Yuki Takano

Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers

Background:We previously conducted gene expression microarray analyses to identify novel indicators for colorectal cancer (CRC) metastasis and prognosis from which we identified PVT-1 as a candidate gene. PVT-1, which encodes a long noncoding RNA, mapped to chromosome 8q24 whose copy-number amplification is one of the most frequent events in a wide variety of malignant diseases. However, PVT-1 molecular mechanism of action remains unclear.Methods:We conducted cell proliferation and invasion assays using colorectal cancer cell lines transfected with PVT-1siRNA or negative control siRNA. Gene expression microarray analyses on these cell lines were also carried out to investigate the molecular function of PVT-1. Further, we investigated the impact of PVT-1 expression on the prognosis of 164 colorectal cancer patients by qRT–PCR.Results:CRC cells transfected with PVT-1 siRNA exhibited significant loss of their proliferation and invasion capabilities. In these cells, the TGF-β signalling pathway and apoptotic signals were significantly activated. In addition, univariate and multivariate analysis revealed that PVT-1 expression level was an independent risk factor for overall survival of colorectal cancer patients.Conclusion:PVT-1, which maps to 8q24, generates antiapoptotic activity in CRC, and abnormal expression of PVT-1 was a prognostic indicator for CRC patients.

Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer.

Background:Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC).Methods:Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT–PCR.Results:Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001).Conclusions:Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.

Up-regulation of NEK2 by microRNA-128 methylation is associated with poor prognosis in colorectal cancer.

BackgroundNIMA-related kinase 2 (NEK2), an enzyme involved in the development and progression of cancer, is abnormally expressed in a wide variety of human cancers, including colorectal cancer (CRC), and is known to have roles in cell division and mitotic regulation through centrosome splitting. We investigated the clinical significance of NEK2 in CRC. In particular, we examined miR-128 expression, which is thought to target NEK2.MethodsWe measured NEK2 mRNA and miR-128 levels in clinical samples by quantitative reverse transcription real-time PCR and analyzed the associations between NEK2 levels, miR-128 levels, clinicopathological factors, and prognoses. Furthermore, we performed in vitro assays using a pre-miR-128 precursor and conducted miR-128 methylation analyses.ResultsMiR-128 inhibited NEK2 expression and cancer cell proliferation via cell cycle arrest. Moreover, miR-128 was silenced by DNA methylation. Increased NEK2 expression was associated with serosal invasion, lymphatic invasion, and peritoneal dissemination. Patients with high NEK2 expression also had significantly poorer prognoses. Multivariate analysis indicated that high NEK2 expression was an independent prognostic factor for survival. Patients with high miR-128 expression had significantly lower NEK2 expression and lower recurrence rates than those with low miR-128 expression.ConclusionsNEK2 may be an independent prognostic factor for CRC and was regulated by miR-128, a microRNA that was subjected to epigenetic regulation. Thus, this miR-128/NEK2 pathway may be a prospective therapeutic target for patients with CRC.

Paired related homoeobox 1, a new EMT inducer, is involved in metastasis and poor prognosis in colorectal cancer

Background:Paired related homoeobox 1 (PRRX1) has been identified as a new epithelial-mesenchymal transition (EMT) inducer in breast cancer. However, the function of PRRX1 in colorectal cancer (CRC) has not been elucidated.Methods:We utilised ectopic PRRX1-expressing cell lines to analyse the function of PRRX1 in CRC. The clinical significance of PRRX1 was also examined on three independent CRC case sets.Results:PRRX1 induced EMT and the stem-like phenotype in CRC cells. In contrast to studies of breast cancer, abundant expression of PRRX1 was significantly associated with metastasis and poor prognosis in CRC.Conclusion:PRRX1 is an indicator of metastasis and poor prognosis in CRC cases. Further investigation is required to uncover the signalling network regulating PRRX1.

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

Gene expression of bone morphogenic protein 8B in the primary site, peripheral blood and bone marrow of patients with gastric cancer

The prognosis for individuals that are diagnosed with gastric cancer remains poor due to the high frequency of metastatic disease. In response to tumor-derived secreted factors, the bone marrow generates a suitable microenvironment for the development of metastasis. However, it is largely unknown whether secreted factors in bone marrow associated with metastatic disease of patients with gastric cancer are present. Secreted factors from the bone marrow of patients with metastatic gastric cancer were identified using a DNA microarray analysis and the mRNA expression levels were investigated in 355 bone marrow, 295 peripheral blood and 144 primary site samples using quantitative PCR (qPCR). Using DNA microarray analysis, the present study identified bone morphogenetic protein 8B (BMP8B) as a secreted signaling molecule in the bone marrow that was associated with the metastatic disease of human gastric cancer. The expression levels of BMP8B in the bone marrow of 355 gastric cancer patients were increased with metastatic disease. A significant correlation was demonstrated between BMP8B mRNA expression in the bone marrow and in the peripheral blood. High BMP8B expression in the bone marrow was associated with the diffuse type of gastric cancer (P=0.009), lymph node metastasis (P=0.009), liver metastasis (P=0.044) and peritoneal dissemination (P<0.001). In the primary site, a multivariate analysis revealed BMP8B mRNA expression as one of the independent prognostic factors of gastric cancer [hazard ratio (HR), 2.066; 95% CI, 1.132–3.772]. This study suggests that BMP8B, a previously unknown secreted factor in cancer progression, has the potential to be used as a prognostic biomarker. The present study may provide insight into a new mechanism that underlies the dissemination of gastric cancer cells.

Circulating exosomal microRNA-203 is associated with metastasis possibly via inducing tumor-associated macrophages in colorectal cancer

A primary tumor can create a premetastatic niche in distant organs to facilitate the development of metastasis. The mechanism by which tumor cells communicate with host cells to develop premetastatic niches is unclear. We focused on the role of microRNA (miR) signaling in promoting metastasis. Here, we identified miR-203 as a signaling molecule between tumors and monocytes in metastatic colorectal cancer (CRC) patients. Notably, high expression of serum exosomal miR-203, a major form in circulation, was associated with distant metastasis and an independent poor prognostic factor, whereas low expression in tumor tissues was a poor prognostic factor in CRC patients. We also found that exosomes carrying miR-203 from CRC cells were incorporated into monocytes and miR-203 could promote the expression of M2 markers in vitro, suggesting miR-203 promoted the differentiation of monocytes to M2-tumor-associated macrophages (TAMs). In a xenograft mouse model, miR-203-transfected CRC cells developed more liver metastasis compared to control cells. In conclusion, serum exosomal miR-203 expression is a novel biomarker for predicting metastasis, possibly via promoting the differentiation of monocytes to M2-TAMs in CRC. Furthermore, we propose the concept of site-dependent functions for miR-203 in tumor progression.

Microarray analysis reveals that high mobility group A1 is involved in colorectal cancer metastasis

Tumor size indicates the extent of cell proliferation in most cases of colorectal cancer (CRC), although there are some advanced small tumors with metastases. Lymph node metastasis is a significant factor that greatly impacts disease prognosis in CRC cases. The underlying factors that cause lymph node metastasis in CRC cells are not fully understood. We investigated the mechanism that might induce CRC metastasis by focusing on smaller sized (<2 cm) invasive tumors. We carried out gene expression array analysis for CRC cases; group 1 consisted of 6 cases with tumors <2 cm with metastases, and group 2 consisted of 65 cases with tumors >2 cm without metastases. Results were validated using gene expression array data from an additional 77 cases and another bulk case set of 172 cases. Gene ontology and pathway analysis using microarray data revealed that anti-apoptotic activity had a crucial role in CRC metastasis. High mobility group A1 (HMGA1) was identified as a biomarker for poor prognosis and metastasis formation. HMGA1 expression levels were higher in lymph node-positive cases than in lymph node-negative cases, even in subgroup analysis of submucosal invasive cases. The present study strongly supports the clinical significance of HMGA1 expression as a predictive indicator of lymph node metastasis in CRC cases, even in submucosal invasive cases which could be cured by local resection.

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma.

Background Few driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes. Methods We searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort. Results We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC. Conclusion Our integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.

Loss of CDCP1 expression promotes invasiveness and poor prognosis in esophageal squamous cell carcinoma.

BackgroundHuman CDCP1 gene, located on chromosome 3p21.3, is a transmembrane glycoprotein widely expressed in epithelial tissues, and its role in cancer remains to be understood.MethodsUsing microarray profiles of gene expression and copy number data from 69 esophageal squamous cell carcinoma (ESCC) samples, we performed informatics analyses to reveal the significance of CDCP1 expression. We also performed migration and invasion assays of siRNA-targeted CDCP1-transfected cells and CDCP1-overexpressing cell in vitro. Moreover, we evaluated the clinical magnitude of CDCP1 expression in esophageal squamous cell cancer cases.ResultsAllelic loss of chromosome 3p was confirmed by copy number analysis. The expression level of CDCP1 in tumor tissue was significantly lower than that in corresponding normal tissue. siRNA targeting of CDCP1 promoted the migratory and invasive abilities of esophageal cancer cell lines, whereas both abilities were reduced in CDCP1-overexpressing cells. Gene set enrichment analysis showed that expression levels of CDCP1 were associated with tumor differentiation and metastasis, consistent with the result of clinicopathologic analyses. Finally, multivariate analysis revealed that the expression level of CDCP1 was an independent prognostic factor for survival.ConclusionsLoss of CDCP1 expression may be a novel indicator for biological aggressiveness in ESCC.