Yohei Masugi

Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis

Objective Accumulating evidence links the intestinal microbiota and colorectal carcinogenesis. Fusobacterium nucleatum may promote colorectal tumour growth and inhibit T cell-mediated immune responses against colorectal tumours. Thus, we hypothesised that the amount of F. nucleatum in colorectal carcinoma might be associated with worse clinical outcome. Design We used molecular pathological epidemiology database of 1069 rectal and colon cancer cases in the Nurses’ Health Study and the Health Professionals Follow-up Study, and measured F. nucleatum DNA in carcinoma tissue. Cox proportional hazards model was used to compute hazard ratio (HR), controlling for potential confounders, including microsatellite instability (MSI, mismatch repair deficiency), CpG island methylator phenotype (CIMP), KRAS, BRAF, and PIK3CA mutations, and LINE-1 hypomethylation (low-level methylation). Results Compared with F. nucleatum-negative cases, multivariable HRs (95% CI) for colorectal cancer-specific mortality in F. nucleatum-low cases and F. nucleatum-high cases were 1.25 (0.82 to 1.92) and 1.58 (1.04 to 2.39), respectively, (p for trend=0.020). The amount of F. nucleatum was associated with MSI-high (multivariable odd ratio (OR), 5.22; 95% CI 2.86 to 9.55) independent of CIMP and BRAF mutation status, whereas CIMP and BRAF mutation were associated with F. nucleatum only in univariate analyses (p<0.001) but not in multivariate analysis that adjusted for MSI status. Conclusions The amount of F. nucleatum DNA in colorectal cancer tissue is associated with shorter survival, and may potentially serve as a prognostic biomarker. Our data may have implications in developing cancer prevention and treatment strategies through targeting GI microflora by diet, probiotics and antibiotics.

Mesenchymal stem cells regulate epithelial–mesenchymal transition and tumor progression of pancreatic cancer cells

Cancer‐associated fibroblasts contribute to cancer progression that is caused by epithelial–mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor‐promoting cancer stroma. Here we report that α‐smooth muscle actin‐positive myofibroblast‐like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC‐derived myofibroblasts function to maintain tumor‐initiating stem cell‐like characteristics, including augmenting expression levels of various stemness‐associated genes, enhancing sphere‐ forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ‐secretase inhibitor and siRNA directed against Jagged‐1 attenuated MSC‐associated E‐cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC‐derived myofibroblasts play important roles in regulating EMT and tumor‐initiating stem cell‐like properties of pancreatic cancer cells through an intermediating Notch signal.

Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility

Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

Molecular pathogenesis of hepatocellular carcinoma: Altering transforming growth factor-β signaling in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) occurs subsequent to liver injury, where regenerative hepatocytes develop into a dysplastic nodule and then early HCC, supporting the multistep hepatocarcinogenesis theory. Molecular alterations such as the p53 mutation, p16 gene silencing, and AKT signaling activation are found in the late stage of HCC progression. The overexpression of some marker molecules is observed at the early stage. Transforming growth factor-β (TGF-β), a potent inhibitor of cell proliferation, is frequently overexpressed in HCC, although the role of TGF-β signaling during HCC development remains controversial. We previously reported that HCC cells show TGF-β receptor-dependent growth inhibition in response to TGF-β. Also, reduced TGF-β receptor II in HCC correlates with intrahepatic metastasis and shorter time-to-recurrence, suggesting a role of TGF-β signaling in tumor suppression. In contrast, TGF-β overexpression in HCC is known to correlate with malignant potential, suggesting a role in tumor promotion. Enhanced formation of stroma is a feature of advanced HCC, and TGF-β also promotes the proliferation of stromal fibroblasts. The microenvironment produced via tumor-stromal interactions may be the key to the modulation of the dual roles of TGF-β signaling in HCC progression.

Candidate Molecular Markers for Histological Diagnosis of Early Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. HCC occurs mainly in chronically diseased livers, e.g. following hepatitis B and C infection. These high-risk patients are closely followed up, and increasing numbers of small equivocal lesions are detected by imaging diagnosis. They are now widely recognized as precursor or early-stage HCCs and are classified as dysplastic nodule or early HCC. These lesions lack typical imaging and histology of ordinary HCC and do not show elevated serum markers of α-fetoprotein and PIVKA-II, for example. Molecular analysis of these lesions would help to develop molecular markers for objective histological diagnosis of early HCC and possibly new serum markers for early detection of HCC. It has been reported that HSP70, CAP2, glypican 3 and glutamine synthetase could serve as molecular markers for early HCC. Further analysis is expected to evaluate their usefulness in routine pathological diagnosis including biopsy diagnosis and also as serum markers for early detection of HCC.

Solitary cell infiltration is a novel indicator of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer.

Pancreatic cancer is one of the most aggressive and lethal human malignancies in the Western world. A wide variety of intratumor glandular differentiation, including solitary infiltrating cancer cells, is a prominent microscopic finding in pancreatic cancer. We reviewed 114 resected cases of pancreatic ductal adenocarcinoma to investigate the prognostic impact of the degree of solitary cell infiltration, defined by the number of solitary infiltrating cancer cells. The clinicopathologic correlation of solitary cell infiltration was further evaluated. Seventy-six (67%) cases showed 7 or more solitary infiltrating cancer cells in 10 high-power fields and were labeled as having a high degree of solitary cell infiltration. A high degree of solitary cell infiltration correlated significantly with poor overall survival, the grade, lymphatic invasion, and lymph node metastasis. Multivariate analysis revealed that the degree of solitary cell infiltration, the grade, and the margin status were independent prognostic factors. Grade 1 and 2 tumors with a high degree of solitary cell infiltration, compared with low infiltration, correlated significantly with poor overall survival. Grade 3 tumors showed a worse overall survival than grade 1 and 2 tumors with either a high or a low degree of solitary cell infiltration. Immunohistochemical analysis showed that a high degree of solitary cell infiltration correlated with reduced E-cadherin and increased vimentin expression. In conclusion, solitary cell infiltration is a significant prognostic indicator and serves as a morphological clue to epithelial-mesenchymal transition in pancreatic cancer.

Bmi-1 gene is upregulated in early-stage hepatocellular carcinoma and correlates with ATP-binding cassette transporter B1 expression

(Cancer Sci 2010; 101: 666–672)

Association of Dietary Patterns With Risk of Colorectal Cancer Subtypes Classified by Fusobacterium nucleatum in Tumor Tissue

Importance Fusobacterium nucleatum appears to play a role in colorectal carcinogenesis through suppression of the hosts’ immune response to tumor. Evidence also suggests that diet influences intestinal F nucleatum. However, the role of F nucleatum in mediating the relationship between diet and the risk of colorectal cancer is unknown. Objective To test the hypothesis that the associations of prudent diets (rich in whole grains and dietary fiber) and Western diets (rich in red and processed meat, refined grains, and desserts) with colorectal cancer risk may differ according to the presence of F nucleatum in tumor tissue. Design, Setting, and Participants A prospective cohort study was conducted using data from the Nurses’ Health Study (June 1, 1980, to June 1, 2012) and the Health Professionals Follow-up Study (June 1, 1986, to June 1, 2012) on a total of 121 700 US female nurses and 51 529 US male health professionals aged 30 to 55 years and 40 to 75 years, respectively (both predominantly white individuals), at enrollment. Data analysis was performed from March 15, 2015, to August 10, 2016. Exposures Prudent and Western diets. Main Outcomes and Measures Incidence of colorectal carcinoma subclassified by F nucleatum status in tumor tissue, determined by quantitative polymerase chain reaction. Results Of the 173 229 individuals considered for the study, 137 217 were included in the analysis, 47 449 were male (34.6%), and mean (SD) baseline age for men was 54.0 (9.8) years and for women, 46.3 (7.2) years. A total of 1019 incident colon and rectal cancer cases with available F nucleatum data were documented over 26 to 32 years of follow-up, encompassing 3 643 562 person-years. The association of prudent diet with colorectal cancer significantly differed by tissue F nucleatum status (P = .01 for heterogeneity); prudent diet score was associated with a lower risk of F nucleatum–positive cancers (P = .003 for trend; multivariable hazard ratio of 0.43; 95% CI, 0.25-0.72, for the highest vs the lowest prudent score quartile) but not with F nucleatum–negative cancers (P = .47 for trend, the corresponding multivariable hazard ratio of 0.95; 95% CI, 0.77-1.17). There was no significant heterogeneity between the subgroups in relation to Western dietary pattern scores. Conclusions and Relevance Prudent diets rich in whole grains and dietary fiber are associated with a lower risk for F nucleatum–positive colorectal cancer but not F nucleatum–negative cancer, supporting a potential role for intestinal microbiota in mediating the association between diet and colorectal neoplasms.

Identification by differential tissue proteome analysis of talin-1 as a novel molecular marker of progression of hepatocellular carcinoma.

Objective: Hepatocellular carcinoma (HCC) is characterized by a multistage process of tumor progression. This study addressed its molecular features to identify novel protein candidates involved in HCC progression. Methods: Using liquid chromatography-tandem mass spectrometry, proteomes of 4 early HCCs and 4 non-HCC tissues derived from 2 cases of liver transplant surgery were compared with respect to the separation profiles of their tryptic peptides. Immunohistochemistry was performed on 106 HCC nodules to confirm the results of the proteomic analysis. Results: Statistical analysis of the profiles selected the peptide peaks differentiating HCC from non-HCC. A database search of the tandem mass spectrometry data from those peptide peaks identified 61 proteins, including a cytoskeletal protein, talin-1, as upregulated in HCC. Talin-1 expression levels in HCC nodules were significantly associated with the dedifferentiation of HCC (p = 0.001). A follow-up survey of the examined clinical cases revealed a correlation between talin-1 upregulation and a shorter time to recurrence after resection (p = 0.039), which may be related to the higher rate of portal vein invasion in HCCs with talin-1 up-regulation (p = 0.029). Conclusions: Proteomic analysis led to identification of talin-1 as a promising HCC marker. Talin-1 upregulation is associated with HCC progression and may serve as a prognostic marker.

Reduced transforming growth factor- β receptor II expression in hepatocellular carcinoma correlates with intrahepatic metastasis

Hepatocellular carcinoma (HCC) occurs mainly in the liver associated with chronic hepatitis and hepatic cirrhosis as a result of prolonged viral infection. Transforming growth factor-β (TGF-β) induces the fibrosis in hepatic cirrhosis, although it is also an inhibitor of hepatocyte proliferation. To understand the role of TGF-β signaling in HCC progression, we analyzed gene expression in HCC cells in relation to TGF-β signaling using a two-way clustering algorithm. By the analysis, five HCC cell lines were classified into two groups according to their metastatic capacity. TGF-β receptor II (TGFBR2) was downregulated in metastatic cells, which did not show a response to TGF-β. Immunohistochemistry demonstrated clear membrane distribution of TGFBR2 in noncancerous hepatocytes, whereas reduced TGFBR2 expression was observed in 34 of 136 HCCs. In clinical cases, reduced TGFBR2 expression correlated with larger tumor size (P<0.001), poor differentiation (P<0.001), portal vein invasion (P=0.002), intrahepatic metastasis (IM) (P<0.001), and shorter recurrence-free survival (P=0.022). In conclusion, reduced TGFBR2 expression was associated with aggressive features of HCC such as IM, and may represent an immunohistochemical biomarker to detect aggressive HCC.