited viability and colony formation capacity of lung adenocarcinoma cell lines and induced cell-cycle arrest at G0/G1 phase in A549 cells and at S phase in H1299 cells. Complementary DNA microarray analysis identified 304 upregulated and 386 downregulated genes induced by RNA-binding motif 10 overexpression, which may be involved in cancer, focal adhesion, peroxisome proliferator-activated receptor-regulated gene pathway, cytokine-cytokine receptor interaction, mitogen-activated protein kinase signaling, complement and coagulation cascades, platelet amyloid precursor protein pathway, extracellular matrix-receptor interaction, and small cell lung cancer-related genes. Expression of FGF2, EGFR, WNT5A, NF-κB, and RAP1A was downregulated, whereas expression of AKT2, BIRC3, and JUN was upregulated. RNA-binding motif 10 messenger RNA and protein were reduced in lung adenocarcinoma tissues, and RNA-binding motif 10 overexpression inhibited lung adenocarcinoma cancer cell malignant behavior in vitro. Molecularly, RNA-binding motif 10 regulates many gene pathways involving in the tumor development or progression.

SPOP promotes SIRT2 degradation and suppresses non-small cell lung cancer cell growth

SIRT2 is a NAD-dependent deacetylase and inhibition of SIRT2 has a broad anticancer activity. Here we report that SPOP binds to SIRT2 and mediates its degradation by the 26S proteasome, which can be blocked by MG132 treatment. We also found that the levels of SPOP significantly decreased, while the levels of SIRT2 significantly increased in non-small cell lung cancer (NSCLC) cell lines, compared to normal bronchial epithelial cell line and NSCLC specimens, compared to the paired non-tumor lung tissue. Furthermore, SPOP can suppress NSCLC cell growth. Notably, mutations in NSCLC inhibit the abilities of SPOP to degrade SIRT2 and suppress NSCLC cell growth. These results reveal a novel regulation of SIRT2 by SPOP mediated degradation, which is important for the growth of lung tumor cells.

P1.02-037 Mutations of EGFR and KRAS Genes in Belorussian Patients With Non-Small Cell Lung Cancer

Mutations of the epidermal growth factor receptor (EGFR) cause increased activation of EGFR and sensitivity of patients with non-small cell lung cancer (NSCLC) to tyrosine kinase inhibitors (TKIs). The effectiveness of TKIs also depends on mutations of the KRAS gene that encodes small GTPase that is activated in response to a signal from EGFR and transmits it to the cascade of tyrosine kinases. Treatment of patients with KRAS mutations by TKIs is inefficient. Therefore, the research of these alterations plays an important role in determining the possibility of additional factors prognosis of NSCLC and adjusting individual tumor therapy. The aim of this study is to identify mutations in the exons 19 and 21 EGFR gene and in the exon 2 KRAS gene in patients with NSCLC. Analysis of mutations in the EGFR and KRAS genes was performed by PCR followed by sequencing DNA, which was extracted from the tumor and non-tumor lung tissues and blood samples of 97 patients with NSCLC (71 men, 26 women). 51 people have an adenocarcinoma (AC) and 46 people - squamous cell carcinoma (SCC). Analysis of mutations in the EGFR gene showed that the frequency of classical mutations is 5,2% of deletions in exon 19 (p.E746_A750del and p.L747_P753>S) and 1,1% of p.L858R mutation in exon 21. All mutations were detected only in the tumor tissue of non-smoking women with AC. Thus, 27,3% of women with AK are carriers of mutations in EGFR gene. These types of mutations were not detected among men. Also in the researched group was identified silent mutation c.2508C>T in exon 21 in the tumor, non-tumor tissue and blood among 3,3% of patients. Analysis of mutations in exon 2 KRAS gene detected 3 types of mutations: p.G12D (1,03%), p.G12C (2,06%) и p.G13C (1,03%). The frequency of all mutations was 4,1% in the total group of patients. The mutations were found only in tumor tissues of men. 75% of mutations carriers are smokers. Analysis of KRAS gene mutations in association with the development of a specific histological type of lung cancer showed that mutations are more common in patients with AC (5,9%) than in patients with SCC (2,2%). Thus, in the researched group of patients mutations in the EGFR gene were found only among non-smoking women with AC, mutations in the EGFR gene were detected only among men independently of histological type of NSCLC.

MicroRNA-300 targets hypoxia inducible factor-3 alpha to inhibit tumorigenesis of human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most deadly human cancers. MicroRNA-300 acts as both tumor promoter and suppressor in different types of cancer. Here, we try to identify the function of microRNA-300 in human NSCLC. We compared MicroRNA-300 levels between tumor tissues versus paired adjacent non-tumor lung tissues from NSCLC patients, and in NSCLC versus normal lung cell lines. Effects of microRNA-300 on cell proliferation, invasion and migration were examined in vitro, and on tumor growth in vivo using a xenograft mouse model. Potential mRNA targets of microRNA-300 were predicted and underlying mechanism was explored. MicroRNA-300 expression was lower in both NSCLC tissues and cell lines. Overexpression of microRNA-300 inhibited proliferation, invasion and migration of NSCLC cells in vitro, and tumor growth in vivo. MicroRNA-300 could directly bind to the 3'-UTR of hypoxia inducible factor-3 alpha (HIF3α) mRNA, and inhibit both its mRNA and protein expressions. Restoring HIF3α expression could rescue the inhibitory effects of microRNA-300 on tumorigenesis of NSCLC both in vitro and in vivo. MicroRNA-300 is a tumor suppressor microRNA in NSCLC by downregulating HIF3α expression. Both microRNA-300 and HIF3α may serve as potential therapeutic targets in NSCLC treatment.

Higher Tissue Levels of Thymidylate Synthase Determined by ELISA Are Associated with Poor Prognosis of Patients with Lung Cancer.

Thymidylate synthase (TS) is essential in thymidylate biosynthesis and DNA replication. Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in pyrimidine catabolism and is important in catabolism of 5-fluorouracil (5-FU). The significance of TS and DPD expressed in lung cancer remains controversial. Here we analyzed the relationship between TS and DPD expression and clinicopathological features of lung cancer. Enzyme-linked immunosorbent assays (ELISAs) were used to measure TS and DPD levels in paired tumor and non-tumor lung tissues obtained from 168 patients (107 adenocarcinomas, 39 squamous cell carcinomas, and 22 others), who had operations at the Shinshu University Hospital from 2004 to 2007 and were followed up for a median of 57.0 months. TS and DPD expression levels were higher in tumor tissues, and TS expression levels were significantly lower in adenocarcinomas than those in other subtypes. In addition, patients with low TS levels survived longer compared with patents with high TS levels. By contrast, DPD expression levels were not correlated with overall patient survival. Importantly, patients with low TS and DPD levels exhibited significantly prolonged survival than those with high TS and DPD. Among the 168 patients, 59 patients were treated with tegafur-uracil (UFT), a DPD-inhibitory fluoropyrimidine, and the UFT-treated patients with high TS and high DPD levels showed worst prognosis. Our study demonstrates a significant correlation between low TS expression levels and long-term prognosis of patients with lung cancer. Thus, ELISA is a clinically useful method to measure TS and DPD expression in lung cancer tissues.

Monoclonal antibodies targeting non-small cell lung cancer stem-like cells by multipotent cancer stem cell monoclonal antibody library

Cancer stem cells (CSCs) are a rare subset of cancer cells that play a significant role in cancer initiation, spreading, and recurrence. In this study, a subpopulation of lung cancer stem-like cells (LCSLCs) was identified from non-small cell lung carcinoma cell lines, SPCA-1 and A549, using serum-free suspension sphere-forming culture method. A monoclonal antibody library was constructed using immunized BLAB/c mice with the multipotent CSC cell line T3A-A3. Flow cytometry analysis showed that 33 mAbs targeted antigens can be enriched in sphere cells compared with the parental cells of SPCA-1 and A549 cell lines. Then, we performed functional antibody screening including sphere-forming inhibiting and invasion inhibiting assay. The results showed that two antibodies, 12C7 and 9B8, notably suppressed the self-renewal and invasion of LCSLCs. Fluorescence-activated cell sorting (FACs) found that the positive cells recognized by mAbs, 12C7 or 9B8, displayed features of LCSLCs. Interestingly, we found that these two antibodies recognized different subsets of cells and their combination effect was superior to the individual effect both invitro and in vivo. Tissue microarrays were applied to detect the expression of the antigens targeted by these two antibodies. The positive expression of 12C7and9B8 targeted antigen was 84.4 and 82.5%, respectively, which was significantly higher than that in the non-tumor lung tissues. In conclusion, we screened two potential therapeutic antibodies that target different subsets of LCSLCs.

Human Lung Tissue Transcriptome: Influence of Sex and Age

BackgroundSex and age strongly influence the pathophysiology of human lungs, but scarce information is available about their effects on pulmonary gene expression.MethodsWe followed a discovery-validation strategy to identify sex- and age-related transcriptional differences in lung.ResultsWe identified transcriptional profiles significantly associated with sex (215 genes; FDR < 0.05) and age at surgery (217 genes) in non-involved lung tissue resected from 284 lung adenocarcinoma patients. When these profiles were tested in three independent series of non-tumor lung tissue from an additional 1,111 patients, we validated the association with sex and age for 25 and 22 genes, respectively. Among the 17 sex-biased genes mapping on chromosome X, 16 have been reported to escape X-chromosome inactivation in other tissues or cells, suggesting that this mechanism influences lung transcription too. Our 22 age-related genes partially overlap with genes modulated by age in other tissues, suggesting that the aging process has similar consequences on gene expression in different organs. Finally, seven genes whose expression was modulated by sex in non-tumor lung tissue, but no age-related gene, were also validated using publicly available data from 990 lung adenocarcinoma samples, suggesting that the physiological regulatory mechanisms are only partially active in neoplastic tissue.ConclusionsGene expression in non-tumor lung tissue is modulated by both sex and age. These findings represent a validated starting point for research on the molecular mechanisms underlying the observed differences in the course of lung diseases among men and women of different ages.

The Prognostic Effect of MAC30 Expression on Patients With Non-Small Cell Lung Cancer Receiving Adjuvant Chemotherapy.

The purpose of this study was to examine the MAC30 expression in non-small cell lung cancer and to evaluate its prognostic value on therapeutic response in patients with non-small cell lung cancer receiving postoperative chemotherapy. Among a total of 218 retrospective Chinese patients with non-small cell lung cancer, 164 patients receiving adjuvant chemotherapy were enrolled in this study. Real-time polymerase chain reaction was performed to confirm the expression of MAC30 messenger RNA in 32 cases of non-small cell lung cancer tumors with the corresponding nontumor lung tissues. The MAC30 protein expression in all specimens was analyzed by immunohistochemical staining. Moreover, we assessed the correlation of MAC30 expression with clinicopathological features, therapeutic response, and survival of patients. Here, we observed the increased expression of MAC30 messenger RNA in patients with non-small cell lung cancer compared to those in control samples. The overexpression of MAC30 was strongly associated with poor tumor differentiation, high tumor-node-metastasis stage, and lymph node metastasis. In addition, we observed that patients with increased MAC30 expression showed gloomy overall survival and disease-free survival. A multivariate analysis explicated that higher MAC30 expression was a valuable independent prognostic factor of poorer tumor differentiation, shorter overall survival, and disease-free survival in patients receiving chemotherapy. MAC30 could be a useful biomarker of tumor differentiation and outcome of patients with non-small cell lung cancer. Overexpression of MAC30 predicts a worse tumor differentiated stage and prognosis in patients with non-small cell lung cancer receiving adjuvant chemotherapy.

SRSF5: a novel marker for small-cell lung cancer and pleural metastatic cancer

ObjectivesSR-splicing factors (SRSFs) play important roles in oncogenesis. However, the expression of SRSF 5–7 proteins in lung cancer (LC) is unclear, and their use in the diagnosis of pleural diseases has never been assessed. We evaluated SRSF 5–7 protein levels in LC and their diagnostic potential for cancer cells in lung and pleural effusion (PE) and, for the dysregulated SRSFs, investigated their neutralization effect on LC. Materials and methodsSRSF 5–7 levels in lung tissue and PE cell lysate samples (n=453) were compared with the results of conventional tumor markers. Knockdown of SRSF gene expression was performed using small interfering RNAs on small-cell LC (SCLC) cell lines. ResultsIn lung tissue analysis, SRSF 5–7 levels were up-regulated in LC samples compared with non-tumoral lung tissue samples; they were markedly higher in SCLC than in adenocarcinoma or squamous cell carcinoma. SRSF5 showed the highest detection accuracy (89%) for total LC, and it was superior to that (74%) of carcinoembryonic antigen [CEA, a commonly used non-SCLC (NSCLC) marker]. Notably, the detection accuracies of the three SRSFs for SCLC were all 100% and higher than that (69%) of a pro-gastrin-releasing peptide (a well-known SCLC marker). In PE cell analysis, the detection accuracy (86%) of SRSF5 for malignant cells was highest among SRSFs and comparable to that (83%) of CEA. SRSF5 additionally detected 70% of CEA-missed non-NSCLC cases. Down-regulation of the SRSFs induced mild (SRSF5 and SRSF7) to remarkably (SRSF6) reduced cell proliferation. ConclusionsOur results demonstrated the up-regulated expression of SRSF 5–7 proteins in LC with much more profound up-regulation in SCLC than in NSCLC and suggest that up-regulation of the SRSFs is related to SCLC proliferation. Moreover, we identified SRSF5 as a novel detection marker for SCLC and pleural metastatic cancer cells.

Osteopontin Induces Epithelial-to-Mesenchymal Transitions in Human Lung Cancer Cells via PI3K/Akt and MEK/Erk1/2 Signaling Pathways

SESSION TITLE: Mechanisms of Lung Cancer SESSION TYPE: Original Investigation Slide PRESENTED ON: Saturday, April 16, 2016 at 02:15 PM - 03:45 PM PURPOSE: A multifunctional protein known as Osteopontin (OPN) has an important, yet poorly understood role in Non-Small Cell Lung Cancer (NSCLC) pathogenesis. OPN was reported to be associated with the epithelial-to-mesenchymal transition (EMT) of cells, promoting tumor cells metastasis, but its mechanism remains unclear. METHODS: The present study investigates genetic variations, molecular functions, and/or biological processes of chromosome 4 genes between subtypes of lung cancer, all-the-while investigating patient survival rate of the selected target gene OPN. mRNA and protein expression of OPN, Vimentin, and E-cadherin were further validated in NSCLC tumors and their adjacent non-tumorous lung tissues, using immunohistochemistry in tissue microarray consisting of 208 cases of NSCLCs. RESULTS: We found that OPN and Vimentin overexpressed in NSCLC tissues and negatively correlated with E-cadherin expression. Furthermore, higher levels of OPN were correlated with lymph node metastasis, postoperative recurrence, or distal site metastasis in NSCLC patients. OPN-induced EMT was accompanied with increased cell proliferation and migration, through Akt and Erk1/2 signaling pathways. Interference to OPN expression resulted in the impaired proliferation and movement of lung cancer cells and the up-regulated expression of E-cadherin protein. Silencing of OPN in mouse xenograft models of human lung cancer significantly inhibited tumor growth. CONCLUSIONS: Our data indicate that OPN may serve as a switch during EMT which could be initiated by an autocrine mechanism or through PI3K/Akt and MAPK/Erk1/2 signaling pathways. CLINICAL IMPLICATIONS: OPN can be a potential prognostic marker to screen patients for unfavorable prognosis, which can be important for future clinical applications and represent a revolution in the target therapy for lung cancer. DISCLOSURE: The following authors have nothing to disclose: Lin Shi, Lin Wang, Xiang Wang No Product/Research Disclosure Information

MiR-99a regulates ROS-mediated invasion and migration of lung adenocarcinoma cells by targeting NOX4.

miR-99a is frequently downregulated in various types of human malignancies including lung adenocarcinoma. Recent studies have reported that miR-99a regulates cell growth and cell cycle progression by targeting mTOR, AKT1 and FGFR3. However, the underlying mechanisms involved in the modulation of invasion and migration by miR-99a remain elusive. In this study, we analyzed the relationship between the expression of miR-99a and clinical stage or metastasis in 90 matched lung adenocarcinoma and adjacent non-tumor lung tissues. Downregulation of miR-99a was significantly associated with advanced stage and tumor metastasis in lung adenocarcinoma patients, and it was found to be a poor prognostic factor in lung adenocarcinoma. Furthermore, functional experiments found that overexpression of miR-99a inhibited the proliferation, migration and invasion of lung adenocarcinoma A549 and Calu3 cells in vitro. We then identified NOX4 as a target gene of miR-99a and NOX4 mediated the inhibition of invasion and migration of lung adenocarcinoma cells by miR-99a. By targeting NOX4-mediated ROS production, miR-99a regulated the invasion and migration of lung adenocarcinoma cells. Moreover, overexpression of miR-99a significantly inhibited tumor growth in vivo. Immunohistochemical staining analysis of the mouse tumor tissues revealed that NOX4 levels were downregulated in the miR-99a treatment group, confirming the in vitro data of NOX4 as a direct target gene of miR-99a. Taken together, these data indicate for the first time that miR-99a directly regulates the invasion and migration in lung adenocarcinoma by targeting NOX4 and that overexpression of miR-99a may become a therapeutic strategy for lung adenocarcinoma.

SIRT2 inhibits non-small cell lung cancer cell growth through impairing Skp2-mediated p27 degradation.

Skp2 is a component of the E3 ubiquitin ligase which promotes the ubiquitination-associated degradation of a cyclin-dependent kinase inhibitor, p27, resulting in increases in non-small cell lung cancer (NSCLC) cell growth. We recently showed that down-regulation of Sirtuin deacetylases 2 (SIRT2) in NSCLC increased cancer cell growth through suppressing p27. However, the underlying mechanisms remain unknown. Here, we examined the relationship between SIRT2 and Skp2 in regulation of NSCLC cell growth through p27. We found that the levels of SIRT2 significantly decreased, while the levels of Skp2 significantly increased in NSCLC specimens, compared to the paired non-tumor lung tissue. The levels of SIRT2 and Skp2 inversely correlated. Low SIRT2 levels were associated with poor patients' survival. Moreover, in several lung cancer cell lines, the SIRT2 levels significantly decreased and the Skp2 levels significantly increased. Overexpression of SIRT2 promoted Skp2 deacetylation and degradation, resulting in increases in p27 and suppression of NSCLC cell growth, whereas knockdown of Skp2 inhibited Skp2 deacetylation and degradation, resulting in decreases in p27 and increases in NSCLC cell growth. The deacetylation of Skp2 by SIRT2 and degradation of p27 by Skp2 were significantly inhibited by histone deacetylase inhibitor and proteasome inhibitor, respectively. Finally, SIRT2 and Skp2 co-immunoprecipitated in NSCLC cells. Together, our data suggest that SIRT2 may induce Skp2 deacetylation and subsequent degradation to abolish the effects of Skp2 on p27 to affect NSCLC cell growth. Thus, re-expression of SIRT2 may be a promising strategy for treating NSCLC.

Prognostic and Predictive Role of Sirtuin1 Expression in Lung Adenocarcinoma.

SIRT1 is the homologue of sir2 in mammals, which is a nicotinamide adenine dinucleotide (NAD+) dependent histone deacetylase. Several studies have reported that the overexpression of Sirtuin1 (SIRT1) was associated with poor prognosis in various human cancers. However, little is known regarding the prognostic value of SIRT1 in lung adenocarcinoma. SIRT 1 expression in tissues of 60 lung adenocarcinoma (LAC) patients was evaluated using immunohistochemistry. The staining results were compared with clinicopathological characteristics and to the outcome of patients. The intensity of SIRT1 staining was remarkably higher in LAC tissues than in matched non-tumor lung tissues; high levels of SIRT1 expression were found in 40 of 60 (66.7%) LAC and 5 of 60 (8.3%) non-tumor tissues (p < 0.05). SIRT1 expression displayed a significant correlation with histological grade (p = 0.01). According to the Kaplan-Meier survival analysis, median OS was 49.0 months (95% confidence interval (CI), 39.139 - 58.861) in the SIRT1-low group and 22.0 months (CI: 14.572 - 29.428) in the SIRT1-high group. Compared with SIRT1-high expression patients, SIRT1-low expression patients had higher 5-year OS, and the difference was significant (p < 0.01). Multivariate analyses revealed that SIRT1-low expression was a significant prognostic factor. Taken together, our findings suggest that high expression of SIRT1 might be a potential unfavorable prognostic factor for patients with LAC.

Loss of MKK3 and MK2 Copy Numbers in Non-Small Cell Lung Cancer.

Identification of genetic alterations in members of the p38 mitogen-activated protein kinase (MAPK) pathway is important as these proteins have dynamic roles in tumor progression and may serve as potential therapeutic targets in cancer. We analyzed tumor and non-tumorous lung tissue of 233 non-small cell lung cancer (NSCLC) patients for the presence of copy number alterations (CNAs) in the MAPK kinase 3 (MKK3) and MAPK-activated kinase 2 (MK2) genes. We report frequent CNAs in MKK3 and MK2 genes in NSCLC. Copy number losses were detected in 31% of NSCLC tumors (odds ratio: 7.08, 95% confidence interval: 3.2-15.6, P<0.001) for the MKK3 gene and in 28% of tumors for the MK2 gene (odds ratio: 3.68, 95% confidence interval: 1.9-7.2, P<0.001). Several of the non-tumorous tissues showed an elevated MKK3 copy number, with a concurrent loss of this in 89% of the paired tumors. MKK3 gene deletions were significantly more frequent in squamous and large cell carcinoma than in adenocarcinoma. These data demonstrate a novel loss of MKK3 and MK2 genomic copy numbers in NSCLC tumors, and suggest these genes as interesting therapeutic candidates in NSCLC.

IMP3 Predicts Invasion and Prognosis in Human Lung Adenocarcinoma

Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with several aggressive and advanced cancers. Whether IMP3 can predict invasion, and prognosis in patients with human lung adenocarcinoma (LAC) remains unclear. Ninety-five LAC and 75 non-tumor lung tissue samples were included in a tissue microarray. IMP3 expression was assessed by immunohistochemical examination. Correlation between IMP3 expression levels, clinicopathological characteristics, and overall prognosis was evaluated. In a separate in vitro study, RNA interference method was applied for knockdown of IMP3 gene in human LAC cell lines. Invasive potential of LAC cells was then evaluated by transwell migration assay. IMP3 immunoreactivity was observed in 39 out of 95 (41.1 %) LAC patients, but not in non-tumor lung tissues. IMP3 expression levels were closely associated with histological grade (P = 0.037), TNM stage (P = 0.034), and lymph node metastasis (P = 0.011). Patients presenting with positive IMP3 expression (P = 0.000), an advanced TNM stage (P = 0.000), and lymph node metastasis (P = 0.001) had a worse overall survival, compared to those lacking these characteristics. Both IMP3 expression (hazard ratio [HR], 2.310; 95 % confidence interval [CI] 1.192-4.476; P = 0.013) and TNM stage (HR 2.338; 95 % CI 1.393-3.925; P = 0.001) were independent predictors of poor prognosis. The invasive potential of LAC cells was significantly inhibited by IMP3 knockdown. IMP3 appears to play an important role in tumor invasion in patients with LAC and may serve as a useful prognostic biomarker in these patients.

MiR-198 targets SHMT1 to inhibit cell proliferation and enhance cell apoptosis in lung adenocarcinoma.

MiR-198 is involved in tumorigenesis, migration, invasion, and metastasis of various malignant cancers. However, the exact expression levels of miR-198 and the molecular mechanism underlying its role in lung adenocarcinoma require further exploration. In this study, quantitative real-time PCR was applied to study miR-198 and serine hydroxymethyltransferase 1 (SHMT1) expression in 47 paired lung adenocarcinoma tissues and adjacent nontumor lung tissues. Clinicopathological characters were analyzed. Pearson's correlation analysis was used to detect the relationship between miR-198 and SHMT1 expression. The function of miR-198 was explored by measuring cell proliferation, cell apoptosis, and the cell-cycle in vitro and in vivo. The target gene of miR-198 was certified using dual luciferase report assay. We found that in lung adenocarcinoma, miR-198 was significantly downregulated and SHMT1 was inversely upregulated. A strong negative correlation was noticed between miR-198 and SHMT1 expression. Further analysis revealed that miR-198 expression was associated with TNM stage and lymph node metastasis. Upregulated miR-198 could inhibit cell proliferation, enhance cell apoptosis, and lead to cell-cycle arrest in lung adenocarcinoma, which showed a more effective alteration than SHMT1 siRNA. Moreover, we identified SHMT1 as a target gene of miR-198. In conclusion, miR-198 suppressed proliferation of lung adenocarcinoma cells both in vitro and in vivo by directly targeting SHMT1. miR-198 may be a potential therapeutic target for lung adenocarcinoma in the near future.

Significance of DEK overexpression for the prognostic evaluation of non-small cell lung carcinoma.

In the present study, we explored the role of DEK expression for the prognostic evaluation of non-small cell lung carcinoma (NSCLC). DEK protein and mRNA expression levels were detected in NSCLC cells and fresh tissue samples of NSCLC paired with adjacent non-tumor tissues, respectively. NSCLC cases (n=196) meeting strict follow-up criteria were selected for immunohistochemical staining of DEK protein. Correlations between DEK expression and clinicopathological features of the NSCLC cases were evaluated using Chi-square tests. Survival rates were calculated using the Kaplan-Meier method, and the relationship between prognostic factors and patient overall survival was analyzed using Cox proportional hazard analysis. Based on the results, the levels of DEK protein and mRNA were significantly upregulated in 6 fresh tissue samples of NSCLC. Immunohistochemical analysis showed that the DEK expression rate was significantly higher in the NSCLC samples compared with either the adjacent non-tumor tissues or normal lung tissues. DEK expression was correlated with poor differentiation and late pathological stage of NSCLC. DEK expression was also correlated with low disease-free survival and overall survival rates. In the early-stage group, disease-free and overall survival rates of patients with DEK expression were significantly lower than those of patients without DEK expression. Further analysis using a Cox proportional hazard regression model revealed that DEK expression emerged as a significant independent hazard factor for the overall survival rate of patients with NSCLC. Consequently, DEK plays an important role in the progression of NSCLC. DEK may potentially be used as an independent biomarker for the prognostic evaluation of NSCLC.

Skp2 regulates non-small cell lung cancer cell growth by Meg3 and miR-3163.

Maternally expressed gene 3 (Meg3) encodes a long non-coding RNA that has been shown to play a role in tumorigenesis. Skp2 is a component of the E3 ubiquitin ligase SCF that specifically promotes the ubiquitination-associated degradation of CDK inhibitor p27, and has been shown to promote cancer cell growth in different types of cancers, including non-small cell lung cancer (NSCLC). Nevertheless, a regulatory relationship between Meg3 and Skp2 has not been acknowledged. Here, we showed that NSCLC specimens had significant higher levels of Skp2 and significantly lower levels of Meg3, compared to paired non-tumor lung tissue. The levels of Meg3 and Skp2 were inversely correlated in NSCLC specimens. Patients with low Meg3 levels had a poor survival. Overexpression of Meg3 decreased Skp2 protein and increased p27 protein, while depletion of Meg3 increased Skp2 protein and decreased p27 protein in NSCLC cells, without altering Skp2 mRNA. These data suggest that the Skp2 may be regulated by Meg3 at post-transcriptional level. Bioinformatics analyses showed that miR-3163 bound to 3'-UTR of Skp2 mRNA in NSCLC cells to inhibit its translation, which was supported by luciferase reporter assay. Meg3 augmented the effects of miR-3163 on Skp2 mRNA, possibly through binding-induced function enhancement, which was supported by the double fluorescent in situ hybridization showing co-localized intracellular Meg3 and miR-3163 signals in NSCLC cells. The miR-3163 levels in NSCLC were not different from in NT, suggesting that the regulation of Skp2 in NSCLC by miR-3163 may require coordination of Meg3. Thus, our data suggest that Meg3 and miR-3163 may coordinate suppression of translation of Skp2 mRNA in NSCLC cells to inhibit NSCLC cell growth.

Regulation of activating protein-4-associated metastases of non-small cell lung cancer cells by miR-144.

Activating protein-4 (AP4) has been recently shown to regulate the cancer metastases in some cancers including non-small cell lung cancer (NSCLC). Specifically, AP4 regulates mTor/p21 and transforming growth factor β (TGFβ) receptor signaling pathway to increase an epithelial-mesenchymal transition process to augment cell invasiveness. Nevertheless, how AP4 is regulated in NSCLC has not been studied. Here, we showed that in the specimens from the NSCLC patients, the levels of miR-144 were significantly decreased and the levels of AP4 were significantly increased, compared to the paired non-tumor lung tissue. The levels of miR-144 and AP4 inversely correlated in patients' specimens. Bioinformatics analyses revealed that miR-144 targeted the 3'-UTR of AP4 mRNA to inhibit its translation, confirmed by luciferase-reporter assay. Moreover, miR-144 overexpression inhibited AP4-mediated cell invasiveness, while miR-144 depletion increased AP4-mediated cell invasiveness in NSCLC cells. Together, our data suggest that miR-144 suppression may be the cause of the increased levels of AP4, as well as the augmented cancer metastases, in NSCLC.

Abstract 1214: TP53 affects SOX2 copy number alterations and expression in non-small cell lung cancer

Abstract Background: Amplifications of the transcription factor, SRY (sex determining region Y)-box 2 (SOX2), are common in non-small cell lung cancer (NSCLC). SOX2-signaling is important in maintaining the stem cell-like phenotype of cancer cells and contributes to the pathogenesis of lung cancer. TP53 is recognized as a critical regulator of stem cell pluripotency, and it is known that TP53 represses many stem cell associated genes following DNA damage. We hypothesized that SOX2 copy number alterations in lung tumors could be correlated to mutational status of TP53 gene in tumors and that TP53 played a role in regulation of SOX2 gene expression. Material and Methods: Early-stage lung cancer cases (n = 258) of Norwegian Caucasians origin were recruited when admitted to Haukeland University Hospital in Bergen, Norway between 1986 and 1994. Samples of adjacent histologically non-tumorous lung tissue were collected in the lobectomi specimens at the time of surgery. Tumor histology was confirmed by an experienced pathologist and samples containing &amp;gt;80% of tumor cells were analyzed in this study. After resection tumor and non-tumorous tissues were snap-frozen in liquid nitrogen and kept at -80oC until further processing. All subjects gave written consent, and the study was approved by the regional ethical committee in accordance with the Helsinki Declaration. SOX2 copy number alterations were evaluated by quantitative RT- PCR using SYBR Green I Technology. The TP53 and SOX2 expression levels were assessed by q-PCR in the tumors where mRNA was available. Furthermore, to understand mechanisms, TP53 and SOX2 genes were knocked down by siRNA in the A427 human lung adenocarcinoma cell line. Additionally, some miRNAs were also analyzed by q-PCR. TP53 mutations in tumors were analyzed by sequencing and PAH-DNA adduct levels were determined in non-tumorous lung tissue by 382P-postlabelling. Results: SOX2 copy number alterations were more frequent in tumor tissues (34%) than in the adjacent non-tumorous tissues (3%) and TP53 mutations were associated with an increased risk of acquiring a SOX2 copy number alteration (OR = 2.08, 95% CI: 1.14-3.79, p = 0.017). SOX2 and TP53 expressions were strongly correlated in lung tumors and reduction in TP53 decreased SOX2 expression in lung adenocarcinoma cells. TP53 knockdown also reduced the miRNA hsa-miR-145, which has previously been shown to regulate SOX2 expression. We propose that SOX2 may be an important target of TP53 signaling in lung cancer and that hsa-miR-145-5p may be one potential driver miRNA involved in this regulation requiring further studies on the mechanisms behind the TP53-induced regulation of SOX2 expression and the possible importance of hsa-miR-145 in lung cancer. Citation Format: Johanna Samulin-Erdem, Vidar Skaug, Per Bakke, Amund Gulsvik, Aage Haugen, Shanbeh Zienolddiny. TP53 affects SOX2 copy number alterations and expression 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 1214. doi:10.1158/1538-7445.AM2015-1214