Sayaka Tsukioka

Synergistic antitumor effect of S-1 and the epidermal growth factor receptor inhibitor gefitinib in non-small cell lung cancer cell lines: role of gefitinib-induced down-regulation of thymidylate synthase

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the therapeutic response to EGFR tyrosine kinase inhibitors (TKI) in patients with advanced non-small cell lung cancer (NSCLC). The response rate to these drugs remains low, however, in NSCLC patients with wild-type EGFR alleles. Combination therapies with EGFR-TKIs and cytotoxic agents are considered a therapeutic option for patients with NSCLC expressing wild-type EGFR. We investigated the antiproliferative effect of the combination of the oral fluorouracil S-1 and the EGFR-TKI gefitinib in NSCLC cells of differing EGFR status. The combination of 5-fluorouracil and gefitinib showed a synergistic antiproliferative effect in vitro in all NSCLC cell lines tested. Combination chemotherapy with S-1 and gefitinib in vivo also had a synergistic antitumor effect on NSCLC xenografts regardless of the absence or presence of EGFR mutations. Gefitinib inhibited the expression of the transcription factor E2F-1, resulting in the down-regulation of thymidylate synthase at the mRNA and protein levels. These observations suggest that gefitinib-induced down-regulation of thymidylate synthase is responsible, at least in part, for the synergistic antitumor effect of combined treatment with S-1 and gefitinib and provide a basis for clinical evaluation of combination chemotherapy with S-1 and EGFR-TKIs in patients with solid tumors. [Mol Cancer Ther 2008;7(3):599–606]

Different histological types of non‐small cell lung cancer have distinct folate and DNA methylation levels

Aberrant DNA methylation is a commonly observed epigenetic change in lung cancer. Folate has been suggested to play a role in the homeostasis of DNA methylation and has also been implicated in cancer chemotherapy. We investigated a possible role for folate in DNA methylation by measuring folate concentrations in tumors and adjacent normal tissues from 72 non‐small cell lung cancer (NSCLC) patients. These were compared to DNA methylation levels and to clinicopathological features. Folate concentrations were determined as the sum of 5,10‐methylenetetrahydrofolate and tetrahydrofolate. The MethyLight assay was used to quantitate methylation in promoter regions of P16(CDKN2A), APC, CDH13, RARB, RASSF1, RUNX3, and MYOD1. Methylation of LINE‐1 repeats was used as a surrogate for global methylation. Folate levels in tumors correlated positively with LINE‐1, CDH13, and RUNX3 methylation. Folate concentrations and methylation of LINE‐1, RASSF1, and RUNX3 were significantly higher in adenocarcinoma compared to squamous cell carcinoma (SCC). Two sets of array‐based data retrieved from the Gene Expression Omnibus consistently showed that expression of FOLR1, a folate transport enzyme, and GGH, an enzyme that prevents folate retention, were higher and lower, respectively, in adenocarcinomas compared to SCC. This was independently validated by quantitative RT‐PCR in 26 adenocarcinomas and 13 SCC. Our results suggest that folate metabolism plays a role in aberrant DNA methylation in NSCLC. The histological subtype differences in folate concentration and DNA methylation observed here were associated with distinct expression patterns for folate metabolizing enzymes. These findings may have clinical applications for histology‐directed chemotherapy with fluoropyrimidine and anti‐folates in NSCLC. (Cancer Sci 2009l; 100: 2325–2330)

In vivo evidence for a significant role of folylpolyglutamate synthase in combined chemotherapy with oral fluoropyrimidine, UFT or S-1, and leucovorin

Combined chemotherapy with 5-fluorouracil and leucovorin (LV) has been widely used for the treatment of patients with colorectal cancer. Given that LV effects are attributable to increased levels of reduced folate in cancer cells, we attempted here to show the in vivo role of folylpolyglutamate synthetase (FPGS), which stabilizes intracellular reduced folate, in the anticancer activities of oral fluoropyrimidines, UFT or S-1, combined with LV. To this end, HCT-15 human colon cancer cells were knocked down for FPGS expression by RNA interference. The cell line stably expressing FPGS shRNA (FPGS shRNA HCT-15) was cloned and transferred subcutaneously into nude mice fed a low-folate diet. FPGS shRNA HCT-15 tumors expressed a significantly lower level of FPGS at protein and mRNA levels than parental HCT-15 cells, and the levels of reduced folate in FPGS shRNA HCT-15 tumors became 57% of those in parent after a single administration of 10 mg/kg of LV. Notably, FPGS downregulation did not affect the tumor growth or sensitivity to fluoropyrimidine. Importantly, we observed that LV given for 14 days failed to enhance the anticancer effects of UFT and S-1 in FPGS shRNA HCT-15. This was in keeping with the results that LV did not increase the ternary complex of TS, FdUMP and reduced folate. In conclusion, the present results provide in vivo evidence that intratumor FPGS plays an important role in the efficacy of oral fluoropyrimidine plus LV therapy for colorectal cancer.

Abstract B87: Preclinical investigation of the potentiation in antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, in combination with irinotecan (CPT-11) in colorectal cancer.

Background: Irinotecan (CPT-11) is a topoisomeraseI (TopoI) inhibitor, which is widely used for the treatment of metastatic colorectal cancer. CPT-11 inhibits DNA religation by TopoI, thus it stabilizes DNA-TopoI complexes and subsequently induces cytotoxicity. TAS-102, a novel oral antitumor agent, consisting of trifluorothymidine(FTD) and thymidine phosphorylase inhibitor, has reported promising clinical results in metastatic colorectal cancer. Several reports indicated that aberrant nucleoside incorporation into DNA, such as with cytarabine or gemcitabine, enhanced DNA-TopoI complexes. FTD, the effector of TAS-102, is a nucleoside which is efficiently incorporated and retained into DNA. Here, we evaluated the potency of TAS-102 in combination with CPT-11 in vitro and in vivo Materials and Methods: The combinational effect of FTD and SN-38 which is the active metabolite of CPT-11 was evaluated with several treatment schedules. Various colorectal cancer cell lines (SW48, SW620, LS174T and HCT116) were used in this assay. In one set, cells were washed after a 24 hour pre-exposure to FTD followed by a 48 hour exposure to SN-38 (FTD pre-exposure schedule); in the other set, cells were washed after a 6 hour pre-exposure to SN-38 followed by a 66 hour exposure to FTD (SN-38 pre-exposure schedule). A combinational intensity was estimated by using a combination index (CI) which indicates synergism (CI 1.2). Additionally, the antitumor effect of TAS-102 in combination with CPT-11 was investigated in a human colorectal cancer xenograft model. TAS-102 was orally administered for 14 days consecutively and CPT-11 was intravenously administered once a week to mice. Results: The combination of SN-38 and FTD showed a synergistic effect (SW48, SW620 and LS174T) or an additive effect (HCT116) in both the FTD pre-exposure schedule as well as the SN-38 pre-exposure schedule. Also, the enhancement of antitumor effect of TAS-102 in combination with CPT-11 was observed in vivo. Furthermore, these cell lines had various genotypes as regards KRAS, PI3CA and other DNA repair enzymes frequently detected in colorectal cancer, indicating that the combination9s efficacy seemed to be independent of genetic status. Conclusion: TAS-102/CPT-11 represents a novel combination strategy for the treatment of advanced colorectal cancer. A synergistic effect was observed in both schedules tested, suggesting that various underlying mechanisms may exist. One potential mechanism is that FTD uptake or incorporation into DNA is increased by TopoI inhibition, and that FTD incorporation then further stabilizes the CPT-11 induced cleavable complex. However, the precise mechanism needs to be elucidated in the future. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B87. Citation Format: Keiji Ishida, Nozomu Tanaka, Sayaka Tsukioka, Kokoro Eshima, Akio Fujioka, Keisuke Yamamura, Satoko Ito, Kenichi Matsuo, Teruhiro Utsugi. Preclinical investigation of the potentiation in antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, in combination with irinotecan (CPT-11) in colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B87.

Abstract B89: Expression of DNA damage repair enzymes determine the efficacy of a novel dUTPase inhibitor, TAS-114.

Background: Deoxyuridine 59-triphosphate nucleotidehydrolase (dUTPase) is a pyrophosphatase, which selectively catalyzes FdUTP and dUTP hydrolysis, and tightly restricts 5-fluorouracil (5-FU) and uracil misincorporation into DNA. TAS-114 is a novel potent inhibitor of dUTPase, and is under clinical development as a fluoropyrimidine enhancer. TAS-114 potentiates the antitumor activity of fluoropyrimidines through increasing misincorporation of 5-FU and uracil into DNA, however, precise mechanisms of cytotoxicity after misincorporation of the aberrant base is unknown. Here, we report relationships between the DNA damage repair function and the efficacy of TAS-114 on the anticancer activity of fluoropyrimidine. Materials and Methods: The antitumor activities of 5-FU or 5-FU/TAS-114 were evaluated in 9 human tumor xenograft models. Intratumor dUTPase protein was measured by Western blotting, and phosphorylation of histone H2AX was detected by immunohistochemistry. Tissue concentrations of FdUMP and dUMP were measured by HPLC and a thymidylate synthase (TS) binding assay, respectively. Suppression of DNA damage repair proteins were performed by RNA interference technology in HeLa cells, and then, the cells were used to assess an antiproliferative activity of FdUrd/TAS-114. Results: Oral administration of TAS-114 inhibited dUTPase and synergistically increased antitumor activity of 5-FU in various human tumor xenograft models. The magnitude of dUTPase inhibition was estimated by the change of dUTPase enzymatic products (FdUMP and dUMP) and it appeared to correlate with dUTPase expression and synergistic effect. These data suggest that 5-FU and uracil misincorporation is a primary mechanism of TAS-114 efficacy. In addition, TAS-114 combination with 5-FU increased phosphorylation of histone H2AX, suggesting a TAS-114 activated DNA damage response pathway. Suppression of DNA damage repair proteins involved in base excision repair (BER) and homologous recombination repair (HR) revealed that these DNA repair pathways were important in TAS-114’s synergistic increase of 5-FU cytotoxicity. Conclusion: Expression levels of DNA damage repair proteins in BER and HR affect TAS-114 sensitivity. DNA repair response appears to be a determinant of TAS-114 efficacy as well as dUTPase expression. These factors will be measured inTAS-114/fluoropyrimidines combination clinical trial. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B89. Citation Format: Sayaka Tsukioka, Wakako Yano, Tatsushi Yokogawa, Takeshi Wakasa, Akio Fujioka, Keisuke Yamamura, Satoko Itoh, Masayoshi Fukuoka, Kenichi Matsuo, Kazuharu Noguchi, Teruhiro Utsugi. Expression of DNA damage repair enzymes determine the efficacy of a novel dUTPase inhibitor, TAS-114. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B89.

Abstract 3858: Synergistic antitumor effects of combination therapy with S-1 and HER2 targeting agents in gastric cancer with HER2 amplification

HER2 amplification is observed in 20-30% of gastric cancer and it has been correlated to poor outcomes and more aggressive disease. Combination therapies with HER2 targeting agents and cytotoxic agents are considered a therapeutic option for patients with HER2 amplified gastric cancer. We now investigated the effects of the combination treatment with S-1(or 5-fluorouracil (5FU)) and either lapatinib or trastuzumab on the growth of gastric cancer cells with or without HER2 amplification in vitro and in vivo. The combination of 5FU and either lapatinib or trastuzumab showed a synergistic antiproliferative effect in HER2 amplified gastric cancer cells. Combined treatment with 5FU and lapatinib or trastuzumab induced synergistic increases in the number of apoptotic cells and in the activity of caspase-3 in HER2 amplified cells, wheareas, such synergistic antiproliferative effect nor the enhancement of apoptosis was observed in cells without HER2 amplification. Thymidylate synthase (TS) is an important target enzyme for 5FU, with a reduced level of TS expression having been associated with a higher rate of response to 5FU-based chemotherapy. To elucidate the molecular mechanism of the synergistic effect by the combination of 5FU and lapatinib or trastuzumab, we examined the effects of lapatinib or trastuzumab on the expression and activity of TS in gastric cancer cells. Both lapatinib and trastuzumab induced down-regulation of TS expression and inhibition of TS activity in HER2 amplified cells, whereas neither lapatinib nor trastuzumab has such effects on TS expression and activity in cells without HER2 amplification. Similar to the effects of lapatinib or trastuzumab, TS depletion by siRNA resulted in the enhancement of 5FU-induced apoptosis, suggesting that TS down-regulation attributes, at least in part, to the synergistic proapoptotic interaction with 5FU. Furthermore, the combination of oral fluoropyrimidine S-1 and lapatinib or trastuzumab synergistically inhibited the growth of gastric cancer cell xenografts with HER2 amplification. Our results thus suggest that the combination of S-1 and lapatinib or trastuzumab has synergistic antitumor effects in HER2 amplified gastric cancer cells and that effect is likely attributable to the down-regulation of TS expression and inhibition of TS activity by lapatinib or trastuzumab. In conclusion, these observations provide a basis for clinical evaluation of combination chemotherapy with S-1 and HER2 targeting agents, lapatinib or trastuzumab, in gastric cancer patients with HER2 amplification. 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 3858.