Eiji Kunii

Significance of c-MET overexpression in cytotoxic anticancer drug-resistant small-cell lung cancer cells

The c‐MET receptor tyrosine kinase is the receptor for hepatocyte growth factor. Recently, activation of the c‐MET/hepatocyte growth factor signaling pathway was associated with poor prognosis in various solid tumors and was one of the mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib. But the link between c‐MET activation and the cytotoxic anticancer drug has not been fully examined. Here, we found that the enhanced expression and activation of c‐MET in cytotoxic anticancer agent‐resistant small‐cell lung cancer cells. Downregulation of c‐MET expression by siRNA against the c‐MET gene or inhibition of c‐MET activation by SU11274, a c‐MET inhibitor, in the resistant cells altered resistance to the cytotoxic anticancer agent. These results indicated that c‐MET overexpression might play an important role in acquired resistance to cytotoxic anticancer drugs. Furthermore, the number of c‐MET gene loci was increased in the resistant cells compared to the parental cells. In conclusion, increased c‐Met expression through an increase in the number of c‐MET gene loci is one of the mechanisms of acquired resistance to cytotoxic anticancer drugs. Our results add a new strategy, the targeting of c‐MET, for overcoming resistance to cytotoxic agents in small‐cell lung cancer.

C609T Polymorphism of NAD(P)H Quinone Oxidoreductase 1 As a Predictive Biomarker for Response to Amrubicin

Introduction: Amrubicin is a promising agent in the treatment of lung cancer, but predictive biomarkers have not yet been described. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme known to metabolize amrubicinol, the active metabolite of amrubicin, to an inactive compound. We examined the relationship between NQO1 and amrubicinol cytotoxicity. Methods: Gene and protein expression of NQO1, amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 were evaluated in 29 lung cancer cell lines: 14 small cell lung cancer (SCLC) and 15 non-SCLC (NSCLC). The involvement of NQO1 in amrubicinol cytotoxicity was evaluated by small interfering RNA against NQO1. Results: A significant inverse relationship between both gene and protein expression of NQO1 and amrubicinol cytotoxicity was found in all cell lines. Treatment with NQO1 small interfering RNA increased amrubicinol cytotoxicity and decreased NQO1 expression in both NSCLC and SCLC cells. Furthermore, cell lines genotyped homozygous for the 609T allele showed significantly lower NQO1 protein expression and higher sensitivity for amrubicinol than those with the other genotypes in both NSCLC and SCLC cells. Conclusions: NQO1 expression is one of the major determinants for amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 could be a predictive biomarker for response to amrubicin treatment.

Involvement of intermediate filament nestin in cell growth of small-cell lung cancer

BACKGROUND Nestin is a class VI intermediate filament protein expressed in stem/progenitor cells during the development of the central nervous system. Nestin is detected in various types of tumors and is involved in malignant processes. This study investigated the expression and function of nestin in small-cell lung cancer (SCLC). METHODS Expression of nestin and achaete-scute homolog 1 (ASH1) was studied in 21 lung cancer cell lines. To assess the function of nestin, a short hairpin RNA (shRNA) targeting nestin was transfected into two SCLC cell lines (DMS53 and SBC3), and cloned cells that showed apparent down-regulation of nestin were obtained. Nestin expression was also studied immunohistochemically in surgically resected SCLC primary tumors and metastatic SCLC tumors obtained from autopsy cases. RESULT Nestin was expressed in nine of 10 SCLC cell lines. The nestin expression level was significantly higher in SCLC cell lines than in NSCLC cell lines (P < 0.01). There was a statistically significant positive correlation between the expression levels of nestin and ASH1 in SCLC cell lines. Nestin knock-down cells created by transfection with shRNA exhibited decreased invasion and cell proliferation capabilities. Furthermore, nestin was detected in SCLC tumor cells and tumor vessels in all clinical tumor specimens. CONCLUSION Nestin is expressed in SCLC in association with neuroendocrine features and participates in malignant phenotypes, including cell growth. Therefore, nestin may be a novel therapeutic target for SCLC.

Efficacy of combined corticosteroid and pirfenidone for acute exacerbation of idiopathic pulmonary fibrosis after surgery for lung cancer: A case report

Hirotsugu Ohkubo, MD, PhD, Eiji Kunii, MD, Satoru Moriyama, MD, PhD, Akio Niimi, MD, PhD Department of Respiratory Medicine, Allergy and Clinical Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan Department of Oncology, Immunology, and Surgery, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan

Organic cation transporter OCT6 mediates cisplatin uptake and resistance to cisplatin in lung cancer

PurposeThe purposes of this study were to determine whether organic cation transporters (OCTs) can mediate platinum uptake, and whether OCT down-regulation confers resistance against cisplatin (CDDP) in cancer cells.MethodsTwo lung cancer cell lines, PC-6 and PC-14, and their CDDP-resistant derivatives, PC-6/CDDP and PC-14/CDDP, were analyzed. OCT expression levels were assayed using quantitative RT-PCR and Western blotting. Additionally, the effect of OCT6 overexpression, induced by transfection of the OCT6 gene SLC22A16 using a forced expression vector, on cellular sensitivity to CDDP and on intracellular platinum accumulation was measured using PC-14/CDDP cells.ResultsBoth gene and protein expression of OCT6 were decreased in both CDDP-resistant cell lines compared with their expression in their respective parental cells. Intracellular accumulation of platinum was decreased in PC-14/CDDP cells compared with the parental cells after CDDP treatment. Furthermore, OCT6 overexpression induced by transfection of the OCT6 gene (SLC22A16) forced expression vector-sensitized PC-14/CDDP cells to CDDP and oxaliplatin (L-OHP) concomitant with increased intracellular concentration of platinum.ConclusionOCT6 is a mediator of platinum uptake in cancer cells, and down-regulation of OCT6 is possibly one of the mechanisms of resistance against cisplatin in lung cancer.

A folylpoly-γ-glutamate synthase single nucleotide polymorphism associated with response to pemetrexed treatment combined with platinum for non-small cell lung cancer.

OBJECTIVES In this study, we investigated whether single nucleotide polymorphisms (SNPs) in folylpoly-γ-glutamate synthase (FPGS), which catalyzes the polyglutamation of pemetrexed (PEM), is related to FPGS expression and the response to PEM in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS We first examined FPGS protein expressions according to FPGS SNPs genotype groups in 15 lung adenocarcinoma cell lines. Next, 101 non-squamous NSCLC patients treated with PEM and platinum drugs were classified into FPGS SNP genotype groups to investigate the relation between FPGS SNP genotypes and treatment outcome. RESULTS When the 15 adenocarcinoma cell lines were classified into FPGS SNP 2572C>T genotype groups, we found that the FPGS protein expression was significantly higher in the CC genotype group than in the TT+CT genotype group (p=0.0022). In contrast, there was no significant difference in FPGS expression when another FPGS SNP was analyzed. We also examined the FPGS SNP 2572C>T genotype in 101 non-squamous NSCLC patients treated with PEM and platinum drugs. Among these 101 patients, response rate was significantly higher in the CC genotype group than in the TT+CT genotype group (p=0.0034). When we examined the patients treated with PEM, platinum drugs and Bev, almost all (29/33) were classified into the TT+CT genotype group. The response rate, progression-free survival, and over-all survival were all significantly better in the patients of the TT+CT genotype group who also received Bev than in those who did not receive Bev (p=0.034, 0.021, 0.018, respectively). CONCLUSION FPGS SNP 2572C>T is a predictive marker of the efficacy of PEM and platinum drugs for NSCLC.

Abstract 2946: Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines

Background: Aldehyde dehydrogenase (ALDH) forms a superfamily of enzymes that catalyze conversion of aldehydes into carboxylic acids, and they are categorized into 19 families in human. Increased ALDH activity was reported to be a potential marker of cancer stem cell (CSC) in various solid tumors including lung cancer. Furthermore, High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs. Recent studies revealed that ALDH7A1 is highly expressed in prostate cancer and associated with recurrence in patients with surgically resected non-small-cell lung cancer. However, there is not a fundamental link between ALDH7A1 expression and malignant phenotype, including drug resistance and colony formation. Material and methods: Anticancer drug resistant cell lines were established from lung cancer cell lines (PC-6, DMS53, PC-14, PC-9, NCI-H23 and NCI-H2228 cells) by exposure to various anticancer drug, including 7-ethyl-10-hydroxycamptothesin (SN-38), gemcitabine (GEM), cisplatin (CDDP), etoposide, paclitaxel (TXL), pemetrexed, amrubicin, erlotinib and crizotinib. Protein expression was determined by western blotting and gene expression was examined by RT-PCR. ALDH enzyme activity was measured by ALDEFLOUR TM assay. Proliferation was measured using MTS assay. ALDH7A1encoding plasmid was stably transfected into PC-14 cell. These transfected cells displayed sphere formation on ultralow binding plates and survived for more than 3 weeks. Results: We found that the levels of ALDH7A1 expression were higher in various anticancer drug-resistant lung cancer cell lines compared with the parental cells. ALDH enzyme activity was about 4-9 fold higher in SN-38, TXL, CDDP and GEM resistant cell lines (PC6/SN38, PC6/TXL, PC14/CDDP and PC14/GEM) than the parents cells. Up-regulation of ALDH7A1 expression by using a forced expression vector in PC14 cell altered cytotoxicity to SN-38 and TXL. Furthermore, overexpression of ALDH7A1in PC-14 cells enhanced sphere formation relative to the cells transfected with control vector. Conclusion: These results suggested that increased ALDH7A1 expression in lung cancer cell lines might be one of the mechanisms of acquired resistance to anticancer agents and enhance colony formation. Citation Format: Yuichi Sakamori, Hiroaki Ozasa, Eiji Kunii, Yoshitaka Yagi, Takahiro Tsuji, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2946.

Abstract 3723: TAS-115, a novel MET + VEGFRs dual inhibitor, decreases the cytotoxic anticancer drug resistance in lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA c-Met is the receptor tyrosine kinase for hepatocyte growth factor (HGF). c-Met/HGF signal stimulates the expression of many downstream genes, which are associated with biological events such as oncogenesis, cancer metastasis and drug resistance. The enhanced c-Met/HGF signal is detected in various cancer cells containing lung cancer. Interestingly, recent studies revealed this is a new mechanism of acquired resistance to anticancer agents, especially epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) gefitinib. TAS-115 [4-[2-fluoro-4-[[[(2-phenylacetyl)amino]thioxomethyl]amino]-phenoxy]-7-methoxy-N-methyl-6-quinolinecarboxamide] is identified as a novel potent c-Met and VEGFR dual inhibitor. In biochemical assay, it was identified to be a potent Adenosine Triphosphate (ATP) competitive inhibitor of both c-Met and VEGFR that is equal or more potent than those of other known inhibitors such as crizotinib. In this study, we investigate whether c-Met inhibition by TAS-115 influence the resistance against cytotoxic anticancer agents in several resistant cell lines harboring c-Met activation. We found that the levels of c-Met expression and activation in various drug resistances, including the 7-ethyl-10-hydroxycamptothesin (SN-38) -resistant lung cancer cell lines (PC-6/SN-38 GEM) and gemcitabine (GEM) -resistant lung cancer cell lines (PC-9/GEM) were enhanced relative to the parental cells by western blotting (WB). We confirmed that TAS-115 dose-dependently inhibits phoshorylation of c-Met and downstream signals, ERK1/2 and AKT, in PC-6/SN-38 cells and PC-9/GEM cells. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay was performed to determine whether certain concentrations TAS-115 inhibit cell growths of PC-6/SN-38 and PC-9/GEM cells. Cell growth of PC-6/SN-38 cells was not affected by treatment with TAS-115 (2 or 4 μM) for 72h. The same results were observed in PC-9/GEM cells that were treated with TAS-115 (4 or 8 μM). These results suggested that c-MET inhibition is not enough to inhibit cell growth when treated with TAS-115 alone. Next, we exposed PC-6/SN-38 cells to DMSO or TAS-115 (2 or 4 μM) in combination with SN-38 (12.5 nM) for 72 h. Although PC-6/SN-38 cells were resistant to SN-38, the combined treatment with TAS-115 resulted in growth inhibition in a dose-dependent manner. We got the same results in PC-9/GEM cells when combined treatment with GEM and TAS-115. These results show that inhibition of c-Met signaling decrease the resistance of cytotoxic anticancer agent resistant cells. In conclusion, TAS-115 might become to overcome the resistance against cytotoxic anticancer agents by c-Met/HGF signal. Citation Format: Eiji Kunii, Hiroaki Ozasa, Tetsuya Oguri, Ken Maeno, Osamu Takakuwa, Takehiro Uemura, Niimi Akio. TAS-115, a novel MET + VEGFRs dual inhibitor, decreases the cytotoxic anticancer drug resistance in lung cancer. [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 3723. doi:10.1158/1538-7445.AM2014-3723

Transformed lymphoplasmacytic lymphoma involving the main carina: A case report

A 41-year-old male was admitted to Nagoya City University Hospital subsequent to experiencing a cough with bloody sputum for a few days. The patient had a 4-year history of lymphoplasmacytic lymphoma (LPL) and had achieved a good partial response to anticancer chemotherapy. Chest computed tomography (CT) showed an endobronchial tumor of the main carina. A bronchoscopy revealed an exophytic tumor at the main carina, and autofluorescence imaging bronchovideoscopy showed that the tumor and surrounding area were magenta in color. The biopsy specimens demonstrated that the endobronchial tumor was composed of large atypical lymphoid cells. The patient was diagnosed with a high-grade transformation of LPL. In addition to describing a rare case of transformed LPL involving the main carina, the present study also summarizes and discusses endobronchial lymphomas, with a brief review of a number of published studies.

Abstract 4360: Involvement of intermediate filament nestin in cell growth of small-cell lung cancer.

Small-cell lung cancer (SCLC) represents 13% of all newly diagnosed cases of lung cancer. Clinical progression of SCLC is rapid and aggressive. Unfortunately, 70% of SCLC cases have already metastasized at the time of diagnosis, and the recent median survival time and two-year survival rate in SCLC patients with extensive-stage disease is only 8-13 months and 5%, respectively. Clinical trials of SCLC treatments have been conducted since the mid-1980s, but have not achieved prolonged survival; this lack of success is likely due to a lack of appropriate target molecules. To improve outcomes for SCLC patients, new therapeutic strategies including novel molecular targets for SCLC are desired. Nestin is a class VI intermediate filament protein expressed in stem/progenitor cells during the development of the central nervous system. Nestin is detected in various types of tumors and reported to be involved in malignant phenotypes. Here, we investigated the expression and function of nestin in SCLC. At the beginning, protein expression of nestin and achaete-scute homolog 1 (ASH1) was studied in 21 lung cancer cell lines. Nestin was expressed in 9 of 10 SCLC cell lines. The nestin expression level in SCLC cell lines was significantly higher than that in non-small-cell lung cancer (NSCLC) cell lines (P Citation Format: Eiji Kunii, Osamu Takakuwa, Ken Maeno, Tetsuya Oguri, Midori Yokoyama, Hisatoshi Hijikata, Takehiro Uemura, Daishi Kasai, Hirotsugu Ohkubo, Mikinori Miyazaki, Akio Niimi. Involvement of intermediate filament nestin in cell growth of small-cell lung cancer. [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 4360. doi:10.1158/1538-7445.AM2013-4360