Ai Sato

miR‐493 induction during carcinogenesis blocks metastatic settlement of colon cancer cells in liver

Liver metastasis is a major lethal complication associated with colon cancer, and post‐intravasation steps of the metastasis are important for its clinical intervention. In order to identify inhibitory microRNAs (miRNAs) for these steps, we performed ‘dropout’ screens of a miRNA library in a mouse model of liver metastasis. Functional analyses showed that miR‐493 and to a lesser extent miR‐493* were capable of inhibiting liver metastasis. miR‐493 inhibited retention of metastasized cells in liver parenchyma and induced their cell death. IGF1R was identified as a direct target of miR‐493, and its inhibition partially phenocopied the anti‐metastatic effects. High levels of miR‐493 and miR‐493*, but not pri‐miR‐493, in primary colon cancer were inversely related to the presence of liver metastasis, and attributed to an increase of miR‐493 expression during carcinogenesis. We propose that, in a subset of colon cancer, upregulation of miR‐493 during carcinogenesis prevents liver metastasis via the induction of cell death of metastasized cells.

Induction of the Stem-like Cell Regulator CD44 by Rho Kinase Inhibition Contributes to the Maintenance of Colon Cancer–Initiating Cells

The difficulty in expanding cancer-initiating cells in vitro is one of major obstacles for their biochemical characterization. We found that Rho kinase (ROCK) inhibitors as well as blebbistatin, a myosin II inhibitor, greatly facilitated the establishment of spheroids from primary colon cancer. The spheroid cells expressed cancer stem cell markers, showed the ability to differentiate, and induced tumors in mice. The spheroids were composed of cells that express various levels of CD44, whereas CD44(high) cells were associated with increased sphere-forming ability, expression of the activating form of β-catenin, and elevated levels of glycolytic genes, CD44(-/low) cells showed increased levels of differentiation markers and apoptotic cells. The spheroid cells expressed variant forms of CD44 including v6, and the induction of the variants was associated with the activating phosphorylation of c-Met. As expected from the predicted hierarchy, CD44(high) cells differentiated into CD44(-/low) cells. Unexpectedly, a fraction of CD44(-/low) cells generated CD44(high) cells, and the ROCK inhibitor or blebbistatin primed the transition by inducing CD44 expression. We propose that the transition from CD44(-/low) to CD44(high) state helps to maintain a CD44(high) fraction and the tumorigenic diversity in colon cancer.

Differential expression of nanog1 and nanogp8 in colon cancer cells

Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications

Recently, many types of in vitro 3‐D culture systems have been developed to recapitulate the in vivo growth conditions of cancer. The cancer 3‐D culture methods aim to preserve the biological characteristics of original tumors better than conventional 2‐D monolayer cultures, and include tumor‐derived organoids, tumor‐derived spheroids, organotypic multicellular spheroids, and multicellular tumor spheroids. The 3‐D culture methods differ in terms of cancer cell sources, protocols for cell handling, and the required time intervals. Tumor‐derived spheroids are unique because they are purposed for the enrichment of cancer stem cells (CSCs) or cells with stem cell‐related characteristics. These spheroids are grown as floating spheres and have been used as surrogate systems to evaluate the CSC‐related characteristics of solid tumors in vitro. Because eradication of CSCs is likely to be of clinical importance due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance, the investigation of tumor‐derived spheroids may provide invaluable clues to fight against cancer. Spheroid cultures have been established from cancers including glioma, breast, colon, ovary, and prostate cancers, and their biological and biochemical characteristics have been investigated by many research groups. In addition to the investigation of CSCs, tumor‐derived spheroids may prove to be instrumental for a high‐throughput screening platform or for the cultivation of CSC‐related tumor cells found in the circulation or body fluids.

Clinical outcome of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly in the rituximab era.

Diffuse large B‐cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma. The incidence of Epstein–Barr virus (EBV)‐positive DLBCL in Asian and Latin American countries ranges from 8 to 10%. The prognosis of patients with EBV‐positive DLBCL is controversial. To compare the clinical outcome of EBV‐positive and EBV‐negative patients with DLBCL in the rituximab era, we analyzed 239 patients with de novo DLBCL diagnosed between January 2007 and December 2011. The presence of EBV in lymphoma cells was detected using EBV‐encoded RNA in situ hybridization, and it was found that 18 (6.9%) of 260 patients with diagnosed DLBCL tested positive. Among the 260 cases, 216 cases were treated with rituximab plus chemotherapy, as were 8 EBV‐positive DLBCL patients. The median overall survival and progression‐free survival times in patients with EBV‐positive DLBCL were 8.7 months and 6.8 months, respectively. The median overall survival and progression‐free survival could not be determined in EBV‐negative DLBCL patients (P = 0.0002, P < 0.0001, respectively). The outcome of patients with EBV‐positive DLBCL remains poor, even in the rituximab era.

MKK7 mediates miR-493-dependent suppression of liver metastasis of colon cancer cells

The prognosis of advanced colon cancer patients is profoundly affected by the presence or absence of liver metastasis. miR‐493 functions as a potent suppressor of liver metastasis, and low‐level miR‐493 expression in human primary colon cancer is associated with an elevated incidence of liver metastasis. We previously showed that IGF1R is a target gene of miR‐493, and that the inhibition of IGF1R partly explains how miR‐493 suppresses liver metastasis. However, major functional targets that mediate the antimetastatic activity of miR‐493 remain elusive. Here, we extended our search for target genes and identified MKK7, a mitogen‐activated protein kinase kinase, as a novel target of miR‐493. miR‐493 inhibits MKK7 expression by targeting the binding site at the 3′‐UTR of the mkk7 gene. MKK7 was expressed in six out of seven colon cancer cell lines examined but not in non‐transformed colon epithelial cells, and its expression was required for the activating phosphorylation of JNK. RNA interference‐mediated inhibition of MKK7 resulted in marked suppression of liver metastasis of colon cancer cells. A significant decrease of metastasized cells by the MKK7 knockdown was observed, even at early stages of the metastatic settlement, in accordance with a time course of the miR‐493‐mediated inhibition of the metastasis. Immunohistochemical examination in human primary colon tumors revealed that the occurrence of liver metastasis is associated with elevated levels of MKK7. Thus, MKK7 is a major functional target of miR‐493, and its suppression thwarts liver metastasis of colon cancer cells.

Single nucleotide polymorphisms of cytarabine metabolic genes influence clinical outcome in acute myeloid leukemia patients receiving high-dose cytarabine therapy

Cytarabine arabinoside (Ara-C) is the most important agent for treating acute myeloid leukemia (AML). Here, we genotyped 11 single nucleotide polymorphisms (SNPs) of seven Ara-C metabolism-related genes in 39 AML patients who had received high-dose Ara-C as a single-agent treatment. Univariate analysis identified three SNPs that were significantly associated with shorter time-to-relapse (TTR): CTPS rs12144160 GG compared to AA/AG, DCTD rs9990999 AG/GG compared to AA, and SLC29A1 rs693955 CC compared to AA/AC. Multivariate analysis of TTR revealed the SLC29A1 rs693955 CC genotype and first induction failure to be significantly associated with a shorter TTR. The DCTD rs9990999 AG/GG and SLC29A1 rs693955 CC genotypes were also significantly associated with shorter duration of neutropenia. The results of our study suggest that SNP analysis can be an important tool in improving drug responsiveness and enabling a better understanding of this condition and the development of tailor-made treatments for AML patients who benefit from consolidated high-dose Ara-C therapy.

Establishment and Characterization of an In Vitro Model of Ovarian Cancer Stem-like Cells with an Enhanced Proliferative Capacity

The establishment of cancer stem-like cell (CSC) culture systems may be instrumental in devising strategies to fight refractory cancers. Inhibition of the Rho kinase ROCK has been shown to favorably affect CSC spheroid cultures. In this study, we show how ROCK inhibition in human serous ovarian cancer (SOC) cells can help establish a CSC system, which illuminates cancer pathophysiology and its treatment in this setting. In the presence of a ROCK kinase inhibitor, spheroid cultures of SOC cells expressed characteristic CSC markers including ALDH1A1, CD133, and SOX2, along with differentiation and tumorigenic capabilities in mouse xenograft models of human SOC. High expression levels of ALDH, but not CD133, correlated with spheroid formation CSC marker expression and tumor forming capability. In clinical specimens of SOC, high levels of ALDH1A1 correlated with advanced stage and poor prognosis. Pharmacologic or genetic blockade of ALDH blocked cell proliferation and reduced expression of SOX2, the genetic ablation of which abolished spheroid formation, whereas SOX2 overexpression inhibited ALDH1A1 expression and blocked spheroid proliferation. Taken together, our findings illustrated a new method to culture human ovarian CSC, and they defined a reciprocal regulatory relationship between ALDH1A1 and SOX2, which impacts ovarian CSC proliferation and malignant progression.

Clinicopathological characteristics and genomic profile of primary sinonasal tract diffuse large B‐cell lymphoma (DLBCL) reveals gain at 1q31 and RGS1 encoding protein; high RGS1 immunohistochemical expression associates with poor overall survival in DLBCL NOS

We aimed to define the clinicopathological characteristics of 29 primary sinonasal diffuse large B cell lymphoma (DLBCLsn) in a series of 240 cases of DLBCL not otherwise specified [DLBCLall (NOS)], including DLBCLsn training set (n = 11) and validation set (n = 18), and DLBCLnon‐sn (n = 211).

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkins lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.