Yasufumi Saito

Chlorinated Water Modulates the Development of Colorectal Tumors with Chromosomal Instability and Gut Microbiota in Apc-Deficient Mice

The gastrointestinal tract is continuously exposed to a variety of chemicals and commensal bacteria. Recent studies have shown that changes in gut microbial populations caused by chlorine or other chemicals in the drinking water influence the development of human colorectal cancer, although the mechanism of tumorigenesis in the gut epithelium is obfuscated by the diversity of microflora and complexity of the tumor microenvironment. In this regard, mouse models that recapitulate human colorectal cancer are an invaluable tool. In this study, we used two conditional adenomatous polyposis coli (Apc) knockout mouse models to investigate the effect of chlorinated water on tumorigenesis in the digestive tract. Mice with colon-specific carcinoma—caused by either chromosomal (CDX2P 9.5-NLS Cre;Apc+/flox, abbreviated to CPC;Apc) or microsatellite (CDX2P9.5-G19Cre;Apcflox/flox and CDX2P9.5-G22Cre;Apcflox/flox) instability, respectively—were administered chlorinated (10.0 mg/L chlorine) or tap (0.7 mg/L chlorine) water and evaluated for colon polyp formation. In CPC;Apc mice given chlorinated drinking water, tumors tended to develop in the colon, whereas in those that drank tap water, tumors were mostly observed in the small intestine. There was no difference in the rate of tumor formation of CDX2P9.5-G19Cre;Apcflox/flox and CDX2P9.5-G22Cre;Apcflox/flox mice consuming chlorinated as compared to tap water, suggesting that microsatellite instability in the Apc gene does not significantly affect tumorigenesis. Chlorinated water altered the enteric environment by reducing the fecal populations of the obligatory anaerobes Clostridium perfringens and C. difficile, as well as species belonging to the Atopobium cluster, including Enterobacteriaceae and Staphylococcus sp., which was associated with colon tumorigenesis in CPC;Apc mice. These results suggest that differences in tumorigenesis among CPC;Apc mice consuming chlorinated versus tap water may be due to differences in gastrointestinal commensal populations.

Submucosal Invasive Micropapillary Carcinoma of the Colon with Massive Lymph Node Metastases: A Case Report

Micropapillary carcinoma was originally reported to be an aggressive variant of breast carcinoma, and it is associated with frequent lymphovascular invasion and a dismal clinical outcome. It has subsequently been found in other organs; however, at present, only a limited number of cases of colorectal micropapillary carcinoma have been reported. We present a case of early colon cancer with extensive nodal metastases in a Japanese patient. An 82-year-old man was found by colonoscopy to have a 20-mm pedunculated polyp in his sigmoid colon. Endoscopic resection of the sigmoid colon tumor was performed, and pathological examination of the resected specimen revealed a poorly differentiated adenocarcinoma component and a micropapillary component. Despite the tumor being confined within the submucosa, massive lymphatic invasion was noted. Thereafter, the patient underwent laparoscopic sigmoidectomy with lymph node dissection, and multiple lymph node metastases were observed. Our case suggests that when a micropapillary component is identified in a pre-operative biopsy specimen, even for early colorectal cancer, surgical resection with adequate lymph node dissection would be required because of the high potential for nodal metastases.

KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer.

KRAS mutations occur in 30–40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apcflox/flox; LSL-KrasG12D and CDX2P-G22Cre;Apcflox/flox mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription–PCR (qRT–PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT–PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin–nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin–NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.

Gasdermin C Is Upregulated by Inactivation of Transforming Growth Factor β Receptor Type II in the Presence of Mutated Apc, Promoting Colorectal Cancer Proliferation

Mutations in TGFBR2, a component of the transforming growth factor (TGF)-β signaling pathway, occur in high-frequency microsatellite instability (MSI-H) colorectal cancer (CRC). In mouse models, Tgfbr2 inactivation in the intestinal epithelium accelerates the development of malignant intestinal tumors in combination with disruption of the Wnt-β-catenin pathway. However, no studies have further identified the genes influenced by TGFBR2 inactivation following disruption of the Wnt-β-catenin pathway. We previously described CDX2P-G19Cre;Apcflox/flox mice, which is stochastically null for Apc in the colon epithelium. In this study, we generated CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice, with simultaneous loss of Apc and Tgfbr2. These mice developed tumors, including adenocarcinoma in the proximal colon. We compared gene expression profiles between tumors of the two types of mice using microarray analysis. Our results showed that the expression of the murine homolog of GSDMC was significantly upregulated by 9.25-fold in tumors of CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice compared with those of CDX2P-G19Cre;Apcflox/flox mice. We then investigated the role of GSDMC in regulating CRC tumorigenesis. The silencing of GSDMC led to a significant reduction in the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GSDMC enhanced cell proliferation. These results suggested that GSDMC functioned as an oncogene, promoting cell proliferation in colorectal carcinogenesis. In conclusion, combined inactivation of both Apc and Tgfbr2 in the colon epithelium of a CRC mouse model promoted development of adenocarcinoma in the proximal colon. Moreover, GSDMC was upregulated by TGFBR2 mutation in CRC and promoted tumor cell proliferation in CRC carcinogenesis, suggesting that GSDMC may be a promising therapeutic target.

Risk Factors for the Development of Desmoid Tumor After Colectomy in Patients with Familial Adenomatous Polyposis: Multicenter Retrospective Cohort Study in Japan.

BackgroundDesmoid tumor (DT) is the primary cause of death in patients with familial adenomatous polyposis (FAP) after restorative proctocolectomy. This study aimed to identify risk factors for DT in a Japanese population.MethodsClinical data for 319 patients with FAP undergoing first colectomy from 2000 to 2012 were reviewed retrospectively.ResultsTwo hundred seventy-seven FAP patients were included in this study. Thirty-nine (14.1 %) patients developed DT. Occurrence sites were the intraperitoneal region in 25 (64.1 %) cases, intraperitoneal region and abdominal wall in three (7.7 %), and abdominal wall in nine (23.1 %). The mean period from surgery to DT development was 26.3 months (range 4–120 months). Gender (female vs. male, p = 0.03), age at surgery (>30 vs. ≤30 years, p = 0.02), purpose of surgery (prophylactic vs. cancer excision, p = 0.01), and surgical procedure (proctocolectomy [ileoanal anastomosis (IAA), ileoanal canal anastomosis (IACA), total proctocolectomy (TPC)] vs. total colectomy [ileorectal anastomosis, partial colectomy]; p = 0.03) significantly influenced the estimated cumulative risk of developing DT at 5 years after surgery. Conversely, approach (laparoscopic vs. open, p = 0.17) had no significant effect on the increased risk of DT occurrence. In multivariate analysis, female gender, with a hazard ratio of 2.2 (p = 0.02,) and proctocolectomy (IAA, IACA, TPC), with a hazard ratio of 2.2 (p = 0.03), were independent risk factors for DT incidence after colectomy.ConclusionsFemale gender and proctocolectomy (IAA, IACA, TPC) were independent risk factors for developing DT after colectomy in patients with FAP.

Increased Calcineurin A Expression Is Associated with a Lower Relapse-Free Survival Rate after Colorectal Cancer Surgery

Objective: Increased expression of calcineurin in colorectal cancer (CRC) has been reported. Although the oncogenic function has been suggested, the clinical relevance is still unclear. We herein studied calcineurin expression as a prognostic biomarker in patients receiving curative surgery for stages I-III CRC. Methods: In 121 patients with stages I-III CRC treated at Hiroshima University between 1997 and 2003, calcineurin A expression was examined using immunohistochemistry (IHC) staining of surgical specimens. Specimens were considered positive for calcineurin A if any IHC-stained cells were observed within the carcinomatous area, and clinicopathological characteristics and survival outcomes were compared between IHC-positive and -negative groups. Results: Calcineurin A was preferentially expressed in the cytoplasm of cancer cells, and a median of 8% of the cells (range: 0-80%; interquartile range: 0-22.5%) were stained within the carcinomatous areas. Of 121 cases, 81 were determined as IHC positive while 40 were determined to be negative. Positive expression of calcineurin A, as well UICC-TNM stage, was associated with low relapse-free survival (RFS) rates in multivariate analyses (hazard ratio = 2.92; 95% CI: 1.27-7.92; p = 0.010). Conclusion: Increased calcineurin A expression is associated with lower RFS rates and may have clinical value in predicting recurrence.

Assessment of cancer pain in a patient with communication difficulties: a case report

BackgroundThe number of patients who have difficulty with mutual understanding has been increasing recently due to an aging society. This emerging issue needs to be addressed. We report an instructive case of a patient who had communication difficulties due to dementia and sequelae of alcoholic encephalopathy.Case presentationA 66-year-old man of Mongolian race presented with coronary arteriosclerosis, spinal canal stenosis, transverse colon cancer, and alcoholic encephalopathy. We had been requested to remove wires that had been used for the closure of his chest in a coronary artery bypass grafting procedure. However, on admission, a tortured expression and abdominal distention were observed, along with emaciation. We diagnosed terminal stage cancer, and palliative care was offered. An abdominal computed tomographic scan revealed rectal cancer with stenosis and invasion to the adjacent tissues. A metallic stent was inserted, leading to reduction of the abdominal distention and an improvement of tachycardia. However, the patient’s tortured expression was not completely relieved; therefore, an assessment of cancer pain was considered. The Abbey Pain Scale was applied. On the basis of the patient’s score, analgesics and an opioid, among other medications, were administered. These led to relief of the patient’s tortured expression and reduced his Abbey Pain Scale score. Following this, the patient’s vital signs continued to be stable, and he was transferred to the referral institution.ConclusionsManagement of cancer pain in elderly patients with mutual understanding difficulties must be performed carefully. In the case of our patient, staff at the referral institution informed us of the patient’s latent torture, and we applied the Abbey Pain Scale. There was some confusion and uncertainty regarding clinical management throughout the patient’s care; however, his condition eventually stabilized. We believe the application of the Abbey Pain Scale assists in the relief of cancer pain. However, accumulation of further cases and experiences to verify this assessment is required.

Total colectomy for multiple metachronous colon cancers in a patient with Lynch syndrome

Lynch syndrome (LS) is a disorder caused by mismatch repair gene mutations, which have been recognized to be associated with an increased frequency of colorectal and extracolorectal tumors. However, it remains controversial as to whether total or segmental colectomy should be performed to treat colorectal cancer in patients with LS. A 58-year-old male underwent total colectomy with ileostomy for advanced transverse colon cancer. He was also found to have LS based on his characteristic family history and the findings of a preoperative examination, including a microsatellite instability analysis of past multiple metachronous cancers. The postoperative histological findings showed mucinous adenocarcinoma without lymph node metastasis, and the loss of the MSH2 protein expression was confirmed on an immunohistochemical examination. The present case provided important information on the clinical management of multiple developing metachronous colorectal cancers in patients with LS.

Abstract 823: Gene expression profiling for oncogenic Kras mutation in mice and human colorectal cancer

Purpose: Oncogenic KRAS mutations are found in 40-50% of human colorectal cancers. However, specific therapeutics has not been yet available. We aimed to search new therapeutic molecular targets or biomarkers in KRAS mutated colorectal cancers. Experimental design: We generated Apcflox/flox; CDX2P9.5-G22Cre and LSL-KrasG12D; Apcflox/flox; CDX2P9.5-G22Cre mice, that had been considered as sporadic colon cancer mouse model with Kras wild and mutant, respectively. We observed the carcinogenesis at the age of 3-4 weeks by necropsy, and harvested the tumors for gene expression profiling. We compared the gene expression by Microarray (GeneChip, Affymetrix) between each 3 tumors from these mice strains. Genes with >5 fold changes between the two groups were selected as candidate genes, and subsequently narrow these down by bibliographic search for further analyses in human colorectal cancers. Result: In macroscopic findings, extensive polyps were generated in cecum and proximal colon, but not in other site of gastrointestinal tract. Tumors occurred in the both strains were considered as well differentiated adenocarcinomas in hematoxylin-eosin staining. It was confirmed that tumors generated in Apcflox/flox; CDX2P9.5-G22Cre mice had Cre-targeted Apc 580D alleles and wild-type Kras, and that tumors generated in LSL-KrasG12D; Apcflox/flox; CDX2P9.5-G22Cre mice had both Cre-targeted Apc 580D alleles and activated oncogenic KrasG12D. In the subsequent gene expression profiling, we identified 31 genes with >5 fold change in Microarray analyses. Of these, we focused on some genes that showed lower expression in Kras mutant tumor than in Kras wild type tumor. We confirmed the lower expressions of these genes in murine tumors by quantitative RT-PCR, and the lower expression in early-staged human colorectal cancers with oncogenic KRAS mutant by immunohistochemistry staining. To analyze the functions, the retroviral vectors that lead overexpression of these genes were constructed and infected to SW480 and RKO CRC cell lines. Proliferation and migration is being analyzed using these cell lines. Conclusion: We identified novel genes associated with oncogenic KRAS mutation, using the analyses of gene expression in colon cancer mouse model. Citation Format: Hiroaki Niitsu, Takao Hinoi, Yasuo Kawaguchi, Kazuhiro Sentani, Naohide Oue, Yusuke Sotomaru, Tomohiro Adachi, Yasufumi Saito, Masashi Miguchi, Masatoshi Kochi, Manabu Shimomura, Wataru Yasui, Hideki Ohdan. Gene expression profiling for oncogenic Kras mutation in mice and human colorectal 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 823. doi:10.1158/1538-7445.AM2015-823

Abstract 2303: Impact of synbiotics administration on tumorigenesis of colon cancer mouse model

Introduction: Previous study showed that Synbiotics might have beneficial effect in human and animal colitis like ulcerative colitis. However, it is not well known that the influence of Synbiotics for tumorigenesis in colon cancer. The aim of present study was to investigate the influence of Synbiotics in colon cancer mouse model and colitis associated cancer mouse model. Methods: Mouse stains carrying colon epithelium-preferential Apc mutation (CPC;Apc mice), that show sporadic colorectal adenomas and carcinomas are used in this study. We also used CPC;Apc mice consumed 1%Dextran Sulfate Sodium (DSS) as colitis associated colon cancer model, developing apploximately fourfold more adenomas and adenocarcinomas than CPC;Apc not consumed. At first, 9 of CPC;Apc mice were administerated symbiotics (Probiotics (Lactobacillus) 0.1g + Prebiotics (Oligosaccharide) 10mg/0.3ml/body) between 7 and 20 age in weeks and 10 was not administerated. Secondaly, using 9 of CPC;Apc consumed 1%DSS was administrated Symbiotics, 8 of not administrated, 7 of only Probiotics, and 7 of only Prebiotics. Body weight was measured weekly. Fecal microbial analysis was performed. At sacrifice in 21 age in weeks, the small and large bowel were histologically assessed. Results: Synbiotics administration group has no differences of tumorigenesis comparing to control group (tumor incidence rate(88.9%(8/9) vs. 62.5% (5 / 8); control vs. Synbiotics; p = 0.21)average tumor number(3.2 vs. 2.1; p = 0.38), average tumor greatest diameter 5.8 vs. 5.1 mm; p = 0.51). Using CPC;Apc consumed 1%DSS, Symbiotics administration group showed inhibiting morbidity (50% (8 / 16) vs. 80% (8 / 10); control vs. Synbiotics; p = 0.04) and weight loss, and less tumor incidence than control (18.8 vs. 9.6; control vs. Synbiotics; p = 0.01). On the other hands, administration of probiotics or prebiotics alone had no significant differences of tumorigeneis. Conclusion: This data suggest that Synbiotics administration may have influence of inhibiting tumorigenesis in the colitis associated colon cancer. Citation Format: Yasufumi Saito, Takao Hinoi, Tomohiro Adachi, Manabu Shimomura, Masashi Miguchi, Hiroaki Niitsu, Masatoshi Kochi, Hideki Ohdan, Kazuhiro Sentani, Naohide Oue, Wataru Yasui. Impact of synbiotics administration on tumorigenesis of colon cancer mouse model. [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 2303. doi:10.1158/1538-7445.AM2015-2303