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Defective DNA mismatch repair creates a strong mutator phenotype, recognized as microsatellite instability (MSI). Various next-generation sequencing-based methods for evaluating cancer MSI status have been established, and NGS-based studies have thoroughly described MSI-driven tumorigenesis. Accordingly, high-frequency MSI (MSI-H) has been detected in 81 tumor types, including those in which MSI was previously underrated. The findings have increased the use of immunotherapy, which is assumed to be efficient in tumors having a high mutation burden and/or neoantigen load. In MSI tumorigenesis, positively and negatively selected driver gene mutations have been characterized in colorectal cancers. Recent advancements in genome-wide studies of MSI-H cancers have developed novel diagnostic and therapeutic approaches, including CXCR2 inhibitor, a synthetic lethal therapy targeting the Werner gene and inhibition of nonsense-mediated mRNA decay. MSI is a predictive marker for chemotherapy as well as immunotherapy. Thus, analyses of MSI status and MSI-related alterations in cancers are clinically relevant. We present an update on MSI-driven tumorigenesis, focusing on a novel landscape of diagnostic and therapeutic approaches.

Oligonucleotide therapeutics, drugs consisting of 10-50 nucleotide-long single- or double-stranded DNA or RNA molecules that can bind to specific DNA or RNA sequences or proteins, include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamers, and decoys. These oligonucleotide therapeutics could potentially become the third pillar of drug development. In particular, ASOs and siRNAs are advanced tools that are widely used to silence gene expression. They are used in clinical trials, as they have high specificity for target mRNAs and non-coding RNAs and limited toxicity. However, their clinical application remains challenging. Although chemotherapy has benefits, it has severe adverse effects in many patients. Therefore, new modalities for targeted molecular therapy against tumors, including oligonucleotide therapeutics, are required, and they should be compatible with diagnosis using next-generation sequencing. This review provides an overview of the therapeutic uses of ASOs, siRNAs, and miRNAs in clinical studies on malignant tumors. Understanding previous research and development will help in developing novel oligonucleotide therapeutics against malignant tumors.

The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2‐step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN‐2B) plus interferon‐β (IFNβ); (ii) SVN‐2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN‐2B plus IFNβ. The primary endpoint was progression‐free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty‐three patients were randomly assigned to receive SVN‐2B plus IFNβ (n = 30), SVN‐2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B‐specific CTLs were found to be increased in the SVN‐2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN‐2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN‐2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN‐2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).

Deficient DNA mismatch repair causes a robust mutator phenotype known as microsatellite instability (MSI). MSI is a feature of Lynch syndrome-related cancers and is also found in approximately 15% of sporadic gastric, colorectal, and endometrial cancers. Epigenetic inactivation of the MLH1 gene is often associated with sporadic MSI cancers. Recent next-generation sequencing (NGS)-based analyses have comprehensively characterized MSI-positive (MSI+) cancers, and several approaches for detecting the MSI phenotype of tumors using NGS have been developed. The FDA has recently granted accelerated approval to an anti-PD-1 antibody, pembrolizumab, for use in pediatric and adult patients with MSI+ solid tumors. Genome-wide analyses using NGS have revealed that hypermutation defined as >10 somatic mutations per megabase appears to be more prevalent than previously estimated, affecting approximately 17% of adult cancers. These results potentially expand the use of immunotherapy, which is thought to be effective in cancers with an increased mutational burden. Therefore, evaluation of MSI and MSI-associated molecular changes in tumors has emerged as clinically important. MSI is a valuable diagnostic marker of Lynch syndrome and a potential predictive marker for chemotherapy and immunotherapy efficacy. Here, we provide an update on MSI-associated cancers, focusing on findings obtained by genome-wide analyses using NGS, and the predictive role of MSI in immune checkpoint immunotherapy.

PRDM14 is highly expressed in several cancers but is not detected in normal tissues. It confers cancer stem cell-like properties, including chemoresistance and distant metastasis, to cancer cells. Herein, we aimed to develop a highly effective therapy against advanced stage cancer based on intravenously delivered PRDM14-targeted siRNA. First, we examined PRDM14 expression and gene amplification in breast and pancreatic tumors and cell lines. PRDM14 was expressed in breast cancer, including the triple-negative subtype, and pancreatic cancer. PRDM14 was amplified in 23.8% of patients with PRDM14+ breast cancer. Next, we investigated the inoculated tumor growth and distant metastasis following PRDM14 depletion by administering mice with PRDM14-specific chimeric siRNA combined with a novel branched PEGylated poly-L-ornithine (PLO)-based intravenous drug delivery system, designated PRDM14 unit polyion complex (uPIC) (n = 6/group). Inhibition of PRDM14 expression with PRDM14 uPIC by systemic intravenous injection effectively reduced tumor size and metastasis in vivo, thereby improving survival. Finally, pharmacokinetic/toxicokinetic analyses were performed on PRDM14 uPIC, which was intravenously administered to rats (n = 10-15/group) and cynomolgus monkeys (n = 3-5/group), twice weekly for 4 weeks. This revealed that PRDM14 uPIC was relatively nontoxic and the siRNA exposure in serum was greater than that predicted by the administered dose ratio when delivered as a uPIC. Taken together, our study indicated that PRDM14 uPIC is highly effective in suppressing malignant features of solid cancers and does not cause severe toxicity, making it a promising therapeutic agent for cancer treatment.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer that is typically diagnosed at a later stage with metastases and is difficult to treat. Therefore, investigating the mechanism of PDAC initiation is important to aid early-stage cancer detection. PRDM14 is a transcription factor that maintains pluripotency in embryonic stem cells and is overexpressed in several cancers. We previously reported that PRDM14 is overexpressed and regulates cancer stem-like phenotypes in PDAC, and herein, we assess whether PRDM14 expression increases prior to tumorigenesis. Through immunohistochemistry analyses of clinical tissues, we detected PRDM14-positive cells in precursor pancreatic intraepithelial neoplasia and chronic pancreatitis, which is a risk factor for PDAC, lesions. PRDM14 staining in chronic pancreatitis was as high as that in PDAC and cancer adjacent tissues. We induced pancreatitis in mouse models by cerulein injection, and observed that PRDM14 expression increased in chronic pancreatitis models but not in control or acute pancreatitis mice. Moreover, cerulein treatment increased PRDM14 expression in PK-1 and AsPC-1 pancreatic cancer cell lines. Our results suggest that inflammation increases the expression of PRDM14, which regulates cancer stem-like phenotypes, and this occurs prior to PDAC initiation and progression.

[][1] The sad news that Ralph A. Reisfeld, PhD, passed away on December 6, 2020 reached us early this year, and we would like to reflect on Ralph's long career and distinctive contribution to biomedical science and in particular to the field of cancer research. Ralph was well known

PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem-like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates-glucose-regulated protein 78 (GRP78) and heat shock protein 90-α (HSP90α)-were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA-MB231). Surface plasmon resonance analysis using GST-PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C-terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase-based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem-like CD24- CD44+ and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation.

Pancreatic cancer is one of the most lethal types of cancer, with aggressive properties characterized by metastasis, recurrence and drug resistance. Cancer stem cells are considered to be responsible for these properties. PRDM14, a transcriptional regulator that maintains pluripotency in embryonic stem cells, is overexpressed in some cancers. Here, we assessed PRDM14 expression and the effects of PRDM14 knockdown on cancer stem-like phenotypes in pancreatic cancer. We observed that PRDM14 protein was overexpressed in pancreatic cancer tissues compared with normal pancreatic tissues. Using lentiviral shRNA-transduced pancreatic cancer cells, we found that PRDM14 knockdown decreased sphere formation, number of side population and cell surface marker-positive cells and subcutaneous xenograft tumors and liver metastasis in mice. This was accompanied by upregulation of some microRNAs (miRNAs), including miR-125a-3p. miR-125a-3p, a tumor suppressor that is down-regulated in pancreatic cancer, has been suggested to regulate the expression of the Src-family kinase, Fyn. In PRDM14-knockdown cells, Fyn was expressed at lower levels and downstream proteins were less activated. These changes were considered to cause suppression of the above cancer phenotypes. In addition, we used small interfering RNA (siRNA)-based therapy targeting PRDM14 in a mouse model of liver metastasis induced using MIA-PaCa2 cells, and this treatment significantly decreased metastasis and in vitro migration. Taken together, these results suggest that targeting the overexpression of PRDM14 suppresses cancer stem-like phenotypes, including liver metastasis, via miRNA regulation and siRNA-based therapy targeting it shows promise as a treatment for patients with pancreatic cancer.

// Hiroaki Taniguchi 1 , Daisuke Hoshino 2 , Chiharu Moriya 1 , Hitoshi Zembutsu 3 , Nobuhiro Nishiyama 4 , Hiroyuki Yamamoto 5 , Kazunori Kataoka 6 and Kohzoh Imai 1, 7 1 The Center for Antibody and Vaccine Therapy, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan 2 Cancer Biology Department, The Kanagawa Cancer Center Research Institute, Kanagawa 241-0815, Japan 3 Division of Genetics, National Cancer Center Research Institute, Tokyo 104-0045, Japan 4 Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, Kanagawa 226-8503, Japan 5 Department of Gastroenterology and Hepatology, School of Medicine, St. Marianna Medical University, Kanagawa 216-0015, Japan 6 Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan 7 The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan Correspondence to: Hiroaki Taniguchi, email: h-tani@ims.u-tokyo.ac.jp Keywords: breast cancer stem cell, cancer stemness, epigenetic alterations, nucleic acid medicine, PRDM14 Received: November 11, 2016 Accepted: March 22, 2017 Published: April 01, 2017 ABSTRACT PR domain zinc finger protein 14 (PRDM14) maintains stemness in embryonic stem cells via epigenetic mechanisms. Although PRDM14 is elevated in several cancers, it is unclear if and how PRDM14 confers stem cell-like properties and epigenetic changes to cancer cells. Here, we examined the phenotypic characteristics and epigenetic and gene expression profiles of cancer cells that differentially express PRDM14, and assessed the potential of PRDM14-targeted cancer therapy. PRDM14 expression was markedly increased in many different cancer types and correlated with poor survival of breast cancer patients. PRDM14 conferred stem cell-like phenotypes to cancer cells and regulated the expression of genes involved in cancer stemness, metastasis, and chemoresistance. PRDM14 also reduced the methylation of proto-oncogene and stemness gene promoters and PRDM14-binding regions were primarily occupied by histone H3 Lys-4 trimethylation (H3K4me3), both of which are positively correlated with gene expression. Moreover, strong PRDM14 binding sites coincided with promoters containing both H3K4me3 and H3K27me3 histone marks. Using calcium phosphate hybrid micelles as an RNAi delivery system, silencing of PRDM14 expression by chimera RNAi reduced tumor size and metastasis in vivo without causing adverse effects. Conditional loss of PRDM14 function also improved survival of MMTV-Wnt-1 transgenic mice, a spontaneous model of murine breast cancer. Our findings suggest that PRDM14 inhibition may be an effective and novel therapy for cancer stem cells.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer by providing new options in addition to existing therapies. However, peptide vaccination therapies still represent an attractive approach, because of the antigen specificity. We identified survivin 2B peptide (SVN‐2B), a 9‐mer antigenic peptide encoded by survivin, and an SVN‐2B peptide vaccine‐based phase II randomized clinical trial targeting unresectable and refractory pancreatic carcinoma was undertaken. The SVN‐2B peptide vaccine did not have any statistically significant clinical benefits in that study. Therefore, we undertook an autopsy study to analyze the immune status of the pancreatic cancer lesions at the histological level. Autopsies were carried out in 13 patients who had died of pancreatic cancer, including 7 who had received SVN‐2B peptide vaccination and 6 who had not, as negative controls. The expression of immune‐related molecules was analyzed by immunohistochemical staining. Cytotoxic T lymphocytes were analyzed by tetramer staining and enzyme‐linked immunospot assay. Histological analysis revealed dense infiltration of CD8+ T cells in some lesions in patients who had received the SVN‐2B peptide vaccine. A high rate of programmed cell death ligand 1 expression in cancer cells was observed in these cases, indicating that CTLs were induced by SVN‐2B peptide vaccination and had infiltrated the lesions. The lack of a significant antitumor effect was most likely attributable to the expression of immune checkpoint molecules. These findings suggest that the combination of a tumor‐specific peptide vaccine and an ICI might be a promising approach to the treatment of pancreatic carcinoma in the future.

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation, drug and radiation resistance, invasive growth, metastasis, and tumor relapse, which are the main causes of cancer-related deaths. Gastrointestinal cancers are the most common malignancies and still the most frequent cause of cancer-related mortality worldwide. Because gastrointestinal CSCs are also thought to be resistant to conventional therapies, an effective and novel cancer treatment is imperative. The first reported CSCs in a gastrointestinal tumor were found in colorectal cancer in 2007. Subsequently, CSCs were reported in other gastrointestinal cancers, such as esophagus, stomach, liver, and pancreas. Specific phenotypes could be used to distinguish CSCs from non-CSCs. For example, gastrointestinal CSCs express unique surface markers, exist in a side-population fraction, show high aldehyde dehydrogenase-1 activity, form tumorspheres when cultured in non-adherent conditions, and demonstrate high tumorigenic potential in immunocompromised mice. The signal transduction pathways in gastrointestinal CSCs are similar to those involved in normal embryonic development. Moreover, CSCs are modified by the aberrant expression of several microRNAs. Thus, it is very difficult to target gastrointestinal CSCs. This review focuses on the current research on gastrointestinal CSCs and future strategies to abolish the gastrointestinal CSC phenotype.

Mutation in K-RAS (K-RAS-MT) plays important roles in both cancer progression and resistance to anti-epidermal growth factor receptor (EGFR) therapy in gastrointestinal tumors. Insulin-like growth factor-1 receptor (IGF-1R) signaling is required for carcinogenicity and progression of many tumors as well. We have previously shown successful therapy for gastrointestinal cancer cell lines bearing a K-RAS mutation using an anti-IGF-1R monoclonal antibody. In this study, we sought to evaluate effects of forced K-RAS-MT expression on gastrointestinal cancer cell lines representing a possible second resistance mechanism for anti-EGFR therapy and IGF-1R-targeted therapy for these transfectants. We made stable transfectants of K-RAS-MT in two gastrointestinal cancer cell lines, colorectal RKO and pancreatic BxPC-3. We assessed the effect of forced expression of K-RAS-MT on proliferation, apoptosis, migration, and invasion in gastrointestinal cancer cells. Then we assessed anti-tumor effects of dominant negative IGF-1R (IGF-1R/dn) and an IGF-1R inhibitor, picropodophyllin, on the K-RAS-MT transfectants. Overexpression of K-RAS-MT in gastrointestinal cancer cell lines led to more aggressive phenotypes, with increased proliferation, decreased apoptosis, and increased motility and invasion. IGF-1R blockade suppressed cell growth, colony formation, migration, and invasion, and up-regulated chemotherapy-induced apoptosis of gastrointestinal cancer cells, even when K-RAS-MT was over-expressed. IGF-1R blockade inhibited the Akt pathway more than the extracellular signal-regulated kinase (ERK) pathway in the K-RAS-MT transfectants. IGF-1R/dn, moreover, inhibited the growth of murine xenografts expressing K-RAS-MT. Thus, K-RAS-MT might be important for progressive phonotype observed in gastrointestinal cancers. IGF-1R decoy is a candidate molecular therapeutic approach for gastrointestinal cancers even if K-RAS is mutated. © 2016 Wiley Periodicals, Inc.

The PRDI-BF1 and RIZ (PR) domain zinc finger protein 14 (PRDM14) is upregulated in approximately 60% of breast cancers, some of which exhibit gene amplification. In contrast, PRDM14 is not expressed in normal, and differentiated tissues. PRDM14+ breast cancer cells are resistant to chemotherapy drugs, are tumorigenic, and metastasize to the lungs. It is commonly assumed that genes that are overexpressed in cancers, such as PRDM14, are effective targets for new therapies that specifically abrogate the expression of these genes. RNA interference of PRDM14, a gene expressed by breast cancer cells, reduced the size of tumors and lung metastases in nude mice. In this chapter, we introduce the concept and methods to develop and apply systematically injected small interfering RNA therapy for breast cancer models in vivo.

Polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that correlates with the regulation of invasion and metastasis and is overexpressed in human cancers such as colorectal cancer. MicroRNA-31 (miR-31) plays an oncogenic role and is associated with BRAF mutation and poor prognosis in colorectal cancer. EZH2 is functionally considered to suppress miR-31 expression in human cancers; however, no study has reported its relationship with colon cancer. We therefore evaluated EZH2 expression using immunohistochemistry and assessed miR-31 and epigenetic alterations using 301 colorectal carcinomas and 207 premalignant lesions. Functional analysis was performed to identify the association between EZH2 and miR-31 using cancer cell lines. In the current study, negative, weak, moderate, and strong EZH2 expressions were observed in 15%, 19%, 25%, and 41% of colorectal cancers, respectively. EZH2 was inversely associated with miR-31 (P < 0.0001), independent of clinicopathological and molecular features. In a multivariate stage-stratified analysis, high EZH2 expression was related to favorable prognosis (P = 0.0022). Regarding premalignant lesions, negative EZH2 expression was frequently detected in sessile serrated adenomas/polyps (SSA/Ps) (76%; P < 0.0001) compared with hyperplastic polyps, traditional serrated adenomas, and non-serrated adenomas (25–36%). Functional analysis demonstrated that the knockdown of EZH2 increased miR-31 expression. In conclusion, an inverse association was identified between EZH2 and miR-31 in colorectal cancers. Our data also showed that upregulation of EZH2 expression may be rare in SSA/Ps. These results suggest that EZH2 suppresses miR-31 in colorectal cancer and may correlate with differentiation and evolution of serrated pathway.

Viable and stable human cancer cell lines and animal models combined with adequate clinical information are essential for future advances in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available; however, they lack detailed information on the patient from which they originate, including disease phenotype and drug sensitivity. Patient-derived xenografts (PDX) with clinical information (so-called 'cancer xenopatients') are a promising advance that may accelerate the development of anticancer therapies. We established 61 PDX lines from 116 surgically removed tumor tissues inoculated subcutaneously into NOG mice (53% success rate). PDX lines were established from various types of epithelial tumors and also from sarcomas, including gastrointestinal stromal tumors and Ewing/PNET sarcomas. The metastatic tumors yielded PDX lines more effectively (65%) than the primary tumors (27%, P

Human gut microbiota is being increasingly recognized as a player in colorectal cancers (CRCs). Evidence suggests that Fusobacterium nucleatum (F. nucleatum) may contribute to disease progression and is associated with CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) in CRCs; however, to date, there are no reports about the relationship between F. nucleatum and molecular features in the early stage of colorectal tumorigenesis. Therefore, we investigated the presence of F. nucleatum in premalignant colorectal lesions. In total, 465 premalignant lesions (343 serrated lesions and 122 non-serrated adenomas) and 511 CRCs were studied. We determined the presence of F. nucleatum and analyzed its association with molecular features including CIMP, MSI and microRNA-31 status. F. nucleatum was detected in 24% of hyperplastic polyps, 35% of sessile serrated adenomas (SSAs), 30% of traditional serrated adenomas (TSAs) and 33% of non-serrated adenomas. F. nucleatum was more frequently detected in CIMP-high premalignant lesions than in CIMP-low/zero lesions (p = 0.0023). In SSAs, F. nucleatum positivity increased gradually from sigmoid colon to cecum (p = 0.042). F. nucleatum positivity was significantly higher in CRCs (56%) than in premalignant lesions of any histological type (p

Recently, bacterial infection causing periodontal disease has attracted considerable attention as a risk factor for pancreatic cancer. Fusobacterium species is an oral bacterial group of the human microbiome. Some evidence suggests that Fusobacterium species promote colorectal cancer development; however, no previous studies have reported the association between Fusobacterium species and pancreatic cancer. Therefore, we examined whether Fusobacterium species exist in pancreatic cancer tissue. Using a database of 283 patients with pancreatic ductal adenocarcinoma (PDAC), we tested cancer tissue specimens for Fusobacterium species. We also tested the specimens for KRAS, NRAS, BRAF and PIK3CA mutations and measured microRNA-21 and microRNA-31. In addition, we assessed epigenetic alterations, including CpG island methylator phenotype (CIMP). Our data showed an 8.8% detection rate of Fusobacterium species in pancreatic cancers; however, tumor Fusobacterium status was not associated with any clinical and molecular features. In contrast, in multivariate Cox regression analysis, compared with the Fusobacterium species-negative group, we observed significantly higher cancer-specific mortality rates in the positive group (p = 0.023). In conclusion, Fusobacterium species were detected in pancreatic cancer tissue. Tumor Fusobacterium species status is independently associated with a worse prognosis of pancreatic cancer, suggesting that Fusobacterium species may be a prognostic biomarker of pancreatic cancer.

The Japan Diabetes Society/Japanese Cancer Association Joint Committee on Diabetes and Cancer published its first report in July 2013 on the epidemiological assessment of the associations of diabetes with cancer risk/prognosis, the common risk factors for diabetes and cancer, and cancer risk associated with diabetes treatment. The Joint Committee continued its work to assess the role of glycemic control in the development of cancer in patients with diabetes. This review shows that high‐quality evidence examining the association between glycemic control and cancer risk is lacking. In 2014, the Japan Diabetes Society (JDS) and the Japanese Cancer Association (JCA) restarted the JDS/JCA Joint Committee on Diabetes and Cancer, which published the second committee report in Japanese [1]. This is the English version of that report. This article has been jointly published in Diabetology International (doi:10.1007/s13340‐016‐0257‐z) and Cancer Science by the Japan Diabetes Society and the Japanese Cancer Association. Members of the JDS/JCA Joint Committee on Diabetes and Cancer. JDS: Mitsuhiko Noda, Kohjiro Ueki, Masato Kasuga, Naoko Tajima, and Ken Ohashi; Editorial collaborators: Atsushi Goto and Hiroshi Noto; JCA: Ryuichi Sakai, Shoichiro Tsugane, Nobuyuki Hamajima, Kazuo Tajima, Kohzoh Imai, and Hitoshi Nakagama.

PRDI-BF1 and RIZ homology (PR) domain zinc finger protein 14 (PRDM14) contains a PR domain related to the SET methyltransferase domain and zinc finger motifs. PRDM14 maintains stemness in embryonic stem cells and primordial germ cells via epigenetic mechanisms. PRDM14, however, is not expressed in normal differentiated tissues. We and other groups previously reported that PRDM14 expression is markedly higher in some types of cancers compared to the corresponding normal tissues. PRDM14 confers stem cell-like characteristics upon cancer cells, such as sphere formation, dye efflux, chemotherapy resistance, proliferation, and distant metastasis. Cancer stem cells (CSCs) are thought to be responsible for tumor initiation, drug and radiation resistance, invasive growth, metastasis, and tumor relapse, which are the primary causes of cancer-related deaths. Because CSCs are also thought to be resistant to conventional therapies, an effective and novel therapeutic approach for CSCs is imperative.RNAi silencing of PRDM14 expressed by breast and pancreatic cancer cells reduced tumor size and distant metastasis of these cells in nude mice. Inhibition of PRDM14 expression by cancer cells may be an effective and radical therapy for solid cancers. In this chapter, we discuss methods for studying CSC-like properties in cancer cells and describe the use of siRNA with a drug delivery system by systemic injection in vivo.

Deficient DNA mismatch repair (MMR) results in a strong mutator phenotype known as microsatellite instability (MSI), which is a hallmark of Lynch syndrome-associated cancers. MSI is characterized by length alterations within simple repeated sequences that are called microsatellites. Lynch syndrome is primarily caused by mutations in the MMR genes, mainly MLH1 and MSH2, and less frequently in MSH6, and rarely PMS2, and large genomic rearrangements account for 5-20 % of all mutations. Germ line hemiallelic methylations of MLH1 or MSH2 are termed as epimutations and have been identified as causative of Lynch syndrome. Moreover, germ line 3' deletions of EPCAM gene is involved in MSH2 methylation. MSI is also observed in about 15 % of sporadic colorectal cancer (CRC), gastric cancer (GC), and endometrial cancer (EC), and at lower frequencies in other cancers, often in association with hypermethylation of the MLH1 gene. Trimethylation of histone H3 on Lys36 (H3K36 me3) is an epigenetic histone mark that was required for DNA MMR in vivo. Thus, mutations in the H3K36 trimethyltransferase SETD2 have been reported as a potential cause of MSI. Genetic, epigenetic, and transcriptomic differences have been identified between cancers with and without MSI. Recent comprehensive molecular characterizations of CRC, EC, and GC by The Cancer Genome Atlas indicate that MSI+ cancers are distinct biological entities. The BRAF V600E mutation is specifically associated with sporadic MSI+ CRCs with methylated MLH1, but is not associated with Lynch syndrome-related CRCs. Accumulating evidence indicates a role of interactions between MSI and microRNA (miRNA) in the pathogenesis of MSI-positive (MSI+) cancer. As another new mechanism underlying MSI, overexpression of miR-155 or miR-21 has been shown to downregulate the expression of the MMR genes. Gene targets of frameshift mutations caused by MSI are involved in various cellular functions, including DNA repair (MSH3 and MSH6), cell signaling (TGFBR2 and ACVR2A), apoptosis (BAX), epigenetic regulation (HDAC2 and ARID1A), and miRNA processing (TARBP2 and XPO5), and a subset of MSI+ CRCs reportedly shows the mutated miRNA machinery phenotype. Moreover, microsatellite repeats in miRNA genes, such as hsa-miR-1273c, may be novel MSI targets for CRC, and mutations in noncoding regulatory regions of MRE11, BAX (BaxΔ2), and HSP110 (HSP110ΔE9) may affect the efficiency of chemotherapy. Thus, analyses of MSI and its related molecular alterations in cancers are increasingly relevant in clinical settings, and MSI is a useful screening marker for identifying patients with Lynch syndrome and a prognostic factor for chemotherapeutic interventions. In this review, we summarize recent advances in the pathogenesis of MSI and focus on genome-wide analyses that indicate the potential use of MSI and related alterations as biomarkers and novel therapeutic targets.

The absence of highly specific markers for malignant mesothelioma (MM) has served an obstacle for its diagnosis and development of molecular-targeting therapy against MM. Here, we show that a novel mucin-like membrane protein, sialylated protein HEG homolog 1 (HEG1), is a highly specific marker for MM. A monoclonal antibody against sialylated HEG1, SKM9-2, can detect even sarcomatoid and desmoplastic MM. The specificity and sensitivity of SKM9-2 to MM reached 99% and 92%, respectively; this antibody did not react with normal tissues. This accurate discrimination by SKM9-2 was due to the recognition of a sialylated O-linked glycan with HEG1 peptide. We also found that gene silencing of HEG1 significantly suppressed the survival and proliferation of mesothelioma cells; this result suggests that HEG1 may be a worthwhile target for function-inhibition drugs. Taken together, our results indicate that sialylated HEG1 may be useful as a diagnostic and therapeutic target for MM.

The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP-high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA-31 (miR-31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP-high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR-31 and epigenetic features in serrated pathway, and hypothesized that miR-31 supports the "colorectal continuum" concept. We evaluated miR-31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non-serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR-31 expression and CIMP-high status in serrated lesions with BRAF mutation (p = 0.0001). In contrast, miR-31 was slightly but insignificantly associated with CIMP status in the cases with wild-type BRAF. miR-31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high-grade dysplasia. The frequency of miR-31, BRAF mutation CIMP-high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR-31 expression was associated with CIMP-high status in serrated lesions with BRAF mutation. Our data also suggested that miR-31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum.

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O6-methylguanine (O6MeG) adducts through O6MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-β signaling because such properties were completely abrogated by absorption of TGF-β under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-β-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease. Stem Cells 2014;32:913–925

Background Dysregulation of microRNAs (miRNAs) has been implicated in bladder cancer (BCa), although the mechanism is not fully understood. Objective We aimed to explore the involvement of epigenetic alteration of miRNA expression in BCa. Design, setting, and participants Two BCa cell lines (T24 and UM-UC-3) were treated with 5-aza-2′-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which their miRNA expression profiles were analyzed using a TaqMan array (Life Technologies, Carlsbad, CA, USA). Bisulfite pyrosequencing was used to assess miRNA gene methylation in 5 cancer cell lines, 83 primary tumors, and 120 preoperative and 47 postoperative urine samples. Outcome measurements and statistical analysis Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic performance of the miRNA gene panel. Results and limitations Of 664 miRNAs examined, 146 were upregulated by 5-aza-dC plus PBA. CpG islands were identified in the proximal upstream of 23 miRNA genes, and 12 of those were hypermethylated in cell lines. Among them, miR-137, miR-124-2, miR-124-3, and miR-9-3 were frequently and tumor-specifically methylated in primary cancers (miR-137: 68.7%; miR-124-2: 50.6%; miR-124-3: 65.1%; miR-9-3: 45.8%). Methylation of the same four miRNAs in urine specimens enabled BCa detection with 81% sensitivity and 89% specificity; the area under the ROC curve was 0.916. Ectopic expression of silenced miRNAs in BCa cells suppressed growth and cell invasion. Conclusions Our results indicate that epigenetic silencing of miRNA genes may be involved in the development of BCa and that methylation of miRNA genes could be a useful biomarker for cancer detection.

Colorectal cancers (CRCs) exhibit multiple genetic alterations, including allelic imbalances (copy number alterations, CNAs) at various chromosomal loci. In addition to genetic aberrations, DNA methylation also plays important roles in the development of CRC. To better understand the clinical relevance of these genetic and epigenetic abnormalities in CRC, we performed an integrative analysis of copy number changes on a genome-wide scale and assessed mutations of TP53, KRAS, BRAF, and PIK3CA and DNA methylation of six marker genes in single glands isolated from 39 primary tumors. Array-based comparative genomic hybridization (array-CGH) analysis revealed that genomic losses commonly occurred at 3q26.1, 4q13.2, 6q21.32, 7q34, 8p12-23.3, 15qcen and 18, while gains were commonly found at 1q21.3-23.1, 7p22.3-q34, 13q12.11-14.11, and 20. The total numbers and lengths of the CNAs were significantly associated with the aberrant DNA methylation and Dukes' stages. Moreover, hierarchical clustering analysis of the array-CGH data suggested that tumors could be categorized into four subgroups. Tumors with frequent DNA methylation were most strongly enriched in subgroups with infrequent CNAs. Importantly, Dukes' D tumors were enriched in the subgroup showing the greatest genomic losses, whereas Dukes' C tumors were enriched in the subgroup with the greatest genomic gains. Our data suggest an inverse relationship between chromosomal instability and aberrant methylation and a positive association between genomic losses and distant metastasis and between genomic gains and lymph node metastasis in CRC. Therefore, DNA copy number profiles may be predictive of the metastatic behavior of CRCs. © 2012 Wiley Periodicals, Inc.

Aberrant DNA methylation is implicated in the epigenetic field defect seen in gastric cancer. Our aim in this study was to identify predictive biomarkers by screening for DNA methylation in noncancerous background gastric mucosa from patients with gastric cancer. Using methylated-CpG island amplification coupled with CpG island microarray (MCAM) analysis, we identified 224 genes that were methylated in the noncancerous gastric mucosa of patients with gastric cancer. Among them, RASGRF1 methylation was significantly elevated in gastric mucosa from patients with either intestinal or diffuse type gastric cancer, as compared with mucosa from healthy individuals (8.3% vs. 22.4%, P < 0.001; 8.3% vs. 19.4%, P < 0.001). RASGRF1 methylation was independent of mucosal atrophy and could be used to distinguish both serum pepsinogen test-positive [sensitivity, 70.0%; specificity, 86.7%; area under the receiver operator characteristic (ROC) curve, AUC, 0.763] and -negative patients with gastric cancer (sensitivity, 72.2%; specificity, 87.0%; AUC, 0.844) from healthy individuals. Ectopic expression of RASGRF1 suppressed colony formation and Matrigel invasion by gastric cancer cells, suggesting it may be involved in gastric tumorigenesis. Collectively, our data suggest that RASGRF1 methylation is significantly involved in an epigenetic field defect in the stomach, and that it could be a useful biomarker to identify individuals at high risk for gastric cancer. Cancer Prev Res; 5(10); 1203–12. ©2012 AACR.

The p53 tumor suppressor belongs to a gene family that includes two other structurally and functionally related members: p73 and p63. The regulation of p53 activity differs significantly from that of p73 and p63. To enhance the tumor suppressive activity of p53, we constructed six recombinant adenoviruses that encode hybrid proteins with three functional domains derived from either p53 or TAp63γ. The potency of these hybrid molecules in suppressing tumorigenesis was evaluated using in vitro and in vivo models. Of the hybrid molecules tested, one hybrid named p63-53O was the most potent activator of apoptosis in human cancer cells. The p63-53O hybrid is composed of the transcriptional activation domain and DNA-binding domain of TAp63γ and the oligomerization domain of p53. The p63-53O hybrid efficiently transactivated p53AIP1. Moreover, silencing of p53AIP1 partially abolished the apoptotic response to p63-53O in human cancer cells. The p53-p63 hybrid molecule is a novel potent anti-proliferative agent for the treatment of cancer.

Recently, intensive laboratory and preclinical studies have identified and validated therapeutic molecular targets in multiple myeloma (MM). The introduction of novel agents such as the proteasome inhibitor bortezomib and the immunomodulatory drugs thalidomide and lenalidomide, which were rapidly translated from preclinical studies at the Dana-Farber Cancer Institute into clinical trials, has changed the treatment paradigm and markedly extended overall survival; MM has therefore become a remarkable example of translational cancer research in new drug development. In this article, with the aim of determining the key factors underlying success in translational research, we focus on our studies of MM at Dana-Farber Cancer Institute as well as at our institutes. The identification of these key factors will help to promote translational cancer research not only in MM but also in other hematologic malignancies and solid tumors, to develop novel therapies, to overcome drug resistance, and to thereby improve the prognosis of cancer patients. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02384.x, 2012)

The Kruppel-like factor (KLF) proteins are multitasked transcriptional regulators with an expanding tumor suppressor function. KLF2 is one of the prominent members of the family because of its diminished expression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic roles. In this study, we show that epigenetic silencing of KLF2 occurs in cancer cells through direct transcriptional repression mediated by the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2). Binding of EZH2 to the 5'-end of KLF2 is also associated with a gain of trimethylated lysine 27 histone H3 and a depletion of phosphorylated serine 2 of RNA polymerase. Upon depletion of EZH2 by RNA interference, short hairpin RNA or use of the small molecule 3-Deazaneplanocin A, the expression of KLF2 was restored. The transfection of KLF2 in cells with EZH2-associated silencing showed a significant anti-tumoral effect, both in culture and in xenografted nude mice. In this last setting, KLF2 transfection was also associated with decreased dissemination and lower mortality rate. In EZH2-depleted cells, which characteristically have lower tumorigenicity, the induction of KLF2 depletion 'rescued' partially the oncogenic phenotype, suggesting that KLF2 repression has an important role in EZH2 oncogenesis. Most importantly, the translation of the described results to human primary samples demonstrated that patients with prostate or breast tumors with low levels of KLF2 and high expression of EZH2 had a shorter overall survival.

Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors. We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC = 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors. Cancer Prev Res; 4(5); 674–83. ©2011 AACR.

BACKGROUND: Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for tumorigenicity and tumor progression of gastrointestinal cancers. The authors previously reported the success of therapy for gastrointestinal cancers using adenoviruses that expressed dominant-negative IGF-IR (IGF-IR/dn). In addition, it has been demonstrated that IGF-IR signaling affects vascular endothelial growth factor (VEGF) expression in some other types of tumors. The objective of the current study was to evaluate this interaction by studying the roles of IGF-IR in tumor angiogenesis and lymphangiogenesis and their implications for targeted therapy in gastric cancer. METHODS: The impact of IGF signals on the expression of VEGF-A and VEGF-C in a human gastric cancer cell, MKN45, and vascular formation were assessed. The effects of IGF-IR/dn with or without bevacizumab on angiogenesis, lymphangiogenesis, and tumor suppression in mouse xenografts were assessed. RESULTS: IGFs induced the expression of VEGF ligands and up-regulated in vitro vascular vessel formation. IGF-IR/dn reduced VEGF expression, reduced the activation of both protein kinase B (Akt) and mitogen-activated protein kinase (MAPK), and reduced vascular formation, indicating that IGF-IR/dn inhibited tumor growth in mice by inhibiting both angiogenesis and lymphangiogenesis. However, IGF-IR/dn did not affect either blood sugar or body weight in these mice. The combination of IGF-IR/dn and bevacizumab was highly effective against these xenograft tumors, and only this combination resulted in the complete regression of 43% of tumors, reduced the expression of VEGF, and induced apoptosis. CONCLUSIONS: The current results indicated that IGF-IR is involved in angiogenesis and lymphangiogenesis through the modulation of VEGF ligand expression in the gastric cancer cell line MKN45. Targeting IGF-IR in combination with agents that block the VEGF pathway may have therapeutic utility for gastric cancer therapy. Cancer 2011. © 2011 American Cancer Society.

Interferon-induced transmembrane protein (IFITM) is reported to be frequently overexpressed in colorectal tumors. This study aimed to determine the usefulness of detecting fecal IFITM messenger RNA (mRNA) by real-time reverse-transcription polymerase chain reaction (RT-PCR) for colorectal cancer (CRC) screening. This pilot study included 21 patients with CRC and 23 healthy controls. Total RNA was isolated from the feces of the patients, and the expression levels of the mRNA of IFITM1, IFITM2 and IFITM3 were measured by real-time RT-PCR to detect CRC. Receiver operating characteristic curves of respective genes were generated, and the area under the curve (AUC), sensitivity and specificity were determined. When the 44 patients were analyzed, the AUCs of fecal IFITM1, IFITM2 and IFITM3 expression analysis were 0.82, 0.80 and 0.65, respectively. The sensitivities were 67% [14/21; 95% confidence interval (CI) 43–85%], 67% (14/21; 95% CI 43–85%) and 71% (15/21; 95% CI 48–89%), respectively; and the specificities were 96% (1/23; 95% CI 78–100%), 96% (1/23; 95% CI 78–100%) and 61% (9/23; 95% CI 39–80%), respectively. When IFITM1 and IFITM2 were combined, the sensitivity was 86% (18/21; 95% CI 64–97%) and the specificity was 96% (1/23; 95% CI 78–100%). The fecal expression analysis of IFITM1 and IFITM2 mRNA by real-time RT-PCR for CRC screening exhibited high specificities, and the sensitivity was further improved by combining IFITM1 and IFITM2.

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.

Interferon regulatory factors (IRFs) are transcription factors known to play key roles in innate and adaptive immune responses, cell growth, apoptosis, and development. Their function in tumorigenesis of gastric cancer remains to be determined, however. In the present study, therefore, we examined epigenetic inactivation of IRF1-9 in a panel of gastric cancer cell lines. We found that expression of IRF4, IRF5, and IRF8 was frequently suppressed in gastric cancer cell lines; that methylation of the three genes correlated with their silencing; and that treating the cells with the demethylating agent 5-aza-2'-deoxycytidine (DAC) restored their expression. Expression of IRF5 in cancer cells was enhanced by the combination of DAC treatment and adenoviral vector-mediated expression of p53, p63, or p73. Interferon-gamma-induced expression of IRF8 was also enhanced by DAC. Moreover, treating gastric cancer cells with DAC enhanced the suppressive effects of interferon-alpha, interferon-beta, and interferon-gamma on cell growth. Among a cohort of 455 gastric cancer and noncancerous gastric tissue samples, methylation of IRF4 was frequently observed in both gastric cancer specimens and noncancerous specimens of gastric mucosa from patients with multiple gastric cancers, which suggests IRF4 methylation could be a useful molecular marker for diagnosing recurrence of gastric cancers. Our findings indicate that epigenetic IRF inactivation plays a key role in tumorigenesis of gastric cancer, and that inhibition of DNA methylation may restore the antitumor activity of interferons through up-regulation of IRFs.

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling plays important parts in both the tumorigenicity and progression of digestive/gastrointestinal malignancies. In this study, we sought to test the effectiveness of a practical approach to blocking IGF-IR signaling using RNA interference delivered by recombinant adenoviruses. We constructed a recombinant adenovirus expressing short hairpin RNA targeting IGF-IR (shIGF-IR) and assessed its effect on signal transduction, proliferation, and survival in digestive/gastrointestinal cancer cell lines representing colorectal, gastric, and pancreatic adenocarcinoma, esophageal squamous cell carcinoma, and hepatoma. We analyzed the effects of shIGF-IR alone and with chemotherapy in vitro and in nude mouse xenografts, as well as on insulin signaling and hybrid receptor formation between IGF-IR and insulin receptor. shIGF-IR blocked expression and autophosphorylation of IGF-IR and downstream signaling by the IGFs, but not by insulin. shIGF-IR suppressed proliferation and carcinogenicity in vitro and up-regulated apoptosis in a dose-dependent fashion. shIGF-IR augmented the effects of chemotherapy on in vitro growth and apoptosis induction. Moreover, the combination of shIGF-IR and chemotherapy was highly effective against tumors in mice. shIGF-IR reduced hybrid receptor formation without effect on expression of insulin receptor. shIGF-IR may have therapeutic utility in human digestive/gastrointestinal cancers, both alone and in combination with chemotherapy.

Disruption of circadian rhythms, daily oscillations in biological processes that are regulated by an endogenous clock, has been linked to tumorigenesis. Normal and malignant tissues often show asynchronies in cell proliferation and metabolic rhythms. Cancer chronotherapy takes biological time into account to improve the therapy. However, alterations of the circadian clock machinery genes have rarely been reported in human cancer. Herein, we show that the BMAL1 gene, a core component of the circadian clock, is transcriptionally silenced by promoter CpG island hypermethylation in hematologic malignancies, such as diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias. We also describe how BMAL1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas BMAL1 depletion by RNA interference in unmethylated cells enhances tumor growth. We also show that BMAL1 epigenetic inactivation impairs the characteristic circadian clock expression pattern of genes such as C-MYC, catalase, and p300 in association with a loss of BMAL1 occupancy in their respective promoters. Furthermore, the DNA hypermethylation–associated loss of BMAL1 also prevents the recruitment of its natural partner, the CLOCK protein, to their common targets, further enhancing the perturbed circadian rhythm of the malignant cells. These findings suggest that BMAL1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting the cellular circadian clock. [Cancer Res 2009;69(21):8447–54]

The cellular origin, in vivo function and fate of donor bone marrow-derived cells residing in the recipient intestinal epithelial cells, pericryptal myofibroblasts or endothelial cells remain obscure. Although 'immunoprivileged' mesenchymal stem cells (MSCs) are prime candidates for cell- and gene-based therapy, their precise role in colitis remains largely undetermined. Using a dextran sulphate sodium (DSS) colitis with busulphan (BU)-induced hypoplastic marrow model, we examined the therapeutic effects of MSC transplantation, focusing on the role of MSCs as both cell providers and immunomodulators. Donor-derived MSCs were detected by eGFP immunofluorescence and fluorescence in situ hybridization for Y-chromosome (Y-FISH) analysis. Western blot analysis of apical-most tight junction proteins was performed with antibodies against claudin-2, -7, -8, -12, -13, -15 and ZO-1. Cytokine and cell cycle profiles were analysed by semi-quantitative RT-PCR and flow cytometry. Susceptibility to DSS colitis was significantly increased by co-existing BU-induced bone marrow hypoplasia and this increase was significantly reduced by enhancing epithelial engraftment of MSCs, an effect depending on restoring epithelial barrier integrity rather than inhibiting host immune responses. We provide evidence that implicates MSCs in maintaining epithelial barrier function by reassembling apical-most tight junction proteins, claudins. The therapeutic efficacy of extrinsic MSCs depends on enhancing epithelial engraftment in damaged crypts by busulphan conditioning. Such a role for the MSC-derived intestinal cells in colitis therapy merits further examination and may offer a promising new treatment for inflammatory bowel disease (IBD).

Pancreatic ductal adenocarcinoma (PDAC) shows the worst mortality among the common malignancies and development of novel therapies for PDAC through identification of good molecular targets is an urgent issue. Among dozens of over-expressing genes identified through our gene-expression profile analysis of PDAC cells, we here report CST6 (Cystatin 6 or E/M) as a candidate of molecular targets for PDAC treatment. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis confirmed over-expression of CST6 in PDAC cells, but no or limited expression of CST6 was observed in normal pancreas and other vital organs. Knock-down of endogenous CST6 expression by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in maintaining viability of PDAC cells. Concordantly, constitutive expression of CST6 in CST6-null cells promoted their growth in vitro and in vivo. Furthermore, the addition of mature recombinant CST6 in culture medium also promoted cell proliferation in a dose-dependent manner, whereas recombinant CST6 lacking its proteinase-inhibitor domain and its non-glycosylated form did not. Over-expression of CST6 inhibited the intracellular activity of cathepsin B, which is one of the putative substrates of CST6 proteinase inhibitor and can intracellularly function as a pro-apoptotic factor. These findings imply that CST6 is likely to involve in the proliferation and survival of pancreatic cancer probably through its proteinase inhibitory activity, and it is a promising molecular target for development of new therapeutic strategies for PDAC.

Activation of the Wnt signaling pathway is frequently observed in hepatocellular carcinoma (HCC), though mutation of three of its components, CTNNB1, AXIN1, and AXIN2, is observed substantially less often.We examined the relationship between Wnt signaling and epigenetic alteration of secreted frizzled-related protein (SFRP) genes in HCC.We frequently detected the active form of beta-catenin and accumulation of nuclear beta-catenin in liver cancer cell lines. We detected methylation of SFRP family genes in liver cancer cell lines (SFRP1, 9/12, 75%; SFRP2, 7/12, 58%; SFRP4, 3/12, 25%; SFRP5, 7/12, 58%) and primary HCCs (SFRP1, 9/19, 47%; SFRP2, 12/19, 63%; SFRP5, 8/19, 42%), though methylation of SFRP4 was not found in primary HCCs. SFRP methylation also was detected in hepatitis B or C virus-associated chronic hepatitis (SFRP1, 6/37, 16%; SFRP2, 14/37, 38%; SFRP5, 5/37, 14%) and liver cirrhosis (SFRP1, 10/28, 36%; SFRP2, 9/28, 32%; SFRP5, 3/28, 11%), suggesting that methylation of these genes is an early event in liver carcinogenesis. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcriptional activity in liver cancer cells, while overexpression of a beta-catenin mutant and depletion of SFRP1 using siRNA synergistically upregulated TCF/LEF transcriptional activity.Our results confirm the frequent methylation and silencing of Wnt antagonist genes in HCC, and suggest that their loss of function contributes to activation of Wnt signaling during hepatocarcinogenesis.

Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy. [Mol Cancer Ther 2008;7(4):779–87]

p73 and p63 are members of the p53 gene family and have been shown to play an important role in development and homeostasis mainly by regulating the transcription of a variety of genes. A subset of these genes encodes secreted proteins and receptors that may be involved in the communication between adjacent cells. We report here that flotillin-2, a major hydrophobic protein on biomembrane microdomain lipid rafts, is a direct transcriptional target of the p53 family member genes. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. We found that the expression of flotillin-2 was specifically up-regulated by either TAp73β or TAp63γ, but not significantly by p53. In addition, flotillin-2 transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we showed that silencing endogenous p73 abolishes the induction of flotillin-2 transcription following cisplatin treatment. Furthermore, we identified a p73/p63-binding site located upstream of the flotillin-2 gene that is responsive to the p53 family members. This response element is highly conserved between humans and rodents. We also found that ectopic expression of TAp73 as well as TAp63 enhances signal transduction by assessing the interleukin-6–mediated phosphorylation of signal transducers and activators of transcription 3. Thus, in addition to direct transactivation, p53 family member genes enhance a set of cellular processes via lipid rafts. (Mol Cancer Res 2008;6(3):395–406)

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight ras-association domain family (RASSF) genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in 6 of 10 gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2'-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. Complementary DNA microarray analysis revealed that RASSF2A suppresses the expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein-Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.

DNA methylation plays an important role in silencing cancer related genes. Recent studies have revealed that genes involved in cell cycle regulation, apoptosis, DNA repair, invasion, metastasis, and immune response, are shown to be aberrantly methylated in cancers. Subsets of cancers show simultaneous methylation of multiple genes, indicating that these tumors have CpG island methylator phenotype (CIMP). Colorectal and gastric cancers with CIMP show distinct clinicopathological features, and appear to be caused by increased rate of de novo methylation. DNA methylation changes can be detected with a high degree of sensitivity, and can potentially be important for diagnosis of cancer. Various types of cancer can be detected by analyzing methylation in DNA obtained from serum, stool, and pancreatic juice. Because DNA methylation is an epigenetic change, expression of silenced genes can be reversed by treating cells with DNA methyltransferase inhibitors such as 5-aza-2'-deoxycytidine (decitabine).

DNA mismatch repair (MMR) deficiency results in a strong mutator phenotype and high-frequency microsatellite instability (MSI-H), which are the hallmarks of tumors arising within Lynch syndrome. MSI-H is characterized by length alterations within simple repeated sequences, microsatellites. Lynch syndrome is primarily due to germline mutations in one of the DNA MMR genes; mainly hMLH1 or hMSH2 and less frequently hMSH6 and rarely hPMS2. Germline hemiallelic methylation of MLH1, termed epimutation, has been reported to be a new cause of Lynch syndrome. MSI-H is also observed in approximately 15% of colorectal, gastric and endometrial cancers and in lower frequencies in a minority of other tumors, where it is associated with the hypermethylation of the promoter region of hMLH1. MSI-H underlies a distinctive tumorigenic pathway because cancers with MSI-H exhibit many differences in genotype and phenotype relative to cancers without MSI-H, irrespective of their hereditary or sporadic origins. Genetic, epigenetic and transcriptomic differences exist between cancers with and those without the MSI-H. The BRAF V600E mutation is associated with sporadic MSI-H colorectal cancers (CRCs) harboring hMLH1 methylation but not Lynch syndrome-related CRCs. The differences in genotype and phenotype between cancers with and those without MSI-H are likely to be causally linked to their differences in biological and clinical features. Therefore, the diagnosis of MSI-H in cancers is thus considered to be of increasing relevance, because MSI-H is a useful screening marker for identifying patients with Lynch syndrome, a better prognostic factor and could affect the efficacy of chemotherapy. This review addresses recent advances in the field of microsatellite instability research.

Hedgehog-interacting protein (HHIP) was identified as a putative antagonist of the Hh pathway and as a target of Hh signalling. Our aim was to clarify the expression profiles and epigenetic alterations of the HHIP gene in gastrointestinal cancer. The expression and promoter epigenetic status of HHIP in cancer cell lines and freshly resected gastrointestinal cancer tissues were examined using RT-PCR, tissue microarray analysis, methylation-specific PCR, and chromatin immunoprecipitation assay. Cells were treated with the demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. WST-8 assays and in vitro invasion assays after treatment with HHIP-specific siRNA were performed. HHIP expression levels were reduced in most of the gastrointestinal cancer cell lines and in a certain subset of cancer tissues, and these were correlated with promoter hypermethylation. A heterochromatic structure characterized by neither acetylated H3 nor acetylated H4, and histone H3 lysine 9 hypermethylation and histone H3 lysine 4 hypomethylation was observed in cancer cells in which the HHIP gene was aberrantly silenced. On the other hand, overexpression of the HHIP gene was also found in some cancer tissues and there were significant correlations between protein expression levels of HHIP and those of Sonic hedgehog (Shh), Indian hedgehog, Patched, and glioma-associated oncogene homologue-1. An association was found between lymph node metastasis and HHIP silencing in colorectal cancer tissues with strong Shh expression and between advanced TNM stage and HHIP silencing in diffuse-type gastric cancer tissues with strong Shh expression. Down-regulation of HHIP expression by siRNA resulted in a significant increase in colon cancer cell growth and invasion in vitro. Silencing of the HHIP gene due to hypermethylation and chromatin remodelling appears to be frequently involved in gastrointestinal tumourigenesis.

Purpose: PR (PRDI-BF1 and RIZ) domain proteins (PRDM) are a subfamily of the kruppel-like zinc finger gene products that play key roles during cell differentiation and malignant transformation. The aim of the present study was to begin to examine the involvement of epigenetic alteration of PRDM expression in gastric and colorectal cancer.Experimental Design: We used real-time PCR to assess expression of PRDM1-17. In addition, we used bisulfite PCR to assess DNA methylation and chromatin immunoprecipitation to assess histone modification in colorectal and gastric cancer cell lines lacking PRDM5 expression.Results: Among the 17 PRDM family genes tested, we found that PRDM5 is the most frequently silenced in colorectal and gastric cancer cell lines. Silencing of PRDM5 was mediated by either DNA methylation or trimethylation of Lys27 of histone H3. Introduction of PRDM5 into cancer cells suppressed cell growth, suggesting that it acts as a tumor suppressor in gastrointestinal cancers. Methylation of PRDM5 was detected in 6.6% (4 of 61) of primary colorectal and 50.0% (39 of 78) of primary gastric cancers but not in noncancerous tissue samples collected from areas adjacent to the tumors.Conclusions: Our data suggest that epigenetic alteration of PRDM5 (e.g., methylation of its 5′-CpG island or trimethylation of Lys27 of histone H3) likely plays a key role in the progression of gastrointestinal cancers and may be a useful molecular marker.

Activation of Wnt signaling has been implicated in tumorigenesis, and epigenetic silencing of Wnt antagonist genes has been detected in various cancers. In the present study, we examined the expression and methylation of DICKKOPF (DKK) family genes in gastrointestinal cancer cell lines. We found that all known DKK genes were frequently silenced in colorectal cancer (CRC) cells (DKK1, 3/9, 33%; DKK2, 8/9, 89%; DKK3, 5/9, 56% and DKK4, 5/9, 56%), but not in normal colon mucosa. DKK1, -2 and -3 have 5' CpG islands, and show an inverse relation between expression and methylation. DKK methylation also was frequently observed in gastric cancer (GC) cell lines (DKK1, 6/16, 38%; DKK2, 15/16, 94% and DKK3, 10/16, 63%), but was seen less frequently in hepatocellular carcinoma and pancreatic cancer cell lines. DKKs also were frequently methylated in primary CRCs (DKK1, 7/58, 12%; DKK2, 45/58, 78% and DKK3, 12/58, 21%) and GCs (DKK1, 15/31, 48%; DKK2, 26/31, 84% and DKK3, 12/31, 39%). Against a background of CTNNB1 or APC mutations, Dickkopfs (Dkks) were less effective inhibitors of Wnt signaling than secreted frizzled-related proteins, though over-expression of Dkks suppressed colony formation of CRC cells with such mutations. Our results demonstrate that DKKs are frequent targets of epigenetic silencing in gastrointestinal tumors, and that loss of DKKs may facilitate tumorigenesis through beta-catenin/T-cell factor-independent mechanisms.