Akihiko Miyanaga

MiR-134/487b/655 Cluster Regulates TGF-β-induced Epithelial-Mesenchymal Transition and Drug Resistance to Gefitinib by Targeting MAGI2 in Lung Adenocarcinoma Cells.

Epithelial–mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non–small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA)-related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in NSCLC. miRNA expression profiles were examined before and after transforming growth factor β1 (TGF-β1) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. miRNA array and real-time quantitative reverse transcription PCR (qRT-PCR) revealed that TGF-β1 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW, and PDZ domain–containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN, was diminished in A549 cells with EMT after the TGF-β1 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-β1–induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-β1–induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, in which suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be a new therapeutic target in patients with advanced lung adenocarcinoma, depending on the EMT phenomenon. Mol Cancer Ther; 13(2); 444–53. ©2013 AACR.

Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model

To ascertain the potential for histone deacetylase (HDAC) inhibitor-based treatment in non-small cell lung cancer (NSCLC), we analyzed the antitumor effects of trichostatin A (TSA) and suberoylanilide hydroxamic acid (vorinostat) in a panel of 16 NSCLC cell lines via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSA and vorinostat both displayed strong antitumor activities in 50% of NSCLC cell lines, suggesting the need for the use of predictive markers to select patients receiving this treatment. There was a strong correlation between the responsiveness to TSA and vorinostat (P < 0.0001). To identify a molecular model of sensitivity to HDAC inhibitor treatment in NSCLC, we conducted a gene expression profiling study using cDNA arrays on the same set of cell lines and related the cytotoxic activity of TSA to corresponding gene expression pattern using a modified National Cancer Institute program. In addition, pathway analysis was done with Pathway Architect software. We used nine genes, which were identified by gene-drug sensitivity correlation and pathway analysis, to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. The prediction performance of the support vector machine model was validated by an additional nine cell lines, resulting in a prediction value of 100% with respect to determining response to TSA and vorinostat. Our results suggested that (a) HDAC inhibitors may be promising anticancer drugs to NSCLC and (b) the nine-gene classifier is useful in predicting drug sensitivity to HDAC inhibitors and may contribute to achieving individualized therapy for NSCLC patients. [Mol Cancer Ther 2008;7(7):1923–30]

Hippo Pathway Gene Mutations in Malignant Mesothelioma: Revealed by RNA and Targeted Exon Sequencing

Introduction: Malignant mesothelioma (MM) is an aggressive neoplasm causatively associated with exposure to asbestos. MM is rarely responsive to conventional cytotoxic drugs, and the outcome remains dismal. It is, therefore, necessary to identify the signaling pathways that drive MM and to develop new therapeutics specifically targeting the molecules involved. Methods: We performed comprehensive RNA sequencing of 12 MM cell lines and four clinical samples using so-called next-generation sequencers. Results: We found 15 novel fusion transcripts including one derived from chromosomal translocation between the large tumor suppressor 1 (LATS1) and presenilin-1 (PSEN1) genes. LATS1 is one of the central players of the emerging Hippo signaling pathway. The LATS1–PSEN1 fusion gene product lacked the ability to phosphorylate yes-associated protein and to suppress the growth of a MM cell line. The wild-type LATS1 allele was undetectable in this cell line, indicating two-hit genetic inactivation of its tumor suppressor function. Using pathway-targeted exon sequencing, we further identified a total of 11 somatic mutations in four Hippo pathway genes (neurofibromatosis type 2 [NF2], LATS2, RASSF1, and SAV1) in 35% (8 of 23) of clinical samples. Nuclear staining of yes-associated protein was detected in 55% (24 of 44) of the clinical samples. Expression and/or phosphorylation of the Hippo signaling proteins, RASSF1, Merlin (NF2), LATS1, and LATS2, was frequently absent. Conclusions: The frequent alterations of Hippo pathway molecules found in this study indicate the therapeutic feasibility of targeting this pathway in patients with MM.

Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4-ALK-rearranged non-small-cell lung cancer harbored coexisting EGFR mutation.

BackgroundThe EML4–ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion oncogene represents a novel molecular target in a small subset of non–small–cell lung cancers (NSCLCs). The EML4–ALK fusion gene occurs generally in NSCLC without mutations in epidermal growth factor receptor (EGFR) and KRAS.Case presentationWe report that a case of EML4–ALK-positive NSCLC with EGFR mutation had a response of stable disease to both an EGFR tyrosine kinase inhibitor (EGFR-TKI) and ALK inhibitor.ConclusionsWe described the first clinical report of a patient with EML4–ALK-positive NSCLC with EGFR mutation that had a response of stable disease to both single-agent EGFR-TKI and ALK inhibitor. EML4–ALK translocation may be associated with resistance to EGFR-TKI, and EGFR signaling may contribute to resistance to ALK inhibitor in EML4–ALK-positive 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 p21waf1/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 p21waf1/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. (Cancer Sci 2010)

Alternative Mammalian Target of Rapamycin (mTOR) Signal Activation in Sorafenib-resistant Hepatocellular Carcinoma Cells Revealed by Array-based Pathway Profiling

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.

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. Mol Cancer Ther; 14(11); 2433–40. ©2015 AACR.

Clinical features, anti-cancer treatments and outcomes of lung cancer patients with combined pulmonary fibrosis and emphysema

BACKGROUND Combined pulmonary fibrosis and emphysema (CPFE) patients may be at significantly increased risk of lung cancer compared with either isolated emphysema or pulmonary fibrosis patients. Acute exacerbation (AE) of interstitial lung disease caused by anticancer treatment is the most common lethal complication in Japanese lung cancer patients. Nevertheless, the clinical significance of CPFE compared with isolated idiopathic interstitial pneumonias (IIPs) in patients with lung cancer is not well understood. METHODS A total of 1536 patients with lung cancer at Nippon Medical School Hospital between March 1998 and October 2011 were retrospectively reviewed. Patients with IIPs were categorized into two groups: (i) CPFE; IIP patients with definite emphysema and (ii) non-CPFE; isolated IIP patients without definite emphysema. The clinical features, anti-cancer treatments and outcomes of the CPFE group were compared with those of the non-CPFE group. RESULTS CPFE and isolated IIPs were identified in 88 (5.7%) and 63 (4.1%) patients respectively, with lung cancer. AE associated with initial treatment occurred in 22 (25.0%) patients in the CPFE group and in 8 (12.7%) patients in the non-CPFE group, irrespective of treatment modality. Median overall survival (OS) of the CPFE group was 23.7 months and that of the non-CPFE group was 20.3 months (P=0.627). Chemotherapy was performed in a total of 83 patients. AE associated with chemotherapy for advanced lung cancer occurred in 6 (13.6%) patients in the CPFE group and 5 (12.8%) patients in the non-CPFE group. Median OS of the CPFE group was 14.9 months and that of the non-CPFE group was 21.6 months (P=0.679). CONCLUSION CPFE was not an independent risk factor for AE and was not an independent prognosis factor in lung cancer patients with IIPs. Therefore, great care must be exercised with CPFE as well as IIP patients when performing anticancer treatment for patients with lung cancer.

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-β1 exposure of A549 cells. Nintedanib was able to reverse TGF-β1-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.

Diagnostic and prognostic significance of the alternatively spliced ACTN4 variant in high-grade neuroendocrine pulmonary tumours

BACKGROUND High-grade neuroendocrine tumours (HGNTs) of the lung manifest a wide spectrum of clinical behaviour, but no method for predicting their outcome has been established. MATERIALS AND METHODS We newly established a monoclonal antibody specifically recognizing the product of the alternatively spliced ACTN4 transcript (namely, variant actinin-4), and used it to examine the expression of variant actinin-4 immunohistochemically in a total of 609 surgical specimens of various histological subtypes of lung cancer. RESULTS Variant actinin-4 was expressed in 55% (96/176) of HGNTs, but in only 0.8% (3/378) of non-neuroendocrine (NE) lung cancers. The expression of variant actinin-4 was significantly associated with poorer overall survival in HGNT patients (P=0.00021, log-rank test). Multivariate analysis using the Cox proportional hazards model showed that the expression of variant actinin-4 was the most significant independent negative predictor of survival in HGNT patients (hazard ratio (HR), 2.15; P=0.00113) after the presence of lymph node metastasis (HR, 2.25; P=0.00023). CONCLUSIONS The expression of variant actinin-4 is an independent prognostic factor for patients with HGNTs. This protein has a high affinity for filamentous actin polymers and likely promotes aggressive behaviour of cancer cells. The present clinical findings clearly support this notion.