Hiroki Ueo

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.

Clinical significance of tissue inhibitor of metalloproteinase expression in gastric carcinoma.

Tissue inhibitor of metalloproteinase (TIMP) has been reported to inhibit tumour invasion through an inactivation of matrix metalloproteinase (MMP) both in vitro and in vivo. Among the TIMP family, TIMP-1 possesses not only proteinase inhibitory activity but also a growth-promoting function. However, the significance of the expression of TIMP-1 in human gastric carcinoma tissue has yet to be clarified. In 50 examined cases of gastric carcinoma, 44 (88%) cases showed a higher expression of TIMP-1 mRNA in the biopsy samples from the tumour tissue (T) than in the biopsy samples from the corresponding normal tissue (N), as determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In a multivariate analysis, the T/N ratio of TIMP-1 mRNA was found to be an independent factor influencing the depth of tumour invasion and was the second most important factor in determining the prognosis of patients. As RT-PCR assay can be performed on biopsy specimens obtained before surgery, an evaluation of the TIMP-1 expression in biopsy specimens by RT-PCR may thus provide useful preoperative information on tumour aggressiveness.

Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation

BackgroundPredictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in the selection of treatment options, including liver transplantation (LT), for HCC. The purpose of this study was to identify specific microRNAs (miRs) in exosomes from the serum of patients with recurrent HCC and to validate these molecules as novel biomarkers for HCC recurrence.MethodsWe employed microarray-based expression profiling of miRs derived from exosomes in the serum of HCC patients to identify a biomarker that distinguishes between patients with and without HCC recurrence after LT. This was followed by the validation in a separate cohort of 59 HCC patients who underwent living related LT. The functions and potential gene targets of the recurrence-specific miRs were analysed using a database, clinical samples and HCC cell lines.ResultsWe found that miR-718 showed significantly different expression in the serum exosomes of HCC cases with recurrence after LT compared with those without recurrence. Decreased expression of miR-718 was associated with HCC tumour aggressiveness in the validated cohort series. We identified HOXB8 as a potential target gene of miR-718, and its upregulation was associated with poor prognosis.ConclusionCirculating miRs in serum exosomes have potential as novel biomarkers for predicting HCC recurrence.

Clinical significance of miR-144-ZFX axis in disseminated tumour cells in bone marrow in gastric cancer cases

Background:We previously reported that bone marrow (BM) was a homing site for gastric cancer (GC) cells leading to haematogenous metastases. There has been little study that microRNAs regulated pathways in malignant cells or host cells in BM, and thereby regulated the progression of GC.Methods:Both microRNA microarray and gene expression microarray analyses of total RNA from BM were conducted, comparing five early and five advanced GC patients. We focused on miR-144-ZFX axis as a candidate BM regulator of GC progression and validated the origin of the microRNA expression in diverse cell fractions (EpCAM+CD45−, EpCAM−CD45+, and CD14+) by magnetic-activated cell sorting (MACS).Results:Quantitative reverse-transcriptase (RT)–PCR analysis validated diminished miR-144 expression in stage IV GC patients with respect to stage I GC patients (t-test, P=0.02), with an inverse correlation to ZFX (ANOVA, P<0.01). Luciferase reporter assays in five GC cell lines indicated their direct binding and validated by western blotting. Pre-miR144 treatment and the resultant repression of ZFX in GC cell lines moderately upregulated their susceptibility to 5-fluorouracil chemotherapy. In MACS-purified BM fractions, the level of miR-144 expression was significantly diminished in disseminated tumour cell fraction (P=0.0005). Diminished miR-144 expression in 93 cases of primary GC indicated poor prognosis.Conclusion:We speculate that disseminated cancer cells could survive in BM when low expression of miR-144 permits upregulation of ZFX. The regulation of the miR-144-ZFX axis in cancer cells has a key role in the indicator of the progression of GC cases.

Elongation factor 1 gamma mRNA expression in oesophageal carcinoma.

Elongation factor 1 gamma (EF1 gamma) is known to be a subunit of EF1, one of the G proteins that mediate the transport of aminoacyl tRNA to 80S ribosomes during translation. As little is known regarding the expression of EF1 gamma in human oesophageal carcinoma, this study looked at its expression using a northern blot analysis. Thirty six cases of oesophageal carcinoma and 15 oesophageal carcinoma cell lines were studied. The EF1 gamma mRNA overexpression at a level of twofold or more was seen in five (14%) of 36 carcinomatous tissues compared with the normal counterparts. All five overexpressed cases showed severe lymph node metastases compared with the non-overexpressed cases, and the difference was significant (p = 0.028). The stage of the disease of these five cases was far advanced compared with the nonoverexpressed cases (p = 0.012). All 15 oesophageal carcinoma cells expressed EF1 gamma mRNA relatively lower than the gastric or pancreatic carcinoma cell lines, in which EF1 gamma was originally isolated. As the expression of EF1 gamma mRNA could be detected even in the biopsy specimens, its overexpression in tumour tissue may provide preoperative useful information for predicting the aggressiveness of tumours.

F-box protein FBXW7 inhibits cancer metastasis in a non-cell-autonomous manner

The gene encoding F-box protein FBXW7 is frequently mutated in many human cancers. Although most previous studies have focused on the tumor-suppressive capacity of FBXW7 in tumor cells themselves, we determined that FBXW7 in the host microenvironment also suppresses cancer metastasis. Deletion of Fbxw7 in murine BM-derived stromal cells induced accumulation of NOTCH and consequent transcriptional activation of Ccl2. FBXW7-deficient mice exhibited increased serum levels of the chemokine CCL2, which resulted in the recruitment of both monocytic myeloid-derived suppressor cells and macrophages, thereby promoting metastatic tumor growth. Administration of a CCL2 receptor antagonist blocked the enhancement of metastasis in FBXW7-deficient mice. Furthermore, in human breast cancer patients, FBXW7 expression in peripheral blood was associated with serum CCL2 concentration and disease prognosis. Together, these results suggest that FBXW7 antagonizes cancer development in not only a cell-autonomous manner, but also a non-cell-autonomous manner, and that modulation of the FBXW7/NOTCH/CCL2 axis may provide a potential approach to suppression of cancer metastasis.

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.