Lung Cancer Tissue Specimens Research Articles

The miR 495-UBE2C-ABCG2/ERCC1 Axis Reverses DDP Resistance via Downregulation of Anti-Drug Genes in Cisplatin Resistant NSCLC Cells

Resistance to DDP has become the leading cause of mortality in NSCLC. The microRNA dysregulation plays a vital role in tumor progression. In our study, we found that miR-495 was significantly downregulated in lung cancer tissue specimens. Our objective was to elucidate the functions, direct target genes, and molecular mechanisms of miR-495 in lung cancer. We found that miR-495 downregulated the expression of its substrate UBE2C by directly binds to the 3′-UTR of UBE2C. UBE2C is a proto-oncogene that is activated in lung cancer; however, its role in drug resistance is unclear. We found here that UBE2C expression was higher in cisplatin resistant NSCLC cells; this was associated with induced cisplatin resistant NSCLC cells proliferation, invasion and cisplatin resistant. EMT was contributed to cisplatin resistance. We also found that UBE2C knockdown downregulated the expression of the vimentin. In contrast, E-cadherin was upregulated. Importantly we found that miR-495 and UBE2C were associated with cisplatin resistance. We performed to evaluate their effects on cell proliferation and cisplatin resistant. We also performed EMT, cell migration and invasion assays in cisplatin resistant NSCLC cells overexpressing miR-495 and under expressing UBE2C. Furthermore, Bioinformatics assays coupled with WB and luciferase assays revealed that UBE2C directly binds to the 5′-UTR of the drug resistance genes, ABCG2 and ERCC1. We also found that miR-495 reduced the expression of ABCG2 and ERCC1 via regulation of UBE2C. Collectively, our results indicated that miR495-UBE2C-ABCG2/ERCC1 axis reverses DDP resistance via downregulation of anti-drug genes and reducing EMT in cisplatin resistant NSCLC cells. Funding Statement: This work was supported by the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067 and ZR2017PH028), and Research Foundation of Binzhou Medical University (BY2015KYQD29 and BY2015KJ14). Competing Interests: The authors declare no competing financial interests. Ethics Approval Statement: The experimental protocol was approved by the Research Ethics Committee of Binzhou Medical University, China (No. 2017-016-01 for human lung cancer specimen and No. 2017-009-09 for mouse experiments in vivo) and the written informed consent was obtained from all subjects. Informed consent was obtained from all individual participants included in the study. All patients were staged based on the criteria of the 7th Edition of the AJCC Cancer Staging Manual: Stomach (2010).

Development of a radionuclide-labeled monoclonal anti-CD55 antibody with theranostic potential in pleural metastatic lung cancer

Decay-accelerating factor (CD55 or DAF) inhibits complement-dependent cytotoxicity. We determined that CD55 is overexpressed in 76.47% of human non-small cell lung cancer tissue specimens. We therefore developed a lutetium-177-labeled chimeric monoclonal antibody against CD55. CD55-specific single-chain variable fragment (scFv) was selected from a naïve chicken scFv phage-display library, converted to IgG, and radiolabeled with lutetium-177 to generate a 177Lu-anti-CD55 antibody. We then charaterized the biodistribution of this antibody in a mouse model of pleural metastatic lung cancer. The 177Lu-anti-CD55 antibody was primarily retained in tumor tissue rather than normal tissue. Treatment of the mice with 177Lu-anti-CD55 reduced the growth of lung tumors and improved median survival in vivo by two-fold compared to controls. Finally, 177Lu-anti-CD55 also enhanced the antitumor activity of cisplatin both in vitro and in vivo. These data suggest 177Lu-anti-CD55 antibody is a promising theranostic agent for pleural metastatic lung cancer.

Aberrant microRNA-137 promoter methylation is associated with lymph node metastasis and poor clinical outcomes in non-small cell lung cancer.

MicroRNA-137 (miR-137) functions as a tumor suppressor and is silenced by aberrant promoter methylation. Previous studies have demonstrated that miR-137 is downregulated in lung cancer. The purpose of the present study was to investigate miR-137 promoter methylation and to assess its prognostic value in non-small cell lung cancer (NSCLC). The expression of miR-137 was analyzed inhuman lung cancer A549 and H1299 cells and normal bronchial epithelial BEAS-2B cells, 10 paired formalin-fixed paraffin-embedded lung cancer and normal tissue samples, and 56 archived paraffin-embedded lung cancer tissues. Quantitative methylation-specific polymerase chain reaction analysis was used to assess the miR-137 methylation status. The associations between miR-137 promoter methylation and the clinicopathological features and prognosis of patients with NSCLC (n=56) were analyzed using analysis of variance. miR-137 was markedly downregulated in lung cancer cells and lung cancer tissue specimens compared with expression in BEAS-2B cells and matched adjacent normal lung tissues. A significant negative correlation between miR-137 expression and miR-137 promoter methylation was observed in human lung cancer tissues (r=−0.343; P=0.01). Smoking, lymph node metastasis and advanced clinical stage were associated with significantly lower expression of miR-137 in variance analysis. High levels of miR-137 promoter methylation were associated with a significantly poorer disease-free survival rate (P=0.034), but were not associated with overall survival, in Kaplan-Meier analysis and univariate analysis. In conclusion, the results of the present study indicated that miR-137 is downregulated and that its promoter is aberrantly methylated in lung cancer, and that high levels of miR-137 promoter methylation may have prognostic value for poor disease-free survival.

MicroRNA-490 regulates lung cancer metastasis by targeting poly r(C)-binding protein 1

Lung cancer remains a leading cause of cancer-related mortality, with metastatic progression remaining the single largest cause of lung cancer mortality. Hence, it is imperative to determine reliable biomarkers of lung cancer prognosis. MicroRNA-490-3p has been previously reported to be a positive prognostic biomarker for hepatocellular cancer. However, its role in human lung cancer has not yet been elucidated. Here, we report that hsa-miR-490-3p expression is significantly higher in human lung cancer tissue specimens and cell line. Gain- and loss-of-function studies of hsa-miR-490-3p showed that it regulates cell proliferation and is required for induction of in vitro migration and invasion-the latter being a hallmark of epithelial to mesenchymal transition. In situ analysis revealed that hsa-miR-490-3p targets poly r(C)-binding protein 1 (PCBP1), which has been previously shown to be a negative regulator of lung cancer metastasis. Reporter assays confirmed PCBP1 as a bona fide target of miR-490-3p, and metagenomic analysis revealed an inverse relation between expression of miR-490-3p and PCBP1 in metastatic lung cancer patients. In fact, PCBP1 expression, as detected by immunohistochemistry, was undetectable in advanced stages of lung cancer patients' brain and lymph node tissues. Xenograft tail vein colonization assays proved that high expression of miR-490-3p is a prerequisite for metastatic progression of lung cancer. Our results suggest that hsa-miR-490-3p might be a potential biomarker for lung cancer prognosis. In addition, we can also conclude that the lung cancer cells have evolved refractory mechanisms to downregulate the expression of the metastatic inhibitor, PCBP1.

Desmoglein-2 is overexpressed in non-small cell lung cancer tissues and its knockdown suppresses NSCLC growth by regulation of p27 and CDK2

Desmoglein-2 (Dsg2) is a cell adhesion protein of the cadherin superfamily. Altered Dsg2 expression is associated with tumorigenesis. This study determined Dsg2 expression in non-small cell lung cancer (NSCLC) tissue specimens for association with clinicopathological and survival data and then assessed the effect of Dsg2 knockdown on regulation of NSCLC cell malignant behaviors in vitro and in nude mouse xenografts. qRT-PCR and Western blot were used to detect Dsg2 expression in 28 paired NSCLC and normal tissue samples. Immunohistochemistry was used to detect Dsg2 expression in 70 cases of paraffin-embedded NSCLC tissues. NSCLC A549, H1703, and H1299 cells were cultured with Dsg2 knockdown performed using Dsg2 siRNA. Cell viability, cell cycle, apoptosis, and colony formation were assessed. siRNA-transfected A549 cells were also used to generate tumor xenografts in nude mice. Both Dsg2 mRNA and protein were highly expressed in NSCLC tissues and associated with NSCLC size, but not with overall survival of patients. Moreover, knockdown of Dsg2 expression reduced NSCLC cell proliferation and arrested them at the G1 phase of the cell cycle, but did not significantly affect NSCLC cell apoptosis. Dsg2 knockdown downregulated cyclin-dependent kinase 2 expression and upregulated p27 expression. Nude mouse xenograft assays showed that Dsg2 knockdown inhibited NSCLC xenograft growth in vivo. This study revealed the importance of Dsg2 in suppression of NSCLC development and progression. Further studies will explore whether restoration of Dsg2 expression is a novel strategy in control of NSCLC.

Abstract 5117: An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens

Abstract Cancers may escape immune surveillance and eradication through the expression of programmed death-ligand 1 (PD-L1) on tumor cells and in the tumor microenvironment. PD-L1 expression has been reported in various cell populations within the tumor, and its expression associated with prognosis for various tumors. Further, clinical studies have shown that this pathway is an important target for immunotherapy and PD-L1 expression on tumor cells and in the tumor microenvironment has been associated with enhanced response. Understanding PD-L1's complex biological function not only on tumor cells but also within the tumor microenvironment requires simultaneous interrogation of multiple biomarkers, ranging from cancer immunology checkpoint markers, tumor infiltrating immune cell markers, and tumor specific markers, etc. Multiplex immunohistochemistry (IHC) allows simultaneous detection of multiple markers to explore the potential cellular composition of immune/stromal/cancer cells in tumor microenvironment. Development of a multiplex IHC assay remains challenging due to antibody species similarity and cross reactivity, stability of fluorophores through multiple rounds of processing, balancing high and low signals and measurement of weakly expressed markers. We present here the development of a fully automated multiplex IHC assay (PD-L1, CD3, CD8, CD68 and FoxP3) using rabbit primary antibodies with a heat deactivation process between each antigen staining cycles on the BenchMark ULTRA automated slide stainer. As part of the technology validation, we compared the 5-plex IHC to the respective single-plex chromogenic IHC assays. Using the automated 5-plex fluorescent IHC assay, we tested a cohort of non-small cell lung (NSCLC) and bladder cancer tissue specimens and characterized PD-L1 and immune marker expression in both tumor and infiltrate immune cells. To provide an objective and reliable readout of the assay, image analysis tools are being developed for automated identification and quantification of the labelled biomarkers and their co-expression on a cell-by-cell basis. This automated multiplex PD-L1 5-Plex IHC assay could be utilized as a tool for further characterizing tumors and its microenvironment and gain a better understanding of which patients may benefit from immune-therapies. Citation Format: Wenjun Zhang, Antony Hubbard, Adriana Racolta, Nick Cummins, Mehrnoush Khojasteh, Liping Zhang, Karl Garsha, Joerg Bredno, Dustin Harshman, Srabani Bhaumik, Tobin Jones, Marcin Kowanetz, Sanjeev Mariathasan, Ian McCaffery, Dustin Smith, J Andrew Williams, Lidija Pestic-Dragovich, Larry Morrison, Lei Tang. An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5117.

Developments for Personalized Medicine of Lung Cancer Subtypes: Mass Spectrometry-Based Clinical Proteogenomic Analysis of Oncogenic Mutations.

Molecular therapies targeting lung cancers with mutated epidermal growth factor receptor (EGFR) by EGFR-tyrosin kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, changed the treatment system of lung cancer. It was revealed that drug efficacy differs by race (e.g., Caucasians vs. Asians) due to oncogenic driver mutations specific to each race, exemplified by gefitinib / erlotinib. The molecular target drugs for lung cancer with anaplastic lymphoma kinase (ALK) gene translocation (the fusion gene, EML4-ALK) was approved, and those targeting lung cancers addicted ROS1, RET, and HER2 have been under development. Both identification and quantification of gatekeeper mutations need to be performed using lung cancer tissue specimens obtained from patients to improve the treatment for lung cancer patients: (1) identification and quantitation data of targeted mutated proteins, including investigation of mutation heterogeneity within a tissue; (2) exploratory mass spectrometry (MS)-based clinical proteogenomic analysis of mutated proteins; and also importantly (3) analysis of dynamic protein-protein interaction (PPI) networks of proteins significantly related to a subgroup of patients with lung cancer not only with good efficacy but also with acquired resistance. MS-based proteogenomics is a promising approach to directly capture mutated and fusion proteins expressed in a clinical sample. Technological developments are further expected, which will provide a powerful solution for the stratification of patients and drug discovery (Precision Medicine).

Expression and clinical significance of SALL4 and LGR5 in patients with lung cancer.

Lung cancer is the most frequent cancer worldwide, in terms of incidence and mortality. Due to challenges in the diagnosis of the disease, the 5-year overall survival rate is only ~16%. Previous studies have suggested that malignant transformations originate from adult stem cells, and malignant lesions may therefore express stem-cell-associated markers. The purpose of the present study is to investigate the expression and clinical significance of the stem cell-associated markers Sal-like protein 4 (SALL4) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) in lung cancer, and to provide novel diagnostic markers and targets for the treatment of lung cancer. The expression of the stem cell-associated markers SALL4 and LGR5 was analyzed by immunohistochemistry performed on 135 human lung cancer tissue specimens and 10 non-cancer lung tissue specimens. The clinical significance of the expression of these markers and correlation between their expression and clinical parameters was also assessed. SALL4 expression was highly upregulated in lung cancer tissues, but was not present in non-cancerous lung tissues, and the sensitivity and specificity of SALL4 reached 88% and 100%, respectively. By contrast, LGR5 demonstrated 97% sensitivity, but the specificity was poor. Therefore, SALL4 may be an extremely useful diagnostic marker for lung cancer, but LGR5 is not as useful.

Expression of the copper transporters hCtr1, ATP7A and ATP7B is associated with the response to chemotherapy and survival time in patients with resected non-small cell lung cancer.

Copper transporter family proteins may regulate the chemoresistance of non-small cell lung cancer (NSCLC) to platinum-based anticancer drugs. The present study aimed to investigate the expression of these proteins in lung cancer tissue specimens for association with clinicopathological data and patient responses to chemotherapy and survival. A total of 54 patients with surgically resected NSCLC that received first-line platinum-based doublet chemotherapy were recruited in the present study, and the paraffin-embedded pre-treatment tumor tissue specimens were subjected to immunohistochemical analysis for the expression of human copper transporter 1 (hCtr1) and copper-transporting p-type adenosine triphosphatase 1 (ATP7A) and 2 (ATP7B). This cohort of patients with NSCLC received platinum-based chemotherapy subsequent to the surgical removal of tumor lesions. ATP7B expression was found to be significantly associated with tumor cell differentiation, while hCtr1 expression was significantly associated with improved chemotherapeutic responses. The median survival time was 20 months in patients possessing tumors with high ATP7A expression, but >66 months in patients possessing tumors with low ATP7A expression at the end of the follow-up (P<0.001). The median survival time at the end of the follow-up was 15 months in patients with low tumor hCtr1 expression, but >66 months in patients with high tumor hCtr1 expression (P<0.001). High hCtr1 and low ATP7A expression were each favorable prognostic factors subsequent to chemotherapy for patients with resected NSCLC. Multivariate analysis revealed that high hCtr1 expression combined with low ATP7A expression, good tumor differentiation and female gender were all favorable independent predictive and prognostic factors for patients with resected NSCLC following chemotherapy. High hCtr1 expression combined with low ATP7A expression was associated with an improved prognosis in patients with resected NSCLC that received platinum-based chemotherapy. Surgery combined with neoadjuvant chemotherapy may improve the survival time of patients with NSCLC.

MicroRNA-26a/b regulate DNA replication licensing, tumorigenesis, and prognosis by targeting CDC6 in lung cancer.

Cancer is characterized by mutations, genome rearrangements, epigenetic changes, and altered gene expression that enhance cell proliferation, invasion, and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are overexpressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their overexpression are largely unknown. One possible mechanism is through miRNA expression differences. Here, it is demonstrated that miR26a and miR26b inhibit replication licensing and the proliferation, migration, and invasion of lung cancer cells by targeting CDC6. Importantly, miR26a/b expression is significantly decreased in human lung cancer tissue specimens compared with the paired adjacent normal tissues, and miR26a/b downregulation and the consequential upregulation of CDC6 are associated with poorer prognosis of patients with lung cancer. These results indicate that miR26a/b repress replication licensing and tumorigenesis by targeting CDC6. The current study suggests that miR26a, miR26b, and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets.

Identification of kinase inhibitor targets in the lung cancer microenvironment by chemical and phosphoproteomics.

A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. While these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib), and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune, and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes, and signaling pathways that are simultaneously engaged by multitargeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments.

Glycoproteomics approach for identifying Glycobiomarker candidate molecules for tissue type classification of non-small cell lung carcinoma.

Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC). LC-MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue.

Reduced expression of liver kinase B1 and Beclin1 is associated with the poor survival of patients with non-small cell lung cancer.

Liver kinase B1 (LKB1) regulates cell polarity and has tumor-suppressor functions, while Beclin1 regulates autophagy and is associated with tumorigenesis. The present study quantified the expression level of LKB1 and Beclin1 in non-small cell lung cancer (NSCLC) tissue specimens and examined the possible association with clinicopathological characteristics and the survival of patients. Quantitative real-time reverse transcriptase‑polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry were used to assess expression of LKB1 and Beclin1 in 142 NSCLC tissue samples. The data revealed that expression levels of both LKB1 and Beclin1 mRNA and protein were significantly reduced in the NSCLC tissues compared to levels in the matched surrounding normal lung tissues, and expression of LKB1 protein was associated with Beclin1 expression in the NSCLC tissues. Reduced expression of LKB1 protein was significantly associated with tumor histology (P<0.001) and poor differentiation (P<0.001), but was not associated with tumor lymph node metastasis, TNM stage or patient gender and age, while the reduced expression of Beclin1 protein was associated with tumor histology (P<0.05) and poor differentiation (P<0.05). Univariate analysis revealed that adenocarcinoma, lymph node metastasis, advanced TNM stage and reduced expression of LKB1 and Beclin1 proteins are associated with the survival of NSCLC patients. Multivariate analysis indicated that adenocarcinoma (P<0.05), lymph node metastasis (P<0.05), advanced TNM stage (P<0.001) and reduced expression of LKB1 (P<0.05) and Beclin1 (P<0.001) are all independent prognostic indicators for the survival of NSCLC patients.

Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P<0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer.

MicroRNA-137 and microRNA-29a expression in non-small cell lung cancer diagnosis and prognostic.

e17501 Background: Current staging methods are inadequate for predicting the outcome of treatment of non–small-cell lung cancer (NSCLC). We investigated whether microRNA expression profiles can predict clinical outcome of NSCLC patients. Methods: We studied frozen specimens of lung-cancer tissue from 76 randomly selected patients who underwent surgical resection of NSCLC. Using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, we chosed U6 as the internal control for real-time RT-PCR because it is invariant in clinical cancer specimens. We obtained microRNA137 and 29a expressions level in 76 NSCLC patients and evaluated diagnoisis value and the association between the level of expression and survival. Results: This microRNA137 is with diagnosis value, microRNA29a expression level signature is an independent predictor of the cancer relapse and survival of NSCLC patients. Conclusions: Our microRNA signature is closely associated with relapse-free and overall survival among patients with NSCLC.

Μolecular impact of bone morphogenetic protein�7, on lung cancer cells and its clinical significance

The aim of this study was to investigate the expression of bone morphogenetic protein 7 (BMP7), in human pulmonary cancer tissues/cells and to evaluate the cellular impact of bone morphogenetic proteins on pulmonary cancer cells. BMP7 expression was determined in human lung cancer cell lines. The invasiveness and growth of cells transfected with BMP7, in vitro, were evaluated using the in vitro invasion assay and in vitro tumour models. Cellular migration was analysed using wounding assays. BMP7-positive tumours correlated with the absence of bone metastasis (P=0.040). In this analysis, we identified that 4 of 4 small cell lung cancer (SCLC) tissue specimens had no BMP7 expression, which illustrated that BMP7 may have no role in SCLC. BMP7 expression was not correlated with the overall survival time in lung cancer patients. Downregulation of BMP7 expression significantly inhibited the invasiveness of SPC-A1 cells (P<0.001) and forced-expression of BMP7 dramatically increased the motility of A549 cells. Overexpression of BMP7 in A549 cells and its knockdown in SPC-A1 cells did not significantly alter proliferation compared with the control cells (P>0.5 respectively). In conclusion, we have demonstrated that BMP7 has an important role in controlling lung cancer cell motility and invasiveness, without affecting the growth process, cell proliferation and cell apoptosis. A higher BMP7 expression may be an indicator for bone metastasis. The therapeutic role of BMP7 warrants further investigation.

TIM-3 Expression Characterizes Regulatory T Cells in Tumor Tissues and Is Associated with Lung Cancer Progression

BackgroundT cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8+ T cells in both chronic infection and tumor. However, the nature of TIM-3+CD4+ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.MethodologyA total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.ConclusionsTIM-3 is highly upregulated on both CD4+ and CD8+ TILs from human lung cancer tissues but negligibly expressed on T cells from patients' peripheral blood. Frequencies of IFN-γ+ cells were reduced in TIM-3+CD8+ TILs compared to TIM-3−CD8+ TILs. However, the level of TIM-3 expression on CD8+ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3+CD4+ TILs expressed FOXP3 and about 60% of FOXP3+ TILs were TIM-3+. Importantly, TIM-3 expression on CD4+ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.

MAGE A1-A6 RT-PCR and MAGE A3 and p16 methylation analysis in induced sputum from patients with lung cancer and non-malignant lung diseases

The melanoma antigen gene (MAGE) A1-A6 RT-PCR system was developed for the detection of lung cancer cells in the sputum. However, we identified MAGE expression in some patients with non-malignant lung diseases. To understand these patterns of MAGE expression, we performed MAGE A3 methylation-specific PCR (MSP) and p16 MSP. We collected 24 biopsy specimens of lung cancer tissue and performed MAGE A1-A6 RT-PCR, MAGE A3 MSP and p16 MSP. RNA and DNA were simultaneously extracted from induced sputum specimens of 133 patients with lung diseases and 30 random sputum specimens of healthy individuals and the 3 molecular analyses were performed. The patients were diagnosed as 65 cases of lung cancer and 68 of benign lung diseases. Positive rates of MAGE A1-A6 RT-PCR, MAGE A3 MSP and p16 MSP were as follows: in lung cancer tissue, 87.5, 58.3 and 70.8%; in the sputum of lung cancer patients, 50.8, 46.2 and 63.1%; benign lung diseases, 10.3, 30.9 and 39.7%; and healthy individuals, 3.3, 6.7 and 3.3%. Of the 40 MAGE-positive cases, 33 were diagnosed with lung cancer and 7 as having benign lung diseases. From the 7 cases of MAGE-positive benign lung diseases, 6 cases showed methylation abnormalities. The MAGE-positive group revealed significantly higher rates of methylation abnormalities. Of the 40 MAGE-positive cases, 39 cases were found to be lung cancer or benign lung diseases with abnormal methylation. Thus, MAGE expression in the sputum suggests the presence of lung cancer cells or pre-cancerous cells.

Abstract 4792: Immune response to a fetal oncoprotein p90/CIP2A in lung cancer

Abstract During a previous study which identified a cytoplasmic RNA-binding protein p62/IMP2 binding to and regulating the expression of IGF-II mRNA, it was noted that autoantibody against an antigen of approximately 90 kDa (p90) was detected with similar frequency in hepatocellular carcinoma (HCC). Full-length cDNA encoding p90 was successfully isolated from a T24 expression library and comprised a sequence coding for a protein of 905 amino acids with predicted molecular mass of 102kDa. Preliminary data showed that anti-p90 was present in 21/160 (13.1%) in HCC, 3/91 (3.3%) in gastric cancer and 1/20 (5.0%) in esophageal cancer. Gastric cancer tissue confirmed cytoplasmic expression of p90. Of interesting finding is that recently a research group showed that p90 could be co-purified with the A subunit of protein phosphatase 2A (PP2A). They showed that p90 was an inhibitor of the tumor suppressor function of PP2A and that this was related to p90 binding c-myc and inhibiting dephosphorylation of S62 by PP2A. They proposed renaming p90, the cancerous inhibitor of PP2A (CIP2A). CIP2A was shown to be overexpressed in low-passaged cell lines from head and neck squamous cell carcinoma. In order to address the possibility whether the p90/CIP2A might be a tumor-associated antigen (TAA) and useful as a biomarker in lung cancer, the full-length recombinant p90/CIP2A protein was used as the antigen in enzyme-linked immunoassay (ELISA) and western blot for the detection of autoantibodies in 105 sera from lung cancer patients. Frequency of autoantibodies to p90/CIP2A was 25.7% (27/105) in lung cancer, showing a significant difference compared to normal individuals (4.9%, 4/82). Positive sera in ELISA were also confirmed by western blot. In further study, the expression of p90/CIP2A in multiple lung cancer tissues (lung cancer tissue array) was evaluated by immunohistochemistry (IHC). Of the 72 lung cancer tissue specimens examined, increased expression of p90/CIP2A was observed in 61 (84.7%) specimens, which is significant higher than in normal lung tissues (14.3%, 9/63). Using confocal microscopy, we were able to confirm our previous finding that p90/CIP2A was predominately localized in the cytoplasm, which was consistent with our IHC data. Depletion of p90/CIP2A via shRNA in A549 lung cancer cell line resulted in the decreased cell proliferation, foci formation and inhibition of anchorage-independent growth. The examination of the dynamic expression of p90/CIP2A during mouse development shows that this protein is mainly expressed during embryo development, and becomes silent after birth. Together with antibodies against other well-validated TAAs such as p53, p62/IMP2 and so on, autoantibody to p90/CIP2A may provide a potential novel marker for lung cancer detection. In addition, our data also support the working hypothesis that autoantibody production in cancer may be directly linked to aberrant expression of proteins involved in tumorigenesis pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4792.

Identification of the reference genes' accuracy for lung tissue specimens by real-time quantitative PCR

Objective To analyze the expression stability of the three widely used reference genes β-glueuronidase (GUSB), glycera]dehydes-3-phosphate dehydrogenase (GAPDH), β2-microglobulin (β2-M) in Chinese lung cancer tissue specimens and normal lung tissue specimens. Methods Gene expression wasmeasured by quantitative real time PCR and expression stability was analyzed with two widely used softwares genorm and normfinder. Results The intra-and inter-group difference of GAPDH is maximum (The intra and inter-group s is 1.07 and 0.93 respectively, |△ Ct|=2.01±1.06; P =0.000). The mean of these three genes' Ct value is the most stable one analyzed by the two softwares. But the t test showed that the mean of Ct value of GUSB and β2-M is the unique combination that had the minimum intra-and inter-group difference, with no statistically significant differences between normal and malignant samples (The intra-and inter-group s is 0.53 and 0.79 respective]y, |△Ct|= 0.73±0.53; P =0.053). Conclusion It is inappropriate to normalize data derived from lung tissue specimens using one of these three housekeeping genes alone. Among the different combinations of these three genes, the mean of the Ct values of GUSB and β2-M is the best choice as the internal control of lung tissue specimens. Key words: Lung neoplasms; Gene; Polymerase chain reaction