miR-200/ZEB axis regulates sensitivity to nintedanib in non-small cell lung cancer cells.

Nintedanib (BIBF1120) is a multi-targeted angiokinase inhibitor and has been evaluated in idiopathic pulmonary fibrosis and advanced non-small cell lung cancer (NSCLC) patients in clinical studies. In the present study, we evaluated the antitumor effects of nintedanib in 16 NSCLC cell lines and tried to identify microRNA (miRNA) associated with sensitivity to nintedanib. No correlations between FGFR, PDGFR and VEGFR family activation and sensitivity to nintedanib were found. The difference in miRNA expression profiles between 5 nintedanib-sensitive and 5 nintedanib-resistant cell lines was evaluated by miRNA array and quantitative RT-PCR analysis (qRT-PCR). Expression of miR-200b, miR-200a and miR-141 belonging to the miR-200 family which contributes to epithelial-mesenchymal transition (EMT), was significantly lower in 5 nintedanib-resistant than in 5 nintedanib-sensitive cell lines. We examined the protein expression of EMT markers in these 10 NSCLC cell lines. E-cadherin expression was lower, and vimentin and ZEB1 expression were higher in 5 nintedanib-resistant cell lines. PC-1 was the most sensitive of the NSCLC cell lines to nintedanib. We established nintedanib-resistant PC-1 cells (PC-1R) by the stepwise method. PC-1R cells also showed decreased expression of miR-200b, miR-141 and miR-429 and increased expression of ZEB1 and ZEB2. We confirmed that induction of miR-200b or miR-141 enhanced sensitivity to nintedanib in nintedanib-resistant A549 and PC1-R cells. In addition, we evaluated the response to gefitinib in combination with nintedanib after TGF-ß1 exposure of A549 cells. Nintedanib was able to reverse TGF-ß1-induced EMT and resistance to gefitinib caused by miR-200b and miR-141 upregulation and ZEB1 downregulation. These results suggested that the miR-200/ZEB axis might be predictive biomarkers for sensitivity to nintedanib in NSCLC cells. Furthermore, nintedanib combined with gefitinib might be a novel therapeutic strategy for NSCLC cells with EMT phenotype and resistance to gefitinib.

Identification of haptoglobin peptide as a novel serum biomarker for lung squamous cell carcinoma by serum proteome and peptidome profiling.

To date, a number of potential biomarkers for lung squamous cell cancer (SCC) have been identified; however, sensitive biomarkers are currently lacking to detect early stage SCC due to low sensitivity and specificity. In the present study, we compared the 7 serum proteomic profiles of 11 SCC patients, 7 chronic obstructive pulmonary disease (COPD) patients and 7 healthy smokers as controls to identify potential serum biomarkers associated with SCC and COPD. Two-dimensional difference gel electrophoresis (2D-DIGE) and mass-spectrometric analysis (MS) using an affinity column revealed two candidate proteins, haptoglobin (HP) and apolipoprotein 4, as biomarkers of SCC, and α-1-antichymotrypsin as a marker of COPD. The iTRAQ technique was also used to identify SCC-specific peptides. HP protein expression was significantly higher in SCC patients than in COPD patients. Furthermore, two HP protein peptides showed significantly higher serum levels in SCC patients than in COPD patients. We established novel polyclonal antibodies for the two HP peptides and subsequently a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of these specific peptides in patient and control sera. The sensitivity of detection by ELISA of one HP peptide (HP216) was 70% of SCC patients, 40% of COPDs patients and 13% of healthy controls. We also measured CYFRA, a cytokeratin fragment clinically used as an SCC tumor marker, in all the 28 cases and found CYFRA was detected in only seven SCC cases. However, when the measurement of HP216 was combined with that of CYFRA, 100% (10 of 10 patients) of SCC cases were detected. Our proteomic profiling demonstrates that the SCC-specific HP peptide HP216 may potentially be used as a diagnostic biomarker for SCC.

Inhibition of ABCB1 Overcomes Cancer Stem Cell-like Properties and Acquired Resistance to MET Inhibitors in Non-Small Cell Lung Cancer.

Patients with non-small cell lung cancer (NSCLC) EGFR mutations have shown a dramatic response to EGFR inhibitors (EGFR-TKI). EGFR T790M mutation and MET amplification have been recognized as major mechanisms of acquired resistance to EGFR-TKI. Therefore, MET inhibitors have recently been used in NSCLC patients in clinical trials. In this study, we tried to identify the mechanism of acquired resistance to MET inhibitors. We analyzed the antitumor effects of two MET inhibitors, PHA-665752 and crizotinib, in 10 NSCLC cell lines. EBC-1 cells with MET amplification were the only cells that were sensitive to both MET inhibitors. We established PHA-665752-resistant EBC-1 cells, namely EBC-1R cells. Activation of KRAS, EGFR, and FGFR2 signaling was observed in EBC-1R cells by FISH and receptor tyrosine kinase phosphorylation antibody arrays. EBC-1R cells also showed overexpression of ATP-binding cassette subfamily B member 1 (ABCB1) as well as phosphorylation of MET. EBC-1R cells grew as cell spheres that exhibited cancer stem cell-like (CSC) properties and epithelial-mesenchymal transition (EMT). The level of miR-138 that targeted ABCB1 was decreased in EBC-1R cells. ABCB1 siRNA and the ABCB1 inhibitor elacridar could reduce sphere numbers and suppress EMT. Elacridar could also reverse resistance to PHA-665752 in EBC-1R cells. Our study demonstrated that ABCB1 overexpression, which was associated with CSC properties and EMT, was involved in the acquired resistance to MET inhibitors. Inhibition of ABCB1 might be a novel therapeutic strategy for NSCLC patients with acquired resistance to MET inhibitors.

Control of the MYC-eIF4E axis plus mTOR inhibitor treatment in small cell lung cancer.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors have anti-tumor effects against renal cell carcinoma, pancreatic neuroendocrine cancer and breast cancer. In this study, we analyzed the antitumor effects of mTOR inhibitors in small cell lung cancer (SCLC) cells and sought to clarify the mechanism of resistance to mTOR inhibitors. METHODS: We analyzed the antitumor effects of three mTOR inhibitors including everolimus in 7 SCLC cell lines by MTS assay. Gene-chip analysis, receptor tyrosine kinases (RTK) array and Western blotting analysis were performed to identify molecules associated with resistance to everolimus. RESULTS: Only SBC5 cells showed sensitivity to everolimus by MTS assay. We established two everolimus resistant-SBC5 cell lines (SBC5 R1 and SBC5 R10) by continuous exposure to increasing concentrations of everolimus stepwise. SPP1 and MYC were overexpressed in both SBC5 R1 and SBC5 R10 by gene-chip analysis. High expression levels of eukaryotic translation initiation factor 4E (eIF4E) were observed in 5 everolimus-resistant SCLC cells and SBC5 R10 cells by Western blotting. MYC siRNA reduced eIF4E phosphorylation in SBC5 cells, suggesting that MYC directly activates eIF4E by an mTOR-independent bypass pathway. Importantly, after reduction of MYC or eIF4E by siRNAs, the SBC5 parent and two SBC5-resistant cells displayed increased sensitivity to everolimus relative to the siRNA controls. CONCLUSION: These findings suggest that eIF4E has been shown to be an important factor in the resistance to everolimus in SCLC cells. Furthermore, a link between MYC and mTOR-independent eIF4E contribute to the resistance to everolimus in SCLC cells. Control of the MYC-eIF4E axis may be a novel therapeutic strategy for everolimus action in SCLC.

MET FISH-positive status predicts short progression-free survival and overall survival after gefitinib treatment in lung adenocarcinoma with EGFR mutation.

BACKGROUND: Lung adenocarcinoma patients with EGFR gene mutations have shown a dramatic response to gefitinib. However, drug resistance eventually emerges which limits the mean duration of response. With that in view, we examined the correlations between MET gene status as assessed by fluorescence in situ hybridization (FISH) with overall survival (OS) and progression-free survival (PFS) in adenocarcinoma patients with EGFR gene mutations who had received gefitinib therapy. METHODS: We evaluated 35 lung cancer samples with EGFR mutation from adenocarcinoma patients who had received gefitinib. Gene copy numbers (GCNs) and amplification of MET gene before gefitinib therapy was examined by FISH. MET protein expression was also evaluated by immunohistochemistry (IHC). RESULTS: FISH assessment showed that of the 35 adenocarcinoma samples, 10 patients (29%) exhibited high polysomy (5 copies≦mean MET per cell) and 1 patient (3%) exhibited amplification (2≦MET gene (red)/CEP7q (green) per cell). IHC evaluation of MET protein expression could not confirm MET high polysomy status. The Eleven patients with MET FISH positivity had significantly shorter progression-free survival (PFS) and overall survival (OS) than the 24 patients who were MET FISH-negative (PFS: p = 0.001 and OS: p = 0.03). Median PFS and OS with MET FISH-positivity were 7.6 months and 16.8 months, respectively, whereas PFS and OS with MET FISH-negativity were 15.9 months and 33.0 months, respectively. Univariate analysis revealed that MET FISH-positivity was the most significant independent factor associated with a high risk of progression and death (hazard ratio, 3.83 (p = 0.0008) and 2.25 (p = 0.03), respectively). CONCLUSIONS: Using FISH analysis to detect high polysomy and amplification of MET gene may be useful in predicting shortened PFS and OS after Gefitinib treatment in lung adenocarcinoma. The correlation between MET gene status and clinical outcomes for EGFR-TKI should be further evaluated using large scale samples.

MiR-379/411 cluster regulates IL-18 and contributes to drug resistance in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence in Japan. In this study, we performed gene and microRNA (miRNA) expression profiling to identify novel therapeutic targets in MPM cells. Based on relative sensitivities to pemetrexed (PEM) and the histone deacetylase (HDAC) inhibitor, vorinostat (SAHA), 211H cells were determined to be the only sensitive MPM cell line out of the 6 tested. On the same series of cell lines, we performed whole genome transcriptomic profiling via DNA microarrays and pathway analysis of the derived data. Of particular note, IL-18 gene expression levels were significantly higher in the cell lines that were either drug resistant or displayed intermediate sensitivity, compared to the sensitive 211H cell line. Pathway analysis revealed IL-18 as an important gene associated with drug sensitivity of MPM cells. A relationship between IL-18 overexpression and drug resistance was also observed following targeted assessment of 10 cytokine genes using quantitative RT-PCR. miRNA expression profiles were evaluated in the MPM cell line panel in order to discern the mechanism of IL-18 induction in the drug-resistant lines. We found that miR-379 and miR-411 belonged to the same cluster of miRNAs located on chromosome 14q32 that commonly target the IL-18 gene. Luciferase reporter assays revealed that miR-379 and miR-411 directly target the IL-18 gene. Introduction of miR-379 plus miR-411, as well as IL-18 silencing, significantly suppressed the invasive capacity of MESO1 cells in vitro. Furthermore, the use of either PEM or SAHA together with miR-379 plus miR-411 mimics mediated increased sensitivity to these drugs in MESO1 cells. These results suggest that the miR-379/411 cluster may provide new therapeutic opportunities for advanced MPM patients, depending on the nature of IL-18 gene expression.

Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed.

Pemetrexed (PEM) is currently recommended as one of the standard anticancer drugs for malignant pleural mesothelioma (MPM). However, the mechanism of the sensitivity of MPM to PEM remains unclear. We analyzed the antitumor effects of PEM in six MPM cell lines by MTS assay. To identify genes associated with drug sensitivity, we conducted gene expression profiling on the same set of cell lines using GeneChips and pathway analysis. Three cell lines were sensitive to PEM. A total fo 18 transcripts and 14 genes identified by GeneChips were significantly correlated with sensitivity to PEM. Pathway analysis revealed that osteopontin (SPP1/OPN) was an important target in PEM sensitivity. Overexpression of SPP1/OPN was observed in the sensitive cells by quantitative PCR and western blot analysis. Introduction of SPP1/OPN by lentiviral vector significantly enhanced the invasion activities of MPM cells. PEM treatment with SPP1/OPN knockdown inhibited the PEM-induced cell growth-inhibitory effect in PEM-sensitive cells. Expression of SPP1/OPN and AKT phosphorylation significantly decreased after PEM treatment of the PEM-sensitive cells. High immunohistochemical expression of SPP1/OPN was observed in two of three MPM patients who had a partial response to PEM-based chemotherapy. PEM has antitumor effects in MPM cells dependent on SPP1/OPN overexpression resulting in AKT activation. Our results suggest that SPP1 may be used as a single predictive biomarker of the effectiveness of PEM treatment in MPM.

MiR-23a regulates TGF-ß-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells.

Transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases including lung cancer. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. MiR-23a/24/27a is a miRNA cluster located in chromosome 19p13.12, which can function as an oncogene in several human cancers. In this study, we analyzed miR-23a/24/27a expression in 10 non-small cell cancer (NSCLC) cell lines by real-time PCR analysis. Correlation between expression of these miRNAs and TGF-ß/Smad signaling was evaluated. We found that miR-23a could be regulated by TGF-ß1 in a Smad-dependent manner in A549 lung adenocarcinoma cells showing the EMT phenomenon. Knockdown of miR-23a partially restored E-cadherin expression under conditions of TGF-ß1 stimulation. In contrast, overexpression of miR-23a could suppress E-cadherin expression and stimulate EMT. Furthermore, A549 cells with overexpressed miR-23a were more resistant to gefitinib compared to the parental cells. These findings suggest that miR-23a regulates TGF-ß-induced EMT by targeting E-cadherin in lung cancer cells and may be useful as a new therapeutic target in NSCLC.

Histone deacetylase inhibitor enhances sensitivity of non-small-cell lung cancer cells to 5-FU/S-1 via down-regulation of thymidylate synthase expression and up-regulation of p21(waf1/cip1) expression.

It is desirable to find more appropriate therapeutic opportunities in non-small-cell lung cancer (NSCLC) due to the current poor prognosis of affected patients. Recently, several histone deacetylase (HDAC) inhibitors, including suberoylanilide hydroxamic acid (SAHA), have been reported to exhibit antitumor activities against NSCLC. S-1, a novel oral fluorouracil anticancer drug, has been developed for clinical use in the treatment of NSCLC in Japan. Using an MTT assay, we analyzed the growth-inhibitory effect of 5-fluorouracil (5-FU), S-1, and SAHA against three NSCLC cell lines, as well as the breast cancer cell line MCF7 which is known to be highly sensitive to 5-FU. Combined treatment with low-dose SAHA enhanced 5-FU- and S-1-mediated cytotoxicity and resulted in synergistic effects, especially in 5-FU-resistant cells. Both the mRNA and protein expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT), which are associated with 5-FU sensitivity/response, were analyzed in the cells undergoing treatment. 5-Fluorouracil-resistant lung cancer cells displayed high expression of TS mRNA and protein. Suberoylanilide hydroxamic acid down-regulated TS mRNA and protein expression, as well as repressed the rapid induction of this factor during 5-FU treatment, in all examined cell types. We also examined the status of the Rb-E2F1 pathway, with SAHA up-regulating p21(waf1/cip1) expression via promoter histone acetylation; this, in turn, blocked the Rb-E2F1 pathway. We conclude that combination therapy with SAHA and S-1 in lung cancer may be promising due to its potential to overcome S-1 resistance via modulation of 5-FU/S-1 sensitivity-associated biomarker (TS) by HDAC inhibitor.

HSP27 modulates epithelial to mesenchymal transition of lung cancer cells in a Smad-independent manner.

Epithelial to mesenchymal transition (EMT) is induced by transforming growth factor-ß1 (TGF-ß1) and is a crucial event for cancer cells to acquire invasive and metastatic phenotypes. However, the signals that induce EMT in cancer cells have yet to be adequately defined. In this study, a proteomic investigation was performed to understand the signaling pathway of the EMT of lung cancer using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. The protein expression profiles of A549 were compared to those of A549 cells treated with TGF-ß1. Of more than 2,000 protein spots shown by 2D-DIGE, 53 were found to be up- or down-regulated upon induction with TGF-ß1. In the 53 protein spots, the protein level of heat shock protein (HSP) 27 was found to increase significantly. HSP27 protein was higher in two different lung cancer cell lines, demonstrating the EMT phenomenon with TGF-ß1. Notably, the silencing of HSP27 enhanced spindle integration, resulting in an additive effect with TGF-ß1-induced EMT. Furthermore, the TGF-ß1-induced HSP27 increase was not affected by the suppression of Smad2 and Smad3 in A549 cells. These results suggest that HSP27 was involved in TGF-ß1-induced EMT in a Smad-independent manner in lung cancer cells and may provide an effective clinical strategy in lung cancer patients whose tumors are dependent on TGF-ß1-induced EMT.