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Constitutive activation of the epidermal growth factor receptor (EGFR) signaling pathway is implicated in the initiation and progression of lung cancer. EGFR tyrosine kinase inhibitor (TKI)-targeted therapy has become the standard treatment for nonsmall cell lung cancer (NSCLC) patients. However, acquired resistance to these agents remains a major obstacle for managing NSCLC. Here, we investigated a novel strategy to overcome EGFR TKI resistance by targeting the stanniocalcin 2 (STC2)-JUN-AXL pathway. We revealed that STC2 was expressed at significantly higher levels in EGFR TKI-resistant cells. Further, clinical analysis showed that STC2 expression was increased after the development of EGFR TKI resistance and that higher levels were correlated with shorter progression-free survival in EGFR TKI-treated lung cancer patients. Moreover, STC2 overexpression in EGFR TKI-sensitive cells resulted in EGFR TKI resistance. Conversely, genetic silencing of STC2 rendered EGFR TKI-resistant cells more sensitive to EGFR TKIs. Mechanically, STC2 enhanced AXL promoter activity by increasing the phosphorylation of c-Jun, which is an indispensable transcription factor that transactivates AXL. STC2 promoted activation of the JUN-AXL-extracellular signal-regulated kinase (ERK) signaling axis in lung cancer cells. Pharmacological or genetic inhibition of AXL-ERK activity inhibited STC2-mediated EGFR TKI resistance. We also demonstrated that PE2988 cells, a C797S-independent osimertinib-resistant primary cancer cell line from a lung cancer patient, responded to combined AXL inhibitor and osimertinib treatment. In conclusion, our research indicates that STC2 overexpression is important for acquired resistance to EGFR TKIs and that STC2-JUN-AXL-ERK signaling might be a potential therapeutic target to overcome resistance to EGFR TKIs.

Background EGFR-mutant lung cancer inevitably develops resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Objective To investigate the clinical relevance of microRNAs (miRNAs) in TKI therapy response and resistance. Methods We performed a miRNA PCR array analysis and used The Cancer Genome Atlas (TCGA) database to identify potential miRNAs related to EGFR TKIs resistance. We then correlated miRNA expression in 70 surgical and 50 malignant pleural effusion specimens with patient outcomes in those with non-small cell lung carcinoma. Molecular manipulation was performed in EGFR mutant lung cancer cells to assess the effect of miR-200c-3p on cell migratory ability and EGFR-TKI sensitivity. Results We identified miR-200c-3p and miR-203a-3p as potential EGFR TKI resistance regulators via their modulation of epithelial-to-mesenchymal transition (EMT). MiR-200c-3p and miR-203a-3p were down-regulated in EGFR TKI-resistant cell lines. Progression-free survival (PFS) with EGFR-TKI treatment of patients with high miR-200c-3p expression, but not miR-203a-3p, in the specimens was significantly longer than that of patients with low expression. MiR-200c-3p overexpression inhibited the EMT process in EGFR TKI resistance cell lines and promoted cell death. MiR-200c-3p silencing in EGFR TKI sensitive cell lines increased drug resistance. Conclusion MiR-200c-3p plays a role in sensitivity to EGFR TKIs via modulating EMT process.

A kinesin family member 5b (KIF5B)-MET proto-oncogene, receptor tyrosine kinase (MET) rearrangement was reported in patients with lung adenocarcinoma but its oncogenic function was not fully evaluated. We used one-step reverse transcription-polymerase chain reaction for RNA samples to screen for the KIF5B-MET fusion in 206 lung adenocarcinoma and 28 pulmonary sarcomatoid carcinoma patients. Genomic breakpoints of KIF5B-MET were determined by targeted next-generation sequencing. Soft agar colony formation assays, proliferation assays, and a xenograft mouse model were used to investigate its oncogenic activity. In addition, specific MET inhibitors were administered to evaluate their anti-tumor activities. A KIF5B-MET fusion variant in a patient with a mixed-type adenocarcinoma and sarcomatoid tumor was identified, and another case was found in a pulmonary sarcomatoid carcinoma patient. Both cases carried the same chimeric gene, a fusion between exons 1–24 of KIF5B and exons 15–21 of MET. KIF5B-MET-overexpressing cells exhibited significantly increased proliferation and colony-forming ability. Xenograft tumors harboring the fusion gene demonstrated significantly elevated tumor growth. Ectopic expression of the fusion gene stimulated the phosphorylation of KIF5B-MET as well as downstream STAT3, AKT, and ERK1/2 signaling pathways. The MET inhibitors significantly repressed cell proliferation; phosphorylation of downstream STAT3, AKT, and ERK1/2; and xenograft tumorigenicity. In conclusion, the KIF5B-MET variant was demonstrated to have an oncogenic function in cancer cells. These findings have immediate clinical implications for the targeted therapy of subgroups of non-small cell lung cancer patients.

Patients with epidermal growth factor receptor (EGFR) mutation-positive lung cancer show a dramatic response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired drug resistance eventually develops. This study explored the novel mechanisms related to TKI resistance. To identify the genes associated with TKI resistance, an integrative approach was used to analyze public datasets. Molecular manipulations were performed to investigate the roles of insulin-like growth factor binding protein 7 (IGFBP7) in lung adenocarcinoma. Clinical specimens were collected to validate the impact of IGFBP7 on the efficacy of EGFR TKI treatment. IGFBP7 mRNA expression in cancer cells isolated from malignant pleural effusions after acquired resistance to EGFR-TKI was significantly higher than in cancer cells from treatment-na&iuml, ve effusions. IGFBP7 expression was markedly increased in cells with long-term TKI-induced resistance compared to in TKI-sensitive parental cells. Reduced IGFBP7 in TKI-resistant cells reversed the resistance to EGFR-TKIs and increased EGFR-TKI-induced apoptosis by up-regulating B-cell lymphoma 2 interacting mediator of cell death (BIM) and activating caspases. Suppression of IGFBP7 attenuated the phosphorylation of insulin-like growth factor 1 receptor (IGF-IR) and downstream protein kinase B (AKT) in TKI-resistant cells. Clinically, higher serum IGFBP7 levels and tumors with positive IGFBP7-immunohistochemical staining were associated with poor TKI-treatment outcomes. IGFBP7 confers resistance to EGFR-TKIs and is a potential therapeutic target for treating EGFR-TKI-resistant cancers.

N-myc down-regulated gene 1 (NDRG1) is a member of the NDRG family which belongs to α/β hydrolase superfamily, but without presenting a hydrolytic catalytic site. NDRG1 gene has been proposed that involved in cellular stress response pathways, including the heavy metal response, hypoxia response, DNA damage response, cellular differentiation, and proliferation. An oncogenic and tumor-promoting role of NDRG1 has been suggested in lung cancer and various malignant cancers. However, the biological functions and downstream molecular mechanisms of NDRG1 gene in lung cancer are still unclear. In this study, we detected the expression of NDRG1 in various lung adenocarcinoma cell lines using quantitative RT-PCR and Western blotting methods. The constitutive NDRG1-expression, and NDRG1-knockdown stable transfectants were established. Over-expression of NDRG1 in lung cancer cells exhibited markedly promoting in the proliferation and anchorage independent growth, whereas reduced of NDRG1 was found to suppress lung cancer cell progression. We also indicated that NDRG1 play an important role in the epithelial-mesenchymal transition (EMT) features, including a morphological change, increased migratory and invasive ability, reduction the epithelial marker expression and up-regulated EMT regulators. In addition, microarray assays were performed to further explore the potential molecular mechanisms of NDRG1 in lung cancer. The candidate NDRG1-regulated genes, including IL11, SOX7, TCF4, IGF2, SLUG and SLIT2, were verified by quantitative RT- PCR. Finally, we demonstrated that NDRG1 induces EMT and invasion process in lung cancer cell through up-regulation SLUG expression. These results will provide information and help us to understand the roles of NDRG1 in lung cancer cells. Citation Format: En-Chu Liu, Hui-Chia Liu, Jeremy J. M. Chen, Meng-Feng Tsai. NDRG1 promotes epithelial-mesenchymal transition in lung cancer cells by upregulation of SLUG expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1539.

The cytoskeleton comprises microtubules, actin microfilaments, and intermediate filaments. The cytoskeleton plays a role in the mechanical function of cell adhesion, motility, and migration. Currently, cancer cell mechanics is an important topic of understanding cancer progression. Atomic force microscopy (AFM) is one of the techniques used to determine the mechanical properties of cells. In this study, AFM was used to measure the force curves of U937 cancer cells treated with two types of anticancer drugs (colchicine and taxol), and alterations in the Young’s modulus were subsequently investigated. The study results indicate that the force curve trend was dissimilar among the three types of cells (cells in the control group and in two types of anticancer drugs). In addition, when using the AFM to analyze the influence of drugs on the mechanical properties of cells, applying a small loading force from the AFM tip (small cell indentation depth) produced more accurate results. At a small cell indentation depth, the Young’s modulus order was E colchicine

// Yi-Nan Liu 1 , Tzu-Hua Chang 1 , Meng-Feng Tsai 2 , Shang-Gin Wu 3 , Tzu-Hsiu Tsai 1 , Hsuan-Yu Chen 4 , Sung-Liang Yu 5 , James Chih-Hsin Yang 6, 7 , Jin-Yuan Shih 1, 8 1 Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan 2 Department of Molecular Biotechnology, Dayeh University, Changhua, Taiwan 3 Department of Internal Medicine, National Taiwan University Hospital, Yun-Lin Branch, Yun-Lin, Taiwan 4 Institute of Statistical Science, Academia Sinica, Taipei, Taiwan 5 Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan 6 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan 7 Graduate Institute of Oncology, Cancer Research Center, National Taiwan University, Taipei, Taiwan 8 Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan Correspondence to: Jin-Yuan Shih, e-mail: jyshih@ntu.edu.tw Keywords: IL-8, EGFR, gefitinib, resistance, stemness Received: October 15, 2014 Accepted: February 15, 2015 Published: March 18, 2015 ABSTRACT Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

To assess the contribution of tumor necrosis factor (TNF)β +252 polymorphisms to risk and prognosis of hepatocellular carcinoma (HCC), we enrolled 150 pairs of sex- and age-matched patients with HCC, patients with cirrhosis alone, and unrelated healthy controls. TNFβ +252 genotypes were determined by polymerase chain reaction with restriction fragment length polymorphism. Multivariate analysis indicated that TNFβ G/G genotype [odds ratio (OR), 3.64; 95%CI, 1.49-8.91], hepatitis B surface antigen (OR, 16.38; 95%CI, 8.30-32.33), and antibodies to hepatitis C virus (HCV) (OR, 39.11; 95%CI, 14.83-103.14) were independent risk factors for HCC. There was an additive interaction between TNFβ G/G genotype and chronic hepatitis B virus (HBV)/HCV infection (synergy index=1.15). Multivariate analysis indicated that factors associated with TNFβ G/G genotype included cirrhosis with Child-Pugh C (OR, 4.06; 95%CI, 1.34-12.29), thrombocytopenia (OR, 6.55; 95%CI, 1.46-29.43), and higher serum α-fetoprotein concentration (OR, 2.53; 95%CI, 1.14-5.62). Patients with TNFβ G/G genotype had poor cumulative survival (p=0.005). Cox proportional hazard model indicated that TNFβ G/G genotype was a biomarker for poor HCC survival (hazard ratio, 1.70; 95%CI, 1.07-2.69). In conclusion, there are independent and additive effects between TNFβ G/G genotype and chronic HBV/HCV infection on risk for HCC. It is a biomarker for poor HCC survival. Carriage of this genotype correlates with disease severity and advanced hepatic fibrosis, which may contribute to a higher risk and poor survival of HCC. Chronic HBV/HCV infected subjects with this genotype should receive more intensive surveillance for early detection of HCC.

Using the Serial Analysis of Gene Expression (SAGE) database from the Cancer Genome Anatomy Project, we identified heparin co-factor II (HCII), which is over-expressed in non-small cell lung cancer (NSCLC). Here, we investigated the clinical significance of HCII and provided molecular evidence to support the suggestion that HCII could enhance cancer metastasis in NSCLC. We found that high HCII expression in tumour tissue was associated with increased cancer recurrence and shorter overall survival times in 75 clinically operable NSCLC patients. High pretreatment plasma concentration of HCII was associated with reduced overall survival in 57 consecutive NSCLC patients. We over-expressed and knocked down HCII expression in lung cancer cell lines and confirmed that HCII could promote cell motility, invasion ability and filopodium dynamics in NSCLC cells in vitro and increased metastatic colonization in an in vivo mouse model. Exogenous treatment of HCII promoted cancer cell migration, and this promigratory effect of HCII was independent of thrombin. We further showed that HCII could up-regulate cancer cell migration through the activation of PI3K, which acts upstream of Rac1 and Cdc42, and this effect could be blocked by heparin. We suggest that HCII is a novel metastasis enhancer and may be used as a prognostic predictor for heparin treatment in NSCLC. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

The prevalence, demographic features, and clinical outcomes of lung adenocarcinoma patients with novel ROS1 oncogenic rearrangement in East Asian populations are not clear. This study aimed to investigate the clinical and prognostic characteristics of lung adenocarcinoma in patients with ROS1 fusion compared with other driver mutations.Multiplex reverse transcription-polymerase chain reaction was used to detect the ROS1 fusion gene in lung adenocarcinoma cases. Immunohistochemistry was used to confirm the expression of ROS1. The demographic data and clinical outcomes of patients with the ROS1 fusion gene were compared with those of patients without the ROS1 fusion gene, including those with the EGFR mutation, EML4-ALK fusion, KRAS mutation, and quadruple-negative patients.Of 492 patients with lung adenocarcinoma, 12 (2.4%) had the ROS1 fusion gene. Their median age was 45.0 years, significantly younger than that of the ROS1 fusion-negative cohorts (p0.001). Acinar (including cribriform) and solid patterns were the two most common histologic subtypes in the ROS1 fusion tumors (7 of 12, 58.3%) and were predominantly seen in CD74-ROS1 fusion tumors (66.7%). There was no significant survival difference between the ROS1 fusion-positive and ROS1 fusion-negative cohorts in surgical group, but ROS1 fusion-positive patients might have worse outcomes than EGFR-mutant patients in the stage IV group.The ROS1 fusion gene can be successfully detected in East Asian patients with lung adenocarcinoma using multiplex reverse transcription-polymerase chain reaction. These patients tend to be younger and have characteristic histologic subtypes. Due to the small number of ROS1 fusion patients, the prognostic value of ROS1 fusion need further studies to confirm.

The pathogenesis of hepatocellular carcinoma (HCC) related to habitual betel quid (BQ) chewing is unclear. Risk of HCCis increased with adverse hepatic fibrosis. This study aimed to assess the impact of chronic viral hepatitis on adverse hepatic fibrosis in HCC related to BQ chewing. This hospital-based case-control study enrolled 200 pairs of age- and gender-matched patients with HCC and unrelated healthy controls. Serologic hepatitis B surface antigen (HBsAg), antibodies to hepatitis C virus (anti-HCV), α-fetoprotein (AFP), and surrogate markers for significant hepatic fibrosis were measured. Information on substance-use habits was obtained with a questionnaire. By analysis of surrogate markers for hepatic fibrosis, the prevalence of significant hepatic fibrosis in patients chewing BQ was between 45.8% and 91.7%, whereas that for patients without BQ chewing was between 18.4% and 57.9%. The difference was significant (P0.05 for each surrogate marker). Multivariate analysis indicated that cirrhosis with Child-Pugh C (odds ratio (OR) = 3.28; 95% confidence interval (CI), 1.29- 8.37), thrombocytopenia (OR = 3.92, 95% CI, 1.77-8.68), AFP400 mg/L (OR = 2.21, 95% CI, 1.05-4.66) and male gender (OR = 4.06, 95% CI, 1.29-12.77) were independent factors associated with habitual BQ chewing. In conclusion, adverse hepatic fibrosis and severe liver damage play important roles in the pathogenesis of BQ- related HCC, which could be aggravated by chronic hepatitis B and hepatitis C. BQ-cessation programs and prevention of chronic HBV/HCV infection are needed to prevent HCC related to BQ chewing.

Pleckstrin 2 (PLEK2), one of the pleckstrin homology domain proteins, have been suggested to modulate actin rearrangement and cell spreading in T lymphocytes. Recently studies also implicated that PLEK2 paly an importance role in human myeloproliferative neoplasms, melanoma, and lung cancers. PLEK2 mRNA genes were related to poor overall survival in lung adenocarcinoma patients. However, the biological functions of PLEK2 in lung cancer is not clear. We detected the expression of PLEK2 in various lung adenocarcinoma cell lines using quantitative RT-PCR methods. Molecular manipulations were performed to investigate the roles of PLEK2 in lung cancer cells. Over-expression of PLEK2 in lung cancer cells exhibited markedly promoting in the proliferation and anchorage independent growth, whereas reduced of PLEK2 was found to suppress lung cancer cell progression. This studies also indicated that PLEK2 may play an important role in the epithelial-mesenchymal transition (EMT) features, including a morphological change, increased invasive and migratory ability, reduction the epithelial marker expression and up-regulated EMT regulators. Our results suggested that PLEK2 expression induce lung cancer cell EMT phenomenon through up-regulation Snail1 expression. These results will provide information and help us to understand the roles of PLEK2 in lung cancer cells. Citation Format: En-Chu Liu, Tsai-Tu Lee, Shey-Lin Wu, Tai-Lin Lee, Meng-Feng Tsai. PLEK2 promotes epithelial mesenchymal transition through increasing Snail1 in lung cancer cells [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 1035.

Introduction Tumor tissue is often not obtainable or suitable for molecular-based epidermal growth factor receptor ( EGFR ) mutational analysis in advanced non–small-cell lung cancer (NSCLC). This retrospective and single-institution study was conducted to evaluate the role of effusion immunocytochemistry using two EGFR mutant-specific antibodies for the detection of relevant EGFR mutations in NSCLC, along with the selection of candidates for first-line therapy with EGFR tyrosine kinase inhibitors (TKIs). Methods Immunocytochemistry using two antibodies binding specifically to the major forms of mutant EGFR, L858R, and E746-A750 deletion (delE746-A750), was performed on cell blocks of malignant pleural effusion (MPE) from 78 patients with lung adenocarcinoma, who received first-line EGFR TKIs. The yield of EGFR -mutation detection and prediction of response rate and progression-free survival to TKI treatment by immunocytochemistry were compared with those by clinical characteristics and EGFR sequencing using cell-derived RNA from MPEs. Results Of the 78 MPE samples, direct sequencing using cell-derived RNA identified L858R mutation in 42 cases, deletions in exon 19 in 12 cases (delE746-A750 in eight cases), other types of mutations in three cases, and wild-type EGFR in 21 cases. Effusion immunocytochemistry with these two mutant-specific antibodies exhibited a sensitivity of 71% and 88% and a specificity of 86% and 96% for identifying predefined L858R and delE746-A750 mutations, respectively. Effusion immunocytochemistry provided a superior prediction of tumor response and progression-free survival to first-line EGFR TKIs than did clinical characteristics like sex and smoking status. Patients whose effusion immunocytochemistry showed a reaction to either of the two antibodies had a comparable TKI response rate (67% versus 72%) to those with EGFR mutations assessed by direct sequencing from cell-derived RNA. Conclusions Effusion immunocytochemistry could be introduced into clinical practice to identify more NSCLC patients likely to have benefit from first-line TKI treatment, especially for those without adequate tissue for molecular-based EGFR analysis.

IntroductionThe echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion represents a novel target in a subset of non-small cell lung cancer, especially adenocarcinoma. EML4-ALK fusion is mutually exclusive with epidermal growth factor receptor (EGFR) mutations. To understand the impact of EML4-ALK on the prognosis of non-small cell lung cancer, we examined EML4-ALK fusion in lung adenocarcinoma from patients with wild-type EGFR and analyzed their clinical treatment outcomes.MethodsLung adenocarcinoma patients with malignant pleural effusions having wild-type EGFR and measurable target lesions were enrolled for EML4-ALK analysis by reverse transcription-polymerase chain reaction and direct sequencing. Demographic data, EML4-ALK status, and survival data were analyzed. We also performed fluorescence in situ hybridization on some available tumor samples to validate the PCR result. In addition, K-ras mutation was analyzed for patients without EML4-ALK fusion genes.ResultsA total of 116 patients with wild-type EGFR sequencing results had complete clinical data for analysis. No patients received ALK inhibitor therapy. There were 39 patients (34%) with the EML4-ALK fusion gene. The concordance rate between reverse transcription-polymerase chain reaction and fluorescence in situ hybridization was 85%. The K-ras mutation rate for patients without EML4-ALK fusion gene was 6.5%. By multivariate analysis, patients who had better performance status (p < 0.001) and EML4-ALK translocation (p = 0.017) had longer overall survival. Comparing patients with tumors harboring variant 1 with those harboring nonvariant 1 EML4-ALK fusion genes, there were no significant differences in clinical factors and survival outcome.ConclusionFor lung adenocarcinoma patients with wild-type EGFR, EML4-ALK translocation is associated with longer overall survival.

Objective Epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma are involved in the tumorigenesis and regulation of cell metabolism via Akt signaling. F-18 fluorodeoxyglucose-positron emission tomography ([18F]FDG PET), a functional imaging modality, can be used to measure tumor cell metabolism. Thus, in this study, we hypothesize that there exist correlations between EGFR mutation status and [18F]FDG uptake of advanced lung adenocarcinoma. Methods From May 2004 to April 2008, patients with stage IIIB or IV lung adenocarcinoma who underwent [18F]FDG PET and EGFR mutation analysis before receiving any treatment were eligible to participate in this study. The association of EGFR mutation status with patient characteristics and the SUVMAX from the [18F]FDG PET was evaluated. Multivariate logistic regression analysis was used to analyze predictors of EGFR mutations. Results Seventy-seven lung adenocarcinoma patients were included in this study. EGFR mutations were identified in 49 (64%) of the patients. The [18F]FDG uptake was significantly higher in EGFR-mutant (mean SUVMAX = 10.5 ± 4.7) than wild-type (8.0 ± 3.3) lung adenocarcinoma patients (P = 0.008). The median SUVMAX was 9.5, and patients with an SUVMAX ≥ 9.5 were more likely to harbor EGFR mutations (P = 0.009). In the multivariate analysis, an SUVMAX ≥ 9.5 remained a statistically significant predictor of EGFR mutations (P = 0.005). Conclusions Among Asian patients with advanced lung adenocarcinoma, those with higher SUVMAX on the [18F]FDG PET are more likely to carry EGFR mutations.

Curcumin (diferuloylmethane) is an active component of the spice turmeric and has a diversity of antitumor activities. In this study, we found that curcumin can inhibit cancer cell invasion and metastasis through activation of the tumor suppressor DnaJ-like heat shock protein 40 (HLJ1). Human lung adenocarcinoma cells (CL1-5) treated with curcumin (1–20 μmol/L) showed a concentration-dependent reduction in cell migration, invasion, and metastatic ability, and this was associated with increased HLJ1 expression. Knockdown of HLJ1 expression by siRNA was able to reverse the curcumin-induced anti-invasive and antimetastasis effects in vitro and in vivo. The HLJ1 promoter and enhancer in a luciferase reporter assay revealed that curcumin transcriptionally up-regulates HLJ1 expression through an activator protein (AP-1) site within the HLJ1 enhancer. JunD, one of the AP-1 components, was significantly up-regulated by curcumin (1–20 μmol/L) in a concentration- and time-dependent manner. Knockdown of JunD expression could partially reduce the curcumin-induced HLJ1 activation and diminish the anti-invasive effect of curcumin, indicating that JunD would seem to be involved in curcumin-induced HLJ1 expression. Curcumin was able to induce c-Jun NH2-kinase (JNK) phosphorylation, whereas the JNK inhibitor (SP-600125) could attenuate curcumin-induced JunD and HLJ1 expression. Activation of HLJ1 by curcumin further leads to up-regulation of E-cadherin and a suppression of cancer cell invasion. Our results show that curcumin induces HLJ1, through activation of the JNK/JunD pathway, and inhibits lung cancer cell invasion and metastasis by modulating E-cadherin expression. This is a novel mechanism and supports the application of curcumin in anti–cancer metastasis therapy. [Cancer Res 2008;68(18):7428–38]

// Shang-Gin Wu 1, 2 , Yi-Nan Liu 3 , Meng-Feng Tsai 4 , Yih-Leong Chang 5 , Chong-Jen Yu 2, 3 , Pan-Chyr Yang 2, 3 , James Chih-Hsin Yang 6 , Yueh-Feng Wen 7 , Jin-Yuan Shih 2, 3 1 Department of Internal Medicine, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin, Taiwan 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 3 Department of Internal Medicine, National Taiwan University Hospital, and College of Medicine, National Taiwan University, Taipei, Taiwan 4 Department of Molecular Biotechnology, Da-Yeh University, Chang-Hua, Taiwan 5 Department of Pathology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan 6 Department of Oncology, National Taiwan University Hospital, and Graduate Institute of Oncology, Cancer Research Center, National Taiwan University, Taipei, Taiwan 7 Department of Internal Medicine, National Taiwan University Hospital Hsinchu Branch, Hsinchu, Taiwan Correspondence to: Jin-Yuan Shih, e-mail: jyshih@ntu.edu.tw Keywords: lung adenocarcinoma, afatinib, T790M, acquired resistance, EGFR TKI Received: October 08, 2015 Accepted: January 23, 2016 Published: February 04, 2016 ABSTRACT Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are associated with favorable response in EGFR mutant lung cancer. Acquired resistance to reversible EGFR TKIs remains a significant barrier, and acquired EGFR T790M-mutation is the major mechanism. Second-generation irreversible EGFR TKI, afatinib, had also been approved for treating EGFR mutant lung cancer patients, but the mechanism of acquired resistance to afatinib has not been well studied. Results: Forty-two patients had tissue specimens taken after acquiring resistance to afatinib. The sensitizing EGFR mutation were all consistent between pre- and post-afatinib tissues. Twenty patients (47.6%) had acquired T790M mutation. T790M rate was not different between first-generation EGFR TKI-naive patients (50%) and first-generation EGFR TKI-treated patients (46.4%) ( p = 0.827). No clinical characteristics or EGFR mutation types were associated with the development of acquired T790M. No other second-site EGFR mutations were detected. There were no small cell or squamous cell lung cancer transformation. Other genetic mutations were not identified in PIK3CA , BRAF , HER2 , KRAS , NRAS , MEK1 , AKT2 , LKB1 and JAK2 . Methods: Afatinib-prescription record of our department of pharmacy from January 2007 and December 2014 was retrieved. We investigated patients with tissue specimens available after acquiring resistance to afatinib. Enrolled patients should have partial response or durable stable disease of treatment response to afatinib. Various mechanisms of acquired resistance to first-generation EGFR TKIs were evaluated. Histology and cytology were reviewed. EGFR , PIK3CA , BRAF , HER2 , KRAS , NRAS , MEK1 , AKT2 , LKB1 and JAK2 genetic alterations were evaluated by sequencing. Statistical analysis was performed using Chi-square test and Kaplan-Meier method. Conclusions: T790M was detected in half of the lung adenocarcinoma after acquiring resistance to afatinib. T790M is still the major acquired resistance mechanism. First-generation EGFR TKI exposure did not influence the prevalence of T790M in lung cancer acquired resistance to afatinib.

Malignant pleural effusion (MPE) is a common clinical problem in non-small cell lung carcinoma (NSCLC) patients; however, the underlying mechanisms are still largely unknown. Recent studies indicate that the frequency of the L858R mutant form of the epidermal growth factor receptor (EGFR-L858R) is higher in lung adenocarcinoma with MPE than in surgically resected specimens, suggesting that lung adenocarcinoma cells harboring this mutation tend to invade the adjacent pleural cavity. The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to investigate the molecular mechanisms involved in the formation of MPE. We found that expression of EGFR-L858R in lung cancer cells resulted in up-regulation of the CXCR4 in association with increased cancer cell invasive ability and MPE formation. Ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We also indicated that Inhibition of CXCR4 with small interfering RNA, neutralizing antibody, or receptor antagonist significantly suppressed the EGFR-L858R–dependent cell invasion. These results suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring a specific type of EGFR mutation.

Lung cancer is the leading cause of cancer-related mortality throughout the world. Non-small cell lung cancer (NSCLC) accounts for 85% of all diagnosed lung cancers. Despite considerable progress in the diagnosis and treatment of the disease, the overall 5-year survival rate of NSCLC patients remains lower than 15%. The most common causes of death in lung cancer patients are treatment failure and metastasis. Therefore, developing novel strategies that target both tumour growth and metastasis is an important and urgent mission for the next generation of anticancer therapy research. Heat shock proteins (HSPs), which are involved in the fundamental defence mechanism for maintaining cellular viability, are markedly activated during environmental or pathogenic stress. HSPs facilitate rapid cell division, metastasis, and the evasion of apoptosis in cancer development. These proteins are essential players in the development of cancer and are prime therapeutic targets. In this review, we focus on the current understanding of the molecular mechanisms responsible for HLJ1's role in lung cancer carcinogenesis and progression. HLJ1, a member of the human HSP 40 family, has been characterised as a tumour suppressor. Research studies have also reported that HLJ1 shows promising dual anticancer effects, inhibiting both tumour growth and metastasis in NSCLC. The accumulated evidence suggests that HLJ1 is a potential biomarker and treatment target for NSCLC.

Recently, mutations in the epidermal growth factor receptor (EGFR) gene in nonsmall cell lung cancer (NSCLC) patients were reported to correlate with gefitinib response. Less than 30% of NSCLC patients are surgically resectable; however, molecular analysis has to rely on nonsurgical diagnostic tissue samples. The objective of this study is to investigate EGFR mutation analysis on needle biopsy/aspiration samples and its correlations with gefitinib response and patients' survival. EGFR mutation was assessed from DNA of 63 paraffin-embedded small needle biopsy/aspiration specimens from 62 patients with NSCLC treated with gefitinib. The peripheral blood lymphocyte DNA of the patients was sequenced to verify the EGFR mutation. EGFR mutations were found in 47% of 62 patients (60% of 20 CT-guided biopsies, 44% of 18 ultrasound-guided biopsies, 31% of 16 endoscopic biopsies and 44% of 9 effusion cell blocks). EGFR mutations were frequently present in females (p = 0.006) and never smokers (p = 0.04). Patients with EGFR mutations had a significantly better response rate compared to that of the nonmutation group (p < 0.001). Multivariate analysis showed that EGFR mutation (p < 0.001) and PS 0-1 (p = 0.02) were independently associated with a better response rate. Cox regression analysis showed that EGFR mutation was the independent prognostic factor for progression-free survival (p = 0.008) and overall survival (p = 0.03). In conclusion, EGFR mutation analysis is feasible in needle biopsy/aspiration paraffin-fixed specimens. EGFR mutation is an independent predictor of gefitinib response and survival in patients of advanced NSCLC treated by gefitinib.

By using microarray and an invasion/metastasis lung cell line model, we identified the DnaJ-like heat shock protein 40, HLJ1, and found that the expression of HLJ1 correlates negatively with cancer cell invasion ability. Overexpression of HLJ1 can suppress cancer cell invasion in vitro. We further characterize the putative promoter region and investigate the transcriptional regulations of human HLJ1. A serial deletion of the 1.2 kb at the 5'-flanking region of the human HLJ1 gene was subcloned into a vector containing reporter gene and transfected into human lung adenocarcinoma cell line CL1-0, followed by luciferase activity assay. The results indicated that the region from -232 to +176 could drive the basal transcriptional activity of the HLJ1 gene. Sequence analysis of the HLJ1 gene promoter region showed absence of a TATA box, but identified an inverted CCAAT box and four YY1 transcriptional factor-binding sites, which may be important in the regulation of HLJ1 expression. Co-transfection of the YY1 and HLJ1 basal promoter regions, site-directed mutagenesis, and electrophoretic mobility shift assay confirmed that YY1 could upregulate HLJ1 basal promoter activity. Furthermore, we also demonstrated that overexpression of YY1 in CL1-0 cells can increase HLJ1 expression and reduce cell invasive capability. The reduction of cancer cell invasive ability is, at least in part, through upregulation of E-cadherin expression. The increase in HLJ1 and E-cadherin expression, as well as the suppression of invasion ability, can be reversed specifically by HLJ1 siRNA.

Purpose Inflammation plays a critical role in cancer progression. In this study we investigate the pro-tumorigenic activities and gene expression profiles of lung cancer cells after interaction with macrophages. Materials and Methods We measured intratumoral microvessel counts and macrophage density in 41 lung cancer tumor specimens and correlated these with the patients' clinical outcome. The interaction between macrophages and cancer cell lines was assessed using a transwell coculture system. The invasive potential was evaluated by in vitro invasion assay. The matrix-degrading activity was assayed by gelatin zymography. The microarray was applied to a large-scale analysis of the genes involved in the interaction, as well as to monitor the gene expression profiles of lung cancer cells responding to anti-inflammatory drugs in cocultures. Results The macrophage density positively correlated with microvessel counts and negatively correlated with patient relapse-free survival (P < .05). After coculture with macrophages, lung cancer cell lines exhibited higher invasive potentials and matrix-degrading activities. We identified 50 genes by microarray that were upregulated more than two-fold in cancer cells after coculture. Northern blot analyses confirmed some gene expression such as interleukin-6, interleukin-8, and matrix metalloproteinase 9. The two-dimensional hierarchical clustering also demonstrated that the gene expression profiles of lung cancer cells responding to various anti-inflammatory drugs in cocultures are distinct. Conclusion The interaction of lung cancer cells and macrophages can promote the invasiveness and matrix-degrading activity of cancer cells. Our results also suggest that a great diversity of gene expression occurs in this interaction, which may assist us in understanding the process of cancer metastasis.

PLEK2 is one of the pleckstrin homology domain (PH domain) proteins. Recently studies suggested that PLEK2 may play a critical role in T lymphocyte migration through by PI3K/AKT dependent and independent pathways. However, the biological functions of PLEK2 in lung cancer is not clear. We detected the expression of PLEK2 in various lung adenocarcinoma cell lines using quantitative RT-PCR methods. The results indicated that PLEK2 expression significantly positive correction with cancer cell migration and invasion ability. Forced expression of PLEK2 in lung cancer cells exhibited markedly promoting in the proliferation rate, anchorage independent growth, cell motility and invasion, whereas knockdown of PLEK2 was found to suppress lung cancer cell progression. In addition, we also reported that PLEK2 expression induce lung cancer cell epithelial-mesenchymal transition (EMT) phenomenon, and involve in chemotherapeutic drug resistance through upregulation NDRG1 gene expression. Overexpression of NDRG1 in PLEK2-knockdown cell line restored cancer cell proliferation, migration, and invasion. These results will provide information and help us to understand the roles of NDRG1 in lung cancer cells. Citation Format: Yi-Pin Hsieh, Hui-Chia Liu, Hao-Yu Huang, Wan-Jiun Tang, Tai-Lin Lee, Meng-Feng Tsai. PLEK2 contributes to lung cancer progression through upregulation of NDRG1 expression [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 1890. doi:10.1158/1538-7445.AM2017-1890

e20572 Background: Lung cancer is a leading cause of cancer death worldwide. The EGFR mutation-positive patients of lung adenocarcinoma had dramatic response to EGFR tyrosine kinase inhibitors (TKIs). However, acquired drug resistance developed invariably after one year treatment course of EGFR TKI. IGFBP-7, a secreted 31 kDa protein, exhibits low affinity for IGF but binds strongly to insulin. No previous studies had been mentioned the correlation between the protein and acquired EGFR TKI resistance. Methods: cDNA microarray was used to screen differentially expressed genes between EGFR TKI-sensitive (PC9 and HCC827) and acquired EGFR TKIs-resistance cell lines (PC9/gef and HCC827/gef). The expression levels of the screened genes were validated by RT -PCR and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of IGFBP-7 on gefitinib resistance in vitro. In addition, clinical specimens were collected to validate the imact of IGFBP-7 on the efficacy of EGFR TKI treatment . Results: IGFBP-7 is over-expressed in gefitinib-resistant cells. The IGFBP-7 mRNA expression in the malignant pleural effusion of patients with acquired resistance to EGFR TKI was significant higher than that in the treatment-naïve patients. Knockdown IGFBP-7 reverses gefitinib resistance in PC9/gef cells. Knockdown IGFBP-7 could increase gefetinib induced-apoptosis through the regulation of BIM. IGFBP-7 affected the mechanism of EGFR TKI resistance resulted from inhibition of IGF-IR.. In clinical practice, low IGFBP-7 serum level is associated with longer progression free survival (PFS) of EGFR TKI as the first line treatment. Furthermore, lower IGFBP-7 level of resected tumor predicts a longer 5-year tumor relapse-free survival. Positive IGFBP-7 immunoihistochemical stain could predict a shorter PFS of the first-line EGFR TKI treatment. Conclusions: IGFBP-7 confers resistance to the EGFR TKI and may be a target for future treatment investigation.

This case–control study aimed to evaluate the diagnostic application of urinary transforming growth factor (TGF) α and serum α-fetoprotein (AFP) in hepatocellular carcinoma (HCC). TGFα and AFP were determined in 90 pairs of age- and gender-matched patients with cirrhotic HCC and patients with cirrhosis alone and 60 healthy controls. The results indicated that TGFα and AFP levels in patients with HCC were higher than in those with cirrhosis alone or healthy controls (each P = 0.0001). Multivariate analysis indicated that TGFα (odds ratio (OR) 1.03, 95 % confidence interval (CI) 1.05–1.16) and AFP (OR 1.03, 95 % CI 1.01–1.06) were closely associated, in a dose-related fashion, with the development of HCC. The optimal cutoff values, determined with the receiver operating characteristic (ROC) curves, were 29 μg/g creatinine for TGFα and 100 ng/ml for AFP, respectively. The areas under ROC curve (AUC) were 0.74 for TGFα and 0.78 for AFP, respectively. Both biomarkers showed the same sensitivity (52.2 %), high specificity, high positive predictive value, and moderate positive likelihood ratio. Determination of both markers in parallel significantly increased the AUC (0.91) and diagnostic accuracy (92.2 %), with a high sensitivity (86.7 %), specificity (97.8 %), positive predictive value (PPV; 97.5 %), and moderate positive likelihood ratio (PLR; 39.4). Among 31 cirrhotic HCC with AFP ≤ 20 ng/ml, the calculated AUC for TGFα was 0.79, with a sensitivity of 64.5 %, specificity of 96.7 %, PPV of 87.0 %, and PLR of 19.5. In conclusion, urinary TGFα and serum AFP are complementary tumor markers for detection of HCC with low AFP production.

Since the symptoms of the diabetic retinopathy, macular degeneration, and many other eye diseases are often associated with the bright spots, dark spots, and vascular abnormalities in retinal images, this study proposes a retinopathy image segmentation (system) to extract the bright spots, dark spots, and blood vessels from retinal images. Moreover, since a high proportion of diabetic patients suffer from the diabetic retinopathy with the most severe one to be the retinal detachment, the retinal image-based tractional retinal detachment diagnosis system is proposed, as well. Two genetic-based parameter detectors are given to provide the most appropriate values for the parameters used by the retinopathy image segmentation system and the tractional retinal detachment diagnosis system. The experimental results illustrate that the retinopathy image segmentation system and the tractional retinal detachment diagnosis system can provide expressive results in segmenting bright spots, dark spots, blood vessels, and in diagnosing tractional retinal detachment. Both systems are helpful for the ophthalmologists in diagnosing eye diseases.

In the era of targeted therapy, the association between lung adenocarcinoma patient survival and malignant pleural effusions (MPEs) remains unclear. This study investigated the clinical characteristics, survival and epidermal growth factor receptor (EGFR) gene (EGFR) mutation status of lung adenocarcinoma patients with MPE. From June 2005 to December 2010, consecutive pleural effusions were collected prospectively. Patient clinical characteristics, EGFR mutation status, and overall survival were analysed. We collected MPEs from 448 patients in stage IV lung adenocarcinoma at initial diagnosis. Median overall survival for patients with MPEs at initial diagnosis and following disease progression were 14.3 months and 21.4 months, respectively (p=0.001). There were 296 (66.1%) patients harbouring EGFR mutations, the mutation rates among patients with an MPE at initial diagnosis and one following disease progression were 68.2% and 56.6%, respectively (p=0.044); the L858R mutation rate was also higher among the former (32.6% versus 18.1%; p=0.009). Multivariate analysis revealed that patients who: developed MPEs following disease progression, harboured EGFR mutations, and received EGFR-tyrosine kinase inhibitor therapy, had longer overall survival. Patients in stage IV lung adenocarcinoma with MPEs at initial diagnosis have shorter overall survival and higher EGFR mutation rate, especially for L858R, than patients who develop MPEs following disease progression.

Molecular targeting therapeutics, such as EGFR tyrosine kinase inhibitors (TKIs), are important treatment strategies for lung cancer. Currently, the major challenge confronting targeted cancer therapies is the development of resistance. Cancer stem cells (CSCs) represent a rare population of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance and spreading. Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs. However, the issue of whether CSCs contribute to EGFR TKI resistance in lung cancer is yet to be established. In the current study, we explored the association of ALDH1A1 expression with EGFR TKI resistance in lung cancer stem cells. ALDH1A1-positive lung cancer cells displayed resistance to gefitinib, compared to ALDH1A1-negative lung cancer cells. Moreover, PC9/gef cells (gefitinib-resistant lung cancer cells) presented a higher proportion of ALDH1A1-positive cells, compared to PC9 cells (gefitinib-sensitive lung cancer cells). Clinical sample studies were consistent with results from cell culture model systems showing that lung cancer cells with resistance to EGFR TKI and chemotherapy drugs contain significantly increased proportions of ALDH1A1-positive cells. These findings collectively suggest that ALDH1A1 positivity in cancer stem cells confers resistance to EGFR TKI in lung cancer.

Epidermal growth factor receptor (EGFR) is a novel target for therapy in subsets of non-small cell lung cancer, especially adenocarcinoma. Tumors with EGFR mutations showed good response to EGFR tyrosine kinase inhibitors (TKIs). We aimed to identify the discriminating capacity of immunohistochemical (IHC) scoring to detect L858R and E746-A750 deletion mutation in lung adenocarcinoma patients and predict EGFR TKIs response. Patients with surgically resected lung adenocarcinoma were enrolled. EGFR mutation status was genotyped by PCR and direct sequencing. Mutation-specific antibodies for L858R and E746-A750 deletion were used for IHC staining. Receiver operating characteristic (ROC) curves were used to determine the capacity of IHC, including intensity and/or quickscore (Q score), in differentiating L858R and E746-A750 deletion. We enrolled 143 patients during September 2000 to May 2009. Logistic-regression-model-based scoring containing both L858R Q score and total EGFR expression Q score was able to obtain a maximal area under the curve (AUC: 0.891) to differentiate the patients with L858R. Predictive model based on IHC Q score of E746-A750 deletion and IHC intensity of total EGFR expression reached an AUC of 0.969. The predictive model of L858R had a significantly higher AUC than L858R intensity only (p = 0.036). Of the six patients harboring complex EGFR mutations with classical mutation patterns, five had positive IHC staining. For EGFR TKI treated cancer recurrence patients, those with positive mutation-specific antibody IHC staining had better EGFR TKI response (p = 0.008) and longer progression-free survival (p = 0.012) than those without. In conclusion, total EGFR expression should be included in the IHC interpretation of L858R. After adjusting for total EGFR expression, the scoring method decreased the false positive rate and increased diagnostic power. According to the scoring method, the IHC method is useful to predict the clinical outcome and refine personalized therapy.

Non-small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown.To examine the role of EMT regulators in resistance to gefitinib.The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription-polymerase chain reaction and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from 44 patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time reverse transcription-polymerase chain reaction.Slug expression, but not that of snail, twist, or zeb-1, was significantly increased in PC9/gef compared with PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib-induced apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim up-regulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naive cancer cells.Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

Background: Mutations of the epidermal growth factor receptor (EGFR) gene in non-small-cell lung cancer (NSCLC) patients predict the patients who will respond to EGFR tyrosine kinase inhibitor (TKI) treatment. A recent study has suggested that 33% of NSCLC showed primary tumor/metastasis discordance of EGFR expression by immunohistochemistry analysis. We intended to find out whether the EGFR mutations of primary lung cancers are concordant to that of corresponding metastatic tumors. Materials and methods: We analyzed EGFR exons 18–21 from paired primary and metastatic tumors in 67 lung cancer patients who had not received TKI before tissues were sampled. Results: Using the direct sequencing method, 9 of 18 (50%) patients with EGFR mutation-positive primary lung tumors had lost the mutations in metastases. For 26 patients who were EGFR mutation positive in the metastatic tumors, 17 (65%) were negative in the primary tumors. We analyzed these paired tissues with discrepant EGFR mutations by the Scorpion Amplified Refractory Mutation System assay. Finally, the discordant rate reached 27% (18 of 67 cases). Conclusion: EGFR mutations in primary lung tumors do not always reflect the same situation in metastases. Analysis of EGFR mutations in the primary lung tumor would be inadequate for planning the use of TKI for advanced NSCLC.

Background.Epidermal growth factor receptor (EGFR) mutations are usually detected in lung adenocarcinoma and are associated with a response to EGFR tyrosine kinase inhibitors (TKIs). However, not all EGFR mutations have similarly high clinical response rates. This study aimed to investigate the clinical characteristics and response to gefitinib in lung adenocarcinoma patients with complex EGFR mutations.Materials and Methods.Three hundred thirty-nine specimens of lung adenocarcinoma from patients treated with gefitinib were collected for EGFR sequencing. Nineteen patients with complex EGFR mutations were enrolled for the study after excluding three patients with the EGFR T790M mutation, which confers resistance to gefitinib.Results.Among the 19 patients, 12 had complex mutations with the classical mutation pattern (L858R or deletion in exon 19). When compared with those without the classical mutation pattern, patients with this mutation pattern had a higher response rate (83% versus 29%), longer progression-free survival duration (median, 12.7 months versus 4.9 months), and longer overall survival time (median, 24.7 months versus 12.3 months) after gefitinib treatment.Comparing patients harboring complex EGFR mutations with a classical mutation pattern with those harboring single classical mutations, there were no statistical differences in the response rate (83% versus 73%), progression-free survival time (median, 12.7 months versus 8.1 months,) or overall survival time (median, 24.7 months versus 16.4 months).Conclusion.Patients with complex EGFR mutations with the classical mutation pattern had the same response rate, progression-free survival duration, and overall survival time as those with single classical mutations. EGFR TKIs may be the choice of treatment for this type of lung adenocarcinoma.

HLJ1 is a novel tumor and invasion suppressor that inhibits tumorigenesis and cancer metastasis. However, the mechanism of HLJ1 activation is currently unclear. Here, we identify an enhancer segment in the HLJ1 gene at −2,125 to −1,039 bp upstream of the transcription start site. A 50-bp element between −1,492 and −1,443 bp is the minimal enhancer segment, which includes the activator protein 1 (AP-1) site (−1,457 to −1,451 bp), an essential regulatory domain that binds the transcriptional factors FosB, JunB, and JunD. Chromatin immunoprecipitation assays confirm that these AP-1 family members bind to a specific site in the HLJ1 enhancer segment in vivo. Overexpression of either YY1 at promoter or AP-1 at enhancer results in a 3-fold increase in the transcriptional activity of HLJ1. We propose a novel mechanism whereby expression of the tumor suppressor, HLJ1, is up-regulated via enhancer AP-1 binding to promoter YY1 and the coactivator, p300, through DNA bending and multiprotein complex formation. The combined expression of AP-1 and YY1 enhances HLJ1 expression by more than five times and inhibits in vitro cancer cell invasion. Elucidation of the regulatory mechanism of HLJ1 expression may facilitate the development of personalized therapy by inhibiting cancer cell proliferation, angiogenesis, and metastasis. [Cancer Res 2007;67(10):4816–26]

Viral infection poses a major problem for public health, horticulture, and animal husbandry, possibly causing severe health crises and economic losses. Viral infections can be identified by the specific detection of viral sequences in many ways. The microarray approach not only tolerates sequence variations of newly evolved virus strains, but can also simultaneously diagnose many viral sequences. Many chips have so far been designed for clinical use. Most are designed for special purposes, such as typing enterovirus infection, and compare fewer than 30 different viral sequences. None considers primer design, increasing the likelihood of cross hybridization to similar sequences from other viruses. To prevent this possibility, this work establishes a platform and database that provides users with specific probes of all known viral genome sequences to facilitate the design of diagnostic chips. This work develops a system for designing probes online. A user can select any number of different viruses and set the experimental conditions such as melting temperature and length of probe. The system then returns the optimal sequences from the database. We have also developed a heuristic algorithm to calculate the probe correctness and show the correctness of the algorithm. (The system that supports probe design for identifying viruses has been published on our web page http://bioinfo.csie.ncu.edu.tw/.)

Non – small-cell lung carcinoma (NSCLC) is the predominant type of lung cancer ( 3 ) . Approximately 30% of patients with NSCLC are diagnosed at an early stage of the disease and receive curative surgery. However, disease will recur within 5 years in approximately 40% of these patients ( 3 – 5 ) . The current clinical staging systems for lung cancer may have reached their limits in providing critical information that may infl uence patient management strategies. It is therefore important to identify patients who are at high risk of recurrence or of treatment failure after surgical resection. Identifi cation of the specifi c genes involved and understanding the molecular pathogenesis in these high-risk patients are urgently needed. Gene expression profi les have been used to identify possible associations with lung cancer behavior or clinical outcome to better predict patient prognosis ( 6 – 10 ) . In a previous study ( 9 ) , we screened lung cancer cell lines with various invasive abilities using microarray analysis of 9600 genes and identifi ed a panel of 589 (6.1%) genes whose expression was associated with invasion and metastasis. We used information from functional databases and hierarchical clustering analyses to categorize many of these genes by function. We then chose 30 candidate genes of unknown function for further characterization, investigated their mechanisms of action, and verifi ed their functions in cancer patients ( 10 , 11 ). Here, Background: We previously identifi ed DnaJ-like heat shock protein (HLJ1) as a gene associated with tumor invasion. Here, we investigated the clinical signifi cance of HLJ1 expression in non – small-cell lung cancer (NSCLC) patients and its role in cancer progression. Methods: We induced HLJ1 overexpression or knockdown in human lung adenocarcinoma CL1 – 5 cells and analyzed cell proliferation, anchorage- independent growth, in vivo tumorigenesis, cell motility, invasion, and cell cycle progression. Expression of genes that act downstream of HLJ1 was examined by DNA microarray analysis, pathway analysis, and western blotting. We measured HLJ1 expression in tumors and adjacent normal tissues of 71 NSCLC patients by quantitative reverse transcription – polymerase chain reaction. Associations between HLJ1 expression and disease-free and overall survival were determined using the log-rank test and multivariable Cox proportional hazards regression analysis. Validation was performed in an independent cohort of 56 NSCLC patients. Loss of heterozygosity (LOH) mapping of the HLJ1 locus was analyzed in 48 paired microdissected NSCLC tumors. All statistical tests were two-sided. Results: HLJ1 ex pression inhibited lung cancer cell proliferation, anchorageindependent growth, tumorigenesis, cell motility, and invasion, and slowed cell cycle progression through a novel STAT1/ P21 WAF1 pathway that is independent of P53 and interferon. HLJ1 expression was lower in tumors than in adjacent normal tissue in 55 of 71 patients studied. NSCLC patients with high HLJI expressing tumors had reduced cancer recurrence (hazard ratio [HR] = 0.47; 95% confi dence interval [CI] = 0.23 to 0.93; P = .03) and longer overall survival (HR = 0.38; 95% CI = 0.16 to 0.89; P = .03) than those with low-expressing tumors. Validation in the independent patient cohort confi rmed the association between HLJ1 expression and patient outcome. LOH mapping revealed high frequencies (66.7% and 70.8%) of allelic loss and microsatellite instability (87.5% and 95.2%) of the HLJ1 locus at chromosome 1p31.1. Conclusions: HLJ1 is a novel tumor suppressor in NSCLC, and high HLJ1 expression is associated with reduced cancer recurrence and prolonged survival of NSCLC patients. [J Natl Cancer Inst 2006;98: 825 – 38 ]

Purpose: In a previous genome-wide gene expression profiling analysis using an invasion cancer cell lines model, we have identified Slug as selectively overexpressed in the highly invasive cancer cells. Here, we investigated the clinical significance of Slug in lung adenocarcinoma and the role of Slug in the process of cancer cell invasion and metastasis.Experimental Design: Real-time quantitative reverse transcription-PCR was used to investigate Slug mRNA in surgically resected lung adenocarcinoma of 54 patients and its correlation with survival. We overexpressed Slug in a lung adenocarcinoma cell line with very low Slug levels and investigated the in vitro and in vivo effects of Slug expression.Results: High expression of Slug mRNA in lung cancer tissue was significantly associated with postoperative relapse (P = 0.03) and shorter patient survival (P < 0.001). The overexpression of Slug enhanced xenograft tumor growth and increased microvessel counts in angiogenesis assay. Both inducible and constitutive overexpression of Slug suppressed the expression of E-cadherin and increased the in vitro invasive ability. Zymography revealed increased matrix metalloproteinase-2 activity in Slug overexpressed cells. ELISA, reverse transcription-PCR, and immunohistochemistry confirmed the increase of matrix metalloproteinase-2 proteins and mRNA in Slug overexpressed cells and xenograft tumors.Conclusions: Slug expression can predict the clinical outcome of lung adenocarcinoma patients. Slug is a novel invasion-promoting gene in lung adenocarcinoma.

BackgroundTheophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline.MethodsMicroarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4.ResultsWe identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (ConclusionOur results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.

Desmocollin-2 (Dsc2), a calcium-dependent transmembrane glycoprotein, is the most widely distributed form of desmocollins that plays a critical role in the maintenance of normal tissue architecture in epithelia. Reduced expression of Dsc2 has been reported in colorectal carcinomas, suggesting that Dsc2 may play a role in the development and progression of colorectal cancer. Recently, down-regulated of Dsc2 expression was also found in breast, esophageal, and lung carcinomas. Dsc2 may act as tumor suppressor in various cancers. However, the role of the Dsc2 in lung cancer cells is still unclear. In this study, we report that knockdown Dsc2 expression using small-hairpin RNA (shRNA) approach can promote lung cancer cell migration and invasion abilities. Consistent with these findings, ectopic expression of Dsc2 inhibited cell migration and invasion in lung cancer cells. Additionally, we also suggested that Dsc2 inhibited lung cancer cells motility and epithelial to mesenchymal transition (EMT) through down-regulate NDRG1 and Slug expression. Citation Format: Yu-Chih Hsu, Po-Chun Ou, Yu-Yi Chen, Meng-Feng Tsai. Desmocollin 2 suppresses lung cancer cell migration and invasion through down-regulation of NDRG1 expression. [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 4989. doi:10.1158/1538-7445.AM2014-4989

Leukemia is a hematopoietic stem cell disorder and leukemia patients suffer their lost in functional blood cell and the ratio of non-function cell increased. K562 cell was isolated from the pleural effusion of the leukemia patient and was used as a model cell line to study the relationship between the blood cell differentiation and signal transduction. In this report, three different inducers, huangqi (Astragalus membranaceus) extract, chemicals Hemin and HMBA were used to induce K562 cell differentiation and several cluster of differentiation (CD) marker were used to identify the cell linage. Two megakaryocyte markers were up-regulated in these treatments. The CD61, a late marker, was highly expressed in HMBA treated cell and CD41, an earlier marker, was identified in huangqi and HMBA treated cell. In former report, the Gi2α and Gsα was induced by the HMBA administration. Since both G proteins affect their downstream effector, adenylate cyclases (ADCYs), the ADCYs that could be participated in the pathway were examined by RT-PCR. The ADCY1 and ADCY6 were up-regulated in the HMBA-induced K562 cell. The ADCY6 was further studied because it coupled to calcium ion channel. The recombinant ADCY6 transfected cells were treated with three inducers, the results showed that all inducers induceed CD61 expression especially in HMBA treated sample. Also, the CD61 gene was 25 times higher expression as the parental cell. The results imply that the signals activated from HMBA and ADCY6 together may participate in the megakaryocyte differentiation. The study suggested that K562 cells can change the cell destination through the ADCY isoforms expression and other signal genes. These results may be used as the reference of the drug discover for leukemia treatment. Citation Format: Shin Hua Jiang, Chiu Yi Ho, Tai Lin Lee, Yun Shiang Chang, Wei Tung Huang, Meng Feng Tsai, Ang Yuan. Analysis of adenylate cyclase 6 in HMBA induced K562 cell megakaryocyte differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2132. doi:10.1158/1538-7445.AM2013-2132

Liver cancer patients could suffer from high saccharide environment due to the malfunction of liver. To mimic this high saccharide environment, HepG2 cells were culture with 30 mM D-riboses. We found that this high concentration of saccharide cause cell death. Cinnamon was report to reduce glucose in model rats. We hypothesized that the administration of cinnamon extracts may benefit liver cancer patients whose metabolism is defected. To test the hypothesis, three different chemical subtypes of cinnamon extracts were added into the culture medium. The adding of all three cinnamon extracts protected HepG2 cells from D-ribose administration and increased around 20% of surviving cells. These protections were even better than the addition of aminoguanidine hemisulfate salt, a chemical used in the treatment of diabetes. The expressions of ghrelin gene and two ghrelin-related genes was monitored by RT-PCR. Ghrelin gene expression was up-regulated in cinnamon treated cells. Also, the ghrelin modify gene, MBOAT4, and ghrelin process enzyme, furin, gene were all detected in HepG2 cells and cinnnamon treated cells. These results demonstrated that if ghrelin expressed in HepG2 cell and maybe liver as well, it can be processed properly. Both ghrelin transcript variants 1 and 3 recombinant plasmid were transfected into HepG2 cells and both transcript variants protected HepG2 cells from the addition of D-ribose without the administration of cinnamon extracts. We conclude that the cinnamon avoids HepG2 cell damage from D-ribose induced oxidative stress by inducing ghrelin gene expression, and through the ghrelin signaling increases cell surviving. Since ghrelin level is abnormally regulated in patients with hepatocellular carcinoma and post-hepatitic liver cirrhosis, the understanding of how ghrelin been regulated in liver may benefit liver disease patients. Citation Format: Jia Huei Shiu, Chin Hao Huang, Tai Lin Lee, Shih Chieh Lee, Shu Ying Liu, Meng Feng Tsai. Ghrelingene expression avoiding the D-ribose caused HepG2 cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2013-583

Lung cancer is the most malignant disease which makes lots of people dead worldwide. More and more evidences showed that cancers might arise from chromosome alteration, including amplification or deletion. Our previous study integrating microarray-based comparative genomic hybridization and affymetrix gene expression profiles identified a potential candidate gene, MAPRE2, in a lung cancer cell line model with different invasion capability. MAPRE2 (Microtubule-associated protein RP/EB family member 2) is usually located on microtubule and interactes with tumor suppress gene APC (adenomatous polyposis coli). MAPRE2 might be involved in microtubule polymerization, cell migration and tumorigenesis of colorectal cancers, but its function is unclear. The purpose of this study is to investigate the effects of MAPRE2 on cancer cell function. The results of real-time PCR and western blotting showed that DNA copy number, RNA and protein level of MAPRE2 were higher in low invasive lung cancer cells than in highly invasive cells. Immunofluorescence assay indicated that the distribution of MAPRE2 protein was predominant in cytoplasm. Furthermore, overexpression of MAPRE2 could inhibit cancer cell proliferation, anchorage- dependent and -independent growth, cell motility, invasion ability in vitro and tumor growth and metastasis in vivo. On the contrary, silencing MAPRE2 enhanced lung cancer cell proliferation and invasion ability. Consider the whole map of MAPRE2-regulated signalling, transcriptomic analysis was performed and the results showed that MAPRE2 could affect 1039 genes (ANOVA, FDR< 0.05) which are dominantly involved in cell cycle and adhesion-related biological signalling, such as MAPK, focal adhesion and tight junction pathways. Our results suggested that MAPRE2 might play a significant role in MAPRE2-mediated pathways to suppress tumorigenesis and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 77. doi:1538-7445.AM2012-77

Propolins, prenylflavanones are isolated and characterized from Taiwanese propolis, have been shown inhibits certain cancer cell lines growth and induced apoptosis. In our previous study, Taiwanese propolis, propolin C and porpolin G, could promote LC3B increased within 6 hours treatment, moreover, and AVOs (acidic vesicular organelles) significantly increased by AO (Acridine orange) staining. The autophagy induced by propolin C or propolin G is accompanied by the inhibition of the PI3K/Akt/mTOR signaling pathway. Flow cytometry assay showed that sub-G1 phase increased significantly in propolin G treatment but not in propolin C treatment. Autophagy pharmacological inhibitors, 3-methyladenine and Chloroquine were used to test whether autophagy blockade could lead to enhance cell death. The results showed that only autophagy inhibitor chloroquine effectively enhanced apoptosis induced by propolin C but not propolin G in A549 cells. The expression of pERK and pAkt was suppressed dramatically by autophagy inhibitor in combination with propolin C. Ectopic expression of mutant Akt (constitutive active) in A549 cells abolished chloroquine sensitization effect of propolin C. Together, autophagy serves a protective role in propolin C-mediated cytotoxicity, and autophagy modulators may be used as adjunctive therapeutic agents for propolin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2264. doi:1538-7445.AM2012-2264

Lung cancer is the most common cause of cancer deaths in the world, and the incidence is increasing each year. Protein tyrosine phosphatases (PTPs) and their associated signaling pathways are crucial for the regulation of numerous cell functions including growth, motility, mitogenesis and metabolism. PTPN2, also known as TCPTP, is an intracellular protein tyrosine phosphatase, which is ubiquitously expressed. There are two splice variants of TCPTP in human cells, TC45 and TC48, which vary at their C-terminal ends. The TC48 isoform is present in the endoplasmic reticulum and also in the nuclear membrane. However, its role and function on human non-small cell lung cancers remain unknown. In this study, we induced ectopic expression of PTPN2 (TC48) in CL1-0 and H1299 cells to investigate its effect on cell migration, invasion, and colony formation. The interacting proteins of PTPN2 are identified by yeast two-hybrid assay. The results showed that PTPN2 inhibited lung cancer cell proliferation, anchorage-dependent and -independent growth, but not migration and invasion. Yeast two-hybrid assay revealed that PTPN2 interacted with CD74 (major histocompatibility complex class II invariant chain) and PRDX6 (Peroxiredoxin 6) which could promote cell proliferation in previous studies. We also found that overexpression of PTPN2 increased the phosphorylation of ERK. Moreover, activation of ERK was enhanced by co-expression of PTPN2 and CD74 or PRDX6. Taken together, we speculated that PTPN2 might modulate ERK activity through interaction with CD74 and PRDX6 and further promoted lung cancer cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4183. doi:1538-7445.AM2012-4183

Metastasis is a major cause leading to mortality for lung cancer patients and gene copy variation is one common mechanism contributing to human malignancies. To identify the novel metastasis-related genes, an approach combined array comparative genomic hybridization and expression microarray was applied in a lung cancer invasion cell model. YWHAZ was identified as a potential candidate, which plays an important role in a wide variety of cellular processes. Nevertheless, its role and underlying mechanism in lung cancer metastasis remain unclear. In this study, we found that YWHAZ promotes cell proliferation, anchorage-independent growth, invasion and migration in vitro, as well as tumorigenesis and metastasis in vivo. It not only increases cell protrusions and branchings but also induces epithelial-mesenchymal transition. Furthermore, YWHAZ associates with β-catenin and enhances slug expression which is regulated by the β-catenin/TCF signaling pathway in lung cancer cells. Our findings demonstrate that YWHAZ plays a critical role in promoting invasion and metastasis of human lung adenocarcinoma cells, which might provide new insight into the future treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3440. doi:10.1158/1538-7445.AM2011-3440

Our previous study disclosed EGFR L858R mutation rate of lung adenocarcinoma with malignant pleural effusions of is higher than that of surgically resected lung adenocarcinoma (Wu et al, Eur Respir J 2008). The molecular mechanism is not clear. The chemokine receptor CXCR4 has recently been shown to mediate the movement of malignant cancer cells to specific organs. Stable clones of wild type and mutant L858R EGFR from lung cancer cell line H1299 were obtained from Dr. Chen (Chen YR et al Oncogene 2006). The Invasive ability of L858R EGFR H1299 cells was higher than that of wild type EGFR H1299 cell. L858R EGFR H1299 cell had higher CXCR4 expression, which was required for L858R EGFR-mediated invasion in vitro. Up-regulation of CXCR4 in L858R EGFR H1299 cell promoted invasion via increased activity of ERK. These results provide a plausible mechanism for L855R EGFR-mediated lung tumor metastasis and establish a functional link between mutant EGFR and CXCR4 signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2011-5238

MicroRNAs (miRNAs) are small non-coding RNAs, approximately 20-23 nucleotides in length. The specific functions of miRNAs involved in several cellular processes, including metabolism, development, differentiation, proliferation, apoptosis and tumorigenesis. In order to identify the invasion/metastasis related miRNA in lung cancer, the cell line model (lowly invasive lung cancer cells, CL1-0, and highly invasive lung cancer cells, CL1-5) and human miRNA microarray were carried out. In this study, we selected one of the invasion/metastasis related miRNAs, miR-127, derived from our microarray results for further functional characterization. Recently reports have indicated that human miR-127 was highly expressed in normal prostate and bladder tissues but was remarkably down-regulated in the corresponding tumors. miR-127 may function as a tumor suppressor in human bladder and prostate cancer cells. However, the role of the miR-127 in lung cancer cell proliferation, migration and invasion ability still remains unclear. The miR-127 expression level in various invasive capability lung cancer cell lines were also verified using stem-loop real time RT-PCR. We have constructed and established a miR-127 constitutive expression system in CL1-0 lung cancer cells using pSilencer3.1 vector, and we also knockdown the miR-127 expression in CL1-5 and A549 lung cancer cells using anti-miR127 modified oligonucleotide. Our results shown that over-expression of miR-127 can increase lung cancer cell proliferation, migration, and invasion ability. Finally, we suggest that miR-127 may be acting like an oncogene in lung adenocarcinoma cells. These results will provide important information and help us to understand the roles of miR127 in lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3947. doi:10.1158/1538-7445.AM2011-3947

The development of hepatocllular carcinoma (HCC) is a multistep process with accumulation of genetic and epigenetic alternations in regulatory genes, leading to activation of oncogenes and inactivation of tumor suppressor genes. For genome-wide DNA methylation study, we used homemade MCGI (mouse CpG islands) microarray to identify many genes that were hypermethylation in the liver tumor tissues of HBx-overexpressd transgenic mice. These selected hypermethylation genes were also confirmed in human hepatocellular carcinoma cell (HCC) lines. In this study, we focused on a methylation-controlled DNAJ family (MCJ) gene, DnaJD1. It has been observed highly frequent DnaJD1 gene methylation and silencing in ovarian cancer, and associated with increased resistance to chemotherapeutic. The DnaJD1 was found hypermethylation in the promoter region and followed reduced mRNA expression in Hep3B hepatoma cells, which contain HBV genome. After treated by 5-aza-2’-deoxycytidine (DAC) in Hep3B cells, the methylation level of DnaJD1 could be reduced and mRNA re-expressed. We over-expressed DnaJD1 in Hep3B cells to study the physiologic effects of DnaJD1 in cell proliferation, cell migration, and invasion ability in vitro. The tumorgenesis in xenotransplanted nude mice were also examined. Data suggested that DnaJD1 re-expressed in Hep3B cells that could affect the cell morphology and lost cell contact insensitive. In these results, we concluded that DnaJD1 could be the one of main role of HCC progression. 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 174.