Oncolytic adenovirus-mediated dual knockdown of survivin and OCT4 improves therapeutic efficacy in esophageal cancer.

Background: Survivin and octamer-binding transcription factor 4 (OCT4) are reportedly up-regulated in esophageal cancer (EC) and have been correlated with high tumor proliferative activity and poor prognosis. Oncolytic viruses encoding specific transgenes have been considered as therapeutic methods to increase therapeutic efficacy in a variety of solid tumors. Methods: In this study, an oncolytic adenovirus carrying short hairpin RNA (shRNA) of survivin (shSRVN) and OCT4 (shOCT4) was constructed to achieve dual knockdown of survivin and OCT4 and to explore the potential effect of the oncolytic adenovirus in EC. Results: The oncolytic adenovirus replicated abundantly in human EC cells, with the replication multiplying by up to 192,085 and 620,055 times in esophageal carcinoma (Eca)-109 cells transfected with purified and completed recombinant adenoviruses called AdSProE1a-dual shRNA (shSRVN + shOCT4) and TE1 cells transfected with AdSProE1a-survivin shRNA (shSRVN) 96 hours after infection, respectively. The shRNAs targeting survivin and OCT4 significantly downregulated the expression levels of survivin and OCT4 in cells, thereby inhibiting the proliferative activity of cancer cells. Furthermore, E-cadherin and vimentin, which are both considered epithelial mesenchymal transition (EMT) markers, were found to be upregulated and downregulated, respectively, in cancer cells after exposure to the viral infection. The interference of survivin and OCT4 also contributed to cell cycle arrest and apoptosis, the half maximal inhibitory concentrations (IC50s) of oncolytic adenovirus loaded with AdSProE1a-shSRVN + shOCT4 in the Eca109 cells and the TE1 cells were 0.7271 and 0.1032 pfu/mL, respectively. Xenograft experiments in vivo showed that oncolytic adenovirus-mediated dual knockdown of survivin and OCT4 effectively inhibited the growth of xenografts and induced cancer cell apoptosis. We concluded that therapies targeting survivin and OCT4 have great potential for improving the therapeutic efficacy in EC. Conclusions: The dual target design strategy ensured the efficacy and safety of the treatment system and provided a novel and effective adjuvant target therapy for EC.

Retraction Notice to: Hypoxic tumor-derived exosomal circular RNA SETDB1 promotes invasive growth and EMT via the miR-7/Sp1 axis in lung adenocarcinoma.

[This retracts the article DOI: 10.1016/j.omtn.2021.01.019.].

Elevated tumor markers in a benign lung disease.

BACKGROUND: This study aimed to determine the underlying pathophysiologic mechanism of elevated carbohydrate antigen 19-9 (CA19-9) in pulmonary sequestration (PS) patients. MATERIALS AND METHODS: Four pulmonary sequestration patients, 12 pneumonia patients and 12 healthy adult volunteers were prospectively studied. Specimens from another 34 pulmonary sequestration patients were retrospectively analyzed. Serum CA19-9 levels of 4 patients were tested before and 1 week, 1 month and 3 months after surgery. The CA19-9 levels of 12 pneumonia patients and 12 healthy adult volunteers were tested as controls. The expression and localization of CA19-9 in diseased lesions and corresponding normal lung tissues were analyzed by Immunohistochemical (IHC). Hematoxylin-eosin (HE) staining was performed to observe the pathological changes in pulmonary sequestration tissues. RESULTS: Serum CA19-9 levels were significantly higher in the 4 patients (797.3 ± 316 IU/ml) than in the pneumonia patients (10.07 ± 5.01 IU/ml) and healthy volunteers (9.85 ± 4.12 IU/ml). In addition, serum CA19-9 levels decreased dramatically after the focus was removed. Positive staining of CA19-9 was found in 70% (24/34) of pulmonary sequestration tissues, and CA19-9 was mainly expressed in the bronchial mucus. In the 4 diseased lesions, deformed alveolar structure and inflammatory cell infiltration were observed, and the degree of damage was positively correlated with serum CA19-9 levels. CONCLUSIONS: CA19-9 could be generated by abnormal columnar epithelia in pulmonary sequestration tissues and was transported into circulation after alveoli damage. CA19-9 could serve as an adjuvant diagnostic marker in pulmonary sequestration.

Potential of the cell-free blood-based biomarker uroplakin 2 RNA to detect recurrence after surgical resection of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer, and ~30% of patients with LUAD develop cancer recurrence after surgery. The present study aimed to identify and validate biomarkers that may be used to monitor recurrence following LUAD surgery. Data from patients with LUAD were downloaded from The Cancer Genome Atlas database and postoperative recurrence samples were selected. Subsequently, weighted gene co-expression network analysis (WGCNA) was subsequently performed to identify key co-expression gene modules. Additionally, enrichment analysis of the key gene modules was performed using the Database for Annotation, Visualization and Integrated Discovery. Furthermore, survival analysis was performed on the most notable biomarker, uroplakin 2 (UPK2), which was downloaded from the Oncomine database, and its effect on prognosis was assessed. WGCNA identified 39 gene modules, of which one was most associated with recurrence. Among them, UPK2, kelch domain containing 3, galanin receptor 2 and tyrosinase-related protein 1 served a central role in the co-expression network and were significantly associated with the survival of patients. A total of 132 blood samples were collected from patients with LUAD with free UPK2 in the plasma. The expression levels of UPK2 relative to GADPH were 0.1623 and 0.2763 in non-relapsed and relapsed patients, respectively. Receiver operating characteristic curve analysis was used to detect free UPK2 mRNA in the blood in order to monitor postoperative recurrence, resulting in an area under the curve of 0.767 and a 95% CI of 0.675-0.858. Patients with high free UPK2 mRNA expression had unfavorable survival outcomes compared with those with low UPK2 expression. Therefore, free UPK2 mRNA expression in the plasma may have the potential to act as an indicator of postoperative recurrence in patients with early stage LUAD.

Hypoxic tumor-derived exosomal circular RNA SETDB1 promotes invasive growth and EMT via the miR-7/Sp1 axis in lung adenocarcinoma.

Hypoxia is a common feature of solid tumors and has been associated with tumor aggressiveness and poor prognosis. Exosomes are involved in mediating cellular-environment interactions. Circular RNAs (circRNAs) are a class of non-coding RNA broadly found in cells and exosomes. However, the functions and regulatory mechanisms of exosomal circRNAs induced by hypoxia remain poorly understood in lung adenocarcinoma (LUAD) development. Differentially expressed circRNAs were identified between exosomes extracted from hypoxic and normoxic conditions through microarray analysis. We focused on hsa-circ-0003439 found on chromosome 1 and derived from SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), and thus we named it circSETDB1. We discovered that exosomes obtained from hypoxic LUAD cells improved the migration, invasion, and proliferation capacity of normoxic LUAD cells. circSETDB1 was found to be significantly upregulated in hypoxia-induced exosomes from LUAD cell lines compared with exosomes in the normal condition. Moreover, knockdown of circSETDB1 significantly inhibited cell malignant growth in vitro. Importantly, we showed that circSETDB1 was upregulated in serum exosomes in LUAD patients, and exosomal circSETDB1 levels were closely associated with disease stage. Finally, using RNA immunoprecipitation (RIP), bioinformatics, and luciferase reporter assays, we elucidated the implication of a circSETDB1/miR-7/specificity protein 1 (Sp1) axis in the development and epithelial-mesenchymal transition (EMT) of lung adenocarcinoma.

SOX12 contributes to the activation of the JAK2/STAT3 pathway and malignant transformation of esophageal squamous cell carcinoma.

As a crucial transcription factor, sex­determining region Y box 12 (SOX12) is closely related with tumorigenesis and malignant transformation in various malignant tumor types. To date, the specific function of SOX12 in esophageal squamous cell carcinoma (ESCC) has remained largely elusive and requires further investigation. The present study aimed to determine whether aberrant expression of SOX12 is associated with malignant development of ESCC. The expression level of SOX12 in ESCC cells and tissues was analyzed by RT­qPCR and western blotting. Short hairpin RNA (shRNA) targeting SOX12 was transfected into ESCC cells to knock down the expression of SOX12. Colony formation and Transwell assays were used to detect viability and mobility of ESCC cells. Signaling pathway­related proteins were assessed using western blot analysis and cellular immunofluorescence. Clinical prognosis data was analyzed by Kaplan­Meier and Cox logistic regression. The present results revealed that SOX12 was overexpressed in ESCC cells and tissues. Knockdown of the expression of SOX12 by shRNA inhibited the colony forming efficiency, migration and invasion capacities of ESCC cells in vitro. Recombinant protein of SOX12 could restore the aggressive phenotype of ESCC cells. Furthermore, knockdown of the expression of SOX12 inhibited the activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway by decreasing the expression of the JAK2/STAT3 signaling pathway. Recombinant protein of SOX12 could recover the activation of the JAK2/STAT3 signaling pathway. Analysis of the clinical data revealed that overexpression of SOX12 indicated shorter overall survival time (OS; P=0.0341) and disease­free survival time (DFS; P=0.04). Univariate and multivariate analysis revealed that overexpression of SOX12 was an independent factor for ESCC. In conclusion, SOX12 was revealed to serve a crucial function in sustaining the viability, as well as enhancing the motility of ESCC cells via activating the JAK2/STAT3 signaling pathway. Thus, SOX12 may potentially serve as a novel biomarker and candidate for the targeted treatment of ESCC.

The influence of thoracic duct ligation on long-term survival of patients with esophageal cancer: a propensity score-matched analysis.

BACKGROUND: Ligation of the thoracic duct (LTD) is known to be a useful way to prevent postoperative chylothorax, but its impact on long-term survival is rare to be assessed. METHODS: Data from 609 patients with esophageal cancer who underwent esophagectomy from September, 2012, to January, 2014, were retrospectively collected. The study cohort was classified into two groups: the thoracic duct ligation group (LG) and the non-ligation group (NLG). Propensity score matching (PSM) was performed to control confounding factors between the two groups. Postoperative complications and length of stay were compared between the two groups. Overall survival was estimated using the Kaplan-Meier method, and compared using the log-rank test. Independent prognostic factors were determined using Cox regression analysis. RESULTS: After PSM, there were 185 patients in each of the two groups. LTD had no significant impact on chylothorax, anastomotic leak, recurrent nerve palsy, pneumonia and length of stay (P>0.05). The 1-, 3- and 5-year survival rates were 87.0%, 64.1%, and 50.9% in the LG, respectively, compared to 85.4%, 59.9%, and 42.3%, respectively, in the NLG. The differences between the 2 groups were not statistically significant (P=0.156). In the multivariable analysis, LTD was not an independent prognostic factor, neither before nor after PSM. CONCLUSIONS: Our study demonstrated that LTD had no significant impact on postoperative complications or long-term survival in patients with esophageal cancer.

Hypoxic Tumor-Derived Exosomal Circ0048117 Facilitates M2 Macrophage Polarization Acting as miR-140 Sponge in Esophageal Squamous Cell Carcinoma.

INTRODUCTION: Hypoxia and tumor-associated macrophage (TAM) are key regulators in remodeling the microenvironment of esophageal squamous cell carcinoma (ESCC). Hypoxia could stimulate tumor cells to secrete more exosomes and activate TAMs to M2 type. Here, we investigated the function and the underlying mechanism of tumor-derived exosomal hsa-circ-0048117 in TAM polarization in ESCC. Collectively, these data indicate that PC cells generate miR-301a-3p-rich exosomes in a hypoxic microenvironment, which then polarize macrophages to promote malignant behaviors of PC cells. METHODS: Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to analyze the physical characteristics of exosomes. High-throughput sequencing and bioinformatic analysis were performed to screen the potential exosomal circRNA. FISH, Ago2 RIP, pull-down and dual-luciferase reporter assay were conducted to figure out the correlation among hsa-circ-0048117, miR-140 and toll-like receptor 4 (TLR4). Flow cytometry and Western blot were used to evaluate their joint effect in macrophages polarization. Then, the invasion and migration ability were evaluated by transwell experiment. At last, serum exo-hsa-circ-0048117 in ESCC patients was compared and the correlation between its expression and T stage, N stage and TNM grades was analyzed. RESULTS: Hsa-circ-0048117 was significantly upregulated and enriched in exosomes secreted by hypoxia pre-challenged tumor cells and contributed to M2 macrophage polarization. Hsa-circ-0048117 depletion in macrophage led to inhibition of M2 polarization while restoration of hsa-circ-0048117 could rescue the process. Moreover, hsa-circ-0048117 could act as sponge of miR-140 by competing with TLR4 to facilitate the M2 macrophage polarization. Exo-hsa-circ-0048117 could be transmitted to macrophages to promote M2 polarization and M2 macrophages could enhance the ability of invasion and migration of tumor cells by secreting Arg1, IL-10 and TGF-ß. Higher serum exo-hsa-circ-0048117 predicted an advanced T and N stage and positively correlated with TNM grade. CONCLUSION: Our findings indicated that ESCC cells generate hsa-circ-0048117-rich exosomes in a hypoxic microenvironment; hsa-circ-0048117 was believed to promote M2 macrophage polarization which favors the malignant behaviors of ESCC cells. These results reminded us that exosomal hsa-circ-0048117 may play a key role in remodeling the microenvironment and modulating progression in ESCC.

HKDC1 promotes the tumorigenesis and glycolysis in lung adenocarcinoma via regulating AMPK/mTOR signaling pathway.

BACKGROUND: Hexokinase domain component 1 (HKDC1) plays an oncogenic role in certain types of cancer, such as lymphoma, liver cancer, and breast cancer. Previous bioinformatics study revealed that HKDC1 was significantly upregulated in lung adenocarcinoma (LUAD). However, its biological functions and potential mechanism in LUAD have not been studied. METHODS: We performed bioinformatics analysis, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and a series of functional assays in vitro and in vivo to investigate the roles of HKDC1 in LUAD. RESULTS: We discovered that HKDC1 was highly expressed in LUAD tissues and cell lines, and the positive expression of HKDC1 was correlated with aberrant clinicopathological characteristics in LUAD patients. Furthermore, HKDC1 could serve as a prognostic predictor for LUAD patients. Overexpression of HKDC1 promoted proliferation, migration, invasion, glycolysis, EMT and tumorigenicity, whereas knockdown of HKDC1 produced the opposite functional effects. Mechanistically, HKDC1 could regulate the AMPK/mTOR signaling pathway to perform its biological function. CONCLUSIONS: Our findings suggest that HKDC1 plays an oncogenic role in LUAD. Targeting this gene may provide a promising therapeutic target to delay LUAD progression.

LEF1/Id3/HRAS axis promotes the tumorigenesis and progression of esophageal squamous cell carcinoma.

Our previous study demonstrated that lymphoid enhancer-binding factor 1 (LEF1) could promote the progression of esophageal squamous cell carcinoma (ESCC). However, the regulatory mechanism of LEF1 was not clear thoroughly. Herein, we continued to explore the downstream mechanism of LEF1 in ESCC. In this study, we applied western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, RNA-Seq analysis, a luciferase reporter assay, chromatin immunoprecipitation (ChIP), bioinformatics analysis, and a series of functional assays in vitro and in vivo. The results demonstrated that LEF1 regulated directly the expression of Id3. Id3 was highly expressed in ESCC tissues and correlated with histologic differentiation (p=0.011), pT stage (p<0.01) and AJCC stage (p<0.01) in ESCC patients. Moreover, Id3 could serve as a prognostic factor of ESCC. By various functional experiments, overexpression of Id3 promoted the proliferation, migration, invasion, EMT, and tumorgenicity. Mechanistically, Id3 could regulate ERK/MAPK signaling pathway via activating HRAS to perform its biological function. Furthermore, activating ERK/MAPK signaling pathway promoted the expression of Id3 gene in turn, indicating that a positive regulatory loop between Id3 and ERK/MAPK pathway may exist in ESCC. In summary, LEF1/Id3/HRAS axis could promote the tumorigenesis and progression of ESCC via activating ERK/MAPK signaling pathway. Targeting this cascade may provide a valid antitumor strategy to delay ESCC progress.

HMGA2 regulates circular RNA ASPH to promote tumor growth in lung adenocarcinoma.

In this study, we identified a circular form of ASPH RNA (circASPH), expression of which was upregulated in lung adenocarcinoma and the human lung adenocarcinoma cell lines. We also found a positive correlation between circASPH level and the T and N stages of lung adenocarcinoma patients. Patients with higher levels of circASPH had a shorter overall survival. Moreover, we demonstrated that circASPH was directly regulated by HMGA2 and Twist1. The direct positive regulation of circASPH by Twist1 was dependent on the presence of HMGA2. Functional assays indicated that circASPH promoted the proliferation, migration, and invasion of lung adenocarcinoma cell lines in vitro. The promoting effect of tumor growth by circASPH was also observed in vivo. Mechanistically, circASPH was identified to act as a molecular sponge for miR-370 and abrogate miR-370-mediated inhibition of HMGA2. Finally, we demonstrated that the oncogenic function of circASPH was HMGA2-dependent. These findings reveal the oncogenic functions of the HMGA2-circASPH-HMGA2 axis and may be useful in developing circRNA-based therapeutic strategies for lung adenocarcinoma.

Removal of tumor thrombus from the azygos vein in an esophageal squamous cell carcinoma patient.

BACKGROUND: Esophageal squamous cell carcinoma with tumor thrombus in the arch of the azygos vein has not been reported to date. Neoadjuvant chemotherapy can decrease the stage in patients with advanced preoperative tumor staging, regaining surgical opportunities and significantly prolonging progression-free survival and overall survival. Herein, we present a case of esophageal squamous cell carcinoma accompanied by tumor thrombus in the arch of the azygos vein, and the patient underwent radical surgery after neoadjuvant chemotherapy. CASE PRESENTATION: A 63-year-old male with esophageal squamous cell carcinoma was found to have tumor thrombus formation in the arch of the azygos vein. Four courses of neoadjuvant chemotherapy with the TP regimen (paclitaxel plus nedaplatin) were given. Reexamination revealed a significant reduction in tumor and tumor thrombus volume. Therefore, McKeown radical resection for esophageal cancer and removal of the tumor thrombus in the arch of the azygos vein were performed. Postoperative pathology suggested complete remission of the esophageal tumor and the presence of small focal cancer tissues in the arch of the azygos vein. CONCLUSION: We report a case of esophageal squamous cell carcinoma with tumor thrombus formation in the azygos vein. We conducted radical resection after 4 rounds of neoadjuvant chemotherapy, and the pathological results revealed complete remission of the tumor. We report our experience addressing this rare case, and we hope to find the underlying mechanism of tumor thrombus formation and whether it has any effects on prognosis in our future study.

MiR-34a-5p Inhibits Proliferation, Migration, Invasion and Epithelial-mesenchymal Transition in Esophageal Squamous Cell Carcinoma by Targeting LEF1 and Inactivation of the Hippo-YAP1/TAZ Signaling Pathway.

Background: Our previous studies reported that lymphoid enhancer-binding factor 1 (LEF1) was upregulated in esophageal squamous cell carcinoma (ESCC) and the positive expression of LEF1 was correlated with aberrant clinicopathological characteristics in ESCC patients. However, the upstream mechanism of regulating LEF1 is not clear fully. In this study, we explored the role of miR-34a-5p in ESCC and the possible regulatory mechanism. Methods: In this study, we applied western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), bioinformatics analysis, a luciferase reporter assay, and a series of functional assays to show the potential role of miR-34a-5p in regulating LEF1 in ESCC. Results: By various functional assays, we demonstrated that LEF1 promoted proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in ESCC cells. By bioinformatics analysis and luciferase reporter assay, miR-34a-5p was identified for directly targeting LEF1. Then we investigated the expression of miR-34a-5p and LEF1 in ESCC. As a result, miR-34a-5p was downregulated while LEF1 was upregulated in ESCC tissue and cell lines. Overexpression of miR-34a-5p could inhibit proliferation, migration, invasion and EMT of ESCC cells. The rescue experiment showed that re-expression of LEF1 reversed the suppressive effect caused by miR-34a-5p. At last, we found that miR-34a-5p could suppress Hippo-YAP1/TAZ signaling pathway in ESCC. Conclusion: Our results indicate miR-34a-5p inhibits proliferation, migration, invasion and EMT in ESCC by targeting LEF1 and suppressing the Hippo-YAP1/TAZ signaling pathway, which may provide a new antitumor strategy to delay ESCC progress.

Expression and Prognostic Value of Id-4 in Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: Our previous study demonstrated that Id-1 may promote the tumorigenicity of esophageal squamous cell carcinoma (ESCC). Id-4 is another member of Id family, which is rare to be studied in ESCC. In this study, we investigated the expression of Id-4 in human ESCC specimens and determined whether Id-4 expression was associated with the clinicopathologic characteristic and the prognosis of ESCC patients. METHODS: We examined Id-4 expression using immunohistochemistry in 92 ESCC tissues and adjacent normal tissues. The association between Id-4 expression and clinical parameters and survival was evaluated by statistical analysis. Cox regression analyses were conducted to identify prognostic factors associated with overall survival (OS). In addition, we explored the functional mechanism of Id-4 in ESCC. RESULTS: Id-4 expression was significantly downregulated in ESCC tissues compared with adjacent normal tissues. The expression of Id-4 was associated negatively with pT stage (p=0.002), AJCC stage (p=0.008) and histologic differentiation (p<0.001). OS was more unfavorable in patients with low expression of Id-4 than those with high expression of ESCC patients (p=0.007). In subgroup analysis, low expression of Id-4 could reveal unfavorable OS of patients with pT1b/T2 stage (p=0.024) or with pN0/N1 stage (p=0.004). By univariate analysis, pT stage and Id-4 expression showed statistically significant associations with OS (p=0.025, p=0.01, respectively). By multivariate analysis, Id-4 expression was an independent prognostic factor in ESCC (p =0.038). In addition, we observed that Id-4 could decrease the levels of the p-Smad2, p-Smad3 and TGF-ß1 in both Eca109 and TE1 cells, indicating Id-4 may inactivate the TGF-ß signaling pathway. CONCLUSION: Low expression of Id-4 suggested unfavorable prognosis for ESCC patients and could identify the prognosis in patients of early-stage tumors. The potential mechanism for Id-4's tumor suppressor role in ESCC may be related to its inhibitory effect on TGF-ß signaling pathway. Thus, we believe that Id-4 may be a promising prognostic marker and a therapeutic target in ESCC.

Impact of thoracic duct ligation on substance metabolism and surgical complications in patients with type-2 diabetes mellitus during esophagectomy / 中国胸心血管外科临床杂志

@#Objective    To investigate the impact of thoracic duct ligation (TDL) on metabolism and postoperative complications during esophagectomy in patients with type-2 diabetes mellitus (T2DM). Methods    We conducted a retrospective clinical data analysis of 230 esophageal carcinoma patients with T2DM who underwent esophagectomy in our hospital from January 2003 to December 2018. Patients were divided into a TDL+ group (n=112), including 78 males and 34 females aged 63.47±7.23 years, and a TDL– group (n=118), including 84 males and 34 females aged 64.38±7.57 years. We compared the blood glucose, liver function parameters and lipid metabolic parameters at different time points before and after surgery. In addition, we compared the postoperative major complications between the two groups. Propensity score-matched (PSM) was used to control the observed confounders. Results    Compared with the TDL–group, patients in TDL+ group had higher blood glucose level (P<0.05, except the fourth postoperative day). The total protein and albumin levels on the first and fourth postoperative days in the TDL+ group were lower than those in the TDL– group (P<0.05). The alanine transaminase (P=0.027) and aspartate transaminase (P=0.007) levels on the fourth postoperative day in the TDL+ group were higher than those in the TDL– group. More pulmonary complications (P=0.014) and anastomotic leaks (P=0.047) were found in the TDL+ group. Conclusion    Given that TDL may aggravate metabolic disorders, increase anastomotic leaks and the pulmonary complications, it is cautious to perform TDL, and prophylactic TDL should not be performed routinely for patients with T2DM.

The transcription factor LEF1 promotes tumorigenicity and activates the TGF-ß signaling pathway in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the most difficult subtype of esophageal cancer to treat due to the paucity of effective targeted therapy. ESCC is believed to arise from cancer stem cells (CSCs) that contribute to metastasis and chemoresistance. Despite advances in diagnosis and treatment, the prognosis of ESCC patients remains poor. METHODS: In this study, we applied western blot, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, RNA-Seq analysis, luciferase reporter assay, Chip-qPCR, bioinformatics analysis, and a series of functional assays to show the potential role of LEF1 in regulating esophageal CSCs. RESULTS: We found that the overexpression of LEF1 was associated with aberrant clinicopathological characteristics and the poor prognosis of ESCC patients. In addition, the elevated expression of LEF1 and OV6 was significantly associated with aberrant clinicopathological features, and poor patient prognosis. Moreover, the overexpression of LEF1 was observed in esophageal CSCs purified by the magnetic sorting of adherent and spheroidal ESCC cells. The increased level of LEF1 in CSCs facilitated the expression of CSC markers, stem cell-like properties, resistance to chemotherapy, and tumorigenicity and increased the percentage of CSCs in ESCC samples. Conversely, the knockdown of LEF1 significantly diminished the self-renewal properties of ESCC. We showed that LEF1 played an important mechanical role in activating the TGF-ß signaling pathway by directly binding to the ID1 gene promoter. A positive association between LEF1 and ID1 expression was also observed in clinical ESCC samples. CONCLUSION: Our results indicate that the overexpression of LEF1 promotes a CSC-like phenotype in and the tumorigenicity of ESCC by activating the TGF-ß signaling pathway. The inhibition of LEF1 might therefore be a novel therapeutic target to inactivate CSCs and inhibit tumor progression.

PRMT1 regulates the tumour-initiating properties of esophageal squamous cell carcinoma through histone H4 arginine methylation coupled with transcriptional activation.

Esophageal squamous cell carcinoma (ESCC) is the most difficult subtype of esophageal cancer to treat due to a paucity of effective targeted therapy. ESCC is believed to arise from tumour initiating cells (TICs), which contribute to metastasis and chemoresistance. In this study, we found that Protein arginine methyltransferase 1(PRMT1) was highly expressed in ESCCs and associated with aberrant clinicopathological characteristics of ESCC patients. In ESCC specimens, the elevated expression of PRMT1 and OV6 was significantly associated with histologic grade, TNM stage and poor patient prognosis. Moreover, overexpression of PRMT1 was observed in esophageal TICs purified by magnetic sorting of adherent and spheroid ECA109/TE1 cells. The increased level of PRMT1 in TICs facilitated the expression of TIC markers, stem cell-like properties, resistance to chemotherapy, tumorigenicity and increased their percentages in ECSS samples. Conversely, knockdown of PRMT1 significantly diminished the self-renewal properties of ESCC. Moreover, we show that PRMT1 can catalyse histone H4R3 asymmetric dimethylation and promote transcription activation of down-stream genes. Further RNA-Seq transcriptome analysis reveals that overexpression of PRMT1 in ESCC cell lines activates Wnt/ß-catenin and Notch signaling pathway. Together, our studies highlight that PRMT1 activates and maintains esophageal TICs by mediating transcription alteration through histone H4 arginine methylation.

Differentiated super-enhancers in lung cancer cells.

Super-enhancers (SEs) are regulatory elements with enriched accumulation of key transcription factors. Few studies were done investigating SEs in lung cancers. Here we analyzed epigenetic profiling data to identify SEs in lung cancer cell lines. Enhancers were classified as SEs and typical enhancers (TEs). Most of the TEs were overlapped between normal cell and cancer cells. A great portion of SEs were differentiated comparing these cells. Analysis of GO terms associated with SEs revealed SE remodeling (lost on some sites while gain on others) between normal and lung cancer cells. By comparing the average number of SEs in each GO term in cancer cells with the number in control cells, surprisingly, no GO terms with significantly increased SE number in cancer condition were observed. On the contrary, in aspects such as "cell-cell adhesion", "receptor activity" and "negative regulation of canonical Wnt signaling pathway", the related SEs were significantly reduced in cancer cells. These findings suggest that in lung cancer, cells may not gain decisive gene expression in the related aspect, instead, they may have lost control of the fateful genes. Taken together, our work with the usability of omics data identified SEs in lung cancer cells and further showed cancer-specific features of SE-related terms.

Prognostic value of association of OCT4 with LEF1 expression in esophageal squamous cell carcinoma and their impact on epithelial-mesenchymal transition, invasion, and migration.

Esophageal squamous cell carcinoma (ESCC) is a malignant disease with poor prognosis. Because of early metastasis prior to diagnosis and therapeutic resistance, ESCC has become one of the leading causes of cancer-related death. Here, we investigated the clinicopathological significance of the association of octamer-binding transcription factor 4 (OCT4) with lymphoid enhancer-binding factor 1 (LEF1) expression and the potential molecular mechanism in the epithelial-mesenchymal transition (EMT), invasion, and migration of ESCC. The expression of OCT4 and LEF1 was detected via immunohistochemistry analysis. High levels of LEF1 expression were observed in 95 ESCC specimens and were obviously associated with aberrant clinicopathological features and poor patient prognosis. Our previous study showed that OCT4 expression level is elevated in ESCC, and statistical analysis showed that the elevated expression of OCT4 and LEF1 in ESCC was significantly associated with histologic grade, lymph node metastasis, TNM stage, and poor patient prognosis. The specific inhibition of OCT4 expression via a lentivirus encoding OCT4-shRNA (LV-shOCT4) in Eca109 cells led to decreased levels of OCT4 and LEF1 in vitro. Additionally, we applied a rescue strategy by infecting LV-shOCT4 Eca109 cells with a LEF1 overexpression plasmid (p-LEF1) and detected changes in EMT, migration, and invasion. Unsurprisingly, the p-LEF1 group exhibited greater EMT, invasion, and migration than did the LV-shOCT4 and negative control groups. This study demonstrates for the first time the relationship between OCT4 and LEF1 expression. The combination of high expression of OCT4 and LEF1 was associated with clinicopathological features of atypical patients, and this combination might be an ideal prognostic factor in ESCC. OCT4 positively regulated LEF1 expression, and LEF1 mediated the effects of OCT4 in cancer cell EMT, invasion, and migration. The data presented here suggest that the inhibition of OCT4-LEF1 signaling may be a new therapeutic target for the treatment of ESCC.

miR-541 suppresses proliferation and invasion of squamous cell lung carcinoma cell lines via directly targeting high-mobility group AT-hook 2.

An increasing number of studies have demonstrated that micro-ribonucleic acids (miRNAs) are important tumor suppressors during carcinogenesis. However, the function of miRNA-541 (miR-541) in malignancies, especially lung cancer, has not been widely reported. In this study, miR-541 expression was significantly decreased in squamous cell lung carcinoma (SCLC) cancerous tissue and SCLC cell lines. To analyze miR-541 function in SCLC, we overexpressed miR-541 in SCLC cell lines (SK-MES-1 and H226). According to the CCK8, wound scratch, and transwell invasion assay results, miR-541 overexpression significantly inhibited SCLC cell proliferation, migration, and invasion ability. Next, using RT-PCR, Western blotting, immunocytochemistry, and luciferase assays, HMGA2 was identified, for the first time, as a direct regulatory target of miR-541 in SK-MES-1 and H226 cells. Furthermore, upregulating HMGA2 expression significantly alleviated the suppressive effects of miR-541 on SK-MES-1 and H226 cell proliferation, migration, and invasion. In summary, our study revealed that miR-541 inhibited SCLC proliferation and invasion by directly targeting HMGA2.