Yuji Toiyama

MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis

Objective Distant metastasis is the major cause of cancer-related death in patients with colorectal cancer (CRC). Although the microRNA-200 (miR-200) family is a crucial inhibitor of epithelial-to-mesenchymal transition (EMT) in human cancer, the role of miR-200 members in the pathogenesis of metastatic CRC has not been investigated. Design Fifty-four pairs of primary CRC and corresponding matched liver metastasis tissue specimens were analysed for expression and methylation status of the miR-200 family members. Functional analysis of miR-200c overexpression was investigated in CRC cell lines, and cells were analysed for proliferation, invasion and migration. Expression of several miR-200c target genes (ZEB1, ETS1 and FLT1) and EMT markers (E-cadherin and vimentin) in CRC cell lines and tissue specimens was validated. Results Liver metastasis tissues showed higher expression of miR-200c (primary CRC=1.31 vs. liver metastasis=1.59; p=0.0014) and miR-141 (primary CRC=0.14 vs. liver metastasis=0.17; p=0.0234) than did primary CRCs, which was significantly associated with hypomethylation of the promoter region of these miRNAs (primary CRC=61.2% vs. liver metastasis=46.7%; p<0.0001). The invasive front in primary CRC tissues revealed low miR-200c expression by in situ hybridization analysis. Transfection of miR-200c precursors resulted in enhanced cell proliferation but reduced invasion and migration behaviours in CRC cell lines. Overexpression of miR-200c in CRC cell lines caused reduced expression of putative gene targets, and resulted in increased E-cadherin and reduced vimentin expression. The associations between miR-200c, target genes and EMT markers were validated in primary CRCs and matching liver metastasis tissues. Conclusions miR-200c plays an important role in mediating EMT and metastatic behaviour in the colon. Its expression is epigenetically regulated, and miR-200c may serve as a potential diagnostic marker and therapeutic target for patients with CRC.

Serum miR-21 as a Diagnostic and Prognostic Biomarker in Colorectal Cancer

BACKGROUND The oncogenic microRNAs (miRNAs) miR-21 and miR-31 negatively regulate tumor-suppressor genes. Their potential as serum biomarkers has not been determined in human colorectal cancer (CRC). METHODS To determine whether miR-21 and miR-31 are secretory miRNAs, we screened expression in medium from 2 CRC cell lines, which was followed by serum analysis from 12 CRC patients and 12 control subjects. We validated expression of candidate miRNAs in serum samples from an independent cohort of 186 CRC patients, 60 postoperative patients, 43 advanced adenoma patients, and 53 control subjects. We analyzed miR-21 expression in 166 matched primary CRC tissues to determine whether serum miRNAs reflect expression in CRC. Patient survival analyses were performed by Kaplan-Meier analyses and Cox regression models. All statistical tests were two-sided. RESULTS Although miR-21 was secreted from CRC cell lines and upregulated in serum of CRC patients, no statistically significant differences were observed in serum miR-31 expression between CRC patients and control subjects. In the validation cohort, miR-21 levels were statistically significantly elevated in preoperative serum from patients with adenomas (P < .001) and CRCs (P < .001). Importantly, miR-21 expression dropped in postoperative serum from patients who underwent curative surgery (P < .001). Serum miR-21 levels robustly distinguished adenoma (area under the curve [AUC] = 0.813; 95% confidence interval [CI] = 0.691 to 0.910) and CRC (AUC = 0.919; 95% CI = 0.867 to 0.958) patients from control subjects. High miR-21 expression in serum and tissue was statistically significantly associated with tumor size, distant metastasis, and poor survival. Moreover, serum miR-21 was an independent prognostic marker for CRC (hazard ratio = 4.12; 95% CI = 1.10 to 15.4; P = .03). CONCLUSIONS Serum miR-21 is a promising biomarker for the early detection and prognosis of CRC.

Correlation of CD133, OCT4, and SOX2 in Rectal Cancer and Their Association with Distant Recurrence After Chemoradiotherapy

BackgroundCancer stem cells are associated with metastatic potential, treatment resistance, and poor patient prognosis. Distant recurrence remains the major cause of mortality in rectal cancer patients with preoperative chemoradiotherapy (CRT). We investigated the role of three stem cell markers (CD133, OCT4, and SOX2) in rectal cancer and evaluated the association between these gene levels and clinical outcome in rectal cancer patients with preoperative CRT.MethodsThirty-three patients with rectal cancer underwent preoperative CRT. Total RNAs of rectal cancer cells before and after CRT were isolated. Residual cancer cells after CRT were obtained from formalin-fixed paraffin-embedded (FFPE) specimens using microdissection. The expression levels of three stem cell genes were measured using real-time reverse-transcription polymerase chain reaction (RT-PCR). The association between these gene levels and radiation was evaluated using colon cancer cell lines. Immunohistochemical staining of these markers after CRT was also investigated.ResultsThere were significant positive correlations among the three genes after CRT. Patients who developed distant recurrence had higher levels of the three genes compared with those without recurrence in residual cancer after CRT. These elevated gene levels were significantly associated with poor disease-free survival. The radiation caused upregulation of these gene levels in LoVo and SW480 in vitro. Immunohistochemically, CD133 staining was observed in not only luminal surface but also cytoplasm.ConclusionsExpression of CD133, OCT4, and SOX2 may predict distant recurrence and poor prognosis of rectal cancer patients treated with preoperative CRT. Correlations among these genes may be associated with tumor regrowth and metastatic relapse after CRT.

Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer.

Objective:To evaluate the ability of epithelial-to-mesenchymal transition–related microRNAs (miRNAs) as serum biomarkers for prognosis and prediction of metastasis in patients with colorectal cancer (CRC). Background:Epithelial-to-mesenchymal transition–related miRNAs drive CRC progression and metastasis. However, their potential as serum biomarkers in CRC has not been studied. Methods:This was a 3-phase study using 446 colorectal specimens. In the first phase, we selected candidate miRNAs associated with metastasis by analyzing the expression of 4 miR-200 family members (miR-200b, -200c, -141, and -429) in serum samples from 12 patients with stage I and IV CRC. The second phase involved independent validation of candidate miRNAs in serum from 182 patients with CRC and 24 controls. Finally, we analyzed expression in matched 156 tumor tissues from 182 patients with CRC and an independent set of 20 matched primary CRC and corresponding liver metastases to identify the source of circulating miRNAs. Results:After initial screening, miR-200c was selected as the candidate serum miRNA best associated with metastasis. Validation analysis revealed that serum miR-200c levels were significantly higher in stage IV than in stage I–III CRCs. High serum miR-200c demonstrated a significant positive correlation with lymph node metastasis, distant metastasis, and prognosis (P = 0.0026, P = 0.0023, and P = 0.0064, respectively). More importantly, serum miR-200c was an independent predictor for lymph node metastasis (odds ratio: 4.81, 95% confidence interval: 1.98–11.7, P = 0.0005) and tumor recurrence (hazard ratio: 4.51, 95% confidence interval: 1.56–13.01, P = 0.005) and emerged as an independent prognostic marker for CRC (hazard ratio: 2.67, 95% confidence interval: 1.28–5.67, P = 0.01). Conclusions:Serum miR-200c has strong potential to serve as a noninvasive biomarker for CRC prognosis and predicting metastasis.

Metastasis-associated long non-coding RNA drives gastric cancer development and promotes peritoneal metastasis

The prognosis of gastric cancer (GC) patients with peritoneal dissemination remains poor, and a better understanding of the underlying mechanisms is critical for the development of new treatments that will improve survival in these patients. This study aimed to clarify the clinical and biological role of two key metastasis-associated long non-coding RNAs (lncRNAs) in GC. We analyzed the expression levels of two lncRNAs-Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and HOX-Antisense Intergenic RNA (HOTAIR)-by real-time reverse transcription PCR in 300 gastric tissues (150 GC and 150 adjacent normal mucosa), and in seven GC cell lines. Functional characterization for the role of HOTAIR in GC was performed by small interfering RNA (siRNA) knockdown, followed by series of in-vitro and in-vivo experiments. Expression of both lncRNAs was significantly higher in cancerous tissues than in corresponding normal mucosa, and higher expression of these lncRNAs significantly correlated with peritoneal metastasis in GC patients. In addition, elevated HOTAIR expression emerged both as an independent prognostic and risk factor for peritoneal dissemination. SiRNA knockdown of HOTAIR in GC cells significantly inhibited cell proliferation, migration and invasion, but concurrently enhanced the anoikis rate in transfected cells. In an in vivo assay, HOTAIR siRNA-transfected MKN45 cells injected into nude mice inhibited the growth of xenograft tumors and peritoneal metastasis compared with controls. Our data provide novel evidence for the biological and clinical significance of HOTAIR expression as a potential biomarker for identifying patients with peritoneal metastasis, and as a novel therapeutic target in patients with gastric neoplasia.

The clinical significance of MiR-148a as a predictive biomarker in patients with advanced colorectal cancer.

Aim Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies. Methods We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival. Results Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93). Discussion MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.

Preoperative C‐reactive protein as a prognostic and therapeutic marker for colorectal cancer

This study aimed to evaluate the significance of preoperative C‐reactive protein (CRP) as a prognostic marker for carcinoembryonic antigen (CEA)‐independent stage I or II colorectal cancer (CRC) patients.

Incidence and Risk Factors

Epidemiological data indicate that inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is the third highest risk condition for the development of colorectal cancer (CRC), namely, colitis-associated cancer (CAC). CD is also associated with an increased risk of small-bowel adenocarcinoma, in response to chronic inflammation of the small intestine. In studies published in the 1990s, the risk for CAC in IBD was approximately 7 % at 20 years after diagnosis. In recent studies, the overall incidence of CAC in IBD is lower, less than 5 % at 20 years. However, several factors, such as the longer duration of colitis, extensive or severer colitis, and coexistent primary sclerosing cholangitis, have continued to be important in the development of CRC in patients with IBD. Despite clinical and experimental investigations, the molecular mechanisms by which chronic inflammation promotes cancer progression are still unknown.

DNA METHYLATION AND MICRORNA BIOMARKERS FOR NONINVASIVE DETECTION OF GASTRIC AND COLORECTAL CANCER

Cancer initiation and progression is controlled by both genetic and epigenetic events. Epigenetics refers to the study of mechanisms that alter gene expression without permanently altering the DNA sequence. Epigenetic alterations are reversible and heritable, and include changes in histone modifications, DNA methylation, and non-coding RNA-mediated gene silencing. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Aberrant epigenetic modifications occur at the earliest stages of neoplastic transformation and are now believed to be essential players in cancer initiation and progression. Recent advances in epigenetics have not only offered a deeper understanding of the underlying mechanism(s) of carcinogenesis, but have also allowed identification of clinically relevant putative biomarkers for the early detection, disease monitoring, prognosis and risk assessment of cancer patients. At this moment, DNA methylation and non-coding RNA including with microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) represent the largest body of available literature on epigenetic biomarkers with the highest potential for cancer diagnosis. Following identification of cell-free nucleic acids in systematic circulation, increasing evidence has demonstrated the potential of cell-free epigenetic biomarkers in the blood or other body fluids for cancer detection. In this article, we summarize the current state of knowledge on epigenetic biomarkers - primarily DNA methylation and non-coding RNAs - as potential substrates for cancer detection in gastric and colorectal cancer, the two most frequent cancers within the gastrointestinal tract. We also discuss the obstacles that have limited the routine use of epigenetic biomarkers in the clinical settings and provide our perspective on approaches that might help overcome these hurdles, so that these biomarkers can be readily developed for clinical management of cancer patients.

MicroRNA-29c mediates initiation of gastric carcinogenesis by directly targeting ITGB1

Objective Gastric cancer (GC) remains difficult to cure due to heterogeneity in a clinical challenge and the molecular mechanisms underlying this disease are complex and not completely understood. Accumulating evidence suggests that microRNAs (miRNAs) play an important role in GC, but the role of specific miRNAs involved in this disease remains elusive. We performed next generation sequencing (NGS)-based whole-transcriptome profiling to discover GC-specific miRNAs, followed by functional validation of results. Design NGS-based miRNA profiles were generated in matched pairs of GCs and adjacent normal mucosa (NM). Quantitative RT-PCR validation of miR-29c expression was performed in 274 gastric tissues, which included two cohorts of matched GC and NM specimens. Functional validation of miR-29c and its gene targets was undertaken in cell lines, as well as K19-C2mE and K19-Wnt1/C2mE transgenic mice. Results NGS analysis revealed four GC-specific miRNAs. Among these, miR-29c expression was significantly decreased in GC versus NM tissues (p<0.001). Ectopic expression of miR-29c mimics in GC cell lines resulted in reduced proliferation, adhesion, invasion and migration. High miR-29c expression suppressed xenograft tumour growth in nude mice. Direct interaction between miR-29c and its newly discovered target, ITGB1, was identified in cell lines and transgenic mice. MiR-29c expression demonstrated a stepwise decrease in wild type hyperplasia-dysplasia cascade in transgenic mice models of GC. Conclusions MiR-29c acts as a tumour suppressor in GC by directly targeting ITGB1. Loss of miR-29c expression is an early event in the initiation of gastric carcinogenesis and may serve as a diagnostic and therapeutic biomarker for patients with GC.