Phenotype Of Lung Cancer Research Articles

Quantitative Secretomic Analysis Identifies Extracellular Protein Factors That Modulate the Metastatic Phenotype of Non-Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths for men and women in the United States, with non-small cell lung cancer (NSCLC) representing 85% of all diagnoses. Late stage detection, metastatic disease and lack of actionable biomarkers contribute to the high mortality rate. Proteins in the extracellular space are known to be critically involved in regulating every stage of the pathogenesis of lung cancer. To investigate the mechanism by which secreted proteins contribute to the pathogenesis of NSCLC, we performed quantitative secretomic analysis of two isogenic NSCLC cell lines (NCI-H1993 and NCI-H2073) and an immortalized human bronchial epithelial cell line (HBEC3-KT) as control. H1993 was derived from a chemo-naïve metastatic tumor, while H2073 was derived from the primary tumor after etoposide/cisplatin therapy. From the conditioned media of these three cell lines, we identified and quantified 2713 proteins, including a series of proteins involved in regulating inflammatory response, programmed cell death and cell motion. Gene Ontology (GO) analysis indicates that a number of proteins overexpressed in H1993 media are involved in biological processes related to cancer metastasis, including cell motion, cell-cell adhesion and cell migration. RNA interference (RNAi)-mediated knock down of a number of these proteins, including SULT2B1, CEACAM5, SPRR3, AGR2, S100P, and S100A14, leads to dramatically reduced migration of these cells. In addition, meta-analysis of survival data indicates NSCLC patients whose tumors express higher levels of several of these secreted proteins, including SULT2B1, CEACAM5, SPRR3, S100P, and S100A14, have a worse prognosis. Collectively, our results provide a potential molecular link between deregulated secretome and NSCLC cell migration/metastasis. In addition, the identification of these aberrantly secreted proteins might facilitate the development of biomarkers for early detection of this devastating disease.

Mechanism of Wnt/β-catenin signaling pathway in enhanced malignant phenotype of non-small cell lung cancer induced by anti-angiogenesis therapy

To study the mechanism of Wnt/β-catenin signaling pathway in the enhanced malignant phenotype of A549 cells of human non-small cell lung cancer induced by the anti-angiogenesis therapy. The siRNA technique was employed to inhibit the expression of vascular endothelial growth factor (VEGF) in A549 cells and simulate the clinical course of anti-angiogenesis therapy. Real-time PCR and western blot were used to study the change in the expression of Wnt/β-catenin signaling molecules at the mRNA and protein level respectively, as well as the effect on the epithelial mesenchymal transition in A549 cells. The proliferation and invasion abilities of tumor cells were detected to discuss the mechanism of Wnt/β-catenin signaling pathway in the enhanced malignant phenotype of non-small cell lung cancer induced by the anti-angiogenesis therapy. The specific siRNA could significantly inhibit the expression of VEGF in cells to simulate the anti-angiogenesis therapy. Under the action of 50nM VEGF siRNA, the proliferation ability of A549 significantly increased (P<0.05). After being treated with VEGF siRNA, the invasion ability of cells increased. Twenty-four hours after the transcription of 50nM siRNA into cells, the number of cells that come through the membrane was 278.3±12.9. Compared with the Ctrl siRNA group, when VEGF was inhibited, the expression of β-catenin and Cyclin D1 increased by 86% and 55% respectively. Meanwhile, the expression of E-cadherin decreased, while the one of vimentin increased. siRNA can significantly inhibit the expression of VEGF. For the anti-angiogenesis therapy, the inhibited expression of VEGF can activate the Wnt/β-catenin signaling pathway to cause the epithelial mesenchymal transition and then the enhanced malignant phenotype of non-small cell lung cancer.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Caveolae as a target to quench autoinduction of the metastatic phenotype in lung cancer.

PurposeMevalonate pathway inhibitors are potentially useful chemotherapeutic agents showing growth inhibition and pro-apoptotic effects in cancer cells. The effects of statins and bisphosphonates on cancer growth are attributed to a reduction in protein isoprenylation. Post-translational modification and activation of GTPase binding Ras superfamily permit the recruitment of these signal proteins to membranes where they mediate the cancer phenotype. Here, the effects of three inhibitors of the mevalonate pathway and one specific inhibitor of sterol regulatory element-binding proteins were studied in both an ER-negative, Ras-inactive breast (MDA-MB-231) and lung adenocarcinoma (CaLu-1) cells in vitro.MethodsTreated cells were subject to genome-wide gene expression profiling. A gene subset was established so that the epithelial to mesenchymal transition (EMT) could be observed and compared with signalling protein shifts.ResultsWithin the subset, some genes normally up-regulated during EMT were asymmetrically reduced by a Δ-24 DHCR inhibitor in the lung cells. Signalling proteins associated with caveolae were down-regulated by this oxidoreductase inhibitor, while those associated with membrane rafts were up-regulated.ConclusionsThis study decouples isoprenylation effects from cholesterol events per se. The data support a hypothesis that caveolae are abolished by Δ-24 DHCR intervention and it is revealed that these microdomains are vital EMT signalling structures for lung cells but not ER- and Ras-negative breast cells. When signalling by extracellular signals is quenched by removal of the hydrophilic conduit provided by caveolae, the transcriptome responds by moving the cellular identity towards quiescence.Electronic supplementary materialThe online version of this article (doi:10.1007/s00432-015-2074-3) contains supplementary material, which is available to authorized users.

Tumor interstitial fluid promotes malignant phenotypes of lung cancer independently of angiogenesis.

Angiogenesis is necessary for cancer progression, but antiangiogenic therapy actually promotes tumor recurrence, progression, and metastasis. This study focused on the contribution of the tumor interstitial fluid (TIF) to lung cancer progression. TIF was isolated and quantified for 10 μg protein/mL. Malignant driver characteristics of TIF were examined by tumor-initiating cells (TIC), self-renewal, epithelial-mesenchymal transition (EMT), autophagy, and apoptosis in vitro. In vivo tumor model was used to investigate the mechanistic roles of TIF in lung cancer progression. In vitro, TIF exhibited distinct malignant driver characteristics, which led to increased numbers of TICs, increased self-renewal and EMT, as well as to decreased autophagy and apoptosis under cell starvation conditions. In vivo, the contribution of TIF was similar, as judged by increased TICs indicated by the cancer stem cell marker Nanog, the proliferation marker proliferating cell nuclear antigen, and the EMT marker N-cadherin; TIF also increased the formation of pulmonary tumors. Interestingly, the blockers of inflammation, Na-K-ATPase, and aldosterone receptor decreased TIF-induced tumor progression but increased angiogenesis. Further, we found that the water content of the tissue was positively correlated with the levels of plasma 5-hydroxyindoleacetic acid or tissue aquaporin-1 but not with CD31. However, vadimezan reduced angiogenesis but promoted TIF-induced tumor progression. Our results suggested that TIF could provide better nutrition to the tumor than angiogenesis and that it could promote the development of malignant phenotypes of lung cancer independently of angiogenesis.

P34: Plasma micro-RNAs as noninvasive biomarkers for diagnostics of lung cancer

Lung cancer (LC) is currently the world’s leading cause of cancer-related mortality with overall mortality to incidence ratio of 0.87. Only 15% of LC patients are diagnosed at early stage of disease and have 5-year survival rate of 54%, whereas 56% of patients have distant metastases and 5-year survival rate of about 4%. Current methods of lung cancer diagnostics are not efficient as screening tools due to high costs (CT), low sensitivity and health risks (radiology), invasiveness (biopsy) or low prevalence in population (genetic alterations). New strategies for preclinical lung cancer screening as well as monitoring of post therapy relapses are required. MiRNAs circulating in blood were shown to reflect the progression of disease and are thus considered as potential biomarkers for cancer diagnostics and theranostics. In this study we investigated seven-miRNA signature circulating in plasma of lung cancer patients and healthy individuals. All seven miRNAs were previously shown to be involved in either regulation of cell cycle and apoptosis and/or tumor development, invasion and vascularization. Blood samples of 50 healthy individuals were obtained from Center of New Medical Technologies (Novosibirsk, Russia) and Novosibirsk Research Institute of Circulation Pathology of E.N. Meshalkin (Novosibirsk, Russia). Samples of 75 lung cancer patients, including 52 patients with squamous cell carcinoma (SCC) and 18 patients with adenocarcinoma (AC) were obtained from Novosibirsk Research Institute of Circulation Pathology of E.N. Meshalkin (Novosibirsk, Russia) and Tomsk Cancer Research Institute RAMS (Tomsk, Russia). None of the patients have undergone surgical treatment or received chemotherapy prior to blood collection. Lung biopsy specimens and imaging techniques were applied to confirm the histopathological features and tumor stages of LC patients. Study was approved by ethical committees of all participating organizations and written informed consent was provided by all participants. Circulating miRNAs were isolated from blood plasma using a single-phase phenol-free protocol (Zaporozhchenko et al., Anal Biochem, 2015; Rus. patent application No. 2014137763, priority date 17.09.2014). Concentrations of miRNAs (miR-21, miR-19b, miR-126, miR-25, miR-205, miR-183, miR-125b) were measured by qRT-PCR and normalized to miR-16. Concentrations of four miRNAs (miR-19b, miR-21, miR-25, miR-183) were significantly different in lung cancer versus healthy individuals (p Receiver Operating Characteristic (ROC) curve analysis of differentially expressed miRNAs in total study population and subtype-based groups showed that miR-19b has highest predictive value for total population and SCC patients, while miR-183 was more effective in discriminating patients with AC. All miRNAs except miR-21 showed strong bias towards one of the subtypes. Presumably a combination of miRNAs with opposite bias should provide a more potent diagnostic tool for cancer detection in total population than individual miRNAs or panels of miRNAs that are specific to one cancer subtype. Indeed, stepwise binary logistic regression has identified the combination of miR-19b and miR-183 to be a strong prediction of disease in total population and yielded a solid increase in AUC: 0,990 (miR-19b+miR-183) versus 0,806 (miR-19b) or 0,924 (miR-183). This combination can be used to identify lung cancer with 94.7% sensitivity and 95.2% specificity. Thus, biological aspects such as tumor genetics and phenotype, stage of disease, response to therapy, and other meaningful tumor properties of such heterogeneous malignancies as LC can interfere with contents and composition of cell-free miRNA pool. Wide profiling of circulating miRNAs will specify biomarkers of LC phenotypes and improve non/less-invasive LC diagnostics.

Combined analysis identifies six genes correlated with augmented malignancy from non-small cell to small cell lung cancer.

With increased malignancy, lung cancer can be classified into adenocarcinoma (ADC), squamous cell carcinoma (SQC), large cell carcinoma (LCC), and the small cell subtype (SCLC); yet, elucidations to this augmented malignancy has not been addressed. In this study, we elucidated the molecular diversity among these subtypes by investigating large-scale sequencing datasets. Among genes upregulated from normal, ADC, SQC, LCC to SCLC, six hub genes were found closely correlated with adverse clinical outcome and were testified on cellular or tissue level with quantitative RT-PCR. Cox regression model was then built to generate a risk signature. The possible linkages among these genes were also explored. Transcript levels of BUB1, E2F1, ESPL1, GTSE1, RAB3B, and U2AF2 were found significantly elevated from normal, ADC, SQC, LCC to SCLC. Overexpression of one or multiple of these genes was correlated with adverse overall survival (OS) and relapse-free survival (RFS) in the whole patient cohort or groups stratified according to clinical variables, while most of all six genes were independent prognostic factors. When used as a six-gene risk signature, patients with high signature score displayed more unfavorable clinical variables and poorer outcome. Tight regulative relationships were found within these genes, while BUB1 and E2F1 were likely to be the drivers. We considered the augmented malignancy from non-small cell lung cancer (NSCLC) to SCLC might be due to the elevation of these six genes. We believe these genes were powerful cancer prognostic markers and potential therapeutic targets in lung cancer; moreover, changes of their level might be correlated with lung cancer phenotype plasticity.

Increased expression of transcription factor EB (TFEB) is associated with autophagy, migratory phenotype and poor prognosis in non-small cell lung cancer.

We investigated the role of lysosomal biogenesis and hydrolase activity in the clinical behavior and postoperative outcome of lung cancer. Using immunohistochemistry we investigated the expression of the transcription factor EB (TFEB) which orchestrates lysosomal biogenesis, the lysosome membrane protein LAMP2a and of the lysosomal hydrolase cathepsin D in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery alone. In vitro experiments with the A549 and H1299 lung cancer cell lines were also performed. Overexpression of TFEB, LAMP2a and Cathepsin D was noted in 47/98 (47.9%), 43/98 (43.9%) and 39/98 (39.8%) cases, respectively, and were significantly correlated with each other and with adenocarcinomas. High LAMP2a was related to high histology grade. Linear regression analysis confirmed significant association of TFEB with BNIP3 (p=0.0003, r=0.35) and LC3A with LAMP2a expression (p=0.0002, r=0.37). An inverse association of Cathepsin D expression with stone-like structures (SLS) was recorded (p=0.02, r=0.22). On univariate analysis all three lyososomal variables were associated with poor prognosis (p=0.05, 0.04 and 0.01, for TFEB, Cathepsin D and LAMP2a, respectively). Multivariate analysis showed that the SLS number (p=0.0001, HR5.37), Cathepsin D expression (p=0.01, HR=2.2) and stage (p=0.01, HR=1.5) were independent prognostic variables. Silencing of TFEB with siRNAs in the A549 and H1299 lung cancer cell lines did not affect proliferation but resulted in reduced migration ability. Lysosomal biogenesis is linked to autophagosomal protein expression in NSCLC and characterizes subgroups of high risk patients after complete surgical lung tumor resection.

MicroRNA-7 inhibits the malignant phenotypes of non-small cell lung cancer in vitro by targeting Pax6

MicroRNA (miR)-7 has been reported to act as a suppressor in several types of cancer, including non-small cell lung cancer (NSCLC). In addition, paired box 6 (Pax6), a highly conserved transcriptional factor, has been implicated in NSCLC. However, the exact role of miR-7, and the association between miR-7 and Pax6 in NSCLC cells remain to be fully elucidated. The present study demonstrated that miR-7 was downregulated and Pax6 was upregulated in NSCLC cell lines. Subsequently, it was demonstrated that overexpression of miR-7 notably inhibited the protein expression of Pax6, while inhibition of miR-7 enhanced the protein expression of Pax6 in NSCLC A549 cells. Further investigation identified Pax6 as a target of miR-7 in A549 NSCLC cells. Ina ddition, the overexpression of miR-7 significantly inhibited A549 cell proliferation and invasion, which was reversed by upregulation of Pax6. Investigation of the underlying molecular mechanism revealed that the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways were downregulated in the miR-7-overexpressed A549 cells, but were activated in the Pax6-overexpressed A549 cells. Based on these findings, it was suggested that miR-7 negatively regulates the protein level of Pax6, which can promote the proliferation and invasion of NSCLC cells via activation of the ERK and MAPK signaling pathways. Therefore, miR-7/Pax6 may offer potential for use as a target for the treatment of NSCLC.

Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/β-catenin signaling pathway.

Maternally expressed gene 3 (Meg3) has been shown to promote tumor progression. However, the role of Meg3 in the development of a chemoresistant phenotype of human lung cancer has remains. Reverse transcription‑quantitative polymerase chain reaction analysis was used to determine the expression of Meg3. Flow cytometric analysis and MTT assay were also used to investigate the cell cycle and apoptosis. The present study detected that the expression levels of Meg3 were significantly lower in cisplatin‑resistant A549/DDP lung cancer cells, compared with those in parental A549 cells. Furthermore, upregulation of Meg3 was able to re‑sensitize the A549/DDP cells to cisplatin in vitro. Whereas downregulation of Meg3, by RNA interference, decreased the sensitivity of A549 cells to cisplatin. The results of the present study also demonstrated that the Meg3‑mediated chemosensitivity enhancement was associated with the induction of cell-cycle arrest and increased apoptosis, through regulation of p53, β‑catenin and survivin, which is a target gene of the WNT/β‑catenin signaling pathway. In conclusion, these results suggested that Meg3 may have a crucial role in the development of cisplatin resistance in non-small cell lung cancer.

Thymidine phosphorylase and its possible correlation with the lung cancer phenotype features

The tumor phenotype investigation is necessary for lung cancer drug therapy optimization. For this purpose is perspective to use the biochemical markers of angiogenesis. Thymidine phosphorylase may be one of them.Aim. The aim of study was to evaluate the correlation between the enzyme activity in tumors and the levels of expression of the vascular endothelial marker CD34.Methods and results. They were examined spectrophotometrically and by the immunohistochemical method, respectively, in 25 samples of lung cancer tissues. It was shown that its activity in tumors is higher as compared to non-neoplastic adjacent tissues (from 30 to150 nmol/min·mg; in 20 cases from total number). The enzyme activity changes were dependent of the levels of the CD 34, a positive correlation between them was shown (the index of Spearman’s rank correlation, i.e. ρ = 0,668; p&lt;0,05).Conclusion. These findings suggest the possibility of using thymidine phosphorylase as a biochemical marker of angiogenesis, its relationship with the lung cancer phenotype features.

Association of chromosome 19 to lung cancer genotypes and phenotypes.

The Chromosome 19 Consortium, a part of the Chromosome-Centric Human Proteome Project (C-HPP, http://www.C-HPP.org ), is tasked with the understanding chromosome 19 functions at the gene and protein levels, as well as their roles in lung oncogenesis. Comparative genomic hybridization (CGH) studies revealed chromosome aberration in lung cancer subtypes, including ADC, SCC, LCC, and SCLC. The most common abnormality is 19p loss and 19q gain. Sixty-four aberrant genes identified in previous genomic studies and their encoded protein functions were further validated in the neXtProt database ( http://www.nextprot.org/ ). Among those, the loss of tumor suppressor genes STK11, MUM1, KISS1R (19p13.3), and BRG1 (19p13.13) is associated with lung oncogenesis or remote metastasis. Gene aberrations include translocation t(15, 19) (q13, p13.1) fusion oncogene BRD4-NUT, DNA repair genes (ERCC1, ERCC2, XRCC1), TGFβ1 pathway activation genes (TGFB1, LTBP4), Dyrk1B, and potential oncogenesis protector genes such as NFkB pathway inhibition genes (NFKBIB, PPP1R13L) and EGLN2. In conclusion, neXtProt is an effective resource for the validation of gene aberrations identified in genomic studies. It promises to enhance our understanding of lung cancer oncogenesis.

Microtubule affinity-regulating kinase 2 is associated with DNA damage response and cisplatin resistance in non-small cell lung cancer.

Microtubule affinity-regulating kinases (MARKs) are involved in several cellular functions but few studies have correlated MARK kinase expression with cancer, and none have explored their role in lung cancer. In this study, we identified MARK2 as frequently disrupted by DNA hypomethylation and copy gain, resulting in concordant overexpression in independent lung tumor cohorts and we demonstrate a role for MARK2 in lung tumor biology. Manipulation of MARK2 in lung cell lines revealed its involvement in cell viability and anchorage-independent growth. Analyses of both manipulated cell lines and clinical tumor specimens identified a potential role for MARK2 in cell cycle activation and DNA repair. Associations between MARK2 and the E2F, Myc/Max and NF-κB pathways were identified by luciferase assays and in-depth assessment of the NF-κB pathway suggests a negative association between MARK2 expression and NF-κB due to activation of non-canonical NF-κB signaling. Finally, we show that high MARK2 expression levels correlate with resistance to cisplatin, a standard first line chemotherapy for lung cancer. Collectively, our work supports a role for MARK2 in promoting malignant phenotypes of lung cancer and potentially modulating response to the DNA damaging chemotherapeutic, cisplatin.

Targeted inhibition of genome-wide DNA methylation analysis in epigenetically modulated phenotypes in lung cancer.

DNA methylation analysis, an epigenetic specification, has been explored for partial determination of cancer cell phenotypes. The development of metastasis in cancerogenesis has led its feasible association with the epigenetic modulations. We generated highly aggressive non-small cell lung cancer cell lines (HTB56 and A549) by using in vivo selection approach. These were, then, subjected to DNA methylation analysis (genome-wide). We also explored the therapeutic effects of azacytidine, an epigenetic agent, on DNA methylation patterns as well as the in vivo phenotypes. During the development of highly aggressive cell lines, we observed widespread modulations in DNA methylation. Reduced representation bisulfite sequencing was used and compared with the less aggressive parental cell lines to identify the differential methylation, which was achieved up to 2.7 % of CpG-rich region. Azacytidine inhibited DNA methyltransferase and reversed the prometastatic phenotype. We found its high association with the preferential loss of DNA methylation from hypermethylated sites. After persisted exposure of azacytidine, we observed that DNA methylation affected the polycomb-binding sites. We found close association of DNA methylome modifications with metastatic capability of non-small cell lung cancer. We also concluded that epigenetic modulation could be used as a potential therapeutic approach to prevent metastasis formation as prometastatic phenotype was reversed due to inhibition of DNA methyltransferase.

Enhancement of tumor initiation and expression of KCNMA1, MORF4L2 and ASPM genes in the adenocarcinoma of lung xenograft after vorinostat treatment.

Cancer stem cells (CSCs) are usually tolerant to chemotherapy and radiotherapy and associated with tumor relapse. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), is currently being used in clinical trials of lung cancer. However, SAHA facilitates the formation of induced pluripotent stem cells from somatic cells. We hypothesized that SAHA would mediate the CSCs properties and subsequently confer a more malignant phenotype in lung cancer. Transfected H1299 lung cancer cells, which stably expresses a triple fused reporter gene (DsRedm-Fluc-tTKsr39) under the control of CMV promoter was used to establish a xenograft mouse model. After the treatment of SAHA, H1299 cell line and tumor xenografts were sorted by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity. We found that SAHA could suppress the growth of xenografted H1299 tumors with decreased proportion of ALDHbr lung cancer cells indicating that SAHA may target CSCs. However, SAHA significantly enhanced the tumor initiating capacity and the expression of malignant genes such as KCNMA1, MORF4L2 and ASPM in the remaining living ALDHbr cells. These findings suggested that SAHA treatment created a more drug-resistant state in residual ALDHbr cells. The in vivo imaging technique may facilitate searching and characterization of CSCs.

Notch1 phenotype and clinical stage progression in non-small cell lung cancer.

BackgroundNotch1 transmembrane receptor is activated through ligand-binding- triggered proteolytic cleavages and, upon release, the intracellular domain (N1-ICD) translocates into the nucleus and modulates target gene transcriptions. Notch activation has been implicated in tumorigenesis in an increasing number of human malignancies including non-small cell lung cancer (NSCLC). However, Notch1 in distinct expression patterns and activation status with tumor progression remains to be defined in NSCLC.MethodsNotch1 and activated Notch1, N1-ICD, were examined by immunohistochemistry in 58 cases of stage I to IV NSCLC tumors. Association between Notch1 or N1-ICD expression and clinicopathological factors was assessed via correlation coefficient r statistics. P-values are two-sided.ResultsDetectable tumor Notch1, predominantly localized to the membrane and cytoplasm, was observed in 29 cases (50%, 95% Blyth-Still-Casella confidence interval 37 – 63%). It was negatively associated with stage (r = - 0.43, P < 0.001) and nodal status (r = - 0.33, P = 0.01), but not tumor size. In contrast, nuclear N1-ICD expression level was low and found in 12% of NSCLC patients, neither significantly associated with stage nor nodal status. Upon Notch1 activation in vitro, a mostly extra-nuclear staining was substantially turned into the nuclear signal in cancer cells.ConclusionsNotch1 in the largely inactivated phenotype is inversely associated with clinical stage progression in NSCLC. Notch1, rather than activated N1-ICD, may be a context-dependent restrictive factor to nodal metastasis.

Proinflammatory Cytokines Increase Vascular Endothelial Growth Factor Expression in Alveolar Epithelial Cells.

Vascular endothelial growth factor (VEGF) is an endothelial permeability mediator that is highly expressed in lung epithelium. In nonlung cells proinflammatory cytokines have been shown to increase VEGF expression, but their effects on lung epithelium remain unclear. We hypothesized that increases in alveolar epithelial cell VEGF RNA and protein expression occur after exposure to proinflammatory cytokines. We tested this using human alveolar epithelial cells (A549) stimulated with 5 proinflammatory cytokines. VEGF RNA expression was increased 1.4–2.7-fold in response to IL-1, IL-6, IL-8, TNF-α, or TGF-β over 6 hours, with TGF-β having the largest response. TNF-α increased VEGF RNA as early as 1 hour. A mix of IL-1, IL-6, and IL-8 had effects similar to IL-1. TNF-α increased protein expression as early as 4 hours and had a sustained effect at 16 hours, whereas IL-1 did not increase protein expression. Only VEGF165 was present in cultured A549 cells, yet other isoforms were seen in human lung tissue. Increased expression of VEGF in alveolar epithelial cells occurs in response to proinflammatory cytokines. Increased VEGF expression likely contributes to the pathogenesis of inflammatory lung diseases and to the angiogenic phenotype of lung cancer, a disease typically preceded by chronic inflammation.

A Generic Data Harmonization Process for Cross-linked Research and Network Interaction. Construction and Application for the Lung Cancer Phenotype Database of the German Center for Lung Research.

Joint data analysis is a key requirement in medical research networks. Data are available in heterogeneous formats at each network partner and their harmonization is often rather complex. The objective of our paper is to provide a generic approach for the harmonization process in research networks. We applied the process when harmonizing data from three sites for the Lung Cancer Phenotype Database within the German Center for Lung Research. We developed a spreadsheet-based solution as tool to support the harmonization process for lung cancer data and a data integration procedure based on Talend Open Studio. The harmonization process consists of eight steps describing a systematic approach for defining and reviewing source data elements and standardizing common data elements. The steps for defining common data elements and harmonizing them with local data definitions are repeated until consensus is reached. Application of this process for building the phenotype database led to a common basic data set on lung cancer with 285 structured parameters. The Lung Cancer Phenotype Database was realized as an i2b2 research data warehouse. Data harmonization is a challenging task requiring informatics skills as well as domain knowledge. Our approach facilitates data harmonization by providing guidance through a uniform process that can be applied in a wide range of projects.

Abstract A53: SmgGDS splice variants interact with K-Ras through association with both the polybasic region and the CAAX motif, and promote the malignant phenotype in lung, breast, and pancreatic cancers

Abstract SmgGDS is a chaperone protein that binds Ras and Rho family members that have a C-terminal polybasic region (PBR). We reported that the splice variants SmgGDS-607 and SmgGDS-558 regulate the prenylation and membrane trafficking of PBR-containing small GTPases. SmgGDS-607 interacts with non-prenylated small GTPases and regulates their entry into the prenylation pathway. In contrast, SmgGDS-558 interacts with prenylated small GTPases and promotes their trafficking to cell membranes. In the current study, we investigated the physical interaction of SmgGDS splice variants with K-Ras, and tested the ability of SmgGDS splice variants to promote the malignant phenotype of cancer cell lines expressing oncogenic K-Ras. Because SmgGDS-607 binds K-Ras before it is prenylated, we hypothesized that SmgGDS-607 recognizes the C-terminal CAAX motif of K-Ras. This CAAX motif consists of the sequence CVIM, and the C-terminal methionine causes newly synthesized K-Ras to interact with the farnesyltransferase and become farnesylated. However, newly synthesized K-Ras will alternatively interact with geranylgeranyltransferase-I and become geranylgeranylated in the presence of farnesyltransferase inhibitors (FTIs), or if the C-terminal methionine is mutated to leucine. To test the recognition of the K-Ras CAAX motif by SmgGDS-607, we examined binding of SmgGDS-607 to K-Ras that has the wildtype CAAX sequence (K-Ras-CVIM) or a mutant CAAX sequence (K-Ras-CVIL). We show that SmgGDS-607 associates more with K-Ras-CVIL than with K-Ras-CVIM in cells, indicating that SmgGDS-607 interacts with non-prenylated K-Ras that is destined to become geranylgeranylated rather than farnesylated. This conclusion is further supported by our finding that FTIs increase binding of wildtype K-Ras-CVIM to SmgGDS-607, indicating that the pharmacological shunting of K-Ras into the geranylgeranylation pathway promotes K-Ras association with SmgGDS-607. These findings indicate that SmgGDS-607 will preferentially interact with non-prenylated K-Ras that is destined to enter the geranylgeranylation pathway caused either by FTIs or mutation of the CAAX motif. In contrast to SmgGDS-607, we found that SmgGDS-558 interacts robustly with both farnesylated and geranylgeranylated K-Ras, as well as with oncogenic K-Ras(G12V). Because SmgGDS-558 interacts with prenylated and oncogenic K-Ras, we hypothesized that SmgGDS-558 plays a greater role than SmgGDS-607 in promoting the malignancy of cancers expressing oncogenic K-Ras. Consistent with this hypothesis, we found that the RNAi-mediated depletion of SmgGDS-558, but not SmgGDS-607, significantly diminishes proliferation, induces a G1 arrest, diminishes expression of the cell cycle promoter Cyclin D1, and increases expression of the cell cycle inhibitor p27 in human cancer cell lines expressing oncogenic K-Ras, including pancreatic (MiaPaCa-2 and PANC-1), lung (NCI-H23), and breast (MDA-MB-231) cancer cell lines. Interestingly, similar responses occurred after depletion of SmgGDS-558 in MCF-7 breast cancer cells but not in NCI-H1703 lung cancer cells, which are both cell lines expressing wildtype K-Ras. These results support our model that SmgGDS-558 promotes malignancy in multiple types of cancers by interacting with prenylated PBR-containing small GTPases, including K-Ras. We are currently investigating how SmgGDS interactions with small GTPases are disrupted by peptides corresponding to the PBR and CAAX motifs of K-Ras and other small GTPases, to further define the mechanisms utilized by SmgGDS to promote malignancy. Citation Format: Nathan J. Schuld, Andrew D. Hauser, Jeffrey S. Vervacke, Ellen L. Lorimer, Mark D. Distefano, Carol L. Williams. SmgGDS splice variants interact with K-Ras through association with both the polybasic region and the CAAX motif, and promote the malignant phenotype in lung, breast, and pancreatic cancers. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A53. doi: 10.1158/1557-3125.RASONC14-A53

Functions of lncRNA HOTAIR in lung cancer.

Long non-coding RNAs (lncRNAs) govern fundamental biochemical and cellular processes. lncRNA HOX transcript antisense RNA (HOTAIR) represses gene expression through recruitment of chromatin modifiers. The expression of HOTAIR is elevated in lung cancer and correlates with metastasis and poor prognosis. Moreover, HOTAIR promotes proliferation, survival, invasion, metastasis, and drug resistance in lung cancer cells. Here we review the molecular mechanisms underlying HOTAIR-mediated aggressive phenotypes of lung cancer. We also discuss HOTAIR’s potential in diagnosis and treatment of lung cancer, as well as the challenges of exploiting HOTAIR for intervention of lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0090-4) contains supplementary material, which is available to authorized users.