Yasunori Akutsu

miR-145, miR-133a and miR-133b: tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma

MicroRNAs (miRNAs), noncoding RNAs 21–25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor‐suppressive function in esophageal squamous cell carcinoma (ESCC) was performed using the miRNA expression signatures obtained from ESCC clinical specimens. A subset of 15 miRNAs was significantly downregulated in ESCC. A comparison of miRNA signatures from ESCC and our previous report identified 4 miRNAs that are downregulated in common (miR‐145, miR‐30a‐3p, miR‐133a and miR‐133b), suggesting that these miRNAs are candidate tumor suppressors. Gain‐of‐function analysis revealed that 3 transfectants (miR‐145, miR‐133a and miR‐133b) inhibit cell proliferation and cell invasion in ESCC cells. These miRNAs (miR‐145, miR‐133a and miR‐133b), which have conserved sequences in the 3′UTR of FSCN1 (actin‐binding protein, Fascin homolog 1), inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR‐145 target sites and 1 miR‐133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss‐of‐function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor‐suppressive miRNAs, miR‐145, miR‐133a and miR‐133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.

Serum microRNA expression profile: miR-1246 as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma

Background:Recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in blood and can serve as useful biomarkers for cancer.Methods:We performed an miRNA array using serum samples obtained from oesophageal squamous cell carcinoma (ESCC) patients or healthy controls. MiR-1246 was the most markedly elevated in ESCC patients. Therefore, miR-1246 was selected as a candidate for further analysis. The serum miR-1246 level in 46 healthy controls and 101 ESCC patients was evaluated and compared among various clinicopathological characteristics. MiR-1246 expressions in tissue, exosomal, and cellular samples were also examined.Results:Serum miR-1246 alone yielded an receiver-operating characteristic curve area of 0.754, with 71.3% sensitivity and 73.9% specificity for distinguishing ESCC patients from healthy controls. Serum miR-1246 was significantly correlated with the TNM stage and showed to be the strongest independent risk factor for poor survival (HR, 4.032; P=0.017). Unlike the tendency shown in previous reports, miR-1246 was not upregulated in ESCC tissue samples. Furthermore, exosomal miR-1246 did not reflect the abundance in the cell of origin.Conclusion:These data support our contention that serum miR-1246 has strong potential as a novel diagnostic and prognostic biomarker in ESCC, and its releasing mechanism is selective and independent of tissue miRNA abundance.

Multicenter, phase II clinical trial of cancer vaccination for advanced esophageal cancer with three peptides derived from novel cancer-testis antigens

BackgroundSince a phase I clinical trial using three HLA-A24-binding peptides from TTK protein kinase (TTK), lymphocyte antigen-6 complex locus K (LY6K), and insulin-like growth factor-II mRNA binding protein-3 (IMP3) had been shown to be promising for esophageal squamous cell carcinoma (ESCC), we further performed a multicenter, non-randomized phase II clinical trial.Patients and methodsSixty ESCC patients were enrolled to evaluate OS, PFS, immunological response employing ELISPOT and pentamer assays. Each of the three peptides was administered with IFA weekly. All patients received the vaccination without knowing an HLA-A type, and the HLA types were key-opened at the analysis point. Hence, the endpoints were set to evaluate differences between HLA-A*2402-positive (24(+)) and -negative (24(−)) groups.ResultsThe OS in the 24 (+) group (n = 35) tended to be better than that in the 24(−) group (n = 25) (MST 4.6 vs. 2.6 month, respectively, p = 0.121), although the difference was not statistically significant. However, the PFS in the 24(+) group was significantly better than that in the 24(−) group (p = 0.032). In the 24(+) group, ELISPOT assay indicated that the LY6K-, TTK-, and IMP3-specific CTL responses were observed after the vaccination in 63%, 45%, and 60% of the 24(+) group, respectively. The patients having LY6K-, TTK-, and IMP3-specific CTL responses revealed the better OS than those not having CTL induction, respectively. The patients showing the CTL induction for multiple peptides have better clinical responses.ConclusionsThe immune response induced by the vaccination could make the prognosis better for advanced ESCC patients.Trial registrationClinicalTrials.gov, number NCT00995358

MicroRNA-133a regulates the mRNAs of two invadopodia-related proteins, FSCN1 and MMP14, in esophageal cancer

Background:FSCN1 and matrix metalloproteinase 14 (MMP14) are both invadopodia-related proteins. We herein elucidate the tumourigenicity of these proteins and identify novel therapeutic agents in esophageal squamous cell carcinoma (ESCC).Methods:FSCN1 and MMP14 were evaluated by immunohistochemistry and quantitative PCR, and microRNA (miR)-133a was also evaluated by PCR in surgical ESCC specimens. The roles of FSCN1, MMP14 and miR-133a were established in ESCC cells.Results:The expression of FSCN1 or MMP14 was an independent poor prognostic factor according to a multivariate analysis of immunohistochemistry, and their co-expression correlated with the poorest overall survival (OS) out of all the examined factors. Additionally, their mRNAs significantly correlated and both inversely correlated with miR-133a in surgical specimens. Transfection of a miR-133a mimic decreased the mRNA and protein levels of both FSCN1 and MMP14 in ESCC cells. The knockdown of FSCN1 or MMP14 and transfection of a miR-133a mimic inhibited the proliferation and invasion of ESCC cells. Patients with a lower miR-133a expression have a significantly poorer OS than those with a higher expression.Conclusion:The combined expression of FSCN1 and MMP14 is associated with a poor prognosis, and miR-133a, which regulates their mRNAs, can serve as a strong tumour suppressor of ESCC.

Inhibition of tumor metastasis by adoptive transfer of IL‐12‐activated Vα14 NKT cells

A unique lymphocyte lineage, the Vα14 NKT cells, expresses both NK1.1 and an invariant antigen receptor encoded by Vα14 and Jα281 gene segments. Vα14 NKT cells play crucial roles in various immune responses, including autoimmune diseases, allergic reactions and anti‐tumor immunity. Vα14 NKT cells were demonstrated to be essential for anti‐tumor effect of IL‐12 in vivo. Here, we report that adoptive transfer of IL‐12‐activated Vα14 NKT cells prevents hepatic metastasis of B16 melanoma. The injection of large amounts of IL‐2, IL‐4, and IFN‐γ, which are cytokines produced by activated Vα14 NKT cells, exhibited no significant inhibition of the metastasis of this melanoma. The cells prepared from the liver of IL‐12‐injected mice expressed a potent cytotoxic activity on B16 melanoma cells in vitro. Although the adoptive transfer of IL‐12‐activated Vα14 NKT cells prevents hepatic metastasis of B16 melanoma, activated NK cells from IL‐12‐injected RAG‐1−/− mice failed to inhibit the metastasis of this melanoma. Thus, the anti‐tumor effect of IL‐12 can be replaced by adoptive transfer of IL‐12‐activated Vα14 NKT cells but not by IL‐12‐activated NK cells, suggesting a minor role of NK cells for the IL‐12‐mediated anti‐tumor effect in this experimental system. Moreover, our studies have suggested the involvement of direct cytotoxic mechanisms rather than cytokine‐mediated immune responses at the effector phase of the Vα14 NKT cell‐mediated anti‐tumor activity.

Pre-operative dental brushing can reduce the risk of postoperative pneumonia in esophageal cancer patients

BACKGROUND The presence of pathogens in dental plaque is a risk factor associated with postoperative pneumonia in esophageal cancer patients. The effectiveness of pre-operative dental brushing to decrease the risk of postoperative pneumonia in esophageal cancer patients was evaluated prospectively. METHODS A total of 86 thoracic esophageal cancer patients who underwent an esophagectomy were investigated. Patients were divided into 2 groups: the control group (41 patients) and the pre-operative dental brushing group (45 patients). The patients in the brushing group were assigned to brush their teeth 5 times a day. After the operation, the frequency of postoperative pneumonia and need for tracheostomy for pulmonary treatment was calculated. RESULTS Postoperative pneumonia was decreased markedly from 32% to 9% (P = .013), and the frequency of postoperative pneumonia requiring tracheostomy decreased from 12% to 0% in the dental brushing group, respectively. Limiting the patients who had positive pathogenic bacteria in their dental plaque on their admission, the frequency of postoperative pneumonia was decreased from 71% (5 of 7 patients) in the control group to 17% (2 of 12 patients) in the dental brushing group (P = .045). CONCLUSION Frequent pre-operative dental brushing is performed easily and seems to prevent postoperative pneumonia in esophageal cancer patients.

The overall prevalence of metastasis in T1 esophageal squamous cell carcinoma: a retrospective analysis of 295 patients.

Objectives:T1 esophageal squamous cell carcinoma (ESCC) has a low, but still present, risk of lymph node (LN) metastasis. Endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) is often applied for T1 ESCC. To achieve successful treatment by EMR/ESD, the risk of LN metastases, LN recurrence, and hematological recurrence need to be better understood. The aim of this study was to determine the precise risk for metastasis in T1 ESCC. Methods:We divided 295 patients with T1 ESCC who underwent surgery and/or ESD/EMR into 6 categories (m1, m2, m3, sm1, sm2, and sm3). Their risks of LN metastasis, LN recurrence, hematological recurrence, and the outcome were determined. Results:The rates of LN metastasis and LN recurrence were 0% in m1 and m2, 9% in m3, 16% in sm1, 35% in sm2, and 62% in sm3 cases. The incidence of hematological recurrence was 0% in m1, m2, m3, and sm1 cases; 9% in sm2 cases; and 13% in sm3 cases. The overall risk of metastasis was 9% in m3, 16% in sm1, 38% in sm2, and 64% in sm3 patients. The 5-year disease-specific survival rates were 100% in m1, m2, and m3; 90.9% in sm1; 78.8% in sm2; and 68.6% in sm3 patients. Statistically, both lymphatic and venous invasion were selected as predictive markers for metastasis. In m3 patients, positivity for either of these had an odds ratio for metastasis of 7.333 (P = 0.093). Conclusions:Our study provides a precise assessment of the comprehensive risk of metastasis and feasible predictive markers for T1 ESCC.

Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma

The aim of this study was to determine whether histone acetylation regulates tumor suppressive microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC) and to identify genes which are regulated by these miRNAs. We identified a miRNA that was highly upregulated in an ESCC cell line by cyclic hydroxamic acid-containing peptide 31 (CHAP31), one of the histone deacetylase inhibitors (HDACIs), using a miRNA array analysis. miR-375 was strongly upregulated by CHAP31 treatment in an ESCC cell line. The expression levels of the most upregulated miRNA, miR-375 were analyzed by quantitative real-time PCR in human ESCC specimens. The tumor suppressive function of miR-375 was revealed by restoration of miR-375 in ESCC cell lines. We performed a microarray analysis to identify target genes of miR-375. The mRNA and protein expression levels of these genes were verified in ESCC clinical specimens. LDHB and AEG-1/MTDH were detected as miR‑375-targeted genes. The restoration of miR-375 suppressed the expression of LDHB and AEG-1/MTDH. The ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene. Knockdown of LDHB by RNAi showed a tumor suppressive function in the ESCC cells. The correlation between gene expression and clinicopathological features was investigated by immunohistochemistry for 94 cases of ESCC. The positive staining of LDHB correlated significantly with lymph node metastasis and tumor stage. It also had a tendency to be associated with a poor prognosis. Our results indicate that HDACIs upregulate miRNAs, at least some of which act as tumor suppressors. LDHB, which is regulated by the tumor suppressive miR-375, may therefore act as an oncogene in ESCC.

Histone Deacetylase Inhibitor FK228 Activates Tumor Suppressor Prdx1 with Apoptosis Induction in Esophageal Cancer Cells

Purpose: The histone deacetylase inhibitor FK228 shows strong activity as a potent antitumor drug but its precise mechanism is still obscure. The purpose of this study is to reveal the effect of FK228 on gene expression in the cell and to determine the mechanism of the antitumor activity of FK228 for further clinical applications. Experimental Design and Results: Microarray analysis was applied to verify the gene expression profiles of 4,608 genes after FK228 treatment using human esophageal squamous cell cancer cell lines T.Tn and TE2. Among them, peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family of antioxidant enzymes having cell growth suppression activity, as well as p21WAF1, were significantly activated by FK288. In addition, FK228 strongly inhibited the cell growth of T.Tn and TE2 by the induction of apoptosis. Further, chromatin immunoprecipitation analysis revealed that FK228 induced the accumulation of acetylated histones H3 and H4 in Prdx1 promoter, including the Sp1-binding site. In mouse xenograft models of T.Tn and TE2 cells, FK228 injection resulted in significant tumor regression as well as activated Prdx1 expression in tumor tissues. Prdx1 suppression by RNA interference hindered the antitumor effect of FK228. Conclusion: Our results indicate that the antitumor effect of FK228 in esophageal cancer cells is shown at least in part through Prdx1 activation by modulating acetylation of histones in the promoter, resulting in tumor growth inhibition with apoptosis induction.

The significance of lymph node status as a prognostic factor for esophageal cancer.

The revision of the TNM Classification of Malignant Tumors, 7th Edition, suggests the lymph node (LN) status to be the most significant risk factor in esophageal cancer. This article reviews the current status of LNs as indicators of prognosis. The significance of the number of metastatic LNs, the number of resected LNs, and a novel index, the “LN ratio” (metastatic LNs/removed LNs) in patients with esophageal cancer, were reviewed. The number of metastatic LNs independently predicted the prognosis of both overall survival and relapse-free survival. The number of positive LNs was also the best predictive marker of survival. Furthermore, overall survival significantly depended on the number of surgically removed LNs, and the LN ratio closely correlated with survival. The LN status is considered to be the most significant information that can be used to predict the prognosis. However, there are many issues that still need to be resolved. Better knowledge of the N-status is therefore needed to effectively utilize this information. Further research should focus on the N-status of patients with esophageal cancer.