Poor Survival In Lung Adenocarcinoma Research Articles

LncRNA LINC01833 is a Prognostic Biomarker and Correlates with Immune Infiltrates in Patients with Lung Adenocarcinoma by Integrated Bioinformatics Analysis.

Due to the absence of accurate tools for early detection and successful treatment, lung adenocarcinoma (LUAD) is one of the most aggressive tumors with high morbidity and mortality globally. It is absolutely necessary to investigate the process behind its development and search for new biomarkers that could aid in the early detection of LUAD. There is a correlation between the immune microenvironment of the tumor and the prognosis of lung cancer as well as the efficacy of immunotherapy. Long noncoding RNAs (lncRNAs) have been identified as potential prognostic biomarkers linked to immunological activities. In this study, we identified 1 downregulated lncRNA and 76 upregulated lncRNAs in LUAD samples from TCGA datasets. Among the 77 dysregulated lncRNAs, our attention focused on lncRNA LINC01833 (LINC01833). When compared with nontumor specimens, the level of expression of LINC01833 was shown to be significantly elevated in LUAD samples. In addition, the data of the ROC study revealed that LUAD patients with high LINC01833 expression had an AUC value of 0.840 (95% confidence interval: 0.804 to 0.876). There was a correlation between high LINC01833 expression and an advanced clinical stage. Patients who had a high expression of LINC01833 were shown to have a lower overall survival rate (p < 0.001) and a lower disease-specific survival rate (p = 0.004) in comparison to patients who were in the low LINC01833 group, according to the data on survival. In addition, the results of the multivariate analysis revealed that high LINC01833 expression was an independent predictor of poor survival in LUAD. Moreover, the immune analysis revealed that we found that the expression of LINC01833 was positively associated with Th2 cells, aDC, and Tgd, while negatively associated with Mast cells, Tcm, Eosinophils, iDC, DC, Tem, Th17 cells, and pDC. Overall, our data point to the possibility that the unique lncRNA LINC01833 might be employed as a diagnostic and prognostic marker, and as a result, it has a significant impact on clinical practice.

Integrative metabolomics and transcriptomics analysis reveals novel therapeutic vulnerabilities in lung cancer.

Non-small cell lung cancer (NSCLC) comprises the majority (~85%) of all lung tumors, with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being the most frequently diagnosed histological subtypes. Multi-modal omics profiling has been carried out in NSCLC, but no studies have yet reported a unique metabolite-related gene signature and altered metabolic pathways associated with LUAD and LUSC. We integrated transcriptomics and metabolomics to analyze 30 human lung tumors and adjacent noncancerous tissues. Differential co-expression was used to identify modules of metabolites that were altered between normal and tumor. We identified unique metabolite-related gene signatures specific for LUAD and LUSC and key pathways aberrantly regulated at both transcriptional and metabolic levels. Differential co-expression analysis revealed that loss of coherence between metabolites in tumors is a major characteristic in both LUAD and LUSC. We identified one metabolic onco-module gained in LUAD, characterized by nine metabolites and 57 metabolic genes. Multi-omics integrative analysis revealed a 28 metabolic gene signature associated with poor survival in LUAD, with six metabolite-related genes as individual prognostic markers. We demonstrated the clinical utility of this integrated metabolic gene signature in LUAD by using it to guide repurposing of AZD-6482, a PI3Kβ inhibitor which significantly inhibited three genes from the 28-gene signature. Overall, we have integrated metabolomics and transcriptomics analyses to show that LUAD and LUSC have distinct profiles, inferred gene signatures with prognostic value for patient survival, and identified therapeutic targets and repurposed drugs for potential use in NSCLC treatment.

Bioinformatic analysis of PLOD family member expression and prognostic value in non-small cell lung cancer.

BackgroundProcollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) are a group of enzymes that can mediate the hydroxylation of lysyl to hydroxylysine and participate in the formation of stabilized collagen. Evidence has demonstrated that PLODs are involved in the steps of tumor progression, including proliferation, invasion, and metastasis. However, limited information is available on the function of PLOD1/2/3 in lung cancer. In this study, we investigated the expression patterns and prognostic values of PLODs in patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC).MethodsThe Oncomine database and UALCAN were used to analyze the mRNA expression levels of PLOD family members in non-small cell lung cancer (NSCLC). The prognostic values of PLODs were investigated by the Kaplan–Meier Plotter database. We collected 33 patients with lung cancer to further verify the expression profiles and prognostic values of PLODs. The Kaplan-Meier method was used to perform survival curves, and the log-rank test was performed to evaluate the differences in survival. According to the GSE31210 databset, univariate and multivariate analyses were performed to identify whether PLODs were independent prognostic indicators for survival. Meanwhile, we investigated the mutations, potential biological functions and immune relevance of PLODs on the basis of the cBioPortal, Metascape and TIMER databases respectively.ResultsWe found that the mRNA and protein expression levels of PLODs in NSCLC tissues were higher than those in normal lung tissues. High PLOD1/2/3 expression had significant relevance to poor survival in LUAD but not in LUSC. In addition, the GSE31210 dataset showed that PLOD1 and PLOD3 were independent risk factors for relapse-free survival and overall survival (OS) in LUAD. We observed a high alteration rate of PLODs in LUSC patients, and the genetic alterations of PLODs had significant relevance to favorable OS. Furthermore, we observed that PLODs were significantly associated with tumor immunity in lung cancer. The enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed that the functions of the PLODs focused on cell cycle, DNA replication, and glycolysis/gluconeogenesis in LUAD.ConclusionsThese results indicated that PLODs were highly expressed in lung cancer and may be suitable prognostic markers.

Decreased HLF Expression Predicts Poor Survival in Lung Adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) is a type of non-small cell carcinoma. Its pathogenesis is being explored and there is no cure for the disease.Material/MethodsThe Gene Expression Omnibus (GEO) was searched to obtain data on expression of messenger RNA. GEO2R, an interactive web tool, was used to calculate the differentially expressed genes (DEGs) in LUAD. All the DEGs from different datasets were imported into VENNY 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) to identify the intersection of the DEGs. An online analysis tool, the Database for Annotation, Visualization, and Integrated Discovery (DAVID), was used to help understand the biological meaning of DEG enrichment in LUAD. Cytoscape 3.7.2 was used to perform centrality analysis and visualize hub genes and related networks. Furthermore, the prognostic value of the hub genes was evaluated with the Kaplan-Meier plotter survival analysis tool.ResultsThe GEO database was used to obtain RNA sequencing information for LUAD and normal tissue from the GSE118370, GSE136043, and GSE140797 datasets. A total of 376 DEGs were identified from GSE118370, 248 were identified from GSE136403, and 718 DEGs were identified from GSE140797. The 10 genes with the highest degrees of expression – the hub genes – were CAV1, TEK, SLIT2, RHOJ, DGSX, HLF, MEIS1, PTPRD, FOXF1, and ADRB2. In addition, Kaplan-Meier survival evaluation showed that CAV1, TEK, SLIT2, HLF, MEIS1, PTPRD, FOXF1, and ADRB2 were associated with favorable outcomes for LUAD.ConclusionsCAV1, TEK, SLIT2, HLF, MEIS1, PTPRD, FOXF1, and ADRB2 are hub genes in the DEG interaction network for LUAD and are involved in the development of and prognosis for the disease. The mechanisms underlying these genes should be the subject of further studies.

LncRNA TUG1 contributes to the tumorigenesis of lung adenocarcinoma by regulating miR-138-5p-HIF1A axis.

Introduction: Increasing evidence indicates that lncRNA TUG1 represents an oncogenic factor in cancer. But, the mechanisms by which lncRNA TUG1 contributes to lung adenocarcinoma (LAC) remain undocumented. Methods: The relationship between lncRNA TUG1/miR-138-5p expression and clinical outcomes in patients with LAC was indicated by qPCR, FISH, and TCGA cohort. Gain- or loss-of-function experiments and in vivo tumorigenesis were used to assess the role of lncRNA TUG1 in LAC. The interplay between TUG1 and miR-138-5p was validated by luciferase gene report and RIP assays. qPCR and Western blot analyses were used to investigate the effects of TUG1 on miR-138-5p/HIF1A axis in LAC cells. Results: We found that upregulation of TUG1 or downregulation of miR-138-5p was associated with lymph node or distant metastasis and indicated a poor survival in LAC. Reduced expression of TUG1 restrained the growth of LAC cells, while restored expression of TUG1 had the opposite effects. TUG1 was identified to negatively regulate miR-138-5p expression, and miR-138-5p reversed TUG1-induced cell proliferation by targeting HIF1A. Elevated expression of HIF1A predicted a poor survival in LAC. Conclusion: Our findings demonstrate that lncRNA TUG1 promotes the growth of LAC by regulating miR-138-5p-HIF1A axis.

EpCAM+CD73+ mark epithelial progenitor cells in postnatal human lung and are associated with pathogenesis of pulmonary disease including lung adenocarcinoma.

Lung injury in mice induces mobilization of discrete subsets of epithelial progenitor cells to promote new airway and alveolar structures. However, whether similar cell types exist in human lung remains unresolved. Using flow cytometry, we identified a distinct cluster of cells expressing the epithelial cell adhesion molecule (EpCAM), a cell surface marker expressed on epithelial progenitor cells, enriched in the ecto-5'-nucleotidase CD73 in unaffected postnatal human lungs resected from pediatric patients with congenital lung lesions. Within the EpCAM+CD73+ population, a small subset coexpresses integrin β4 and HTII-280. This population remained stable with age. Spatially, EpCAM+CD73+ cells were positioned along the basal membrane of respiratory epithelium and alveolus next to CD73+ cells lacking EpCAM. Expanded EpCAM+CD73+ cells give rise to a pseudostratified epithelium in a two-dimensional air-liquid interface or a clonal three-dimensional organoid assay. Organoids generated under alveolar differentiation conditions were cystic-like and lacked robust alveolar mature cell types. Compared with unaffected postnatal lung, congenital lung lesions were marked by clusters of EpCAM+CD73+ cells in airway and cystic distal lung structures lined by simple epithelium composed of EpCAM+SCGB1A1+ cells and hyperplastic EpCAM+proSPC+ cells. In non-small-cell lung cancer (NSCLC), there was a marked increase in EpCAM+CD73+ tumor cells enriched in inhibitory immune checkpoint molecules CD47 and programmed death-ligand 1 (PD-L1), which was associated with poor survival in lung adenocarcinoma (LUAD). In conclusion, EpCAM+CD73+ cells are rare novel epithelial progenitor cells in the human lung. Importantly, reemergence of CD73 in lung adenocarcinoma enriched in negative immune checkpoint molecules may serve as a novel therapeutic target.

MiR-629-3p-induced downregulation of SFTPC promotes cell proliferation and predicts poor survival in lung adenocarcinoma

The long-term prognosis of patients with lung cancer remains poor and thus it is imminent to further elucidate the molecular mechanism for the oncogenesis of lung cancer. In this study, we observed that surfactant protein C (SFTPC) expression was downregulated in human lung adenocarcinoma tissues and cell lines, and low SFTPC expression correlated with poor overall survival of lung adenocarcinoma patients. Moreover, we found that overexpression of SFTPC could inhibit lung cancer cell proliferation in vitro and in vivo, but downregulation of SFTPC showed the opposite results. Besides, it was observed that miR-629-3p expression was upregulated in human lung adenocarcinoma tissues and cell lines. More importantly, we found that miR-629-3p could downregulate SFTPC expression by directly binding to the SFTPC 3'-UTR and inhibit the regulatory effect of SFTPC on lung adenocarcinoma cell proliferation. In conclusion, these data suggested that miR-629-3p-meditated downregulation of SFTPC may promote lung adenocarcinoma progression.

Papillary predominant histological subtype predicts poor survival in lung adenocarcinoma.

This study aims to investigate whether papillary predominant histological subtype can predict poor survival in lung adenocarcinoma. Between January 2005 and December 2016, a total of 80 patients with papillary predominant subtype lung adenocarcinoma (70 males, 10 females; mean age 60.7 years; range, 42 to 79 years) operated in our clinic were included in the study. These patients were compared with those having lepidic, acinar, and mucinous subtypes. Overall and five-year survival rates were evaluated. Five-year survival was 40.5% in papillary predominant histological subtype, while this rate was 70.9%, 59.0%, and 66.6% in lepidic, acinar, and mucinous subtypes, respectively. Papillary subtype showed significantly poor survival compared to lepidic (p=0.002), acinar (p=0.008), and mucinous subtypes (p=0.048). In Stage 1 disease, it was more evident (papillary, 47.5%, lepidic 86.9% [p=0.001], acinar 69.3% [p=0.040], and mucinous 90.0% [p=0.050]). Our study results suggest that papillary predominant subtype predicts poor survival in lung adenocarcinoma and these cases may be candidates for adjuvant treatment modalities even in the earlier stages of disease.

Abstract 2799: Targeting CXCR2-mediated neutrophil recruitment to lung cancer

Abstract With recent advances in immunotherapy, it is evident that targeting the tumor microenvironment (TME) is an effective strategy to treat lung cancer (LC), however, more than half LC patients are still resistant to therapy. Limited attention was given to the relevance of the innate immune system despite its critical role in triggering adaptive responses. Neutrophils (PMNs) are the predominant circulating leukocyte in humans. PMNs are associated with developing lesions and are the main immune component of primary non-small cell LC (NSCLC). Multiple studies support the notion that PMNs promote tumor progression, however, the exact mechanisms in which these PMNs are recruited to the primary and metastatic lung TMEs remain unclear. To this end, we examined available genomic databases of &amp;gt; 1,000 NSCLC primary adenocarcinoma (ADC) patients and observed that high expression of all CXCR2 ligands (CXCL1-8 and MIF) correlate with poor survival in lung ADC. Lung ADC patients display one of the highest fold increases of these ligands as compared to all other cancers. We then performed shRNA knock down (KD) of CXCL1 and MIF in A549 and tested the migration of PMNs towards treated and control cell lines using the novel microfluidic device. We observe 3-fold increase of PMN migration towards A549 compared to control. This increase was significantly inhibited in MIF and CXCL1 KDs as well as using MIF and CXCL1 neutralizing antibodies (NA) as compared to controls. PMN migration was higher to A549 then to PC9EN, and treatment of PMNs with a CXCR2 NA led to a decrease in their migration to A549 while unaffecting their migration to PC9EN. Due to the lack of similar genomic databases on LC metastasis, we profiled liver homogenates of mice intrasplenically injected with liver-metastatic Lewis lung carcinoma (LLC) and observed that Cxcl1 was the most overexpressed gene as compared to non-tumor bearing mice (non-TBM). We then KD CXCL1 from the liver metastatic LLC cell line and compared its capacity to recruit PMNs in live mice using intravital microscopy. We observe a decrease in the number of PMNs around developing CXCL1 KD LLC tumors compared to control LLC. We also observe a decrease in PMN migration toward the CXCL1 KD LLC tumors as compared the control LLC. This resulted in a significant decrease in liver metastasis of the CXCL1 KD LLC as compared to control LLC injected mice. Altogether, our data highlight the importance of CXCR2-mediated PMN migration in primary LC and the establishment of liver metastasis from LC. Thus, inhibiting CXCR2 represents a promising strategy to impede primary tumor growth and metastatic dissemination of LC. Citation Format: Roni F. Rayes, Jack G. Mouhanna, Claire Wang, Simon Milette, Carson Wong, Mariana Usatii, Betty Giannias, France Bourdeau, Rachel Mot, Arvind Chandrasekaran, Christopher Moraes, Sidong Huang, Daniela Quail, Logan Walsh, Veena Sangwan, Nicholas Bertos, Pierre-Olivier Fiset, Jonathan Cools-Lartigue, Lorenzo E. Ferri, Jonathan D. Spicer. Targeting CXCR2-mediated neutrophil recruitment to lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2799.

Trophinin-associated protein expression is an independent prognostic biomarker in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide, with lung adenocarcinoma (LAC) representing the most common subtype. Trophinin-associated protein (TROAP) is a cytoplasmic protein first identified to mediate the process of embryo transplantation, which has been recently found to be involved in microtubule regulation. However, limited information about the role of TROAP in LAC is available. We evaluated the relationship of TROAP expression in LAC tissues with clinical pathologic parameters and the survival time in LAC patients based on a statistical analysis of The Cancer Genome Atlas (TCGA) lung cancer data (N=528). Differences in survival between high and low expression groups (median expression cutoff) from the Cox univariate/multivariate regression analysis were then compared. According to the Chi-square tests, we found high TROAP expression correlated with younger age (≤60) (P=0.047), male sex (P<0.005), an earlier T-stage (P=0.011), N-stage (P=0.017), M-stage (P=0.022), TNM (P=0.007), and a longer smoking history (>30 pack-year) (P<0.001). A Kaplan-Meier analysis demonstrated that high TROAP expression may correspond with poor overall survival of LAC patients in T3 stage (P=0.0013), N0 stage (P=0.014), and M0 stage (P=0.0023). Multivariate analysis confirmed that TROAP expression was related to overall survival in LAC patients independently [hazard ratio (HR): 1.784, 95% confidence interval (CI): 1.072-2.968, P=0.026]. Our results suggested that TROAP is an independent prognostic biomarker of poor survival in LAC.

LDHA upregulation independently predicts poor survival in lung adenocarcinoma, but not in lung squamous cell carcinoma.

To investigate the potential prognostic value of LDHA in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Molecular, clinicopathological and survival data in Cancer Genome Atlas-Lung Cancer were obtained for secondary analysis. LDHA expression was significantly upregulated in both LUAD and LUSC compared with normal lung tissues. LUSC tissues had even higher LDHA expression compared with LUAD tissues. Increased LDHA expression was an independent prognostic indicator in terms of overall survival (hazard ratio: 1.547, 95% CI: 1.253-1.911; p<0.001) and recurrence-free survival (hazard ratio: 1.486, 95% CI: 1.161-1.900; p=0.002) in LUAD, but not in LUSC. LDHA expression might only serve as an independent prognostic indicator of unfavorable overall survival and recurrence-free survival in LUAD, but not in LUSC.

Abstract 3447: Down-regulation of long non-coding RNA MALL-2 is associated with metastasis and poor survival in lung adenocarcinoma

Abstract Lung adenocarcinoma accounts for ~40% of lung cancers and is among the deadliest cancers worldwide. Patients with metastatic lung adenocarcinoma exhibit poor outcome with the average 5-year survival of ~1%. Long non-coding RNAs (lncRNAs) have recently emerged as having critical roles in tumorigenesis. While a subset of lncRNAs have been shown to promote tumor invasion and metastasis, little is known about the role of lncRNAs in metastatic lung adenocarcinoma. Therefore, we analyzed publicly available microarray and RNA-Seq gene expression datasets that include primary and metastatic tissues. Through this meta-analysis we discovered 98 metastasis associated lncRNAs that were dysregulated in metastatic tumors compared to non-metastatic tumors (MALLs). Notably, only two lncRNAs (MALL-2 and MALL-36) were found to be down-regulated in primary tumors, relative to normal tissue, and even further down-regulated in metastatic tumors relative to primary tumors. Gene set enrichment analysis revealed that MALL-2, the most down-regulated lncRNA in metastasis, was coexpressed with protein-coding genes enriched with biological concepts associated with repressing tumor growth and metastasis. Furthermore, we found that FOXA2, a metastasis repressor gene, was highly coexpressed with MALL-2 in lung cancer. A pan-cancer analysis using ~7,000 TCGA RNA-Seq tumor samples across 20 cancer types revealed that MALL-2 was also down-regulated and coexpressed with FOXA2 in multiple cancers suggesting the conserved regulatory relationship between the two genes. We confirmed that FOXA2 expression was decreased upon knock-down of MALL-2 and silencing MALL-2 resulted in a significant increase in cellular migration in lung adenocarcinoma cell lines. Last, we found that low expression of MALL-2 was consistently associated with poor overall survival across multiple independent lung adenocarcinoma cohorts. Taken together, our integrative analysis has revealed MALL-2 that acts as a repressor of lung adenocarcinoma metastasis by regulating FOXA2 and could serve as a biomarker of lung adenocarcinoma patient outcome. Citation Format: Ha X. Dang, Nicole M. White, Emily B. Rozycki, Christopher A. Maher. Down-regulation of long non-coding RNA MALL-2 is associated with metastasis and poor survival 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 3447. doi:10.1158/1538-7445.AM2017-3447

High expression of C-X-C chemokine receptor 4 and Notch1 is predictive of lymphovascular invasion and poor prognosis in lung adenocarcinoma

C-X-C chemokine receptor 4 and Notch1 have been shown to play oncogenic role individually. This study aimed to determine the combinatorial role of C-X-C chemokine receptor 4 and Notch1 in lung adenocarcinoma. Expression of C-X-C chemokine receptor 4 and Notch1 was detected in resected tumor samples from 185 patients with lung adenocarcinoma at stage I-IIIa by immunohistochemistry. Correlations of their immunoscores with clinicopathological characteristics and disease-specific survival were retrospectively investigated. A three-dimensional capillary-like sprouting model was established to assess the effects of C-X-C chemokine receptor 4 and Notch1 on angiogenesis in vitro. The results revealed that expression of C-X-C chemokine receptor 4 and Notch1 was elevated in lung adenocarcinoma tissues. The high co-expression of C-X-C chemokine receptor 4 and Notch1 was significantly correlated with tumor size, tumor status, nodal status, tumor stage, and lymphovascular invasion, as well as decreased disease-specific survival. Multivariate analysis showed that lymphovascular invasion (hazard ratio: 0.205, 95% confidence interval: 0.086-0.491, p < 0.0001) and co-expression of C-X-C chemokine receptor 4 and Notch1 (hazard ratio: 0.293, 95% confidence interval: 0.168-0.510, p < 0.0001) were independent indicators of poor prognosis in lung adenocarcinoma. Furthermore, Notch1 enhanced the effects of C-X-C chemokine receptor 4 to promote angiogenesis by regulating Flt1 and Flt4 in vitro. In conclusion, co-expression of C-X-C chemokine receptor 4 and Notch1 is associated with tumor progression and lymphovascular invasion and is an independent indicator of poor survival in lung adenocarcinoma. In lung adenocarcinoma patients with high C-X-C chemokine receptor 4 and Notch1 expression, simultaneous inhibition of both factors might be an effective treatment strategy.

SCD1 is associated with tumor promotion, late stage and poor survival in lung adenocarcinoma.

The discovery of Warburg effect opens a new era in anti-cancer therapy. Aerobic glycolysis is regarded as a hallmark of cancer cells and increasing literatures indicates that metabolic changes are critical for the maintenance and progression of cancer cells. Besides aerobic glycolysis, increased fatty acid synthesis is also required for the rapid growth of cancer cells, and is considered as one of the most typical metabolic symbols of cancer either. Thus, targeting fatty acid metabolism may provide a potential avenue for the diagnosis and therapeutic treatment of cancer. In this study, we have identified Sterol-CoA desaturase-1 (SCD1) which is the rate-limiting enzyme of unsaturated fatty acid synthesis, universally and highly expressed in lung adenocarcinoma and was required for the cell proliferation, migration and invasion. Both in vitro and in vivo studies demonstrated that high expression of SCD1 remarkably enhanced the ability of tumor formation and invasion, while knockdown of SCD1 significantly repressed tumorigenesis and induced cell apoptosis. Clinical association study suggested that high expression of SCD1 is more frequently observed in late stage patients and presents poor prognosis. Taken together, our results suggested that SCD1 is a potentially novel biomarker of lung adenocarcinoma, and targeting SCD1 may represent a new anti-cancer strategy.

Axl Receptor Axis: A New Therapeutic Target in Lung Cancer

Abstract The Axl receptor tyrosine kinase belongs to the TAM (Tyro3, Axl, Mer) family of proteins and is upregulated in multiple types of cancers, including non-small cell lung cancer. In normal tissues, TAM receptor tyrosine kinases contribute to immune regulation. In cancer, Axl expression is associated with poor outcomes and is controlled through various transcriptional and epigenetic mechanisms. Preclinical studies have shown that Axl is involved in cancer cell migration, growth, survival and resistance to chemotherapy through activation of multiple downstream signaling pathways, such as Ras/MAPK, PI3K and Rac1. Axl is also expressed on endothelial cells and plays a role in angiogenesis. In preclinical models, Axl inhibition decreases cancer growth and impedes lung cancer metastases. Small molecule Axl inhibitors have been developed and are currently being used as monotherapy or in combination with cytotoxic chemotherapy or anti-EGFR therapy in early clinical trials. Here, we review Axl structure, functions, regulation, and preclinical and clinical studies in lung cancer.

Overexpression of major CDKN3 transcripts is associated with poor survival in lung adenocarcinoma.

Background:The cyclin-dependent kinase inhibitor 3 (CDKN3) has been perceived as a tumour suppressor. Paradoxically, CDKN3 is often overexpressed in human cancer. It was unclear if CDKN3 overexpression is linked to alternative splicing variants or mutations that produce dominant-negative CDKN3.Methods:We analysed CDKN3 expression and its association with patient survival in three cohorts of lung adenocarcinoma. We also examined CDKN3 mutations in the Cancer Genome Atlas (TCGA) and the Moffitt Cancer Center's Total Cancer Care (TCC) projects. CDKN3 transcripts were further analysed in a panel of cell lines and lung adenocarcinoma tissues. CDKN3 mRNA and protein levels in different cell cycle phases were examined.Results:CDKN3 is overexpressed in non small cell lung cancer. High CDKN3 expression is associated with poor overall survival in lung adenocarcinoma. Two CDKN3 transcripts were detected in all samples. These CDKN3 transcripts represent the full length CDKN3 mRNA and a normal transcript lacking exon 2, which encodes an out of frame 23-amino acid peptide with little homology to CDKN3. CDKN3 mutations were found to be very rare. CDKN3 mRNA and protein were elevated during the mitosis phase of cell cycle.Conclusions:CDKN3 overexpression is prognostic of poor overall survival in lung adenocarcinoma. CDKN3 overexpression in lung adenocarcinoma is not attributed to alternative splicing or mutation but is likely due to increased mitotic activity, arguing against CDKN3 as a tumour suppressor.

Significant role of Psf3 expression in non-small-cell lung cancer.

The GINS complex associates with cell division cycle (Cdc) protein 45 and mini-chromosome maintenance (Mcm) proteins 2-7 to form the Cdc45-Mcm-GINS (CMG) complex, which is essential for DNA duplication. One member of the GINS complex is Psf3. We previously found that increased Psf3 expression was strongly associated with poor survival in lung adenocarcinoma. Here, we investigated the role of Psf3 expression in non-small-cell lung cancer (NSCLC). We verified Psf3 expression in human NSCLC tissues (180 patients) and cell lines. Immunohistochemical analysis revealed that the overexpression of Psf3 was significantly associated with vessel invasion (P = 0.016), lymphatic invasion (P = 0.002), and pleural invasion (P = 0.036). The overall survival rate in patients with Psf3 overexpression was significantly lower than that in patients without Psf3 overexpression (P = 0.006). Multivariate survival analysis revealed Psf3 expression to be an independent risk factor for an unfavorable outcome (P = 0.049). A proximal ligation assay showed interactions between Psf3 and other CMG components (such as Mcm2 and Cdc45) in both NSCLC specimens and cell lines, indicating that Psf3 acted as the CMG complex, which could lead to excessive proliferation. Knockdown of Psf3 inhibited the proliferation of both cell lines by delaying the S phase, which revealed that Psf3 played an important role in cancer proliferation. Thus, Psf3 acted as the CMG complex, promoting excessive proliferation. These results suggest that Psf3 inhibition might be a therapeutic target for NSCLC with Psf3 overexpression.

Abstract 1149: The role of the novel Notch1-Sox9 signaling axis in NSCLC progression and EMT

Abstract 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 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 to induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling. 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. 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. 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 downstream of Notch1 in lung adenocarcinoma cell lines. By ChIP we determined that Sox9 is a direct target of Notch1 and using luciferase reporter assays, we located the previously unidentified human RBP-Jκ 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 and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Loss of Notch1 expression led to an induced MET phenotype, characterized by decreased cell invasion/migration, MET-like morphological changes, and increased E-cadherin expression, which were rescued by Sox9 overexpression. Our data also demonstrate that Sox9 contributes to Notch1-induced EMT in lung adenocarcinoma. These results establish Sox9 as a key Notch1 target gene mediating Notch1-induced EMT independent of TGF-β, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen M. Capaccione, Xuehui Hong, Katherine M. Morgan, Thaddeus D. Allen, Gregory D. Miles, Elke K. Markert, J. Michael Bishop, Sharon R. Pine. The role of the novel Notch1-Sox9 signaling axis in NSCLC progression and EMT. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1149. doi:10.1158/1538-7445.AM2014-1149

Abstract 1597: Transcriptomic architecture of the field of cancerization in the adjacent normal-appearing airway: Early mechanisms in lung carcinogenesis

Abstract Increasing our understanding of early events in the pathogenesis of lung cancer is crucial for identification of new targets for prevention and treatment of this malignancy. Earlier work has shown that seemingly normal cells adjacent to the tumor carry specific molecular alterations that are characteristic of the tumor itself suggestive of a field of cancerization. By sampling and studying normal-appearing tissue, the molecular field of cancerization provides biological insights into early phases in cancer development. In this study, we sought to characterize molecular field effects in the normal-appearing airway that are most representative of the nearby lung tumor, and thus, are most likely to denote early events in lung carcinogenesis. To achieve this, we performed genome-wide expression profiling of resected field cancerization specimens (n=20 patients) comprised of matched early-stage non-small cell lung cancers (NSCLCs), cytologically normal airways with varying spatial distance from the tumors and distant (relative to location of tumors) normal lung tissues (n=194 samples). Using ordinal logistic regression, we identified 422 genes that were progressively modulated in expression in normal-appearing airways by spatial distance from tumors. Notably, when examined in paired NSCLC and normal lung tissues, these genes were found to recapitulate tumor expression profiles. We then sought to examine the role of lysosomal protein transmembrane 4 beta (LAPTM4B), a putative oncogene that was found to be up-regulated in airways by shorter spatial distance from tumors, in lung oncogenesis. LAPTM4B was significantly elevated in NSCLC tissues compared to paired distant normal lung and was predictive of poor survival in lung adenocarcinoma. Moreover, LAPTM4B promoted anchorage-dependent and -independent lung cancer cell growth and was crucial for cellular survival and the autophagy response under nutrient- and serum-deprived conditions. In addition, pathways analysis of a LAPTM4B-dependent gene expression profile revealed decreased activation of the canonical nuclear factor erythroid 2-like 2 (NRF2)-mediated pathway following LAPTM4B knockdown. Further, we found that LAPTM4B augmented the expression and nuclear translocation of the NRF2 transcription factor following serum deprivation pointing to the probable role of the novel LAPTM4B/NRF2 signaling axis in promoting lung cancer cell survival. All in all, our study points to molecular field of cancerization profiles in the normal-appearing airway that highly signify the nearby lung tumor and comprise early mechanisms (e.g. LAPTM4B) in lung carcinogenesis. Citation Format: Yuho Maki, Junya Fujimoto, Suk-Young Yoo, Melinda Garcia, Adam Gower, Li Shen, Chi-Wan Chow, Carmen Behrens, Neda Kalhor, Cesar Moran, Jing Wang, Avrum Spira, Kevin R. Coombes, Ignacio I. Wistuba, Humam Kadara. Transcriptomic architecture of the field of cancerization in the adjacent normal-appearing airway: Early mechanisms in lung carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1597. doi:10.1158/1538-7445.AM2014-1597

The elevated level of ERα36 is correlated with nodal metastasis and poor prognosis in lung adenocarcinoma

IntroductionERα36 is a recently cloned variant of estrogen receptor-alpha which has been proved to play an active role in a series of malignant diseases. MethodERα36 expression was examined using immunohistochemical methods with sections from 126 resected NSCLC specimens. The immunoreactivity of ERα66 was also studied as a comparison. Kaplan–Meier method and multivariable Cox proportional hazards regression analyses were used to examine the relationship between ERα36 and survival. ResultERα36 was more highly expressed in NSCLC patients compared to ERα66. ERα36 expression has a strong correlation with histology (AC: 53/70, SCC: 16/56, P<0.000) and had a significantly positive correlation with lymphatic metastasis (P=0.014) in adenocarcinoma. High ERα36 expression was correlated with poorer overall survival (OS) (P=0.020) and disease-free survival (DFS) (P=0.024) in adenocarcinoma. Furthermore, ERα36 status was a significant independent prognostic factor of OS (P=0.018, HR: 3.142, 95% CI: 1.215–8.128) and DFS (P=0.024, HR: 2.720, 95% CI: 1.141–6.486) in lung adenocarcinoma patients. ConclusionERα36 had a high expression mainly in adenocarcinoma and the high expression of ERα36 was strongly correlated with more advanced regional lymph node metastasis and poor survival in lung adenocarcinoma.