Michiie Sakamoto

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.

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.

Molecular Diagnosis of Multistage Hepatocarcinogenesis

Human hepatocellular carcinoma is recognized as a good model for multistage carcinogenesis, as the malignant steps from chronic liver disease through to advanced human hepatocellular carcinoma are relatively clear. We address the activation of different molecular pathways during hepatocarcinogenesis that is especially useful in the diagnosis of pathological multistage human hepatocellular carcinoma. In chronic liver disease, the gene-expression signature as well as the degree of liver fibrosis could help us to predict the development of human hepatocellular carcinoma or survival outcome after treatment for human hepatocellular carcinoma. Several genes, such as HSP70, CAP2 and GPC3, have been identified as potential biomarkers for early human hepatocellular carcinoma. Classical oncogenes or tumor suppressor genes, such as beta-catenin and p53, are mutated during the progression from early to advanced human hepatocellular carcinoma. Also, the presence of hepatoblastic feature like CK19 in advanced human hepatocellular carcinoma can be used as a predictor of aggressive human hepatocellular carcinoma. Although many advances have been made in the diagnosis of multistage hepatocarcinogenesis, we still need further useful markers to more precisely evaluate each step of hepatocarcinogenesis for better treatment choices, and that will promote future molecular-targeted therapy.

OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma

BACKGROUND & AIMSnIn the current era of emerging molecular targeted drugs, it is necessary to identify before treatment the specific subclass to which a tumour belongs. Gadoxetic acid is a liver-specific contrast agent that is preferentially taken up by hepatocytes. Therefore, gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) should provide precise molecular information about hepatocellular carcinomas (HCCs). The aim of this study was to investigate the transporters of gadoxetic acid in HCC comprehensively and to analyse the molecular regulatory mechanism of such transporters.nnnMETHODSnExpression levels of transporters, transcriptional factors and Wnt target genes in clinical samples were examined by quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry. LiCl treatment of the HCC cell line KYN-2 was conducted in vitro to assess the effects of Wnt signalling activity.nnnRESULTSnComprehensive analyses of transporter mRNAs and protein expressions revealed that the organic anion transporting polypeptide 1B3 (OATP1B3) had the strongest correlation with tumour enhancement in hepatobiliary-phase images of EOB-MRI. Association analysis with OATP1B3 expression revealed significant correlation with the expression of Wnt/β-catenin target genes. Further, LiCl treatment induced OATP1B3 mRNA expression in KYN-2 cells, indicating a strong association between OATP1B3 expression and Wnt/β-catenin signalling. The sensitivity and specificity to predict Wnt/β-catenin-activated HCC using tumour enhancement in EOB-MRI were 78.9% and 81.7%, respectively.nnnCONCLUSIONSnOATP1B3 was confirmed as the most important transporter mediating HCC enhancement in EOB-MRI. OATP1B3 expression showed a strong association with the expression of Wnt/β-catenin target genes, therefore, OATP1B3-upregulated HCC likely represents a specific subclass of Wnt/β-catenin-activated HCC.

Involvement of hepatocellular carcinoma biomarker, cyclase-associated protein 2 in zebrafish body development and cancer progression

Cyclase-associated protein 2 (CAP2) is a conserved protein that is found up-regulated in hepatocellular carcinoma (HCC). By using zebrafish, combined with HCC cell lines, we further investigated the role of CAP2. The zebrafish CAP2 sequence was 60% identical to human CAP2 with 77% homology in the C-terminal actin-binding domain, and 58% in the N-terminal cyclase-binding domain. CAP2 expression was observed during zebrafish development and was preferentially expressed in the skeletal muscle and heart. Knockdown using two different morpholinos against CAP2 resulted in a short-body morphant zebrafish phenotype with pericardial edema. CAP2 was observed co-localized with actin in zebrafish skeletal muscle, and in the leading edge of lamellipodium in HCC cell lines. CAP2 silencing resulted in a defect in lamellipodium formation and decreased cell motility in HCC cell lines. Strongly positive expression of CAP2 was observed in 10 of 16 (63%) poorly, 30 of 68 (44%) moderately, and 2 of 21 (10%) well differentiated HCC. CAP2 expression was significantly associated with tumor size, poor differentiation, portal vein invasion, and intrahepatic metastasis. Our results indicate that an important conserved function of CAP2 in higher vertebrates may be associated with the process of skeletal muscle development. CAP2 also played an important role in enhancing cell motility, which may promote a more invasive behavior in the progression of HCC. These findings highlight the link between development and cancer.

Immunohistochemical molecular analysis indicates hepatocellular carcinoma subgroups that reflect tumor aggressiveness

Histopathologic parameters and molecular markers are widely accepted as useful predictors of tumor aggressiveness in hepatocellular carcinoma (HCC). However, few studies have analyzed immunohistochemical profiles comprehensively in one series, a fact that has resulted in fragmentation of information that could be applied in clinical practice. We conducted immunohistochemical expression analysis of biliary/stem cell markers (cytokeratin 19, sal-like protein 4, epithelial cell adhesion molecule, and CD133), Wnt/β-catenin signaling-related molecules (β-catenin and glutamine synthetase), p53, and cell proliferation markers (Ki-67 and mitosis) in 162 HCCs surgically resected from 142 patients and analyzed the results with respect to clinicopathological features. Immunohistochemical analysis broadly identified 3 groups: the biliary/stem cell marker-positive group, the Wnt/β-catenin signaling-related marker-positive group, and the biliary/stem cell marker-negative and Wnt/β-catenin signaling-related marker-negative group. p53 was frequently positive in the biliary/stem cell marker-positive group, but it was rarely positive in the Wnt/β-catenin signaling-related marker-positive group. The biliary/stem cell marker-positive group exhibited poor tumor differentiation, increased frequency of portal vein invasion and/or intrahepatic metastasis, and highly proliferative activity. In contrast, the biliary/stem cell marker-negative and Wnt/β-catenin signaling-related marker-negative group exhibited better tumor differentiation, a decreased frequency of portal vein invasion and/or intrahepatic metastasis, and less proliferative activity. The Wnt/β-catenin signaling-related marker-positive group showed neither tendency. The biliary/stem cell marker-positive group had the shortest time to recurrence among the 3 groups. Immunohistochemical profiling of HCC reflects tumor aggressiveness and suggests the potential efficacy of immunohistochemistry-based subclassification of HCC.

Elastin Fiber Accumulation in Liver Correlates with the Development of Hepatocellular Carcinoma

Background & Aims The fibrosis stage, which is evaluated by the distribution pattern of collagen fibers, is a major predictor for the development of hepatocellular carcinoma (HCC) for patients with hepatitis C. Meanwhile, the role of elastin fibers has not yet been elucidated. The present study was conducted to determine the significance of quantifying both collagen and elastin fibers. Methods We enrolled 189 consecutive patients with hepatitis C and advanced fibrosis. Using Elastica van Gieson-stained whole-slide images of pretreatment liver biopsies, collagen and elastin fibers were evaluated pixel by pixel (0.46 μm/pixel) using an automated computational method. Consequently, fiber amount and cumulative incidences of HCC within 3 years were analyzed. Results There was a significant correlation between collagen and elastin fibers, whereas variation in elastin fiber was greater than in collagen fiber. Both collagen fiber (p = 0.008) and elastin fiber (p < 0.001) were significantly correlated with F stage. In total, 30 patients developed HCC during follow-up. Patients who have higher elastin fiber (p = 0.002) in addition to higher collagen fiber (p = 0.05) showed significantly higher incidences of HCC. With regard to elastin fiber, this difference remained significant in F3 patients. Furthermore, for patients with a higher collagen fiber amount, higher elastin was a significant predictor for HCC development (p = 0.02). Conclusions Computational analysis is a novel technique for quantification of fibers with the added value of conventional staging. Elastin fiber is a predictor for the development of HCC independently of collagen fiber and F stage.

Comprehensive Immune Profiling of Lung Adenocarcinomas Reveals Four Immunosubtypes with Plasma Cell Subtype a Negative Indicator

Lung cancer is a complex disease with variable outcomes. Immune cells from 114 cases were quantified immunohistochemically, identifying four immunosubtypes of lung adenocarcinoma (CD8, mast cell, macrophage/DC, and plasma cell) that could potentially be useful for therapy selection. Neoplastic cancer cells and cancer stroma (including infiltrating immune cells) determine the biology and prognosis of cancer. Various types of adaptive and innate immune cells are known to infiltrate the cancer stroma. However, the patterns and spatial distribution of immune cell infiltration as well as its association with tumor histology remain poorly understood. To address these issues, we comprehensively analyzed the infiltrating immune cells present in lung adenocarcinoma. The principal types of both adaptive and innate infiltrating immune cells were immunohistochemically evaluated in the predominant histologic components of 111 lung adenocarcinomas. The same analysis was also carried out on 143 samples of histologic subtypes making up more than 20% of tumors. As a result, plasma cells and B cells with interfollicular distribution were almost exclusively observed in invasive histologic subtypes, while an increased number of mast cells were observed in noninvasive histologic subtypes. Cluster analysis revealed four distinct immunosubtypes (CD8, mast cell, macrophage/dendritic cell, and plasma cell subtypes) based on the infiltrating immune cell profiles. These immunosubtypes correlated with histologic subtypes, and univariate and multivariate analyses identified the plasma cell subtype as an independent negative prognostic factor. These plasma cells may be one of the major producers of the immunosuppressive cytokine IL35 in cancer stroma. Cancer Immunol Res; 4(3); 234–47. ©2016 AACR.

Pathological findings of nonalcoholic steatohepatitis and nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease that has become increasingly common in the AsiaPacific region. The prevalence of NAFLD is reportedly as high as 30% in the Japanese population. Consequently, improved awareness and proper diagnosis of this disease are important. NAFLD can be broadly divided into nonalcoholic fatty liver (NAFL, also called simple steatosis), and nonalcoholic steatohepatitis (NASH). The distinction and diagnosis of NASH is important because NASH generally has more progressive features than NAFL. NASH is also different from other chronic liver diseases such as hepatitis C, hepatitis B, and alcoholic liver disease (ALD) which link directly to the etiology of the diseases. NASH may be caused by multiple environmental or endogenous factors including genetic backgrounds and may overlap with other liver diseases. Although the diagnosis of NASH can be supported by several investigative modalities, histopathology findings remain the gold standard. However, in addition to typical cases that are relatively easy to identify histologically, there are many borderline cases that are difficult to evaluate. This fact can result in discrepancies in diagnosis between pathologists. Inconsistent diagnostic criteria, different interpretations of major features, and the lack of standardized integrating scoring and staging system make the evaluation of NASH more difficult. The histopathological features of NASH include ‘steatosis’, ‘lobular inflammation’, ‘hepatocyte ballooning’, ‘MalloryDenk bodies’ and ‘fibrosis’. NASH is characterized by zone 3 accentuation of lesions. Among these features, ‘hepatocyte ballooning’, ‘Mallory-Denk bodies’ and ‘fibrosis’ are important for the interpretation of NASH and NAFL and to predict the progression of NAFLD. Hepatocyte ballooning’ is a ballooning degeneration, characterized by swelling and vacuolization with clear cytoplasm accompanied by the loss of the normal hexagonal shape of liver lobules. Ballooning degeneration and Mallory-Denk bodies represent liver cell injury and their presence is critical in establishing the diagnosis of NASH. Because degeneration refers to a continuous and reversible change of morphology commonly seen in injured cells, mild, and moderate to severe pathological changes can be observed contemporaneously in different regions of the liver. This characteristic may also cause inconsistency in recognizing and evaluating the presence of ballooning degeneration. Mallory-Denk bodies are irregular eosinophilic inclusion bodies typically seen as caterpillar-like aggregates. Their presence in NASH is generally less prominent than it is in alcoholic steatohepatitis (ASH). Their appearance may overlap with that of finely reticular or granular cytoplasm observed in ballooning degeneration. Burt et al. published an update on grading, staging systems, and histopathological features for the diagnosis and assessment of NAFLD in 2015. Also in Japan, the Japan Society of Hepatology organized a working group of liver pathologists to establish a consensus regarding the pathological findings of NASH and NAFLD. As a result, the Japan Society of Hepatology published a clinical guidebook illustrating the typical histology of definitive hepatocyte ballooning and Mallory-Denk bodies. This guidebook may help to improve and unify the histological interpretations in diagnosing NASH. Here, we report the pathological findings of steatosis, hepatocyte ballooning, Mallory-Denk bodies, and fibrosis, and integrate them into the current grading and staging systems of NASH.

Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase‐associated protein 2 (CAP2), which is a well‐conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.