Shigeo Hisamori

Single-cell dissection of transcriptional heterogeneity in human colon tumors.

Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.

CDX2 as a Prognostic Biomarker in Stage II and Stage III Colon Cancer

Background The identification of high-risk stage II colon cancers is key to the selection of patients who require adjuvant treatment after surgery. Microarray-based multigene-expression signatures derived from stem cells and progenitor cells hold promise, but they are difficult to use in clinical practice. Methods We used a new bioinformatics approach to search for biomarkers of colon epithelial differentiation across gene-expression arrays and then ranked candidate genes according to the availability of clinical-grade diagnostic assays. With the use of subgroup analysis involving independent and retrospective cohorts of patients with stage II or stage III colon cancer, the top candidate gene was tested for its association with disease-free survival and a benefit from adjuvant chemotherapy. Results The transcription factor CDX2 ranked first in our screening test. A group of 87 of 2115 tumor samples (4.1%) lacked CDX2 expression. In the discovery data set, which included 466 patients, the rate of 5-year disease-free survival was lower among the 32 patients (6.9%) with CDX2-negative colon cancers than among the 434 (93.1%) with CDX2-positive colon cancers (hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). In the validation data set, which included 314 patients, the rate of 5-year disease-free survival was lower among the 38 patients (12.1%) with CDX2 protein-negative colon cancers than among the 276 (87.9%) with CDX2 protein-positive colon cancers (hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003). In both these groups, these findings were independent of the patients age, sex, and tumor stage and grade. Among patients with stage II cancer, the difference in 5-year disease-free survival was significant both in the discovery data set (49% among 15 patients with CDX2-negative tumors vs. 87% among 191 patients with CDX2-positive tumors, P=0.003) and in the validation data set (51% among 15 patients with CDX2-negative tumors vs. 80% among 106 patients with CDX2-positive tumors, P=0.004). In a pooled database of all patient cohorts, the rate of 5-year disease-free survival was higher among 23 patients with stage II CDX2-negative tumors who were treated with adjuvant chemotherapy than among 25 who were not treated with adjuvant chemotherapy (91% vs. 56%, P=0.006). Conclusions Lack of CDX2 expression identified a subgroup of patients with high-risk stage II colon cancer who appeared to benefit from adjuvant chemotherapy. (Funded by the National Comprehensive Cancer Network, the National Institutes of Health, and others.).

Hepatic stellate cells promote liver metastasis of colon cancer cells by the action of SDF-1/CXCR4 axis.

BackgroundIt has been determined that the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1) regulate several key processes in a wide variety of cancers. However, the function and mechanism of the SDF-1/CXCR4 system in the metastasis of colorectal cancer remain controversial.MethodsImmunohistochemistry was performed to examine quantitatively the expression of CXCR4 in 40 human samples of colorectal cancer and liver metastasis. The functions of SDF-1 on HCT116 colon cancer cells were investigated in vitro. We subcutaneously inoculated HCT116 cells with hepatic stellate cells (HSCs) expressing SDF-1. The CXCR4 inhibitor AMD3100 was tested in vitro and in vivo.ResultsBy quantitatively counting the number of cells, it was shown that there are more CXCR4-positive cells at the metastatic site in the liver compared with the primary sites. We demonstrated the effect of SDF-1 on the invasion and antiapoptosis of HCT116 cells in vitro. In mouse experiment of liver metastasis, intraperitoneal administration of AMD3100 blocked the metastatic potential of HCT116 cells. Furthermore, we found that α-smooth muscle actin (α-SMA)-positive myofibroblasts derived from HSCs, surrounding the liver metastasis foci, secreted SDF-1. The subcutaneous inoculation of HCT116 cells with HSCs promoted the tumor initiation in nude mice, indicating the importance of the direct interaction between these cells in vivo.ConclusionThese results suggest that HSCs play important role in liver metastasis of colon cancer cells by the action of SDF-1/CXCR4 axis and provide preclinical evidence that blockade of the axis is a target for antimetastasis therapy.

Thalidomide Prevents Bleomycin-Induced Pulmonary Fibrosis in Mice

Pulmonary fibrosis in humans can occur as a result of a large number of conditions. In idiopathic pulmonary fibrosis (IPF), pulmonary function becomes progressively compromised resulting in a high mortality rate. Currently there are no proven effective treatments for IPF. We have recently reported that IL-6 and TGF-β1 plays an important role in proliferation and differentiation of lung fibroblasts, and all-trans-retinoic acid (ATRA) prevented bleomycin-induced lung fibrosis through the inhibition of these cytokines. Thalidomide (Thal) has been used in the treatment of multiple myeloma through the inhibitory effect on IL-6-dependent cell growth and angiogenesis. In this study, we examined the preventive effect of Thal on bleomycin-induced pulmonary fibrosis in mice. We performed histological examinations and quantitative measurements of IL-6, TGF-β1, collagen type Iα1 (COL1A1), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in bleomycin-treated mouse lung tissues with or without the administration of Thal. Thal histologically ameliorated bleomycin-induced fibrosis in mouse lung tissues. Thal decreased the expressions of IL-6, TGF-β1, VEGF, Ang-1 Ang-2, and COL1A1 mRNA in mouse lung tissues. In addition, Thal inhibited angiogenesis in the lung. In vitro studies disclosed that Thal reduced 1) production of IL-6, TGF-β1, VEGF, Ang-1, and collagen synthesis from human lung fibroblasts, and 2) both IL-6-dependent proliferation and TGF-β1-dependent transdifferentiation of the cells, which could be the mechanism underlying the preventive effect of Thal on pulmonary fibrosis. These data may provide a rationale to explore clinical use of Thal for the prevention of pulmonary fibrosis.

miR-142 regulates the tumorigenicity of human breast cancer stem cells through the canonical WNT signaling pathway

MicroRNAs (miRNAs) are important regulators of stem and progenitor cell functions. We previously reported that miR-142 and miR-150 are upregulated in human breast cancer stem cells (BCSCs) as compared to the non-tumorigenic breast cancer cells. In this study, we report that miR-142 efficiently recruits the APC mRNA to an RNA-induced silencing complex, activates the canonical WNT signaling pathway in an APC-suppression dependent manner, and activates the expression of miR-150. Enforced expression of miR-142 or miR-150 in normal mouse mammary stem cells resulted in the regeneration of hyperproliferative mammary glands in vivo. Knockdown of endogenous miR-142 effectively suppressed organoid formation by BCSCs and slowed tumor growth initiated by human BCSCs in vivo. These results suggest that in some tumors, miR-142 regulates the properties of BCSCs at least in part by activating the WNT signaling pathway and miR-150 expression. DOI: http://dx.doi.org/10.7554/eLife.01977.001

All-trans-retinoic acid ameliorates carbon tetrachloride-induced liver fibrosis in mice through modulating cytokine production.

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis.

miR-137 Regulates the Tumorigenicity of Colon Cancer Stem Cells Through the Inhibition of DCLK1

miRNAs have important roles in regulating cancer stem cell (CSC) properties and are considered to be potential therapeutic targets. However, few studies have focused on miRNAs which are specifically related to colon CSCs. Here, a PCR-based miRNA profiling analysis of normal colon stem cells (NCSC) and colon CSCs (EpCAM+/CD44+/CD66a−) identified miRNAs which regulate colon CSC properties. Interestingly, miRNA-137 (miR-137) expression was downregulated in the colon CSCs compared with NCSCs, while doublecortin-like kinase 1 (DCLK1) mRNA was highly expressed in the colon CSCs but low in the NCSCs. In fact, DCLK1-positive cancer cells were widely distributed in clinically resected colon cancer specimens, while DCLK1-positve epithelial cells were rarely detected in normal colon tissues including the crypt bottoms. Luciferase assay and immunoblot analysis revealed that miR-137 regulated DCLK1 gene expression. Transduction of exogenous miR-137 suppressed the development of colon cancer organoids in vitro and the tumorigenicity of colon cancer cells in vivo without affecting the growth of normal intestinal organoids. Furthermore, the suppression of miR-137 enhanced the organoid development of normal colon cells. These data demonstrate that miR-137 has the capacity to suppress the tumorigenicity of colon CSCs and that maintained expression of miR-137 in NCSCs contributes to suppressing uncontrolled cell proliferation through the inhibition of DCLK1 expression. Implications: The miR-137/DCLK1 axis as an important regulator in NCSCs and colon CSCs; further understanding of this axis may foster the development of potential gene therapeutic strategies targeting colon CSCs. Mol Cancer Res; 14(4); 354–62. ©2016 AACR.

Sarcopenia as a predictor of pulmonary complications after esophagectomy for thoracic esophageal cancer

Sarcopenia or loss of skeletal muscle mass has been identified as a poor prognostic factor for a wide variety of diseases and conditions. We investigated whether preoperative sarcopenia is associated with postoperative complications in patients undergoing esophagectomy for thoracic esophageal cancer.

Is laparoscopic total gastrectomy a safe operation? A review of various anastomotic techniques and their outcomes

Laparoscopic distal gastrectomy is an accepted option for gastric cancer surgery; however, laparoscopic total gastrectomy (LTG) is not widely performed. There is concern about the safety of the operation due to the difficulty of extracorporeal reconstruction through a mini-laparotomy. Efforts have been made to establish an intracorporeal anastomotic technique for esophagojejunostomy. This article reviews the current techniques available for laparoscopic esophagojejunostomy and their surgical outcomes. Several different techniques using either circular or linear staplers have been reported; however, the apparent superiority of any particular method has not been confirmed. The incidence of anastomosis-related complications varied among studies, but different techniques all successfully achieved excellent outcomes. The overall complication rate of LTG was similar to that of open total gastrectomy, suggesting that LTG is a safe and feasible option. However, the feasibility of LTG with D2 lymph node dissection for advanced upper gastric cancer needs to be confirmed in further studies, because most of the patients included in the LTG studies were diagnosed with early stages of disease.

All-trans-Retinoic Acid Attenuates Radiation-Induced Intestinal Fibrosis in Mice

BACKGROUND Intestinal fibrosis leading to severe bowel dysmobility or obstruction is a troublesome adverse effect of abdominal or pelvic radiation therapy. We have recently reported that all-trans-retinoic acid (ATRA) prevents radiation- or bleomycin-induced lung fibrosis. Here, we examined the impact of ATRA on the mouse model of radiation-induced intestinal fibrosis. MATERIALS AND METHODS We evaluated the histology of late radiation fibrosis in surgical samples. We then performed histological examinations and quantitative measurements of mRNA of interleukin-6 and transforming growth factor-beta(1) in intestinal tissues of irradiated mice with or without intraperitoneal administration of ATRA and investigated the effect of ATRA on the transdifferentiation and the production of collagen of irradiated human intestinal fibroblasts. RESULTS Human samples of late radiation enteritis showed thickened submucosa and serosa, consistent with mouse model. Administration of ATRA attenuated irradiation-induced intestinal fibrosis and reduced mRNA of interleukin-6 and transforming growth factor-beta(1). In vitro studies disclosed that ATRA suppressed the transdifferentiation of irradiated intestinal fibroblasts and diminished the production of collagen from the cells. CONCLUSIONS Our findings indicate that ATRA ameliorates irradiation-induced intestinal fibrosis. ATRA could be a novel approach in the treatment of fibrosis associated with chronic radiation enteritis.