Daishi Kasai

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.

Over-expression of MDR1 in amrubicinol-resistant lung cancer cells

PurposeAmrubicin, a totally synthetic 9-aminoanthracycline anticancer drug, has shown promising activity for lung cancer, but little is known about the mechanism of resistance for this agent. This study was aimed to clarify the role of P-glycoprotein (P-gp) in amrubicinol, an active metabolite of amrubicin, resistance in lung cancer cells.MethodsAmrubicinol-resistant cell line PC-6/AMR-OH was developed by continuously exposing the small-cell lung cancer cell line PC-6 to amrubicinol. Gene expression level of MDR1, which encodes P-gp, and intracellular accumulation of amrubicinol were evaluated by PC-6 and PC-6/AMR-OH cells. The involvement of MDR1 in amrubicinol resistance was evaluated by treatment with P-gp inhibitor verapamil and small interfering RNA (siRNA) against MDR1. Also, expression levels and single-nucleotide polymorphisms (SNPs) of MDR1 in 22 lung cancer cell lines were examined, and the relationships between these factors and sensitivity to amrubicinol were evaluated.ResultsThe MDR1 gene was increased approximately 4,500-fold in PC-6/AMR-OH cells compared with PC-6 cells, and intracellular accumulation of amrubicinol in PC-6/AMR-OH cells was decreased to about 15 percent of that in PC-6 cells. Treatment with verapamil and siRNA against MDR1 significantly increased the sensitivity to amrubicinol in PC-6/AMR-OH cells with increased cellular accumulation of amrubicinol. Meanwhile, neither MDR1 gene expression levels nor SNPs of the gene were associated with amrubicinol sensitivity.ConclusionsResults of this study indicate that increased MDR1 expression and P-gp activity confer acquired resistance to amrubicinol. In contrast, neither expression level nor SNPs of MDR1 are likely to be predictive markers for amrubicin activity.

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.

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

Abstract 886: OCT6 expression is associated with the resistance to cisplatin in lung cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Organic cation transporters (OCTs) of the solute carrier family 22 have been identified as uptake transporters for the charge state of a substrate such as cationic drugs. The OCTs have an isoform of OCT1, OCT2, OCT3, and OCT6. Previously OCT1-3 have shown to related with intracellular uptake of anticancer platinum drugs such as cisplatin (CDDP) and oxaliplatin; however an association of OCT6 and platinum drugs is not well elucidated. We found decreased gene and protein expressions of OCT6 in CDDP resistant PC14/CDDP cells compared to those in parental lung adenocaricinoma PC14 cells. In contrast, the expression levels of OCT1-3 were not changed between PC14 and PC-14/CDDP cells. The intracellular concentration of CDDP also decreased in PC14/CDDP compared with the parental cell. To elucidate the OCT6 for uptake of CDDP, we established a forced expression of OCT6 cell line by a transfection an OCT6 vector to PC14/CDDP cells. We found that intracellular CDDP concentration are increased concomitant with decreased resistance to CDDP in OCT6 overexpressed PC14/CDDP cells. These results indicate that OCT6 is related with uptake of CDDP, and that the expression of OCT6 could be one of the resistant factors against CDDP in lung cancer cells. Citation Format: Daishi Kasai, Tetsuya Oguri, Takehiro Uemura, Hisatoshi Hijikata, Midori Yokoyama, Eiji Kunii, Osamu Takakuwa, Hirotsugu Ohkubo, Mikinori Miyazaki, Ken Maeno, Akio Niimi. OCT6 expression is associated with the resistance to cisplatin in 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 886. doi:10.1158/1538-7445.AM2013-886

Abstract 1719: Significance of thymidylate synthase copy numbers for resistance to pemetrexed in lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pemetrexed is a multitargeted antifolate with promising clinical activity in non-squamous lung cancer. It targets the folate-dependent enzymes thymidylate synthase (TS), reduced folate carrier, and folylpolygamma-glutamate synthetas, all of which are involved in the de novo biosynthesis of thymidine and purine nucleotides.The primary mechanism of the action of pemetrexed is inhibition of TS, which results in a decrease in the available thymidine necessary for DNA synthesis. We have previously established pemetrexed -resistant lung cancer cell line, and have shown that overexpression of TS resulted in increased pemetrexed resistance. We have also examined that TS copy numbers by quantitative real-time PCR, were increased in pemetrexed-resistant cells. Therefore we investigated TS copy numbers in lung cancer cell lines and clinical samples to examine whether TS copy numbers are the biomarker for pemetrexed sensitivity. First, we examined the correlation of TS copy numbers and 50% cell survival of pemetrexed for 14 lung adenocarcinoma cell lines, and a meaningful relationship was accepted. In addition, we examined TS copy number in 20 bronchoscopic specimens of non-squamous lung cancers, and found that the correlation was also confirmed between TS copy numbers and clinical response. Our study suggests that TS copy number may be a predictive marker for pemetrexed sensitivity in lung cancer. 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 1719. doi:1538-7445.AM2012-1719