KDM6B promotes ESCC cell proliferation and metastasis by facilitating C/EBPß transcription.

BACKGROUND: As an H3K27me3 demethylase and counteracts polycomb-mediated transcription repression, KDM6B has been implicated in the development and malignant progression in various types of cancers. However, its potential roles in esophageal squamous cell carcinoma (ESCC) have not been explored. METHODS: The expression of KDM6B in human ESCC tissues and cell lines was examined using RT-qPCR, immunohistochemical staining and immunoblotting. The effects of KDM6B on the proliferation and metastasis of ESCC were examined using in vitro and in vivo functional tests. RNA-seq and ChIP-seq assay were used to demonstrate the molecular biological mechanism of KDM6B in ESCC. RESULTS: We show that the expression level of KDM6B increased significantly in patients with lymph node metastasis. Furthermore, we confirmed that KDM6B knockdown reduces proliferation and metastasis of ESCC cells, while KDM6B overexpression has the opposite effects. Mechanistically, KDM6B regulates TNFA_SIGNALING_VIA_NFκB signalling pathways, and H3K27me3 binds to the promoter region of C/EBPß, leading to the promotion of C/EBPß transcription. Besides, we show that GSK-J4, a chemical inhibitor of KDM6B, markedly inhibits proliferation and metastasis of ESCC cells. CONCLUSIONS: The present study demonstrated that KDM6B promotes ESCC progression by increasing the transcriptional activity of C/EBPß depending on its H3K27 demethylase activity.

CLDN1 induces autophagy to promote proliferation and metastasis of esophageal squamous carcinoma through AMPK/STAT1/ULK1 signaling.

Tight junction is a structural constitution in cell-cell adhesion and play an important role in the maintenance of permeability and integrity of normal epithelial cell barrier. The protein encoded by Claudin 1 (CLDN1), a member of the claudin family, is an integral membrane protein and a component of tight junction strands. CLDN1 has been proved to regulate the proliferation and metastasis of multiple tumors, but little is known about its role in esophageal squamous cell carcinoma (ESCC). Here, we found that CLDN1 was aberrantly increased in ESCC tissues and cell lines, and mainly distributed in the nucleus of tumor cells. Furthermore, we confirmed that CLDN1 promoted the proliferation and metastasis of ESCC by triggering autophagy both in vitro and in vivo. Mechanically, we validated that CLDN1-induced autophagy via increasing Unc-51 like autophagy activating kinase 1 (ULK1) expression through AMP-activated protein kinase (AMPK)/signal transducer and activator of transcription 1 (STAT1) signaling pathway in ESCC cells. Taken together, our findings demonstrated that aberrant expression and distribution of CLDN1 promoted the proliferation and metastasis of esophageal squamous carcinoma by triggering autophagy through AMPK/STAT1/ULK1 signaling pathway.

[Effect of Epithelial-to-mesenchymal Transition on Biological Activity of NK Cells in Esophageal Squamous Cell Carcinoma].

OBJECTIVE: To investigate the effect of epithelial-mesenchymal transition (EMT) on biological activity of natural killer (NK) cells in esophageal squamous cell carcinoma (ESCC). METHODS: Western blot selected EMT EC9706 and non EMT KYSE150 from six esophageal cancer cell lines. NK cells were collected from 10 cases of healthy volunteers. According to different co-culture conditions, the medium was divided into 1640 normal medium stimulus-NK group (NC group), EC9706 cell supernatant stimulus-NK group (EC9706 group) and KYSE150 cell supernatant stimulus-NK group (KYSE150 group). The expression of NK cells surface/intracellular molecules was detected by flow cytometry after co-culture of NK cells with different conditioned medium for 72 h. 10 ng/mL transforming growth factor-ß (TGF-ß) treated KYSE150 cell line for 7 d to induce EMT. KYSE150 EMT and KYSE150 supernatant were collected and co-cultured with NK cells, respectively. 72 h later, flow cytometry was used to detect the expression of surface/intracellular molecules of NK cells, degranulation ability of CD107a, killing effect of target cell K562, proliferation and apoptosis of NK cells. RESULTS: Two EMT-treated and four non-EMT-treated esophageal squamous cell carcinoma lines were selected from the six strains, and one EC9706 and one KYSE150 respectively were selected for subsequent experiments. The purity of NK cells was more than 90% and Tcells <1%. After the supernatant of esophageal squamous cell carcinoma cells was stimulated, the surface activation type and inhibitory type receptors of NK cells in the three groups were stimulated. The effector molecule results showed that: compared with NC group and KYSE150 group, the expressions of activated type receptors NKP30, NKG2D and NKP44 in EC9706 group were decreased, and the release of granulozyme was decreased ( P<0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( P<0.05). NKP46, CD226, CD16 expressions and perforin release showed no statistically significant difference among the three groups. Compared with KYSE150 supernatant stimulation, the expressions of activated receptors NKP30, NKG2D and NKP44 decreased after KYSE150 EMT supernatant stimulation, and perforin release decreased. The degranulation of CD107a and the killing effect of target cell K562 were decreased, and the proliferation index of NK cell Ki67 was decreased ( P<0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( P<0.05). The expressions of NKP46, CD226 and CD16, granulozyme release and apoptosis of NK cells were not statistically significant. CONCLUSION: EMT of esophageal cancer cells can escape the immune surveillance by inhibit the activity of NK cells and reduce the release of effective molecules.

[Expression of CLDN1 and Its Nuclear Distribution in Esophageal Squamous Carcinoma].

OBJECTIVE: To determine the expression of tight junction protein CLDN1 in esophageal squamous carcinoma (ESCC) and to explore the effect of CLDN1 on the biological function of ESCC TE-11 cells. METHODS: This study collected 30 ESCC tissues,30 adjacent normal tissues and 30 distal esophageal tissues to detected the expression of CLDN1 in tissue microaary by immunohistochemistry method. Western blot was employed to determine the expression of CLDN1 in 15 ESCC tissues and cell lines,and then the distribution of CLDN1 in tumor cells and normal esophageal epithelial cells was analyzed. Lentivirus containing the sequence of CLDN1 shRNA was constructed and transfected into TE-11 cells,the transfection efficiency and intracellular distribution was determined by Western blot and flow cytometry. Cell proliferation was detected by CCK-8,the invasion and migration ability of cells was determined with Transwell. Cytoskeleton changes was observed by laser scanning confocal microscope. RESULTS: Immunohistochemical results showed no significant difference between the cancer tissues and adjacent tissues in term of the positive rate of CLDN1 ( P>0.05). CLDN1 was highly expressed in highly and moderately differentiated ESCC tissues,but its expression was significantly decreased in poorly differentiated cancer tissues. Western blot revealed that CLDN1 was mainly distributed in the nucleus of TE-11 cells. The proliferation of TE-11 cells,as well as invasion and migration ability,were declined obviously when the expression of CLDN1 being down-regulated,while the cytoskeleton protein fluorescence intensity deceased. CONCLUSION: The expression of CLDN1 in ESCC tissue is associated with its differentiation and may promote carcinogenesis in TE-11 cells.

IL-6 and IL-8 secreted by tumour cells impair the function of NK cells via the STAT3 pathway in oesophageal squamous cell carcinoma.

BACKGROUND: Recurrence and metastasis are the leading causes of tumour-related death in patients with oesophageal squamous cell carcinoma (ESCC). Tumour-infiltrating natural killer cells (NK cells) display powerful cytotoxicity to tumour cells and play a pivotal role in tumour therapy. However, the phenotype and functional regulation of NK cells in oesophageal squamous cell carcinoma (ESCC) remains largely unknown. METHODS: Single cell suspensions from blood and tissue samples were isolated by physical dissociation and filtering through a 70 µm cell strainer. Flow cytometry was applied to profile the activity and function of NK cells, and an antibody chip experiment was used to identify and quantitate cytokine levels. We studied IL-6 and IL-8 function in primary oesophageal squamous carcinoma and NK cell co-cultures in vitro and by a xenograft tumour model in vivo. Western blotting was used to quantitate STAT3 (signal transducer and activator of transcription 3) and p-STAT3 levels. Finally, we performed an IHC array to analyse IL-6/IL-8 (interleukin 6/interleukin 8) expression in 103 pairs of tumours and matched adjacent tissues of patients with ESCC to elucidate the correlation between IL-6 or IL-8 and clinical characteristics. RESULTS: The percentages of NK cells in both peripheral blood and tumour tissues from patients with ESCC were significantly increased in comparison with those in the controls and correlated with the clinical characteristics. Furthermore, the decrease in activating receptors and increase in inhibitory receptors on the surface of tumour-infiltrating NK cells was confirmed by flow cytometry. The level of granzyme B, the effector molecule of tumour-infiltrating NK cells, was also decreased. Mechanistically, primary ESCC cells activated the STAT3 signalling pathway on NK cells through IL-6 and IL-8 secretion, leading to the downregulation of activating receptors (NKp30 and NKG2D) on the surface of NK cells. An ex vivo study showed that blockade of STAT3 attenuated the IL-6/IL-8-mediated impairment of NK cell function. Moreover, the expression of IL-6 or IL-8 in tumour tissues was validated by immunohistochemistry to be positively correlated with tumour progression and poor survival, respectively. CONCLUSIONS: Tumour cell-secreted IL-6 and IL-8 impair the activity and function of NK cells via STAT3 signalling and contribute to oesophageal squamous cell carcinoma malignancy.

miR-503 suppresses the proliferation and metastasis of esophageal squamous cell carcinoma by triggering autophagy via PKA/mTOR signaling.

MicroRNA (miR)-503 is involved in the regulation of the malignant phenotype in multiple tumor types, and has been proven to be a novel diagnostic and therapeutic target; however, its function and mechanisms of action have not yet been fully elucidated in esophageal squamous cell carcinoma (ESCC). In the current study, we detected miR­503 expression by RT­qPCR and found that miR­503 expression was increased in ESCC, but negatively correlated with lymph node metastasis, TNM stage and tumor differentiation. Functionally, we confirmed that miR­503 inhibited the proliferation and metastasis of ESCC cells by triggering cellular autophagy. Mechanistically, we confirmed that miR­503 exerted its biological effects by targeting protein kinase CAMP­activated catalytic subunit alpha (PRKACA) in ESCC by dual luciferase reporter assay. Moreover, miR­503 was found to trigger autophagy in ESCC cells through the protein kinase A (PKA)/mammalian target of rapamycin (mTOR) pathway. Taken together, our results demonstrate that miR­503 suppresses the proliferation and metastasis of ESCC via the activation of autophagy, mediated by the PKA/mTOR signaling pathway.

MACC1 induces autophagy to regulate proliferation, apoptosis, migration and invasion of squamous cell carcinoma.

Metastasis-associated colon cancer-1 (MACC1) plays an important role in cancer development, but the role and mechansim of MACC1 in squamous cell carcinoma (ESCC) remain unclear. In this study, we found that MACC1 expression was increased in ESCC, and correlated with lymph node metastasis. MACC1 knockdown suppresed ESCC cell proliferation, metastasis and enchanced cell apoptosis. Moreover, MACC1 knockdown inhibited ESCC cell autophagy, and 3-methyladenine was able to rescue MACC1-induced malignant phenotype of ESCC cells. Furthermore, MACC1 knockdown inactivated AMPK-ULK1 signaling pathway, and metformin could rescue MACC1-induced autophagy in ESCC cells. Collectively, this study found that upregulation of MACC1 in ESCC was associated with lymph node metastasis of patients, and MACC1 regulated ESCC cell proliferation, apoptosis, migration and invasion mainly through AMPK-ULK1 induced autophagy.

[Small interfering RNA targeting C-erbB-2 gene increases the radiosensitivity of lung adenocarcinoma cells in vitro].

OBJECTIVE: To investigate the effect of small interfering RNA (siRNA) targeting C-erbb-2 oncogene on the radiosensitivity of C-erbb-2-overexpressing lung adenocarcinoma cell line. METHODS: Four pairs of siRNA targeting the coding sequence of C-erbb-2 mRNA were synthesized and their interference effects were evaluated using quantitative real-time fluorescence PCR. The siRNA with the best interference effects was transfected into Calu-3 cells, which were then exposed to 2 or 5 Gy irradiation, with the cells with transfection or irradiation alone as the control groups. The cell apoptosis after the treatment was detected using annexin V-FITC Kit. RESULTS: The apoptosis rate of the Calu cells was 7.767-/+0.551 in the blank group, 14.400-/+1.114 in the interference group, 11.867-/+0.737 in 2 Gy irradition group, 23.000-/+1.664 in 2 Gy irradiation + interference group, 16.100-/+0.624 in 5 Gy irradiation group, and 27.900-/+1.709 in 5 Gy irradiation+interference group. CONCLUSION: The siRNA targeting C-erbb-2 gene can enhance the radiosensitivity of Calu-3 cells to gamma-ray and increase their apoptosis rate following gamma-ray exposure in vitro.