Downregulation of SMOC1 is associated with progression of colorectal traditional serrated adenomas.

BACKGROUND: Aberrant DNA methylation is prevalent in colorectal serrated lesions. We previously reported that the CpG island of SMOC1 is frequently methylated in traditional serrated adenomas (TSAs) and colorectal cancers (CRCs) but is rarely methylated in sessile serrated lesions (SSLs). In the present study, we aimed to further characterize the expression of SMOC1 in early colorectal lesions. METHODS: SMOC1 expression was analyzed immunohistochemically in a series of colorectal tumors (n = 199) and adjacent normal colonic tissues (n = 112). RESULTS: SMOC1 was abundantly expressed in normal colon and SSLs while it was significantly downregulated in TSAs, advanced adenomas and cancers. Mean immunohistochemistry scores were as follows: normal colon, 24.2; hyperplastic polyp (HP), 18.9; SSL, 23.8; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), 15.8; TSA, 5.4; TSA with high grade dysplasia (HGD)/EIC, 4.7; non-advanced adenoma, 21.4; advanced adenoma, 11.9; EIC, 10.9. Higher levels SMOC1 expression correlated positively with proximal colon locations and flat tumoral morphology, reflecting its abundant expression in SSLs. Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components. CONCLUSION: Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 expression may be a biomarker for diagnosis of serrated lesions and risk prediction in colorectal tumors.

Serum amyloid A1 recruits neutrophils to the invasive front of T1 colorectal cancers.

BACKGROUND AND AIM: The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs. METHODS: Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n = 49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression. RESULTS: Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils. CONCLUSIONS: Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion.

Activated macrophages promote invasion by early colorectal cancer via an interleukin 1ß-serum amyloid A1 axis.

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA-sequencing (RNA-seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues and detected significant upregulation of serum amyloid A1 (SAA1) in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Coculture experiments using CRC cell lines and THP-1 cells suggested that interleukin 1ß (IL-1ß) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL-1ß. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1-like/M2-like macrophages at SAA1-positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of matrix metalloproteinase-9 in macrophages. Our data suggest that tumor-associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1 and that SAA1 may be a predictive biomarker and a useful therapeutic target.

Upregulation of adipocyte enhancer-binding protein 1 in endothelial cells promotes tumor angiogenesis in colorectal cancer.

Tumor angiogenesis is an important therapeutic target in colorectal cancer (CRC). We aimed to identify novel genes associated with angiogenesis in CRC. Using RNA sequencing analysis in normal and tumor endothelial cells (TECs) isolated from primary CRC tissues, we detected frequent upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in TECs. Immunohistochemical analysis revealed that AEBP1 is upregulated in TECs and stromal cells in CRC tissues. Quantitative RT-PCR analysis showed that there is little or no AEBP1 expression in CRC cell lines, but that AEBP1 is well expressed in vascular endothelial cells. Levels of AEBP1 expression in Human umbilical vein endothelial cells (HUVECs) were upregulated by tumor conditioned medium derived from CRC cells or by direct coculture with CRC cells. Knockdown of AEBP1 suppressed proliferation, migration, and in vitro tube formation by HUVECs. In xenograft experiments, AEBP1 knockdown suppressed tumorigenesis and microvessel formation. Depletion of AEBP1 in HUVECs downregulated a series of genes associated with angiogenesis or endothelial function, including aquaporin 1 (AQP1) and periostin (POSTN), suggesting that AEBP1 might promote angiogenesis through regulation of those genes. These results suggest that upregulation of AEBP1 contributes to tumor angiogenesis in CRC, which makes AEBP1 a potentially useful therapeutic target.

DOT1L inhibition blocks multiple myeloma cell proliferation by suppressing IRF4-MYC signaling.

Epigenetic alterations play an important role in the pathogenesis in multiple myeloma, but their biological and clinical relevance is not fully understood. Here, we show that DOT1L, which catalyzes methylation of histone H3 lysine 79, is required for myeloma cell survival. DOT1L expression levels were higher in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in normal plasma cells. Treatment with a DOT1L inhibitor induced cell cycle arrest and apoptosis in myeloma cells, and strongly suppressed cell proliferation in vitro The anti-myeloma effect of DOT1L inhibition was confirmed in a mouse xenograft model. Chromatin immunoprecipitation-sequencing and microarray analysis revealed that DOT1L inhibition downregulated histone H3 lysine 79 dimethylation and expression of IRF4-MYC signaling genes in myeloma cells. In addition, DOT1L inhibition upregulated genes associated with immune responses and interferon signaling. Myeloma cells with histone modifier mutations or lower IRF4/MYC expression were less sensitive to DOT1L inhibition, but with prolonged treatment, anti-proliferative effects were achieved in these cells. Our data suggest that DOT1L plays an essential role in the development of multiple myeloma and that DOT1L inhibition may provide new therapies for myeloma treatment.

Subtypes of the Type II Pit Pattern Reflect Distinct Molecular Subclasses in the Serrated Neoplastic Pathway.

BACKGROUND: Colorectal serrated lesions (SLs) are important premalignant lesions whose clinical and biological features are not fully understood. AIMS: We aimed to establish accurate colonoscopic diagnosis and treatment of SLs through evaluation of associations among the morphological, pathological, and molecular characteristics of SLs. METHODS: A total of 388 premalignant and 18 malignant colorectal lesions were studied. Using magnifying colonoscopy, microsurface structures were assessed based on Kudo's pit pattern classification system, and the Type II pit pattern was subcategorized into classical Type II, Type II-Open (Type II-O) and Type II-Long (Type II-L). BRAF/KRAS mutations and DNA methylation of CpG island methylator phenotype (CIMP) markers (MINT1, - 2, - 12, - 31, p16, and MLH1) were analyzed through pyrosequencing. RESULTS: Type II-O was tightly associated with sessile serrated adenoma/polyps (SSA/Ps) with BRAF mutation and CIMP-high. Most lesions with simple Type II or Type II-L were hyperplastic polyps, while mixtures of Type II or Type II-L plus more advanced pit patterns (III/IV) were characteristic of traditional serrated adenomas (TSAs). Type II-positive TSAs frequently exhibited BRAF mutation and CIMP-low, while Type II-L-positive TSAs were tightly associated with KRAS mutation and CIMP-low. Analysis of lesions containing both premalignant and cancerous components suggested Type II-L-positive TSAs may develop into KRAS-mutated/CIMP-low/microsatellite stable cancers, while Type II-O-positive SSA/Ps develop into BRAF-mutated/CIMP-high/microsatellite unstable cancers. CONCLUSIONS: These results suggest that Type II subtypes reflect distinct molecular subclasses in the serrated neoplasia pathway and that they could be useful hallmarks for identifying SLs at high risk of developing into CRC.

Epigenetic silencing of diacylglycerol kinase gamma in colorectal cancer.

Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGKγ, was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.

Relationship Between Noncoding RNA Dysregulation and Epigenetic Mechanisms in Cancer.

Epigenetic alterations, including aberrant DNA methylation and histone modification, play key roles in the dysregulation of tumor-related genes, thereby affecting numerous cellular processes, including cell proliferation, cell adhesion, apoptosis, and metastasis. In recent years, studies have demonstrated that short and long noncoding RNAs (ncRNAs) are key players in the initiation and progression of cancer, and epigenetic mechanisms are deeply involved in their dysregulation. Indeed, the growing list of microRNA (miRNA) genes aberrantly methylated in cancer suggests that a large number of miRNAs act as tumor suppressors or oncogenes. In addition, emerging evidence suggests that dysregulation of long ncRNAs (lncRNAs) plays critical roles in tumorigenesis. And because ncRNAs are involved in regulating gene expression through interaction with epigenetic modifiers, their dysregulation appears causally related to epigenetic alterations in cancer. Dissection of the interrelationships between ncRNAs and epigenetic alterations has the potential to reveal novel approaches to the diagnosis and treatment of cancer.

Biological significance of the CpG island methylator phenotype.

Cancers exhibiting the CpG island methylator phenotype (CIMP) are found among a wide variety of human malignancies and represent a subclass of tumors showing concurrent hypermethylation of multiple CpG islands. These CIMP-positive tumors often exhibit characteristic molecular and clinicopathological features, suggesting CIMP represents a distinct carcinogenic pathway. However, marker genes to define CIMP have been largely inconsistent among studies, which has caused results to vary. Nonetheless, recent advances in genome-wide methylation analysis have enabled the existence of CIMP to be confirmed, and large-scale cancer genome analyses have begun to unravel the previously unknown molecular basis of CIMP tumors. CIMP is strongly associated with clinical outcome, suggesting it may be a predictive biomarker.

Association between genomic alterations and metastatic behavior of colorectal cancer identified by array-based comparative genomic hybridization.

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.

Molecular dissection of premalignant colorectal lesions reveals early onset of the CpG island methylator phenotype.

The concept of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) is widely accepted, although the timing of its occurrence and its interaction with other genetic defects are not fully understood. Our aim in this study was to unravel the molecular development of CIMP cancers by dissecting their genetic and epigenetic signatures in precancerous and malignant colorectal lesions. We characterized the methylation profile and BRAF/KRAS mutation status in 368 colorectal tissue samples, including precancerous and malignant lesions. In addition, genome-wide copy number aberrations, methylation profiles, and mutations of BRAF, KRAS, TP53, and PIK3CA pathway genes were examined in 84 colorectal lesions. Genome-wide methylation analysis of CpG islands and selected marker genes revealed that CRC precursor lesions are in three methylation subgroups: CIMP-high, CIMP-low, and CIMP-negative. Interestingly, a subset of CIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway genes. Analysis of mixed lesions containing both precancerous and malignant components revealed that most aberrant methylation is acquired at the precursor stage, whereas copy number aberrations are acquired during the progression from precursor to malignant lesion. Our integrative genomic and epigenetic analysis suggests early onset of CIMP during CRC development and indicates a previously unknown CRC development pathway in which epigenetic instability associates with genomic alterations.

TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells.

Long noncoding RNAs (lncRNAs) play pivotal roles in tumor development. To identify dysregulated lncRNAs in gastric cancer (GC), we analyzed genome-wide trimethylation of histone H3 lysine 4 (H3K4me3) to screen for transcriptionally active lncRNA genes in the non-tumorous gastric mucosa of patients with GC and healthy individuals. We found that H3K4me3 at TM4SF1-AS1 was specifically upregulated in GC patients and that the expression of TM4SF1-AS1 was significantly elevated in primary and cultured GC cells. TM4SF1-AS1 contributes to GC cell growth in vitro and in vivo, and its oncogenic function is mediated, at least in part, through interactions with purine-rich element-binding protein α (Pur-α) and Y-box binding protein 1 (YB-1). TM4SF1-AS1 also activates interferon signaling in GC cells, which is dependent on Pur-α and RIG-I. Chromatin isolation by RNA purification (ChIRP)-mass spectrometry demonstrated that TM4SF1-AS1 was associated with several stress granule (SG)-related proteins, including G3BP2, RACK1, and DDX3. Notably, TM4SF1-AS1 promoted SG formation and inhibited apoptosis in GC cells by sequestering RACK1, an activator of the stress-responsive MAPK pathway, within SGs. TM4SF1-AS1-induced SG formation and apoptosis inhibition are dependent on Pur-α and YB-1. These findings suggested that TM4SF1-AS1 contributes to tumorigenesis by enhancing SG-mediated stress adaptation.

CXCL12 is expressed by skeletal muscle cells in tongue oral squamous cell carcinoma.

BACKGROUND: The CXCL12/CXCR4 axis plays a pivotal role in the progression of various malignancies, including oral squamous cell carcinoma (OSCC). In this study, we aimed to clarify the biological and clinical significance of CXCL12 in the tumor microenvironment of OSCCs. METHODS: Publicly available single-cell RNA-sequencing (RNA-seq) datasets were used to analyze CXCL12 expression in head and neck squamous cell carcinomas (HNSCC). Immunohistochemical analysis of CXCL12, α-smooth muscle antigen (α-SMA), fibroblast activation protein (FAP) and CD8 was performed in a series of 47 surgically resected primary tongue OSCCs. Human skeletal muscle cells were co-cultured with or without OSCC cells, after which CXCL12 expression was analyzed using quantitative reverse-transcription PCR. RESULTS: Analysis of the RNA-seq data suggested CXCL12 is abundantly expressed in stromal cells within HNSCC tissue. Immunohistochemical analysis showed that in grade 1 primary OSCCs, CXCL12 is expressed in both tumor cells and muscle cells. By contrast, grade 3 tumors were characterized by disruption of muscle structure and reduced CXCL12 expression. Quantitative analysis of CXCL12-positive areas within tumors revealed that reduced CXCL12 expression correlated with poorer overall survival. Levels of CXCL12 expression tended to inversely correlate α-SMA expression and positively correlate with infiltration by CD8+ lymphocytes, though these relations did not reach statistical significance. CXCL12 was significantly upregulated in muscle cells co-cultured with OSCC cells. CONCLUSION: Our results suggest that tongue OSCC cells activate CXCL12 expression in muscle cells, which may contribute to tumor progression. However, CXCL12 is reduced in advanced OSCCs due to muscle tissue destruction.

ACLP Activates Cancer-Associated Fibroblasts and Inhibits CD8+ T-Cell Infiltration in Oral Squamous Cell Carcinoma.

We previously showed that upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in vascular endothelial cells promotes tumor angiogenesis. In the present study, we aimed to clarify the role of stromal AEBP1/ACLP expression in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis showed that ACLP is abundantly expressed in cancer-associated fibroblasts (CAFs) in primary OSCC tissues and that upregulated expression of ACLP is associated with disease progression. Analysis using CAFs obtained from surgically resected OSCCs showed that the expression of AEBP1/ACLP in CAFs is upregulated by co-culture with OSCC cells or treatment with TGF-ß1, suggesting cancer-cell-derived TGF-ß1 induces AEBP1/ACLP in CAFs. Collagen gel contraction assays showed that ACLP contributes to the activation of CAFs. In addition, CAF-derived ACLP promotes migration, invasion, and in vivo tumor formation by OSCC cells. Notably, tumor stromal ACLP expression correlated positively with collagen expression and correlated inversely with CD8+ T cell infiltration into primary OSCC tumors. Boyden chamber assays suggested that ACLP in CAFs may attenuate CD8+ T cell migration. Our results suggest that stromal ACLP contributes to the development of OSCCs, and that ACLP is a potential therapeutic target.

A novel pit pattern identifies the precursor of colorectal cancer derived from sessile serrated adenoma.

OBJECTIVES: Sessile serrated adenomas (SSAs) are known to be precursors of sporadic colorectal cancers (CRCs) with microsatellite instability (MSI), and to be tightly associated with BRAF mutation and the CpG island methylator phenotype (CIMP). Consequently, colonoscopic identification of SSAs has important implications for preventing CRCs, but accurate endoscopic diagnosis is often difficult. Our aim was to clarify which endoscopic findings are specific to SSAs. METHODS: The morphological, histological and molecular features of 261 specimens from 226 colorectal tumors were analyzed. Surface microstructures were analyzed using magnifying endoscopy. Mutation in BRAF and KRAS was examined by pyrosequencing. Methylation of p16, IGFBP7, MLH1 and MINT1, -2, -12 and -31 was analyzed using bisulfite pyrosequencing. RESULTS: Through retrospective analysis of a training set (n=145), we identified a novel surface microstructure, the Type II open-shape pit pattern (Type II-O), which was specific to SSAs with BRAF mutation and CIMP. Subsequent prospective analysis of an independent validation set (n=116) confirmed that the Type II-O pattern is highly predictive of SSAs (sensitivity, 65.5%; specificity, 97.3%). BRAF mutation and CIMP occurred with significant frequency in Type II-O-positive serrated lesions. Progression of SSAs to more advanced lesions was associated with further accumulation of aberrant DNA methylation and additional morphological changes, including the Type III, IV and V pit patterns. CONCLUSIONS: Our results suggest the Type II-O pit pattern is a useful hallmark of the premalignant stage of CRCs with MSI and CIMP, which could serve to improve the efficacy of colonoscopic surveillance.

Characteristics and surgical outcome of HCC patients with low platelet count.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) patients often have low platelet count (LPC). The aim of this study was to determine unique features of HCC patients with LPC. METHODOLOGY: HCC patients who underwent surgery were divided into two groups: LPC group (platelet count ≤100,000/mm³, n=84) and control group (platelet count >100,000/mm³, n=240). Surgical outcomes, risk factors for postoperative complications and prognostic factors were retrospectively compared. RESULTS: HCC patients with LPC had poorer liver function, smaller tumors, less anatomical resection and more frequent postoperative liver failure than control group patients. Postoperative survival was not different between the two groups. Tumor invasion to the main branch or trunk of portal vein (Vp3, 4) was the only risk factor for postoperative substantial complications in the LPC group. Postoperative survival was worse in patients with tumor diameter ≥4 cm or multiple tumors and in those who underwent preoperative transcatheter arterial chemoembolization (TACE) in the LPC group by multivariate analysis. Among them, preoperative TACE were not prognostic factors in the control group. CONCLUSIONS: In HCC patients with LPC, Vp3, 4 patients should be carefully monitored after surgery and preoperative TACE is not recommended for long-term postoperative survival.

Role of hilar resection in the treatment of hilar cholangiocarcinoma.

BACKGROUND/AIMS: The aim of this study was to clarify the role of bile duct resection without hepatectomy (hilar resection) in hilar cholangiocarcinoma. METHODOLOGY: We retrospectively compared surgical results for hilar cholangiocarcinoma between 8 patients treated with hilar resection and 21 patients treated with hepatectomy. RESULTS: All hilar resections were performed for Bismuth type I or II tumors with T2 or less lesions, whereas hepatectomy was done for type III or IV tumors excluding one type II tumor. R0 resection was equally achieved in both groups (62.5% in hilar resection group and 76.2% in hepatectomy group, p=0.469) and overall 5-year survival rates were comparable (21.9% vs. 23.6%, p=0.874). With respect to gross tumor appearance, R0 resection was achieved in all patients with papillary tumor in both groups with the excellent 5-year survivals (100% vs. 100%). In patients with nodular and flat tumors, R0 resection was achieved less frequently in the hilar resection vs. hepatectomy group (50% vs. 77.8%) mainly due to failure to clear the proximal ductal margin, resulting in poorer 5-year survival (0% vs. 18.7%). CONCLUSIONS: Hilar resection may be indicated for papillary T1 or 2 tumors in Bismuth type I or II cholangiocarcinoma.

Nuclear expression of thioredoxin-1 in the invasion front is associated with outcome in patients with gallbladder carcinoma.

BACKGROUND: Multifunctional redox protein human thioredoxin (TRX-1) is reduced by thioredoxin reductase (TRX-R). The aim of the present study was to examine the distribution of TRX-1 and TRX-R expressions in gallbladder carcinoma (GBC) to clarify their usefulness as prognostic factors after surgical resection. METHODS: Immunohistochemical staining for TRX-1 and TRX-R was performed in GBC tissue from 38 patients who underwent surgical resection, and TRX-1/TRX-R localization in relation to outcome was examined. RESULTS: TRX-1 protein levels were significantly higher in GBC samples than in cholecystolithiasis samples (P = 0.0174). TRX-1 expression was observed in 100% (38/38) of tumour samples and in the nucleus in 76% (29/38), with nuclear expression in the invasion front observed in 45% (13/29). TRX-R expression was only detected in the cytoplasm of cancer cells and in the invasion front in 28 samples. In all of the samples, the depth of tumour invasion, lymph node metastasis, surgical margin, curability and nuclear expression of TRX-1 in the invasion front were significant prognostic factors by univariate analysis. In 27 selected patients who underwent curative resection, both TRX-1 nuclear expression and TRX-R cytoplasmic expression in the invasion front was a significantly prognostic factor. CONCLUSION: TRX-1 nuclear expression in the GBC invasion front is a significant prognostic marker. Patients with both TRX-1 nuclear expression and TRX-R cytoplasmic expression in the tumour invasion front should be observed carefully even if after curative resection.

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination.

Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. siRNA for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair.

Dual EZH2 and G9a inhibition suppresses multiple myeloma cell proliferation by regulating the interferon signal and IRF4-MYC axis.

Epigenetic mechanisms such as histone modification play key roles in the pathogenesis of multiple myeloma (MM). We previously showed that EZH2, a histone H3 lysine 27 (H3K27) methyltransferase, and G9, a H3K9 methyltransferase, are potential therapeutic targets in MM. Moreover, recent studies suggest EZH2 and G9a cooperate to regulate gene expression. We therefore evaluated the antitumor effect of dual EZH2 and G9a inhibition in MM. A combination of an EZH2 inhibitor and a G9a inhibitor strongly suppressed MM cell proliferation in vitro by inducing cell cycle arrest and apoptosis. Dual EZH2/G9a inhibition also suppressed xenograft formation by MM cells in vivo. In datasets from the Gene Expression Omnibus, higher EZH2 and EHMT2 (encoding G9a) expression was significantly associated with poorer prognoses in MM patients. Microarray analysis revealed that EZH2/G9a inhibition significantly upregulated interferon (IFN)-stimulated genes and suppressed IRF4-MYC axis genes in MM cells. Notably, dual EZH2/G9a inhibition reduced H3K27/H3K9 methylation levels in MM cells and increased expression of endogenous retrovirus (ERV) genes, which suggests that activation of ERV genes may induce the IFN response. These results suggest that dual targeting of EZH2 and G9a may be an effective therapeutic strategy for MM.