Normal Esophageal Cells Research Articles

LncRNA SNHG16 predicts poor prognosis in ESCC and promotes cell proliferation and invasion by regulating Wnt/β-catenin signaling pathway.

Increasing evidence indicated that small nucleolar RNA host gene 16 (SNHG16) acted as a key regulator in the proliferation and metastasis of several cancers, including esophageal squamous cell carcinoma (ESCC). In this research, we aimed to explore biological functions, clinical significance and the underlying molecular mechanisms of SNHG16 in ESCC. qRT-PCR was performed to examine the expression of SNHG16 in ESCC cell lines and clinical ESCC tissue samples. The association of SNHG16 expression with clinicopathological factors and prognosis was statistically analyzed. Cell Counting Kit-8, flow cytometry, and transwell invasion assays were performed to determine the effect of SNHG16 in the regulation of biological behaviors of ESCC cells. Luciferase assay and Western blot were performed to determine the activation of Wnt/β-catenin signaling pathway RESULTS: We observed that SNHG16 expression levels were significantly upregulated in ESCC tissues and cell lines compared with the corresponding normal tissues and normal esophageal cell line, respectively. In addition, increased expression of SNHG16 were strongly linked to tumor stage (p = 0.019), lymph nodes metastasis (p = 0.007) and clinical stage (p = 0.026). Kaplan-Meier assay showed that the survival time of patients with high SNHG16 expression was significantly shorter than those with low SNHG16 expression (p = 0.0017). Univariate and multivariate analyses showed that high SNHG16 expression in ESCC was an independent predictor of poor survival. Loss-of-function experiments revealed that knockdown of SNHG16 suppressed proliferation and invasion and induced apoptosis of ESCC cells. Mechanistically, Wnt/β-catenin signaling pathways were actively modulated by SNHG16 in ESCC cells. Our findings reveal that SNHG16 plays an important role in ESCC proliferation/metastasis via modulating Wnt/β-catenin signaling pathways and could represent a novel biomarker for predicting poor survival as well a promising therapeutic target.

Expression of BRD4 in squamous cell carcinoma and its effects on cell proliferation and invasion ability

Objective: To investigate the expression of BRD4 in squamous cell carcinoma (SCC) tissues and cells, and the effects of its expression on cell proliferation and invasion ability. Methods: Immunohistochemistry was used to detect BRD4 protein expression in SCC tissues and paired normal esophageal squamous epithelial tissues. The expression of BRD4 protein was detected in different SCC cell lines and normal esophageal squamous epithelial cells by Western blot. BRD4 siRNA and control siRNA were used to transfect SCC Eca109 cells, and experiments were divided into three groups: untreated group, control siRNA group and BRD4 siRNA group. Western blot was employed to investigate the expression of BRD4 protein in the three groups of SCC Eca109 cells. CCK-8 kit was utilized to detect cell proliferation ability, and Transwell chamber was used to examine cell invasion ability. Finally, Western blot was used to detect the expression of MMP2 and MMP9 proteins. Results: The positive rate of BRD4 protein expression in SCC tissues was significantly higher than that of normal squamous epithelial tissues. The expression of BRD4 protein in 4 SCC cell lines was higher than that in normal esophageal cell Het-1A. BRD4 siRNA obviously downregulated the expression of BRD4 protein in Eca109 cells, and its downregulation contributed to the suppression of cell proliferation and invasion ability in Eca109 cells (all P<0.05), coupled with the decreases of MMP2 and MMP9 proteins. Conclusion: BRD4 may be closely associated with the proliferation and invasion of SCC, and it thus may be a potential therapeutic target for SCC.

Sa1655 - Forkhead Box F1 (Foxf1) can not only Induce a Barrett's Esophagus Like Metaplastic Change but also Induce Malignant Behavior in Normal Esophageal Cells

Sa1655 - Forkhead Box F1 (Foxf1) can not only Induce a Barrett's Esophagus Like Metaplastic Change but also Induce Malignant Behavior in Normal Esophageal Cells

Expression of the desmosome-related molecule periplakin is associated with advanced stage and poor prognosis of esophageal squamous cell carcinoma

Background: Our previous report showed that periplakin (PPL), a member of the plakin family of proteins, was expressed in all the normal esophageal squamous cells, except the Ki67+ basal cell layer; however, PPL-negative cells were observed in esophageal squamous cell carcinoma (ESCC) tissue samples and the proportion of PPL-positive areas in tumors showed considerable variation. In this study, we analyzed the relationships between PPL expression in tumors and the clinicopathological features of ESCC.

Helicobacter pylori induces caudal-type homeobox protein 2 and cyclooxygenase 2 expression by modulating microRNAs in esophageal epithelial cells.

Dysregulation of microRNAs (miRNAs) has been linked to virulence factors of Helicobacter pylori. The role of H. pylori in esophageal disease has not been clearly defined. We previously reported that H. pylori esophageal colonization promotes the incidence of Barrett's esophagus and esophageal adenocarcinoma in vivo. Here, we studied the direct effects of H. pylori on the transformation of esophageal epithelial cells, with particular focus on whether H. pylori exerts its effects by modulating miRNAs and their downstream target genes. The normal human esophageal cell line HET‐1A was chronically exposed to H. pylori extract and/or acidified deoxycholic acid for up to 36 weeks. The miRNA profiles of the esophageal epithelial cells associated with H. pylori infection were determined by microarray analysis. We found that chronic H. pylori exposure promoted acidified deoxycholic acid‐induced morphological changes in HET‐1A cells, along with aberrant overexpression of intestinal metaplasia markers and tumorigenic factors, including caudal‐type homeobox protein 2 (CDX2), mucin 2, and cyclooxygenase 2 (COX2). Helicobacter pylori modified the miRNA profiles of esophageal epithelial cells, particularly aberrant silencing of miR‐212‐3p and miR‐361‐3p. Moreover, in biopsies from Barrett's esophagus patients, esophageal H. pylori colonization was associated with a significant decrease in miR‐212‐3p and miR‐361‐3p expression. Furthermore, we identified COX2 as a target of miR‐212‐3p, and CDX2 as a target of miR‐361‐3p. Helicobacter pylori infection of esophageal epithelial cells was associated with miRNA‐mediated upregulation of oncoprotein CDX2 and COX2. Our observations provide new evidence about the molecular mechanisms underlying the association between H. pylori infection and esophageal carcinogenesis.

Expression of the desmosome-related molecule periplakin is associated with advanced stage and poor prognosis of esophageal squamous cell carcinoma

Background: Our previous report showed that periplakin (PPL), a member of the plakin family of proteins, was expressed in all the normal esophageal squamous cells, except the Ki67+ basal cell layer; however, PPL-negative cells were observed in esophageal squamous cell carcinoma (ESCC) tissue samples and the proportion of PPL-positive areas in tumors showed considerable variation. In this study, we analyzed the relationships between PPL expression in tumors and the clinicopathological features of ESCC. Methods: PPL expression was evaluated by immunostaining ESCC samples from 70 patients who underwent surgery and we classified the samples into PPL-negative and PPL-positive groups based on the proportion of PPL-positive areas in tumors, and the relationships among PPL expression, clinical features, and the ESCC prognosis were analyzed. ESCC cell line KYSE270 cells were stably transfected with the PPL expression vector and their growth was assessed by colony formation assay and subcutaneous xenografts in athymic mice. Results: As found in our previous study, decreased PPL staining intensity was observed in all of the cancer tissues analyzed, compared with that observed in paired normal esophageal mucosae; however, the PPL-positive group (the percentage of PPL-positive tumor cells was >20% on immunostaining) exhibited positive associations with the pT classification (larger primary tumor) (P=0.029), pN classification (lymph node metastasis) (P=0.0462), and advanced stages of cancer (P=0.0253). High PPL expression was observed in all of 26 lymph node metastases analyzed. Furthermore, patients with PPL-positive tumors showed poor postoperative prognosis compared to those with PPL-negative ones. Forced expression of PPL in the KYSE270 ESCC cell line induced a stratified structure and colony formation. In addition, PPL-transfected cells formed larger tumors in nude mice than mock-transfected cells, suggesting that relatively high PPL expression in tumors may facilitate cell-cell adhesion by desmosome formation and eventual cell growth in vivo. Conclusions: PPL expression was generally reduced in ESCC compared with paired non-cancer tissue; however, relatively high levels of PPL expression in tumors correlated with tumor progression, lymph node metastasis, advanced stage cancer, and a poor prognosis.

Efficacy of Shikonin against Esophageal Cancer Cells and its possible mechanisms in vitro and in vivo.

Increasing evidences indicate that shikonin can suppress the tumor growth. However, the mechanisms remain elusive. In the present study, we investigated the effects and mechanisms of shikonin against esophageal cancer. The expression of hypoxia inducible factor 1α (HIF1α) and pyruvate kinase M2 (PKM2) in esophageal cancer tissues and cells was detected by immunohistochemistry and Western blot. CCK-8 was used to examine the esophageal cancer cell viability. Apoptosis and cell cycle were analyzed by flow cytometry. The expression of EGFR, PI3K, Akt, p-AKT, mTOR, HIF1α and PKM2 was detected by Western blot. EC109/pkm2 was established by lentivirus transducer. Ec109 tumor model was founded to observe the antitumor effect of shikonin in vivo.We found that HIF1α and PKM2 protein expression levels were higher in esophageal cancer tissues and cells than normal esophageal tissues and cells. Shikonin reduced esophageal cancer cells viability and induced cell cycle arrest and apoptosis. Shikonin decreased EGFR, PI3K, p-AKT, HIF1α and PKM2 expression. Overexpression of PKM2 could enhance resistance of esophageal cancer cells to shikonin. In vivo we found that shikonin reduced tumor burden, inducing cell arrest and apoptosis. Taken together, shikonin has a significant antitumor effect in the esophageal cancer by regulating HIF1α/PKM2 signal pathway.

111In-labelled polymeric nanoparticles incorporating a ruthenium-based radiosensitizer for EGFR-targeted combination therapy in oesophageal cancer cells.

Radiolabelled, drug-loaded nanoparticles may combine the theranostic properties of radionuclides, the controlled release of chemotherapy and cancer cell targeting. Here, we report the preparation of poly(lactic-co-glycolic acid) (PLGA) nanoparticles surface conjugated to DTPA-hEGF (DTPA = diethylenetriaminepentaacetic acid, hEGF = human epidermal growth factor) and encapsulating the ruthenium-based DNA replication inhibitor and radiosensitizer Ru(phen)2(tpphz)2+ (phen = 1,10-phenanthroline, tpphz = tetrapyridophenazine) Ru1. The functionalized PLGA surface incorporates the metal ion chelator DTPA for radiolabelling and the targeting ligand for EGF receptor (EGFR). Nanoparticles radiolabelled with 111In are taken up preferentially by EGFR-overexpressing oesophageal cancer cells, where they exhibit radiotoxicity through the generation of cellular DNA damage. Moreover, nanoparticle co-delivery of Ru1 alongside 111In results in decreased cell survival compared to single-agent formulations; an effect that occurs through DNA damage enhancement and an additive relationship between 111In and Ru1. Substantially decreased uptake and radiotoxicity of nanoparticles towards normal human fibroblasts and oesophageal cancer cells with normal EGFR levels is observed. This work demonstrates nanoparticle co-delivery of a therapeutic radionuclide plus a ruthenium-based radiosensitizer can achieve combinational and targeted therapeutic effects in cancer cells that overexpress EGFR.

BMI-1 suppression increases the radiosensitivity of oesophageal carcinoma via the PI3K/Akt signaling pathway.

B-cell‑specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose-and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/Mphase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclinD2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of BMI-1 knockdown, while the kinase agonist IGF-1 reversed the effects of BMI-1 knockdown on cell viability and radiosensitivity. Taken together, BMI-1 knockdown induces radiosensitivity in ESCC and significantly inhibits cell viability, which may contribute to an increased proportion of cells in the G0/G1phase and cell apoptosis via suppression of the PI3K/Akt signalling pathway.

MicroRNA-615-5p targets insulin-like growth factor 2 and exerts tumor-suppressing functions in human esophageal squamous cell carcinoma

To investigate the expression pattern, clinical significance and functional roles of microRNA (miR)-615-5p in human esophageal squamous cell carcinoma(ESCC), , quantitative real-time PCR was performed to detect expression levels of miR‑615‑5p in ESCC tissues and cell lines. Associations between miR‑615‑5p expression and various clinicopathological features of ESCC patients were also statistically evaluated. The candidate targets of miR‑615‑5p were identified by integrating bioinformatics miRNA target prediction, western blot analysis and luciferase reporter assay. Moreover, the functions of miR‑615‑5p in ESCC cell migration and invasion were determined using the transfection of miRNA mimics, or co-transfected with miRNA mimics and the expression vector of its target gene. As a result, miR‑615‑5p expression in ESCC tissues and cells were markedly lower than those in non-cancerous esophageal mucosa and human normal esophageal cells, respectively (both P<0.001). miR‑615‑5p downregulation was significantly associated with advanced tumor-node-metastasis stage, positive lymph node metastasis and moderate-poor differentiation. Functionally, the re-expression of miR‑615‑5p suppressed the invasion and migration of ESCC cells invitro. Interestingly, insulin-like growth factor 2 (IGF2) was identified as a direct target gene of miR‑615‑5p, and the inhibitory effects of miR‑615‑5p in ESCC cell motility were reversed by the restoration of IGF2 expression. In conclusion, miR‑615‑5p downregulation may be an underlying molecular mechanism of development and progression of ESCC, and may function as a potential therapeutic target of this malignancy. Also, we illustrate that the miR‑615‑5p/IGF2 axis may bring important contributions to cell motility of human ESCC.

Expression of microRNA-935 in esophageal squamous cell carcinoma tissues and cells and its effects on cell proliferation and invasion

Objective To investigate the expression of microRNA (miRNA, miR)-935 in esophageal squamous cell carcinoma (ESCC) tissues and cells and explore its effects on proliferation and invasion ability of ESCC cells. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was employed to detect the expression of miR-935 in ESCC tissues and cells as well as normal esophageal tissues and cells. MiR-935 mimic, miR-935 inhibitor and negative control miRNA were utilized to transfect ESCC EC1 cells, and FQ-PCR was utilized to detect the expression of miR-935 48 h after transfection. Furthermore, cell proliferation and invasion ability was analyzed by cell counting kit-8 (CCK-8) and Transwell chamber after transfection. Results Expression of miR-935 in ESCC tissues and cells was significantly higher than that in normal esophageal tissues and cells, and the differences were statically significant (t=8.349, P=0.000). In addition, expression of miR-935 in metastatic ESCC patients were evidently higher than that in non-metastatic ESCC patients, and the differences were statically significant (t=4.264, P=0.000). MiR-935 mimic markedly upregulated the expression level of miR-935 in EC1 cells, whereas miR-935 inhibitor significantly downregulated the expression level of miR-935 in EC1 cells. Further investigation revealed that miR-935 upregulation promoted the proliferation and invasion ability of EC1 cells, conversely, miR-935 downregulation significantly inhibited the proliferation and invasion ability of EC1 cells. Conclusion MiR-935 plays an essential role in the occurrence and development of ESCC, and thus may be an underlying molecular target for the patients with ESCC. Key words: MicroRNA-935; Esophageal squamous cell carcinoma; Cell proliferation; Invasion; Metastasis

Expression of kinesin family protein 14 in esophageal squamous cell carcinoma and its effects on cell proliferation and cell cycle

Objective To investigate the expression of Kinesin family protein 14 (KIF14) in esophageal squamous cell carcinoma (ESCC), and explore its effects of its downregulation on cell proliferation and cell cycle. Methods Immunohistochemistry was used to investigate the expression of KIF14 in 87 cases of ESCC tissues and paired normal esophageal tissues, and the correlations between KIF14 expression and clinic pathological features were analyzed. Western blotting was employed to detect the expression KIF14 protein in various ESCC cells and normal esophageal cells, and KIF14 small interfering RNA (siRNA) and control siRNA were utilized to transfect to ESCC Eca109 cells, and expression of KIF14 protein was examined after transfection. Furthermore, cell counting kit-8 (CCK-8) and Flow cytometry were used to investigate the effects of KIF14 downregulation on cell proliferation and cell cycle of ESCC Eca109 cells. Results The positive expression ratio of KIF14 in ESCC tissues (62.3%) was significantly higher than that in normal esophageal tissues (21.8%), and the differences were statistical significance (χ2=30.474, P=0.000). KIF14 protein expression was tightly associated with histological differentiation, clinic staging and lymph node metastasis (P=0.000, 0.026, 0.007), but not related to gender and age (P=0.821, 0.644). In addition, expression of KIF14 in ECSS cells was significantly higher than that in normal esophageal cell Het-1A, and KIF14 siRNA markedly downregulated the expression of KIF14 protein in Eca109 cells. Most notably, KIF14 downregulation obviously suppressed cell proliferation and arrested cell cycle in G0/G1 phase in Eca109 cells. Conclusion KIF14 may play a pivotal role in the occurrence and development of ESCC, and thus may be an underlying molecular target. Key words: Kinesin family protein 14; Esophageal squamous cell carcinoma; Cell proliferation; Cell cycle

Abstract 5482: Constitutive overexpression of nrf2 in esophageal adenocarcinoma protects cancer cells from bile salts-induced DNA damage and favors cancer cell survival

Abstract Background: Esophageal adenocarcinoma (EAC) is the major type of malignant cancer of esophagus in the USA. The incidence rate has increased 4-10% per year among men since 1976 in the USA, more rapidly than for any other type of cancer. EAC is known to originate from premalignant Barrett’s esophagus (BE) through BE-dysplasia-EAC process. Normal cells have intact anti-oxidative mechanisms, among which NFE2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular response to various stimuli. Cancer cells have high levels of reactive oxygen species and oxidative stress due to activation of oncogenes, inflammatory microenvironment, and dysfunction of anti-oxidative mechanisms. However, the role of NRF2 in Barrett’s related esophageal carcinogenesis is barely known. Methods and Results: We have found that Nrf2 protein expression is significantly upregulated in EAC cell lines as compared to BE cell lines (CPA, BAR10T) and normal esophageal squamous cell lines. We detected a similar overexpression of NRF2 in primary EAC tissue samples. Using luciferase reporter assay, we demonstrated significant induction of NRF2 transcription activity in EAC cells in response to exposure to acidic bile acids, as compared to controls (P&amp;lt;.01). We detected significant increase in the expression of Heme Oxygenase 1 (HO-1) and Glutathione Reductase (GR) in these cells (P&amp;lt;.01). Knockdown of NRF2 by siRNA or crisp/cas9, significantly increased cellular ROS level, oxidative DNA damage (8-oxoguanine), and double strand DNA breaks (p-H2AX). Knockdown of NRF2 sensitized cells to apoptosis following exposure to bile acids and CDDP. Concordant with these results, the use of NRF2 inhibitor, Brusatol, sensitized EAC cells to CDDP treatment with a significant increase in cell death, as compared to single agent treatment (P&amp;lt;.01). Conclusion: These results indicate that constitutive overexpression of NRF2 in EAC cells protects cancer cells from high level of ROS and promotes cancer cell survival. Inhibition of NRF2 through its specific inhibitor may have therapeutic value in EAC. Citation Format: Dunfa Peng, Tianling Hu, Shoumin Zhu, Wael El-Rifai. Constitutive overexpression of nrf2 in esophageal adenocarcinoma protects cancer cells from bile salts-induced DNA damage and favors cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5482. doi:10.1158/1538-7445.AM2017-5482

Abstract 2540: miR-196b as a potential biomarker for human esophageal cancer

Abstract Esophageal cancer is the sixth most common cause of cancer related death. Although multiple genetic and epigenetic alterations have been detected in esophageal cancer, molecular markers for early diagnosis and prediction of prognosis or treatment responses are quite limited. microRNA (miRNA) is a class of small-regulatory non-coding RNA, acting as either a tumor suppressor or oncogene by regulating gene expression through pairing with complementary seed of the targeted messenger RNAs (mRNA). A number of miRNA expression profiling studies have been conducted in esophageal. In our current study, we analyzed the most recent studies on miRNAs expression profiles in esophageal cancer. Consistently dysregulated miRNAs have been identified in esophageal cancer, including upregulation of miR-196b, miR-135a, and downregulation of miR-141, miR-200a-5p, miR-200b-3p, miR-27b, miR-210. We first assessed expression of miRNAs in esophageal cancer cell lines and primary esophageal cancer tissues by real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Significantly overexpressed miR-196b was observed in human esophageal squamous cell carcinoma KYSE-70 and KYSE-180 compared with normal esophageal squamous cell line, HET-1A. Furthermore, overexpression of miR-196b was detected in 12 out of 14 (86%) cancer tissues compared to the matched normal tissues. Forced expression of miR-196b promotes the proliferation and invasion in esophageal cancer cell lines. TargetScanS and miRanda were used to identify the target genes of miR-196b. A list of targets genes were predicted, including GATA6, ARHGAP28, EPS15 and EPHA7. Forced expression of miR-196b resulted in significant downregulated EPS15 and EPHA7 expression in esophageal cancer cell lines. Our present work suggested that miR-196b acts as an oncomiR by regulating EPS15 and EPHA7. Therefore, miR-196b could be used as a biomarker for esophageal cancer. Citation Format: Xiaohui Tan, Tao Chen, Robert Siegel, Sidney Fu. miR-196b as a potential biomarker for human esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2540. doi:10.1158/1538-7445.AM2017-2540

RNA editing of SLC22A3 drives early tumor invasion and metastasis in familial esophageal cancer

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.

Expressions of HPV 16-E6 in esophageal carcinoma and its clinical significance

Aim: The aim of the study was to explore the association between HPV16-E6 protein and esophageal squamous cell carcinoma.Methods: SP immunohistochemical method was used to examine the expression of HPV 16-E6 in 50 cases of esophageal squamous cell carcinoma, 10 cases of normal esophageal squamous cell and 10 cases of adjacent tissue.Result: The expressions of HPV 16-E6 was significantly higher in esophageal carcinoma than in normal esophageal mucosa and in adjacent tissue. The expressions of HPV 16-E6 had a significant correlation with invasive depth (P&lt;0.05), but not with the patient age, lymph node metastasis and tumor size (P&gt;0.05).Conclusion: HPV 16-E6 can promote the growth and metastasis of esophageal squamous cell carcinoma and can be a prognostic factor of esophageal squamous cell carcinoma.

Arenobufagin activates p53 to trigger esophageal squamous cell carcinoma cell apoptosis in vitro and in vivo.

Esophageal squamous cell carcinoma (ESCC) is often diagnosed at late incurable stage and lacks effective treatment strategy. Bufadienolides are cardiotonic steroids isolated from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor with novel anticancer activity. However, there is little information about the effects and action mechanisms of bufadienolides on ESCC cells. In this study, the in vitro and in vivo anti-ESCC activities of bufadienolides, including bufalin (Bu) and arenobufagin (ArBu), were examined and the underlying molecular mechanisms were elucidated. The results showed that ArBu exhibited higher anticancer efficacy than Bu against a panel of five ESCC cells, with IC50 values ranging from 0.8 μM to 3.6 μM. However, ArBu showed lower toxicity toward Het-1A human normal esophageal squamous cells, indicating its great selectivity between cancer and normal cells. Moreover, ArBu effectively induced ESCC cell apoptosis mainly by triggering caspase activation through intrinsic and extrinsic pathways. Treatment of ESCC cells also significantly activated p53 signaling by enhancing its phosphorylation. Interestingly, transfection of cells with p53 small interfering RNA significantly inhibited the ArBu-induced p53 phosphorylation and the overall apoptotic cell death. Furthermore, ArBu also demonstrated novel in vivo anticancer efficacy by inhibiting the tumor growth through activation of p53 pathway. Taken together, these results demonstrate the p53-targeting therapeutic potential of bufadienolides against ESCC.

A novel signaling role for miR-451 in esophageal tumor microenvironment and its contribution to tumor progression.

We evaluated miR-451 expression in serum and tissue samples of esophageal squamous cell carcinoma (ESCC) patients. Then, we examined a secretory role of miR-451 in esophageal tumor microenvironment. miR-451 expression was evaluated in 39 serum samples from esophageal SCC patients compared to 39 normal individuals as well as 26 pairs of fresh-frozen tumor and adjacent normal tissues from patients with ESCC, using qRT-PCR. In a co-culture system of human normal fibroblasts (HFSF-PI3) and esophageal cancer cell line (KYSE-30), we evaluated exosomal miR-451 secretion into the conditioned medium (CM) of both cell lines. Then, we analyzed the effect of primiR-451-transfected fibroblasts on the migration potency of their neighboring KYSE-30 cells. We detected miR-451 over-expression in serum samples of esophageal cancer patients compared to the normal group (P=0.005). Interestingly, fresh-frozen tumor tissues from the same patients showed miR-451 down-regulation compared to their adjacent normal counterparts (P=0.043). Co-culturing the KYSE-30 cell line with normal fibroblasts significantly induced miR-451 exosomal secretion into the CM. Moreover, co-culture of KYSE-30 cell line with miR-451-over-expressing fibroblasts significantly induced migration tendency in KYSE-30 cell line compared to the mock-transfected fibroblasts (P<0.0001). In this system, MIF expression (a validated target of miR-451) in the KYSE-30 cell line was increased although this alteration was not statistically significant (fold change=4.44). Our data suggest that cancer-associated fibroblasts use exosomal miR-451 as a signaling molecule to provide a favorable niche for tumor cell migration and cancer progression. Our findings provide new insights into the stromal role of miR-451 in the esophageal tumor microenvironment as a communicatory molecule and suggest a signaling role for miR-451 in extracellular matrix cross-talks.

Identification of reference genes and miRNAs for qRT-PCR in human esophageal squamous cell carcinoma.

It is important to select an appropriate reference gene and miRNA when using quantitative real-time polymerase chain reaction (qRT-PCR) to analyze gene and miRNA expression. However, many commonly used reference genes and miRNAs are not stably expressed and therefore not suitable for normalization or quantification of qRT-PCR data. This study aims to identify appropriate reference genes and miRNAs for use in human esophageal squamous carcinoma qRT-PCR analysis. Using data provided by The Cancer Genome Atlas, we identified DDX5, LAPTM4A, P4HB, RHOA, miR-28-5p, miR-34a-5p, and miR-186-5p as candidate reference genes and miRNAs. We used qRT-PCR to verify the expression levels of these candidates and another seven commonly used reference genes and miRNAs. A set of 50 paired human normal esophageal tissues and squamous cell carcinoma samples were used in the analysis. We then used geNorm and NormFinder to analyze the results. DDX5, LAPTM4A, RHOA, ACTB, RNU48, miR-28-5p, miR-34a-5p, and miR-186-5p were stably expressed, indicating they are suitable for used as references in qRT-PCR analysis of esophageal squamous cell carcinoma. However, expression levels of 18s rRNA, GAPDH, P4HB, 5s rRNA, U6, and RNU6B varied greatly between esophageal normal and squamous cell carcinoma samples, indicating that they are not suitable for use as references in the qRT-PCR analysis of esophageal squamous cell carcinoma.

Association Between Germline Mutation in VSIG10L and Familial Barrett Neoplasia.

Esophageal adenocarcinoma and its precursor lesion Barrett esophagus have seen a dramatic increase in incidence over the past 4 decades yet marked genetic heterogeneity of this disease has precluded advances in understanding its pathogenesis and improving treatment. To identify novel disease susceptibility variants in a familial syndrome of esophageal adenocarcinoma and Barrett esophagus, termed familial Barrett esophagus, by using high-throughput sequencing in affected individuals from a large, multigenerational family. We performed whole exome sequencing (WES) from peripheral lymphocyte DNA on 4 distant relatives from our multiplex, multigenerational familial Barrett esophagus family to identify candidate disease susceptibility variants. Gene variants were filtered, verified, and segregation analysis performed to identify a single candidate variant. Gene expression analysis was done with both quantitative real-time polymerase chain reaction and in situ RNA hybridization. A 3-dimensional organotypic cell culture model of esophageal maturation was utilized to determine the phenotypic effects of our gene variant. We used electron microscopy on esophageal mucosa from an affected family member carrying the gene variant to assess ultrastructural changes. Identification of a novel, germline disease susceptibility variant in a previously uncharacterized gene. A multiplex, multigenerational family with 14 members affected (3 members with esophageal adenocarcinoma and 11 with Barrett esophagus) was identified, and whole-exome sequencing identified a germline mutation (S631G) at a highly conserved serine residue in the uncharacterized gene VSIG10L that segregated in affected members. Transfection of S631G variant into a 3-dimensional organotypic culture model of normal esophageal squamous cells dramatically inhibited epithelial maturation compared with the wild-type. VSIG10L exhibited high expression in normal squamous esophagus with marked loss of expression in Barrett-associated lesions. Electron microscopy of squamous esophageal mucosa harboring the S631G variant revealed dilated intercellular spaces and reduced desmosomes. This study presents VSIG10L as a candidate familial Barrett esophagus susceptibility gene, with a putative role in maintaining normal esophageal homeostasis. Further research assessing VSIG10L function may reveal pathways important for esophageal maturation and the pathogenesis of Barrett esophagus and esophageal adenocarcinoma.