Erratum: TGF-ß1-Induced Upregulation of MALAT1 Promotes Kazakh's Esophageal Squamous Cell Carcinoma Invasion by EMT: Erratum.

[This corrects the article DOI: 10.7150/jca.48426.].

CXCL6 fuels the growth and metastases of esophageal squamous cell carcinoma cells both in vitro and in vivo through upregulation of PD-L1 via activation of STAT3 pathway.

CXCL6, contraction of C-X-C motif chemokine ligand 6, whose biological roles have been rarely described in esophageal squamous cell carcinoma (ESCC). To understand the clinicopathological and biological roles played by CXCL6 in the growth and metastasis of ESCC, immunohistochemistry was used to detect the expression of CXCL6 in ESCC tissues, totaling 105 cases; and the correlation was statistically analyzed between CXCL6 expression and clinicopathological parameters. The role mediated in migration and invasion was evaluated using wound-healing and Transwell assays. MTT and flow cytometry were used to assay the proliferative variation. In vivo, tail vein injection model was established in nude mice xenografted with human ESCC cell lines whose CXCL6 were artificially manipulated. It was found that relative to normal control, CXCL6 was profoundly higher in ESCC; upregulated CXCL6 only significantly correlated with differentiation degree. In vitro, CXCL6 was found to promote the proliferation, migration, and invasion of ESCC cells; which was fully corroborated by nude mice experiment that CXCL6 can promote the growth and metastases of ESCC cells in vivo. Mechanistically, CXCL6 was discovered to be capable of promoting epithelial-mesenchymal transition and upregulating PD-L1 expression through activation of the STAT3 pathway. Collectively, all the data we showed here demonstrate that CXCL6 can enhance the growth and metastases of ESCC cells both in vivo and in vitro.

TGF-ß1-Induced Upregulation of MALAT1 Promotes Kazakh's Esophageal Squamous Cell Carcinoma Invasion by EMT.

Transforming growth factor ß1 (TGF-ß1) plays an important role in tumor initiation and development by inducing epithelial-mesenchymal Transition (EMT). Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a long noncoding RNA (lncRNA) that contributes to the invasion and metastasis of tumors, including esophageal squamous cell carcinoma (ESCC). The aim of the present study was to explore the underlying mechanisms implicated in EMT and to clarify whether TGF-ß1 regulates MALAT1 expression, thereby promoting the invasion of ESCC. Expression of TGF-ß1, MALAT1 and EMT-related markers, including E-cadherin and Vimentin, was detected in clinical samples of Kazakh's ESCC. The role of TGF-ß1 in the regulation of MALAT1 in ESCC invasion was evaluated at the ESCC cell line level. High TGF-ß1 expression was significantly associated with poor survival among patients with Kazakh's ESCC. Additionally, the expression of Vimentin was upregulated, and the expression of E-cadherin was downregulated and varied. The expression of MALAT1 positively correlated with the expression of TGF-ß1 both in vivo and in vitro. Furthermore, knockdown of MALAT1 inhibited TGF-ß1-induced EMT. Our data indicate that MALAT1 is heavily involved in EMT induced by TGF-ß1. MALAT1 may be a therapeutic target in the suppression of metastasis and invasion of ESCC.

SUMOylation of HSP27 regulates PKM2 to promote esophageal squamous cell carcinoma progression.

A previous proteomic screening of differentially expressed biomarkers between Kazakh patients with esophageal squamous cell carcinoma (ESCC) and normal adjacent tissues demonstrated that heat shock protein 27 (HSP27) and pyruvate kinase isoenzyme M2 (PKM2) were both highly expressed in ESCC samples compared with normal controls. However, the regulatory association between HSP27 and PKM2 in ESCC remains elusive. In the present study, immunohistochemistry and immunoblotting were adopted to examine the expression of HSP27, PKM2 and other relevant biomarkers involved in epithelial­to­mesenchymal transition in clinical tissue samples. The interactions between proteins were detected by co­immunoprecipitation (Co­IP) assay and further confirmed by immunofluorescence assay. The growth and motility of ESCC cells were examined by MTT, Transwell and wound healing assays. Overexpression of HSP27 was found to be significantly associated with T­cell classification, lymph node metastasis and poor prognosis in ESCC. In addition, HSP27 expression was significantly correlated with PKM2 expression in ESCC specimens. Functionally, knockdown of HSP27 inhibited the growth and motility of ESCC cells. Moreover, HSP27 was found to directly interact with small ubiquitin­related modified protein 2/3 (SUMO2/3) in ESCC cell lines, as evidenced by Co­IP and laser confocal imaging. In addition, downregulation of HSP27 was shown to decrease PKM2 and E­cadherin expression. Knockdown of SUMO2/3 was observed to reduce the expression of HSP27, PKM2 and EMT­related biomarkers. The results of the present study indicated that the SUMOylation of HSP27 enhances the proliferation, invasion and migration of ESCC cells via PKM2.

Widely targeted metabolomic analyses unveil the metabolic variations after stable knock-down of NME4 in esophageal squamous cell carcinoma cells.

NME4, also designated nm23-H4 or NDPK-D, has been known for years for its well-established roles in the synthesis of nucleoside triphosphates, though; little has been known regarding the differential metabolites involved as well as the biological roles NME4 plays in proliferation and invasion of esophageal squamous cell carcinoma (ESCC) cells. To understand the biological roles of NME4 in ESCC cells, lentiviral-based short hairpin RNA interference (shRNA) vectors were constructed and used to stably knock down NME4. Then, the proliferative and invasive variations were assessed using MTT, Colony formation and Transwell assays. To understand the metabolites involved after silencing of NME4 in ESCC cells, widely targeted metabolomic screening was taken. It was discovered that silencing of NME4 can profoundly suppress the proliferation and invasion in ESCC cells in vitro. Metabolically, a total of 11 differential metabolites were screened. KEGG analyses revealed that Tryptophan, Riboflavin, Purine, Nicotinate, lysine degradation, and Linoleic acid metabolism were also involved in addition to the well-established nucleotides metabolism. Some of these differential metabolites, say, 2-Picolinic Acid, Nicotinic Acid and Pipecolinic Acid were suggested to be associated with tumor immunomodulation. The data we described here support the idea that metabolisms occurred in mitochondrial was closely related to tumor immunity.

NME4 modulates PD-L1 expression via the STAT3 signaling pathway in squamous cell carcinoma.

NME4, also named Nm23-H4, is a contraction of NME/NM23 Nucleoside Diphosphate Kinase 4, whose major role is the synthesis of nucleoside triphosphates. However, its association with programmed death ligand 1 (PD-L1) remains far from understood. Herein, it was discovered that silencing NME4 can lead to the marked downregulation of PD-L1, with phosphorylated STAT3 at the 705th serine being inactivated in vitro in esophageal squamous cell carcinoma (ESCC) cell lines. To further validate the association between NME4 and PD-L1 that was observed in cell lines, Pearson correlation analysis was performed on the data regarding the transcriptomic RNA sequencing of NME4 and PD-L1 in cervical squamous cell carcinoma (CSCC), which pathologically highly resembles ESCC in terms of tumor origin, obtained from the GEPIA database. It was demonstrated that their correlation was significant but negative between NME4 and PD-L1 in CSCC. To the best of our knowledge, this is the first report describing a modulation exerted by NME4 over PD-L1 in the background of squamous cell carcinoma, strongly suggestive of the underlying role of NME4 working to exclude CD8 T cells from infiltrating into the squamous cell carcinoma microenvironment.

Let-7b-5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377.

KIAA1377 has been found to be linked with lymph node metastasis in esophageal squamous cell carcinoma (SCC) in our previous study; however, the regulation of KIAA1377 remains far from understood. Herein, to understand the regulation of KIAA1377 from the angle of microRNA (miRNA)-messenger RNA (mRNA) modulation in the setting of SCC cells, the basal level of KIAA1377 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis in KYSE-150 and HeLa cells; biological roles of KIAA1377 contributing in the proliferation, migration, and invasion were evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), wound-healing and Transwell assays, respectively, after KIAA1377 was knocked out mediated by the CRISPR-Cas9 system. Bioinformatic prediction revealed that let-7b-5p was a putative miRNA regulating KIAA1377, which was ensuingly validated by the luciferase reporter assay; after which, variation of KIAA1377 expression was further verified by qRT-PCR and western blot analysis. Moreover, the biological roles of let-7b-5p in proliferation, migration, and invasion of KYSE-150 and HeLa cells were also evaluated. It was exhibited that KIAA1377 was able to promote the proliferation and motility of both KYSE-150 and HeLa cells, which can be reverted by re-expression of let-7b-5p. The luciferase reporter assay verified that let-7b-5p can diametrically target KIAA1377. Collectively, our data demonstrated that let-7b-5p can directly but negatively regulate KIAA1377 in SCC cell lines, Ecal109, and HeLa cells.