Tatsunari Sasada

CDX2 Regulates Multidrug Resistance 1 Gene Expression in Malignant Intestinal Epithelium

The caudal-related homeobox transcription factor CDX2 has a key role in intestinal development and differentiation. CDX2 heterozygous mutant mice develop colonic polyps, and loss of CDX2 expression is seen in a subset of colon carcinomas in humans. Ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression is frequently detected in intestinal metaplasia in the stomach and esophagus. We sought to define CDX2-regulated genes to enhance knowledge of CDX2 function. HT-29 colorectal cancer cells have minimal endogenous CDX2 expression, and HT-29 cells with ectopic CDX2 expression were generated. Microarray-based gene expression studies revealed that the Multidrug Resistance 1 (MDR1/P-glycoprotein/ABCB1) gene was activated by CDX2. Evidence that the MDR1 gene was a direct transcriptional target of CDX2 was obtained, including analyses with MDR1 reporter gene constructs and chromatin immunoprecipitation assays. RNA interference-mediated inhibition of CDX2 decreased endogenous MDR1 expression. In various colorectal cancer cell lines and human tissues, endogenous MDR1 expression was well correlated to CDX2 expression. Overexpression of CDX2 in HT-29 cells revealed increased resistance to the known substrate of MDR1, vincristine and paclitaxel, which was reversed by an MDR1 inhibitor, verapamil. These data indicate that CDX2 directly regulates MDR1 gene expression through binding to elements in the promoter region. Thus, CDX2 is probably important for basal expression of MDR1, regulating drug excretion and absorption in the lower gastrointestinal tract, as well as for multidrug resistance to chemotherapy reagent in CDX2-positive gastrointestinal cancers.

Preservation of peritoneal fibrinolysis owing to decreased transcription of plasminogen activator inhibitor-1 in peritoneal mesothelial cells suppresses postoperative adhesion formation in laparoscopic surgery

BACKGROUND Postoperative adhesion formation is regulated by peritoneal fibrinolysis, which is determined by tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). This study compared peritoneal fibrinolysis and adhesion formation after laparoscopic surgery (LAP) and open surgery (OP). METHODS We divided 154 male rats into 3 groups after cecal cauterization: Control, no treatment; LAP, CO2 pneumoperitoneum at 5 mmHg for 60 minutes; and OP, laparotomy for 60 minutes. Adhesions were quantified at day 7. The activity and mRNA level of tPA and PAI-1 were determined by enzyme-linked immunosorbent assay in plasma and peritoneal lavage and by real-time polymerase chain reaction in peritoneal mesothelial cells from omentum. We also examined peritoneal fibrinolysis in human gastric cancer patients treated with LAP (n = 14) or OP (n = 10). RESULTS In the animal study, adhesion scores, PAI-1 activity in peritoneal lavage fluid, and PAI-1 mRNA levels in peritoneal mesothelium were significantly greater in the OP group than the control and LAP groups. In the human study, postoperative PAI-1 mRNA levels were significantly greater in the OP group than the LAP group. Additionally, PAI-1 mRNA levels and subsequent adhesion formation were induced by prolonged operative time in the OP group, but not the LAP group. CONCLUSION Preservation of peritoneal fibrinolysis owing to decreased PAI-1 expression at the transcriptional level in peritoneal mesothelial cells is associated with suppression of postoperative adhesion formation in LAP. PAI-1 mRNA levels and subsequent adhesion formation were not induced by prolonged operative time in LAP. These results highlight the less invasiveness nature of LAP.

The Efficacy and Safety of Preoperative Chemotherapy With Triweekly Abraxane and Cyclophosphamide Followed by 5-Fluorouracil, Epirubicin, and Cyclophosphamide Therapy for Resectable Breast Cancer: A Multicenter Clinical Trial

BACKGROUND It has been reported that tri-weekly Abraxane therapy has better outcomes in recurrent breast cancer than tri-weekly Cremophor-based taxol therapy, and that cyclophosphamide combined with taxane shows an enhanced antitumor effect. We conducted a phase II clinical trial of preoperative chemotherapy with a combination of TRI-ABC. PATIENTS AND METHODS From September 2011 to September 2013, 4 cycles of preoperative chemotherapy with TRI-ABC followed by 4 cycles of FEC were administered in patients with resectable breast cancer. In patients with HER2-positive breast cancer, tri-weekly Trastuzumab was administered with TRI-ABC. The primary end point was the pathological complete response (pCR) rate in the breasts and lymph nodes. RESULTS The treatment outcomes and safety were evaluated in 54 patients who received at least 1 dose of chemotherapy. All patients underwent radical surgery, and the overall pCR rate of 37% (20 of 54) was achieved. The pCR rates according to each subtype were 8% (2 of 24) in hormone receptor (HR)-positive HER2-negative breast cancer, 56% (5 of 9) in HR-positive HER2-positive breast cancer, 63% (5 of 8) in HR-negative HER2-positive breast cancer, and 62% (8 of 13) in triple-negative breast cancer. Multivariate analysis revealed that HR negativity and HER2 positivity were predictive factors of pCR. Clinical response was observed in 49 patients (91%). The safety profile was acceptable. CONCLUSION Preoperative chemotherapy with TRI-ABC followed by FEC showed high efficacy and excellent safety. Further clinical studies should be conducted to compare the efficacy of TRI-ABC followed by FEC with conventional taxane-anthracycline regimens.

Memorial Sloan-Kettering Cancer Center Nomogram to predict the risk of non-sentinel lymph node metastasis in Japanese breast cancer patients

PurposeAxillary lymph node dissection (ALND) remains the standard procedure for breast cancer patients with sentinel lymph node (SLN) metastasis; however, additional nodal metastasis is detected in completion ALND in only about 50% of these patients. To identify the risk of non-SLN metastasis, the Memorial Sloan-Kettering Cancer Center (MSKCC) developed a nomogram. Many validation studies have been performed to evaluate the accuracy of the nomogram in Western populations, but not in Asians. We conducted this study to establish the accuracy of the nomogram in a Japanese population.MethodsThe accuracy of the MSKCC nomogram for predicting non-SLN status was tested in 116 consecutive SLN-positive patients in our hospital. We then compared the findings of the source MSKCC study with those of our study. A receiver operating characteristics (ROC) curve was plotted, and the area under the curve (AUC) was calculated to assess the discriminative power.ResultsDespite the differences between our patients and the source population in many respects, the area under the ROC curve was 0.73, which was comparable to that obtained in the study on the source population.ConclusionsThe MSKCC nomogram provides a fairly accurate predicted probability for the likelihood of non-SLN metastases. Accordingly, it served as a useful tool for our Japanese patients with SLN metastases.

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.

Technical Feasibility and Cosmetic Advantage of Hybrid Endoscopy-Assisted Breast-Conserving Surgery for Breast Cancer Patients

BACKGROUND We developed a new procedure called hybrid endoscopy-assisted breast-conserving surgery (EBCS), which consists of a combination of plastic surgery and endoscopic surgery techniques. The purpose of this study was retrospectively to analyze the clinical outcome of hybrid EBCS and compare the cosmetic outcomes between hybrid EBCS and conventional breast-conserving surgery (CBCS). PATIENTS AND METHODS We reviewed medical records of patients who had undergone hybrid EBCS (n=73) or CBCS (n=90) between May 2005 and April 2011 and had been followed up in our department until March 2012. The clinical outcomes and cosmetic outcomes of these two groups were compared. The safety of hybrid EBCS was also analyzed by confirming its complications and pathological surgical margin. RESULTS In the hybrid EBCS group, operation time was longer by 30-50 minutes. Blood loss was not significantly different between the two groups. The surgical margin of hybrid EBCS was as follows: 1 patient (1.4%) had a positive margin, 4 patients (5.5%) had a margin of <2 mm, in 9 patients (12.3%) the margin was ≥2 mm and <5 mm, and in 59 patients (80.8%) it was ≥5 mm. Seven cases (9.6%) of postoperative complications occurred in 6 hybrid EBCS patients. To date, no local recurrence has been observed in hybrid EBCS patients (postoperative observation period, 18.1±5.6 months). Compared with the CBCS group, the hybrid EBCS group had better cosmetic results, especially with a less noticeable operative scar (P<.01). CONCLUSIONS Hybrid EBCS can provide sufficient free margin, and its surgical curability is acceptable. Additionally, this method is superior to CBCS in terms of cosmetic outcome.

Abstract P1-15-03: Prevention of letrozole–induced bone loss using risedronate in postmenopausal women with hormone receptor positive breast cancer: A multicenter randomized clinical trial:

Background Prevention of letrozole–induced bone loss using oral risedronate has not been proved in the Japanese women. The aim of this study was to assess the effect of risedronate 17.5mg/week on bone mineral density (BMD) in postmenopausal, early breast cancer patients scheduled to receive adjuvant letrozole. Patients and Methods Postmenopausal women with hormone receptor–positive early breast cancer were assigned to one of two strata according to their baseline BMD T-score as being at low and high risk of osteoporosis. Patients with low risk (-2.5 ≤ T score) were randomly assigned to letrozole and risedronate (L+R) or to letrozole alone (L). Patients with high risk (-2.5 > T score) received letrozole and risedronate (L+R). Letrozole was given at a dosage of 2.5 mg/day while oral risedronate was given at 17.5mg/week. The primary end point was the change in lumbar spine (LS) BMD at 12 months. The secondary end points included change in total hip (HP) BMD and bone turnover markers. Results In the low risk group (N=103), treatment with L+R resulted in a significant increase in BMD at LS and at HP compared to treatment with L only at 12 months (1.8% vs -2.2%, P Four patients (14.3%) improved from osteoporotic region to the osteopenic region with L+R treatment. Letrozole and risedronate were well tolerable and there was no serious adeverse event including osteonecrosis of jaw. Conclusions At 12 months, 17.5mg/week risedronate therapy prevented bone loss in postmenopausal women with breast cancer who were receiving adjuvant letrozole, of which results were compatible with previous findings of western populations. Citation Format: Kadoya T, Masumoto N, Shigematsu H, Emi A, Kajitani K, Kobayashi Y, Funakoshi M, Kawabuchi Y, Ohara M, Matsuura K, Noma M, Sasada T, Okada M. Prevention of letrozole–induced bone loss using risedronate in postmenopausal women with hormone receptor positive breast cancer: A multicenter randomized clinical trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-15-03.

Abstract 2716: Oncogenic K-ras mutation associates with GLUT1 expression in mice and human colorectal cancer.

Background and aims: Somatic APC defects are present in 75% of sporadic colorectal cancer. K-ras gene mutations found in 40-50%, occur early in the progression to carcinoma, but their functional contribution remains incompletely understood. Previous study reported that GLUT1, encoding glucose transporter-1, was one of the genes consistently up-regulated in colorectal cancer cell lines with K-RAS or BRAF mutations. In this study, we have analyzed tumors from genetically modified mice carrying either mutation in the Apc gene or in combination with another mutation in the K-ras G12D allele. Subsequently, we evaluated whether K-RAS gene mutations associate GLUT1 expression in human colorectal cancer. Methods: Mice carrying transgenes regulated by CDX2 homeobox gene promoter (CDX2P9.5) showed tightly restricted transgene expression in the colon in adult tissues. Cre recombinase transgenes with 22 guanine nucleotides (G22Cre) introduced downstream of the initiating ATG codon, and CDX2P9.5-G22Cre transgenic mice homozygous for the Apc flox allele lived only for 10-27 d after birth due to the tumor in the proximal colon. Mice carrying loxP-flanked Apc alleles homozygously (Apc loxP/loxP , 580S) were intercrossed with Lox-Stop-Lox K-ras conditional mice strain (referred to as LSL-K-ras G12D) to generate CDX2P9.5-G22Cre;Apc flox/flox ;LSL-K-ras G12D with K-ras G12D expression (CPC;Apc 580D;Lox-K-ras G12D). Gene expression analyses on the 3 samples from CPC;Apc 580D;Lox-K-ras G12D mice and CPC;Apc 580D;K-ras WT mice were performed with high-density oligonucleotide arrays (GeneChip Mouse Genome 430 2.0 Array). Results: Glut1 was found to be up-regulated significantly in CPC;Apc 580D;Lox-K-ras G12D mice. Immunohistochemical staining revealed that Glut1 protein was up-regulated in CPC;Apc 580D;Lox-K-ras G12D mice. 47 samples of colorectal cancer patients with K-RAS mutations in 18 patients (38.3%) showed GLUT1 expression in 12 patients of 18 K-RAS mutated patients and 8 patients of 29 K-RAS wild-type patients. Conclusions: Using mice carrying conditional solely mutant in the Apc gene or in combination with the K-ras G12D allele, we assessed the relevance of oncogenic K-ras with Glut1 in colorectal cancer. Microarray analysis, quantitative PCR, and immunohistochemistry suggested the association of the K-ras with the Glut1 (Glut1). Our clinical analysis showed K-RAS gene mutations significantly associate GLUT1 expression in human colorectal cancer. Citation Format: Yasuo Kawaguchi, Takao Hinoi, Tatsunari Sasada, Tomohiro Adachi, Yasufumi Saito, Shinji Miguchi, Hiroaki Niitsu, Hideki Ohdan. Oncogenic K-ras mutation associates with GLUT1 expression in mice and human colorectal 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 2716. doi:10.1158/1538-7445.AM2013-2716

Abstract 5457: Chlorinated drinking water alters carcinogenesis in a mice model of colon cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: The gastrointestinal tract is constantly exposed to vast numbers of commensal bacteria and inflammatory product. The diet and enteric environment has been implicated in the pathogenesis of enterocolitis and colon cancer. Probiotic Lactbacilli modify the enteric microflora and are thought to have a beneficial effect on enterocolitis, eventually on colon cancer. We conducted this study to evaluate the effects of chlorinated drinking water which was used in general institutions. Experimental design: Mouse stains carrying colon epithelium-preferential Apc mutation (CPC;Apc mice), that show sporadic colorectal adenomas and carcinomas are used in this study. Nine of Eighteen CPC;Apc mice were consumed chlorinated drinking water (chlorinate concentrations of 10.0 mg/L) and 9 consumed tap water (chlorinate concentrations of 0.7 mg/L) for 25 week. Body weight was measured weekly. Faecal microbial analysis was performed. At sacrifice, the small and large bowel were histologically assessed. Results: Chlorinated drinking water strongly prevented intestinal tumorigenesis in terms of polyps formation in distal portion of small intestine. However, in colon we observed an increase in numbers and volumes of tumors. Body weight gain was more favorable by chlorine treated. Conclusions: These results show for the first time the efficacy and associated mechanisms of chlorinated drinking water feeding against spontaneous intestinal tumorigenesis in the CPC;Apc mice suggesting its chemopreventive potential against intestinal cancers. 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 5457. doi:1538-7445.AM2012-5457

Abstract 1520: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The intestine-specific caudal-related homeobox transcription factor CDX2 is important for intestinal development and differentiation. CDX2 heterozygous mice form colonic polyps, and loss of CDX2 expression is seen in some poorly differentiated colon carcinoma in humans. Conversely, ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression is often seen in stomach and esophageal metaplasia. We sought to define CDX2-regulated genes to enhance the knowledge of CDX2 function. HT-29 colorectal cancer cells with minimal endogenous CDX2 expression were generated to express exogenous CDX2, and the gene expression changes were assessed by microarrays. Activation of CDX2 rapidly induced Multidrug Resistance 1 (MDR1/P-glycoprotein/ABCB1) gene expression in HT-29 cells, even in the presence of protein synthesis inhibitor, and RNA interference-mediated inhibition of CDX2 decreased MDR1 expression. In other colorectal cancer cell lines and human tissues, endogenous MDR1 expression was well correlated to CDX2 expression. Analysis with MDR1 reporter gene constructs and chromatin-immunoprecipitation assays suggested that CDX2 directly regulates MDR1 transcription. Overexpression of CDX2 in HT-29 cells revealed increased resistance to MDR1s known substrate, vincristine and paclitaxel, which was reversed by an MDR1 inhibitor, verapamil. These data indicate that CDX2 directly regulates MDR1 gene expression via binding to elements in the promoter region. Thus, CDX2 is probably important for basal expression of MDR1, regulating drug excretion and absorption in the lower gastrointestinal tract, as well as for multidrug resistance to chemotherapy reagent in CDX2-positive gastrointestinal cancers. 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 1520.