Factor In Lung Adenocarcinoma Research Articles

Integrated TCGA analysis implicates lncRNA CTB-193M12.5 as a prognostic factor in lung adenocarcinoma

BackgroundLung cancer is a malignant tumor with the highest incidence and mortality around the world. Recent advances in RNA sequencing technology have enabled insights into long non-coding RNAs (lncRNAs), a previously largely overlooked species in dissecting lung cancer pathology.MethodsIn this study, we used a comprehensive bioinformatics analysis strategy to identify lncRNAs closely associated with lung adenocarcinoma, using the RNA sequencing datasets collected from more than 500 lung adenocarcinoma patients and deposited at The Cancer Genome Atlas (TCGA) database.ResultsDifferential expression analysis highlighted lncRNAs CTD-2510F5.4 and CTB-193M12.5, both of which were significantly upregulated in cancerous specimens. Moreover, network analyses showed highly correlated expression levels of both lncRNAs with those of differentially expressed protein-coding genes, and suggested central regulatory roles of both lncRNAs in the gene co-expression network. Importantly, expression of CTB-193M12.5 showed strong negative correlation with patient survival.ConclusionsOur study mined existing TCGA datasets for novel factors associated with lung adenocarcinoma, and identified a largely unknown lncRNA as a potential prognostic factor. Further investigation is warranted to characterize the roles and significance of CTB-193M12.5 in lung adenocarcinoma biology.

Isoform specific gene expression analysis of KRAS in the prognosis of lung adenocarcinoma patients

BackgroundAberrant mutations in KRAS play a critical role in tumor initiation and progression, and are a negative prognosis factor in lung adenocarcinoma (LUAD).ResultsUsing genomic analysis for K-Ras isoforms (K-Ras4A and K-Ras4B) and large-scale multi-omics data, we inspected the overall survival (OS) and disease-free survival (DFS) of LUAD patients based on the abundance of transcript variants by analyzing RNA expression and somatic mutation data from The Cancer Genome Atlas (n = 516). The expression of the minor transcript K-Ras4A and its proportion were positively correlated with the presence of KRAS mutations in LUAD. We found that both K-Ras4A abundance measures (expression and proportion) have a strong association with poor OS (p = 0.0149 and p = 3.18E-3, respectively) and DFS (p = 3.03E-4 and p = 0.0237, respectively), but only in patients harboring KRAS mutations. A Cox regression analysis showed significant results in groups with low expression (hazard ratio (HR) = 2.533, 95% confidence interval (CI) = 1.380−4.651, p = 2.72E-3) and low proportion (HR = 2.549, 95% CI = 1.387−4.684, p = 2.58E-3) of K-Ras4A.ConclusionsBased on the above results, we report the possible use of abundance measures for K-Ras4A for predicting the survival of LUAD patients with KRAS mutations.

The ratio of cancer cells to stroma within the invasive area is a histologic prognostic parameter of lung adenocarcinoma

ObjectivesThis study evaluated whether the proportion of cancer cells to non-cancerous stroma within the invasive area is associated with the prognosis of patients with lung adenocarcinoma. Materials and methodsA total of 127 patients with lung adenocarcinomas with tumors larger than 3 cm in total size were enrolled in this study. We classified the tumors according to the ratio of area occupied by cancer cells within the invasive area (Type A: more than 50% of the invasive area, Type B: 10–50%, and Type C: less than 10%) and analyzed the clinicopathological differences between Types A, B, and C. ResultsThe invasive size of Type A tumors (n = 35) was significantly larger than those of the other two tumor types; however, there was no significant difference in the invasive size between Types B (n = 65) and C (n = 25) tumors. The recurrence-free survival time of patients with Type C tumors was significantly longer than those of patients with Type A and B (P < .001) tumors. Multivariate analysis revealed that Type C tumor was an independent favorable prognostic factor (P = .037) but that invasive size was not. The invasive area of Type C tumor was composed of a significantly higher proportion of collapsed elastic fibers than the invasive areas of Type A and B tumors (P < .001). ConclusionA lower cancer cell to stroma ratio within the invasive area could be a significant prognostic factor in lung adenocarcinoma, suggesting that not only the invasive size but also the invasive character might be an important histologic prognostic parameter.

Limb-Bud and Heart Attenuates Growth and Invasion of Human Lung Adenocarcinoma Cells and Predicts Survival Outcome

Background/Aims: The transcription cofactor limb-bud and heart (LBH) is involved in embryonic development. However, its role in human lung cancer, especially lung adenocarcinoma (LUAD), remains unclear. Methods: A public database and tissue microarray (TMA) were used to compare differences in LBH expression and its relationship with clinical characteristics. Tissue from an additional 70 LUAD patients with follow-up records was used to explore the correlation of LBH expression with prognosis. Cellular and molecular studies validated the role of LBH in LUAD growth and invasion. Results: LBH was significantly down-regulated in lung cancer tissue samples and was correlated with the prognosis and clinical characteristics of lung cancer patients based on a public database and TMA. Survival analysis revealed that LBH-negative expression was associated with poor overall survival of LUAD patients (P = 0.021). Cox regression analysis showed that LBH expression status was a favorable independent prognostic factor (hazard ratio = 0.120, 95% confidence interval = 0.016–0.894, P = 0.039). LBH knockdown accelerated LUAD cell proliferation, migration, and invasion. Furthermore, bioinformatics analysis indicated that LBH was significantly related to the cell adhesion pathway. Western blot analysis confirmed that LBH could regulate the expression of integrin family members (integrin-α1, integrin-α2, integrin-α4, integrin-αv, and integrin-β4). Conclusion: Our data suggest that LBH plays an important role in lung cancer. Importantly, LBH is an independent prognostic factor in LUAD and can attenuate cell growth and invasion. LBH may be a potential prognostic biomarker in LUAD patients.

P3.16-013 Prognostic Effect of EGFR Gene Mutation for Recurrence in Completely Resected Lung Adenocarcinoma

Prognostic effect of EGFR gene mutation in disease progression which affecting recurrence pattern and recurrence dynamics after complete resection of lung adenocarcinoma has not been well established. We investigated the relationship between EGFR gene mutation and recurrence dynamics in completely resected lung adenocarcinoma. We retrospectively review 527 patients who underwent curative surgery for lung adenocarcinoma from January 2006 to December 2009. Demographics, clinic-pathologic data, and prognosis data were obtained by review of medical records. The EGFR gene mutation was analyzed by nested polymerase chain reaction followed by bidirectional direct sequencing in case of recurrence. Adenocarcinoma patients who experienced recurrence and had data of EGFR mutation were included in the analysis. Pathologic stage IV were excluded. Patients were divided into M group (mutant EFGR gene) or W group (wild-type EGFR gene). Sites of recurrence and disease-free times (DFT) of two groups were compared. Median follow-up duration was 72 months. Overall 5-year survival rate was 80.1%. Recurrence was detected in 153 patients and 5-year disease-free rate was 58.2%. EGFR gene sequencing was performed in 118 (77.1%) patients. There were 38 (32.2%) loco-regional recurrences and 80 (67.8%) distant metastases. Any mutation of EGFR gene was detected in 66 (59.9%) patients (M group) and 52 patients had wild-type EGFR gene (W group). Sites of recurrence in M group were loco-regional in 25 (37.9%) and distant metastasis in 41 (62.1%) patients. Site of recurrence in W group were loco-regional in 13 (25%) and distant metastasis in 39 (75%) patients. Sites of recurrence of both groups were not significantly different. (p=0.14) Median DFT of two groups were significantly different (M = 20.3 months vs W = 15.1 months, p=0.039). Visceral pleura invasion (p=0.045) and EGFR gene mutation (p=0.039) were prognostic factor for DFT in univariable analysis. In multivariable analysis, EGFR gene mutation was the only prognostic factor for DFT. (HR – 0.676, 95% CI – 0.371 ∼ 0.986, p=0.042) No significant factor for DFT was identified in loco-regional recurrence. EGFR gene mutation, however, was the only significant prognostic factor for DFT of distant metastasis (HR – 0.561, 95% CI – 0.356 ∼ 0.885, p=0.013) in univariable and multivariable analysis. In recurrent lung adenocarcinoma, EGFR gene mutation group showed longer DFT than wild-type EGFR group. EGFR gene mutation is a prognostic factor in lung adenocarcinoma and slow-growing nature of adenocarcinoma with EGFR gene mutation should be considered in surveillance after surgery.

Long noncoding RNA TUG1 is a diagnostic factor in lung adenocarcinoma and suppresses apoptosis via epigenetic silencing of BAX.

Lung cancer is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel diagnostic markers and therapeutic targets. Increasing evidences have indicated that long non-coding RNAs (lncRNAs) play an important role in initiation and progression of lung cancer. However, the role of lncRNA Taurine upregulated 1 (TUG1) in lung adenocarcinoma (LAD) progression is not well known. In this study, we determined the diagnostic value of TUG1 in LAD patients, and further uncovered the underlying functional mechanism. Our results showed that TUG1 was significantly upregulated in LAD cells and serum samples. Receiver operator characteristic (ROC) analysis suggested a relatively higher area under the curve (AUC) of TUG1 (0.756) contrast to cyfra21-1 (0.619). In addition, high TUG1 level was associated with enhanced tumor size, degree of differentiation, lymph node metastases, distant metastasis and TNM stage. Cell functional assays showed that knockdown of TUG1 suppressed LAD cell viability and promoted cell apoptosis. We then sought to reveal the underlying regulatory mechanism, and the pro-apoptotic protein BAX was then identified as the downstream target of TUG1. Gain and loss functional assays showed that inhibition of BAX reversed the induced apoptosis by TUG1 knockdown. Finally, RNA immunoprecipitation and Chromatin immunoprecipitation revealed that TUG1 suppressed BAX expression through physically interacting with EZH2. In conclusion, lncRNA TUG1 is a promising diagnostic marker for LAD patients and suppression of TUG1 levels could be a future direction to promote the prognosis of LAD patients.

Integrated analysis reveals candidate genes and transcription factors in lung adenocarcinoma.

Lung adenocarcinoma is the most common type of non‑small cell lung cancer in Asia. Therefore, it is important to improve understanding of the underlying transcriptional regulatory mechanisms involved. The present study aimed to identify potential candidate genes and transcription factors (TFs) associated with the disease. Four gene expression profiles were downloaded from the Gene Expression Omnibus database, which included 141 lung adenocarcinoma patients and 191healthy controls. The differentially expressed genes (DEGs) were screened out and functional annotation was performed. In addition, TFs were identified and a global transcriptional regulatory network was constructed. Integrated analysis gave rise to a total of 1,238 DEGs in lung adenocarcinoma when compared with healthy tissues, including 970 upregulated and 268 downregulated DEGs. The six overexpressed outlier genes of ceruloplasmin, heparan sulfate 6‑O‑sulfotransferase 2, transmembrane protease serine4, anillin actin binding protein, cellular retinoic acid binding protein 2 and cystatin SN may serve important roles in the development of lung adenocarcinoma. In addition, the downregulation of carbonic anhydrase 4 and S100 calcium binding protein A12 may render these effective diagnostic biomarkers. The results of the transcriptional regulatory network demonstrated that the hub nodes were sex determining region Y‑box10, Spi‑B transcription factor and nuclear receptor subfamily 4 group A member 2. The four TFs, forkhead box D1, E74‑like ETS transcription factor 5, homeobox A5 and kruppel‑like factor 5, may warrant future investigations into their function in disease development. In conclusion, the present study provided for further studies a list of candidate genes and TFs for the detection and treatment of lung adenocarcinoma.

SMOKING STATUS AFFECTING SURVIVAL OF ADENOCARCINOMA LUNG CANCER PATIENTS IN KUALA LUMPUR, MALAYSIA

Objective: Adenocarcinoma (AC) of the lung is now the most common histologic type of non-small cell lung cancer (NSCLC) worldwide since the past 20 years. This study was conducted to investigate survival difference among smoker and non-smoker lung AC patients.Methods: A retrospective observational study was conducted for 81 advanced NSCLC adult Malaysian patients in Radiotherapy and Oncology Clinic at Hospital Kuala Lumpur, Malaysia. A total of adult 30 Malaysian smokers and 51 non-smokers with lung AC were included. Ex-smokers were not included in the study. Demographic and clinical data were collected and described. For survival analysis, Kaplan–Meier test and log-rank test were used to calculate overall survival (OS) and analyse the difference in the survival curve. Cox proportional hazard model was used to identify prognostic significance of smoking status.Results: Non-smokers showed a significant association with female gender and Stage IV NSCLC. The median OS was higher for non-smokers (493 days) as compared to smokers (230 days). The Cox proportional hazard model showed higher hazard ratio for smokers.Conclusion: Non-smoking is an independent positive prognostic factor in lung AC.

Abstract 4840: SNP rs1052566 (Ala273Val) in BRMS1 variant2 promotes metastasis in lung adenocarcinoma

Abstract Background: Metastatic lung cancer kills over 180,000 people in the United States annually. Family history is one risk factor for lung adenocarcinoma (LUAD), suggesting a putative genetic predisposition to the development of LUAD. Single nucleotide polymorphisms (SNPs) are the most common genetic variations in the human genome and major determinants of variations in disease susceptibility, prognosis, response to medication, and toxicity. Recent studies indicate that SNPs play important roles in the progression of LUAD. We have previously shown that breast cancer metastasis suppressor-1 (BRMS1) plays a critical role in inhibiting the progression of lung cancer. We observed that SNP rs1052566 homo-variant in breast cancer metastasis suppressor-1 (BRMS1) variant 2 (v2) causes an A273V mutation. However, the role of this SNP in the progression of lung cancer remains to be determined. Methods: We utilized next-generation DNA sequencing and Taqman SNP assays to detect the prevalence of rs1052566 in 40 surgically resected LUAD specimens. Next, we generated a retroviral expressional system (pBabe puro) encoding V5-tagged BRMS1v2 wild-type (WT) and A273V mutant and created NSCLC H1299 and A549 cell lines that stably express V5-tagged BRMS1v2 WT, A273V, or vector control. The metastatic capabilities of these stable cells were evaluated by invasion chamber assays in vitro and a tail-vein injection metastasis model in vivo. To explore the mechanism(s) through which BRMS1v2 A273V promotes metastasis, we performed RT profiler PCR assay. Results: We determined that SNP rs1052566 is present in 45% of LUAD patients. Of these, the homo-variant occurs in 7.5% of patients (3 of 40). Importantly, BRMS1v2 A273V significantly promotes metastasis of NSCLC cells in vitro and in vivo. Additionally, in our mouse model, A273V was associated with decreased survival, compared with BRMS1v2 WT or control. On RT profiler PCR array, 7 genes, including L1CAM, were found to be significantly differentially regulated by BRMS1v2 WT and A273V. Confirmatory RT-PCR and immunoblots proved that BRMS1v2 A273V increases both the transcript and protein levels of L1CAM. Furthermore, we found that either c-Jun or c-Fos promotes the transcription of L1CAM. However, BRMS1v2 A273V specifically enhances c-Fos-, but not c-Jun-, induced L1CAM transcription. Conclusion: We observed that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1 and promotes metastasis. Up-regulation of c-Fos-induced L1CAM transcription is a putative mechanism of BRMS1v2 A273V-induced metastasis. Our data suggest that SNP rs1052566, especially its homo-variant, is associated with an increased risk of metastasis in LUAD. Note: This abstract was not presented at the meeting. Citation Format: Neel P. Chudgar, Yuan Liu, Joseph Montecalvo, Marty W. Mayo, Prasad S. Adusumilli, David R. Jones. SNP rs1052566 (Ala273Val) in BRMS1 variant2 promotes metastasis in lung adenocarcinoma [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 4840. doi:10.1158/1538-7445.AM2017-4840

Overexpression of MAGEA2 has a prognostic significance and is a potential therapeutic target for patients with lung cancer.

Melanoma-associated antigens (MAGE) are expressed in different type of cancers including lung cancer and have been shown to be functionally related to p53 tumor suppressor gene. Little is known about the relationship between MAGE genes and p53 aberrant expression in lung cancer. The aims of this study were to observe the expression of MAGEA2, examine the role of MAGEA2 in lung cancer survival, investigate its correlation between MAGEA2 and p53, and explore its clinicopathologic significance as a prognostic marker. Quantitative reverse transcription-polymerase chain reaction was performed to detect the expression of MAGEA2 using 36primary tumors and 31 metastatic lymph nodes from patients with lung cancer. The role of MAGEA2 in cancer cell growth and in the regulation of p53 downstream genes were examined using small interfering RNA. The expression of MAGEA2 and p53 were analyzed immunohistochemically using tissue microarray from 353 resected lung specimens. High-level expression of MAGEA2 (High-MAGEA2) was confirmed in lung tumors with high frequency. Inhibiting MAGEA2 expression effectively suppressed cancer cell growth and decreased the expression of p53 downstream target genes invitro. In adenocarcinoma, High-MAGEA2 was strongly associated with aberrant p53 expression (P<0.001) and was associated with worse clinical outcomes (5-year OS, 87.1% in low vs. 74.1% in high, P=0.014). Aberrant p53 expression was also significant worse prognostic factor (P=0.029). Among the adenocarcinoma patients with wild-type p53, High-MAGEA2 had poorer prognosis than low-level MAGEA2 groups (5-year OS, 90.1% vs. 72.1%, P=0.037), whereas had no difference in p53 aberrant tumors. On multivariate analysis, MAGEA2 was independently associated with survival (hazard ratio; 2.12, P=0.030). In conclusion, suppression of MAGEA2 in lung cancer cells significantly reduced the growth/survival of cancer cells. High-MAGEA2 was identified as an independent prognostic factor in lung adenocarcinoma. Specific inhibition of MAGEA2 may be a promising therapeutic strategy for patients with lung cancer.

Prognostic alternative mRNA splicing signature in non-small cell lung cancer

Alternative splicing provides a major mechanism to generate protein diversity. Increasing evidence suggests a link of dysregulation of splicing associated with cancer. Genome-wide alternative splicing profiling in lung cancer remains largely unstudied. We generated alternative splicing profiles in 491 lung adenocarcinoma (LUAD) and 471 lung squamous cell carcinoma (LUSC) patients in TCGA using RNA-seq data, prognostic models and splicing networks were built by integrated bioinformatics analysis. A total of 3691 and 2403 alternative splicing events were significantly associated with patient survival in LUAD and LUSC, respectively, including EGFR, CD44, PIK3C3, RRAS2, MAPKAP1 and FGFR2. The area under the curve of the receiver-operator characteristic curve for prognostic predictor in NSCLC was 0.817 at 2000 days of overall survival which were also over 0.8 in LUAD and LUSC, separately. Interestingly, splicing correlation networks uncovered opposite roles of splicing factors in LUAD and LUSC. We created prognostic predictors based on alternative splicing events with high performances for risk stratification in NSCLC patients and uncovered interesting splicing networks in LUAD and LUSC which could be underlying mechanisms.

The frequency and clinical impact of HER2 alterations in lung adenocarcinoma.

Human epidermal growth factor receptor 2 (HER2 or ErbB2) can be overexpressed, amplified and/or mutated in malignant tumors, and is a candidate for therapeutic targeting. However, molecular associations and clinical significances of these alterations were controversial in lung cancer. In this study, we investigated the frequency and clinicopathological significance of HER2 dysregulation in patients with lung adenocarcinoma. HER2 protein overexpression, gene amplification, and gene mutation were evaluated by immunohistochemistry (IHC), silver in situ hybridization, and direct sequencing, respectively. The H-scoring method and American Society of Clinical Oncology/College of American Pathologists breast cancer guidelines were used to interpret IHC results. Genetic analyses of EGFR and KRAS mutations, and of ALK and ROS1 rearrangements, were also performed. Of the 321 adenocarcinoma patients identified, HER2 overexpression (H-score ≥200) and gene amplification were found in 6 (1.9%) and 46 (14.3%), respectively. HER2 overexpression was correlated with papillary predominant histology; furthermore, it indicated poor overall survival and was an independent prognostic factor. HER2 amplification was associated with pleural invasion and showed a tendency towards shorter overall and disease-free survival. High-level gene amplification (HER2/CEP17 ratio ≥5 or copy number ≥10) was a poor prognostic factor for disease-free survival. HER2 mutations were detected in 6.7% (7 of 104) of driver oncogene-negative adenocarcinomas. Our study suggests that HER2 overexpression or amplification is a poor prognostic factor in lung adenocarcinoma, although the frequency of such events is low. Since molecular targeted agents are being tested in clinical trials, awareness of the specific HER2 status can influence the prognostic stratification and treatment of patients with molecularly defined subsets of lung adenocarcinoma.

PUB117 Beta Estrogen Receptor as Potential Prognostic Factor in Lung Adenocarcinoma

The greater lung cancer incidence in the female population has been primarily correlated to the increasingly present smoking habit among females all over the world. The hormonal estrogen status has been reported as another possible factor contributing to the increasing incidence of lung cancer in females, due to the fact that still a lower percentage of smoking women is affected by lung cancer than smoking men. There are two tipes of estrogen receptors (ER) - alfa and beta. ER- beta expression is excessively higher in lung cancer than ER- alfa expression. The objectives of this study were to establish the treatment response of lung adenocarcinoma patients depending on the presence of ER-beta in the tumor tissue; the treatment response and survival differences between the sexes depending on the ER-beta status, and finally whether the ER-beta status may be a prognostic factor in these patients. The study included 200 patients with diagnosis of lung adenocarcinoma (diagnosis were established during 2010 according to 5-year survival rate). Neither was statistically significant difference registered in the time passing till the onset of the disease progression in the overall examined females, in whom it was 16.5 and 9.5 months with ER-beta + and ER-beta - tumor status respectively making statistically significant difference (p=0.041). The analysis of the cumulative survival depending on patients' sex and receptor status (males ER-beta -, males ER-beta +, females ER-beta -, females ER-beta +) has revealed that ER-beta + females had a significantly longer survival than other three subgroups. The obtained results suggest a conclusion that the hormonal receptor status is one of many factors in lung cancer carcinogenesis and may be establish as a prognostic factor in lung cancer, as well as a potential factor in multimodal (hormonal anti-estrogen) treatment of malignancy.

Recurrence Pattern of Pathologic Stage I Lung Adenocarcinoma With Visceral Pleural Invasion

Visceral pleural invasion (VPI) is well known to be a poor prognostic factor in lung adenocarcinoma. There were few studies reporting postoperative recurrence pattern in lung adenocarcinoma with VPI. This study was to evaluate the clinical effect of VPI on recurrence pattern in pathologic stage I lung adenocarcinoma after curative resection. Among 574 patients with pathologic stage I lung adenocarcinoma after complete resection between 2003 and 2012, the clinicopathologic characteristics of 89 patients (16%) who had recurrence were retrospectively reviewed. The pathologic findings, postrecurrence survival, and patterns of recurrence were compared between patients with VPI and without VPI. Median follow-up duration was 53.6 months. The VPI was found in 43 patients (group I) and not found in 46 patients (group II). Both total tumor size and invasive size were larger in group I (3.2 versus 2.7 cm and 2.5 versus 2.1 cm; p < 0.05). The median duration of overall survival was 58 months in group I and 76 months in group II. As patterns of recurrence, pleural seeding was found in 25 patients, and the percentage of pleural seeding was significantly higher in group I than group II (44.2% versus 13.0%; p= 0.001). In group I, bilateral lung metastasis was significantly common (39.5% versus 13.0%; p= 0.004), and increasing percentage of pleural seeding was observed as the invasive tumor size grows. The presence of VPI was a significant predictable factor for pleural seeding and bilateral lung metastasis as patterns of recurrence after complete resection in pathologic stage I lung adenocarcinoma.

Increased lysyl oxidase-like 2 associates with a poor prognosis in non-small cell lung cancer.

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and is associated with invasiveness and metastasis in breast cancer. However, its relevance in non-small cell lung cancer (NSCLC) remained largely unknown. LOXL2 protein levels in a cohort of NSCLC and adjacent normal lung tissues were evaluated and analyzed their clinicopathologic and prognostic significance. It was found that cytoplasmic and nuclear LOXL2 levels were higher in lung adenocarcinoma (AD) and squamous cell carcinoma (SCC) tissues than in paired adjacent normal tissues. High LOXL2 levels were associated with p-TNM stage, and cytoplasmic, but not nuclear, LOXL2 levels were an independent prognostic factor in lung AD and SCC patients. These results demonstrate that elevated LOXL2 levels are positively associated with poor prognosis in NSCLC patients. LOXL2 might, therefore, serve as a novel prognostic biomarker and potential therapeutic target in NSCLC patients.

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.

Notch-1 Confers Chemoresistance in Lung Adenocarcinoma to Taxanes through AP-1/microRNA-451 Mediated Regulation of MDR-1.

We previously demonstrated that expression of Notch-1 is associated with poor prognosis in lung adenocarcinoma (LAD) patients. The aim of this study is to reveal whether Notch-1 was associated with Taxanes-resistant LAD and, the underlying mechanisms. We collected 39 patients of advanced LAD treated with Taxanes and found that positive Notch-1 expression is closely related to LAD lymph node metastasis, recurrence and poorer prognosis, and Notch-1 acts as an independent poor prognostic factor in LAD by multivariate analysis with Cox regression model. Then, by using the Docetaxel (DTX)-resistant LAD cell lines that we established previously, we found that Notch-1 contributes to resistance of LAD cells to DTX in vitro, and inhibition of Notch-1 sensitizes LAD to DTX in vivo. We further demonstrated that Notch-1 mediates chemoresistance response and strengthens proliferation capacity in LAD cells partially through negative regulation of miR-451 by transcription factor AP-1. Moreover, we found that MDR-1 is a direct target of miR-451 and influences chemoresistance of LAD cells. Taken together, our data revealed a novel Notch-1/AP-1/miR-451/MDR-1 signaling axis, and suggested a new therapeutic strategy of combining DTX with Notch inhibitors to treat DTX-resistant LAD.

The association of intravascular stromal cells with prognosis in high-grade neuroendocrine carcinoma of the lung.

Histological vascular invasion (VI) is major predictor of recurrence after surgery for several cancer types. We previously reported that VI with abundant stromal cell infiltrates was a negative prognostic factor in lung adenocarcinoma. This study examined whether stromal cell infiltrates within VI are associated with prognosis in high-grade neuroendocrine carcinoma (HGNEC) of the lung. We investigated the relationship between the frequency and density of VI and recurrence-free survival (RFS) of 60 resected HGNEC patients without adjuvant chemotherapy. We examined prognostic effects of CD204 (+) macrophages, CD34 (+) microvessels and α-smooth muscle actin (+) myofibroblasts within VI. Correlation between expressions of stem cell-related molecules (ALDH-1, Notch 1, CD44) and epithelial-mesenchymal transition-related molecules (E-cadherin, S100A4) in cancer cells within VI and RFS was also analyzed. Among 60 cases, 51 cases showed VI. Although the presence of VI was a significant predictor for worse RFS (P = 0.04), the frequency and density of VI were not correlated with RFS. The number of CD204 (+) macrophage, CD34 (+) microvessels and α-smooth muscle actin (+) myofibroblasts within VI did not influence on the RFS. On the contrary, cases with higher Notch 1 expression in cancer cells in the VI displayed significantly shorter RFS than lower expression group (7.6 vs. 29.4 months, P = 0.02). The current study revealed the presence of stromal cells within VI was not significant predictor for recurrence in HGNEC. This suggests that in HGNEC, unlike adenocarcinoma, intravascular stromal cells are not major contributors to metastasis.

Dimethylarginine dimethylaminohydrolase 2 promotes tumor angiogenesis in lung adenocarcinoma.

Although embryonal proteins have been used as tumor marker, most are not useful for detection of early malignancy. In the present study, we developed mouse monoclonal antibodies against fetal lung of miniature swine, and screened them to find an embryonal protein that is produced at the early stage of malignancy, focusing on lung adenocarcinoma. We found an antibody clone that specifically stained stroma of lung adenocarcinoma. LC-MS/MS identified the protein recognized by this clone as dimethylarginine dimethylaminohydrolase 2 (DDAH2), an enzyme known for antiatherosclerotic activity. DDAH2 was found to be expressed in fibroblasts of stroma of malignancies, with higher expression in minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma than in adenocarcinoma in situ (AIS). Moreover, tumors with high stromal expression of DDAH2 had a poorer prognosis than those without. In vitro analysis showed that DDAH2 increases expression of endothelial nitric oxide synthase (eNOS), inducing proliferation and capillary-like tube formation of vascular endothelial cells. In resected human tissues, eNOS also showed higher expression in invasive adenocarcinoma than in AIS and normal lung, similarly to DDAH2. Our data indicate that expression of DDAH2 is associated with invasiveness of lung adenocarcinoma via tumor angiogenesis. DDAH2 expression might be a prognostic factor in lung adenocarcinoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-015-1863-z) contains supplementary material, which is available to authorized users.

Fibulin-1 functions as a prognostic factor in lung adenocarcinoma.

Fibulin-1 is a member of the fibulin gene family, characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. Fibulin-1 plays important roles in a range of cellular functions including morphology, growth, adhesion and mobility. It acts as a tumor suppressor gene in cutaneous melanoma, prostate cancer and gastric cancer. However, whether fibulin-1 also acts as a tumor suppressor gene in lung adenocarcinoma remains unknown. We also determined the association of fibulin-1 expression with various clinical and pathological parameters, which would show its potential role in clinical prognosis. We investigated and followed up 140 lung adenocarcinoma patients who underwent lung resection without pre- and post-operative systemic chemotherapy at the Affiliated Hospital of Nantong University from 2009 to 2013. Western blot assay and immunohistochemistry were used to evaluate the expression of fibulin-1 in lung adenocarcinoma tissues. We then analyzed the correlations between fibulin-1 expression and clinicopathological variables as well as the patients' overall survival rate. Both western blot assay and immunohistochemistry demonstrated that the level of fibulin-1 was downregulated in human lung adenocarcinoma tissues compared with that of normal lung tissues. Fibulin-1 expression significantly correlated with histological differentiation (P = 0.046), clinical stage (P< 0.01), lymph node status (P = 0.038) and expression of Ki-67 (P = 0.013). More importantly, multivariate analysis revealed that fibulin-1 was an independent prognostic marker for lung adenocarcinoma, and high expression of fibulin-1 was significantly associated with better prognosis of lung adenocarcinoma patients. The results supported our hypothesis that fibulin-1 can act as a prognostic factor in lung adenocarcinoma progression.