Naohiro Nishida

Clinical relevance of circulating cell-free microRNAs in cancer.

Efficient patient management relies on early diagnosis of disease and monitoring of treatment. In this regard, much effort has been made to find informative, blood-based biomarkers for patients with cancer. Owing to their attributes—which are specifically modulated by the tumour—circulating cell-free microRNAs found in the peripheral blood of patients with cancer may provide insights into the biology of the tumour and the effects of therapeutic interventions. Moreover, the role of microRNAs in the regulation of different cellular processes points to their clinical utility as blood-based biomarkers and future therapeutic targets. MicroRNAs are optimal biomarkers owing to high stability under storage and handling conditions and their presence in blood, urine and other body fluids. In particular, detection of levels of microRNAs in blood plasma and serum has the potential for an earlier cancer diagnosis and to predict prognosis and response to therapy. This Review article considers the latest developments in the use of circulating microRNAs as prognostic and predictive biomarkers and discusses their utility in personalized medicine.

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Plastin3 Is a Novel Marker for Circulating Tumor Cells Undergoing the Epithelial–Mesenchymal Transition and Is Associated with Colorectal Cancer Prognosis

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial-mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n = 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMT-induced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n = 381) and the validation set [n = 330; HR = 2.17; 95% confidence interval (CI) = 1.38-3.40 and HR = 3.92; 95% CI = 2.27-6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR = 4.07; 95% CI = 1.50-11.57) and Dukes C (HR = 2.57; 95% CI = 1.42-4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value.

Downregulation of miR-144 is associated with colorectal cancer progression via activation of mTOR signaling pathway

The mammalian target of rapamycin (mTOR) is a downstream integrator of essential pathways. mTOR signaling is frequently dysregulated in a variety of human cancers, and in silico analysis has revealed two miR-144 binding sites in the mTOR 3 untranslated region. We investigated the clinicopathologic magnitude of the mTOR pathway regulating microRNA, miR-144 in colorectal cancer (CRC) cases. The regulation of mTOR by miR-144 was examined with inhibitor miR-144-transfected cells. We also investigated changes in sensitivity to the mTOR inhibitor, rapamycin, in inhibitor miR-144-transfected cells. Quantitative RT-PCR was used to evaluate the clinicopathologic significance of miR-144 expression in 137 CRC. Furthermore, we assessed the correlation between CRC prognosis and the expression of 16 genes in the Akt/mTOR pathway. In vitro assays showed that mTOR is a direct target of miR-144, and downregulation of miR-144 facilitated proliferation of CRC cell line, HT29. In addition, the viability of HT29 cells with downregulated miR-144 expression was significantly reduced with rapamycin treatment. Low expression levels of miR-144 were associated with enhanced malignant potential such as venous invasion (P = 0.0013), liver metastasis (P = 0.08), liver recurrence (P = 0.0058) and poor prognosis (P = 0.0041). Multivariate analysis indicated that low miR-144 expression was an independent prognostic factor for survival. Among many genes consisting of the mTOR pathway, only high expression of Rictor was associated with poor prognosis of CRC. miR-144 is a meaningful prognostic marker. Downregulation of miR-144 leads to poor prognosis of CRC patients via activation of the mTOR signaling pathway.

MicroRNA miR-125b is a prognostic marker in human colorectal cancer

MicroRNAs (miRNAs) are small, non-coding RNAs that can function as oncogenes or tumor suppressors in human cancer. Recent reports have highlighted the oncogenic aspects of microRNA miR-125b. However, the clinical significance of miR-125b in gastrointestinal cancers has not been sufficiently investigated. To this end, we analyzed miR-125b expression in colorectal cancer cases. Quantitative RT-PCR was used to evaluate miR-125b expression in 89 colorectal cancer cases to determine the clinicopathological significance of miR-125b expression. The high miR-125b expression group showed a greater incidence of advanced tumor size and tumor invasion compared to the low miR-125b expression group (P<0.05). In addition, the high miR-125b expression group had a significantly poorer prognosis compared to the low expression group (P<0.05). Multivariate analysis indicated that high miR-125b expression was an independent prognostic factor for survival. Our analysis of miR-125b focused on the miR-125b/p53 pathway. In vitro assays revealed that overexpression of miR-125b repressed the endogenous level of p53 protein in human colorectal cancer cells. These data show that miR-125b is directly involved in cancer progression and is associated with poor prognosis in human colorectal cancer. Our findings suggest that miR-125b could be an important prognostic indicator for colorectal cancer patients.

Down-regulation of miR-125a-3p in human gastric cancer and its clinicopathological significance

Recent reports have demonstrated that another strand of mature microRNA (miRNA), called microRNA* or 3p (5p) strand, which is generated from the same precursor miRNA (Pre-miR), has a crucial role in cellular function. We previously reported the tumor suppressive effect of miR-125a-5p in gastric cancer. The current study was designed to examine the function and clinical significance of miR-125a-3p, a partner strand of miR-125a-5p, in human gastric cancer. Quantitative RT-PCR was used to evaluate miR-125a-3p expression in 70 gastric cancer cases to determine the clinicopathologic significance of miR-125a-3p expression. In addition, the effect of miR-125a-3p on the proliferation of gastric cancer cells was investigated. Low expression levels of miR-125a-3p were associated with indicators of enhanced malignant potential such as tumor size (p=0.0002), tumor invasion (p=0.0149), lymph node metastasis (p=0.018), liver metastasis (p=0.016), peritoneal dissemination (p=0.03), advanced clinical stage (p=0.0037) and poor prognosis (p=0.0083). Multivariate analysis indicated that low miR-125a-3p expression was an independent prognostic factor for survival, while in vitro assays demonstrated that miR‑125a-3p suppressed the proliferation of gastric cancer cells. MiR-125a-3p is a potent prognostic marker in gastric cancer. The clinical significance and tumor suppressive effect of miR‑125a-3p, as well as previously reported miR-125a-5p, suggest that the functional role of another strand of the mature form miRNA cannot be ignored, at least in miR-125a biogenesis.

HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named ‘hypoxia-induced noncoding ultraconserved transcripts’ (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.

MicroRNA-372 Is Associated with Poor Prognosis in Colorectal Cancer

Objective: MicroRNA-372 (miR-372) is reportedly shown to be an oncogene in human testicular germ cell tumors and gastric cancers, but its expression in colorectal cancer (CRC) is not yet determined. This study investigated the clinical significance of miR-372 expression in CRC. Methods: qRT-PCR was used to evaluate miR-372 in 144 CRC patients, and large tumor suppressor 2 (LATS2) expression was also examined as the likely target gene of miR-372. In vitroassays were performed to evaluate the biological function of miR-372. Results: Multivariate analysis indicated that high miR-372 expression was an independent prognostic factor (p = 0.006). High miR-372 expression was associated with synchronous liver metastasis (p = 0.035). We found an inverse relationship between miR-372 and LATS2 by qRT-PCR (p = 0.007) and immunohistochemistry (p = 0.042) using CRC tissue samples. Furthermore, pre-miR-372 led to a decrease in the LATS2 protein and an increase in proliferative activity of LoVo cells. We also found a significant association between low LATS2 expression and liver metastasis (p = 0.042). Conclusions: This study suggested that miR-372 was a novel independent prognostic factor in CRC. Our data suggest that LATS2 may serve as one of the target genes of miR-372 in clinical CRC tissues.

Tumour-suppressive function of SIRT4 in human colorectal cancer.

Background:SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers.Methods:We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry.Results:SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis.Conclusions:SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.

Kinesin 18A expression: Clinical relevance to colorectal cancer progression

Kif18A, a member of the kinesin superfamily of molecular motor proteins, is a microtubule depolymerase and a key regulator of chromosome congregation. Kif18As role in cancer progression has not been well defined. Our hypothesis is that Kif18A has a role in the progression of colorectal cancer (CRC). To investigate this expression of Kif18A, mRNA was assessed by quantitative real‐time PCR in 113 operative specimens of primary CRC. Kif18A was overexpressed and significantly (p < 0.0001) higher in CRC than in normal colon tissue. Kif18A overexpression in CRC significantly correlated with clinicopathologic factors such as tumor stage (p < 0.0001), lymphatic invasion (p = 0.001), lymph node metastasis (p = 0.01), venous invasion (p = 0.002) and peritoneal dissemination (p = 0.02), suggesting that it has a key role in CRC progression. In multivariate analysis, high Kif18A expression had independent significance for poorer overall survival after resection of CRC (p = 0.037). To demonstrate Kif18As role in CRC progression, we performed translational and in situ studies. Using in vitro studies on CRC cell lines, we evaluated Kif18As role in proliferation, migration and invasion. CRC cells transfected with Kif18A cDNA demonstrated significant enhanced migration (p < 0.01) and invasion (p = 0.018) compared to mock‐transfected cells. When Kif18A was targeted with specific small interfering RNA, CRC cells had significantly reduced proliferation (p < 0.01), migration (p < 0.01) and invasion (p < 0.05). The in vitro and translational studies demonstrated that Kif18A expression is related to events of metastasis and is a significant factor for CRC progression.