Etsuko Sakamoto

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.

ARHGEF15 overexpression worsens the prognosis in patients with pancreatic ductal adenocarcinoma through enhancing the motility and proliferative activity of the cancer cells

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases, associated with a remarkably poor prognosis. However, the molecular mechanisms underlying the development of PDAC remain elusive. The aim of this study was to identify genes whose expressions are correlated with a poor prognosis in PDAC patients, and to unravel the mechanisms underlying the involvement of these genes in the development of the cancer.MethodsGlobal gene expression profiling was conducted in 39 specimens obtained from Japanese patients with PDAC to identify genes whose expressions were correlated with a shorter overall survival. The effect of gene silencing or overexpression of ARHGEF15 in pancreatic cancer cell lines was examined by introducing siRNAs of ARHGEF15 or the ARHGEF15 expression vector. After assessing the effect of ARHGEF15 deregulation on the Rho-family proteins by pull-down assay, wound healing, transwell and cell viability assays were carried out to investigate the cellular phenotypes caused by the perturbation.ResultsThe global mRNA expression profiling revealed that overexpression of ARHGEF15, a Rho-specific GEF, was significantly associated with a poor prognosis in patients with PDAC. We also found that the depletion of ARHGEF15 by RNA interference in pancreatic cancer cell lines downregulated the activities of molecules of the Rho signaling pathway, including RhoA, Cdc42 and Rac1. Then, we also showed that ARHGEF15 silencing significantly reduced the motility and viability of the cells, while its overexpression resulted in the development of the opposite phenotype in multiple pancreatic cancer cell lines.ConclusionThese data suggest that upregulation of ARHGEF15 contributes to the development of aggressive PDAC by increasing the growth and motility of the pancreatic cancer cells, thereby worsening the prognosis of these patients. Therefore, ARHGEF15 could serve as a novel therapeutic target in patients with PDAC.

Muscle RAS oncogene homolog (MRAS) recurrent mutation in Borrmann type IV gastric cancer

The prognosis of patients with Borrmann type IV gastric cancer (Type IV) is extremely poor. Thus, there is an urgent need to elucidate the molecular mechanisms underlying the oncogenesis of Type IV and to identify new therapeutic targets. Although previous studies using whole‐exome and whole‐genome sequencing have elucidated genomic alterations in gastric cancer, none has focused on comprehensive genetic analysis of Type IV. To discover cancer‐relevant genes in Type IV, we performed whole‐exome sequencing and genome‐wide copy number analysis on 13 patients with Type IV. Exome sequencing identified 178 somatic mutations in protein‐coding sequences or at splice sites. Among the mutations, we found a mutation in muscle RAS oncogene homolog (MRAS), which is predicted to cause molecular dysfunction. MRAS belongs to the Ras subgroup of small G proteins, which includes the prototypic RAS oncogenes. We analyzed an additional 46 Type IV samples to investigate the frequency of MRAS mutation. There were eight nonsynonymous mutations (mutation frequency, 17%), showing that MRAS is recurrently mutated in Type IV. Copy number analysis identified six focal amplifications and one homozygous deletion, including insulin‐like growth factor 1 receptor (IGF1R) amplification. The samples with IGF1R amplification had remarkably higher IGF1R mRNA and protein expression levels compared with the other samples. This is the first report of MRAS recurrent mutation in human tumor samples. Our results suggest that MRAS mutation and IGF1R amplification could drive tumorigenesis of Type IV and could be new therapeutic targets.

Abstract 2975: Comprehensive analysis of genetic alterations and DNA copy number in Borrmann type IV gastric cancer

The prognosis of patients with Borrmann type IV gastric cancer (Type IV) is extremely poor. Thus, there is an urgent need to elucidate the molecular mechanisms underlying the oncogenesis of Type IV and to identify new therapeutic targets. Although previous studies using whole-exome and whole-genome sequencing have elucidated genomic alternations in gastric cancer, no reports have focused on the comprehensive genetic analysis in Type IV. To find cancer relevant genes in Type IV, we performed whole exome sequencing and genome-wide copy number analysis on 14 patients of Type IV. The exome sequencing identified a total of 182 somatic mutations which cause amino-acid substitutions or splice-site alterations. Previously reported mutations in gastric cancer were detected, such as ARID1A, CDH1, TP53 and RHOA. Moreover, our study has found novel mutations that have not been reported in gastric cancer and other tumor types. Among the newly identified genetic alterations, a mutation in muscle RAS oncogene homolog (MRAS) was predicted to cause molecular dysfunction. MRAS belongs to the Ras subgroup of small G proteins, including the prototypic RAS oncogene, KRAS, HRAS, and NRAS. The MRAS mutation discovered by whole-exome sequencing was validated by mutation-specific PCR. To investigate the frequency of mutation in MRAS, we performed Sanger sequencing of additional 46 Type IV samples. A total of 9 non-synonymous mutations were discovered with a mutation frequency of 17%, showing that MRAS is recurrently mutated in Type IV. The genome-wide copy number analysis also showed that copy number was aberrant in 41 genes, including Insulin-like growth factor 1 receptor (IGF1R). The samples with IGF1R amplification showed remarkably higher expression levels of mRNA and protein compared with other samples. In conclusion, this is the first research to discover mutation in MRAS among human tumor samples, although introduction of artificially mutated MRAS is known to have oncogenic activity. In addition, these results suggested that MRAS mutation and IGF1R amplification could drive tumorigenesis of Type IV and could be a new therapeutic target for Type IV. Citation Format: Makiko Yasumoto, Sachiko Ogasawara, Jun Akiba, Hironori Kusano, Akiko Sumi, Taro Isobe, Junya Kizaki, Yoshito Akagi, Takuji Torimura, Etsuko Sakamoto, Hiraku Itadani, Tsutomu Kobayashi, Shinji Mizuarai, Shinji Oie, Hirohisa Yano. Comprehensive analysis of genetic alterations and DNA copy number in Borrmann type IV gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2975. doi:10.1158/1538-7445.AM2015-2975

Abstract 514: ARHGEF15 is associated with a poor prognosis in patients with pancreatic ductal adenocarcinoma through a mechanism involving the enhancement of cellular motility and proliferation

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and is associated with an extremely poor prognosis, since it is difficult to diagnose pancreatic cancer at an early stage despite its remarkably aggressive features. However, the molecular mechanism underlying the development of aggressive PDAC remains elusive. Rho guanine nucleotide exchange factors (ARHGEFs) are known to activate the RhoA signaling pathway in several types of tumor, although its association with the outcome of PDAC has not been reported. Here, we report that a higher expression of ARHGEF15 is significantly associated with a poor prognosis among PDAC patients, and an increase in ARHGEF15 expression plays pivotal roles in the enhancement of cellular motility and the proliferation of PDAC cells. Firstly, we found that a higher level of ARHGEF15 was significantly correlated with a poor prognosis among 39 Japanese patients with PDAC using global gene expression profiling with an Affymetrix microarray. We then showed that RNA interference-mediated gene silencing of ARHGEF15 inactivated the RhoA signaling pathway when examined using a RhoA pull-down assay. Next, the assessment of cellular migration using both a transwell chamber assay and a wound healing assay showed that ARHGEF15 silencing down-regulated cellular migration and invasive ability in pancreatic cancer cell lines with high levels of endogenous ARHGEF15 expression. Since the RhoA signaling pathway is involved in cell proliferation, we also examined the effect of ARHGEF15-silencing on cell viability and observed that ARHGEF15-knockdown markedly retarded cellular growth in the pancreatic cell lines. In parallel with the gene-silencing studies, the effect of ARHGEF15-overexpression was also investigated in pancreatic cell lines with low levels of endogenous ARHGEF15 expression. We observed opposite phenotypes to those observed in the gene-silencing studies: increased cellular motility and viability, compared with the controls, was observed as a result of overexpression. This is the first study to suggest that a higher expression of ARHGEF15 was correlated with a poor prognosis among patients with pancreatic cancer. In addition, these data suggested that the overexpression of ARHGEF15 contributed to a poor prognosis among PDAC patients by enhancing tumor motility and proliferation, suggesting that ARHGEF15 could be a prognostic biomarker as well as a novel therapeutic target for PDAC. Citation Format: Hiroto Fukushima, Makiko Yasumoto, Sachiko Ogasawara, Jun Akiba, Yuhei Kitasato, Yoshiki Naito, Masamichi Nakayama, Yoshinobu Okabe, Masafumi Yasunaga, Hiroyuki Horiuchi, Etsuko Sakamoto, Hiraku Itadani, Shinji Mizuarai, Shinji Oie, Hirohisa Yano. ARHGEF15 is associated with a poor prognosis in patients with pancreatic ductal adenocarcinoma through a mechanism involving the enhancement of cellular motility and proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2015-514