IFIT3 accelerates the progression of head and neck squamous cell carcinoma by targeting PD-L1 to activate PI3K/AKT signaling pathway.

BACKGROUND: Emerging evidence has shown interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) may be predicted to be a candidate oncogene and involved in the onset and progression of cancer, but IFIT3's potential role in cancer, particularly in head and neck squamous cell carcinoma (HNSC), is not well recognized. This study aims to reveal the role of IFIT3 in HNSC and the underlying molecular mechanism. METHODS: Bioinformatics analysis, immunohistochemical staining, RT-PCR, and Western blotting analysis were used to detect IFIT3 expression in HNSC. CCK-8 assays, colony formation assays, wound-healing assays, transwell assays, and sphere formation were used to explore proliferative, migratory, and invasive activities and cancer stemness of HNSC cells after IFIT3 knockdown and over-expressed. The alterations of EMT markers and PI3K/AKT pathway were detected by Western blotting. Animal studies were performed to analyze the effect of IFIT3 on tumor growth and metastasis of HNSC in vivo. RESULTS: In this study, we observed that IFIT3 was highly expressed in HNSC, and its higher expression contributed to poorer survival of patients with clinical stage IV or grade 3. Function assay indicated that IFIT3 promoted malignant behaviors in vitro, as well as tumor growth and lung metastasis in vivo. Meanwhile, PD-L1 knockdown or over-expressed reversed cancer cell stemness, migration, invasion, and PI3K/AKT signaling pathway which were regulated by IFIT3. CONCLUSIONS: Our results reveal that IFIT3 promotes EMT and cancer stemness by targeting PD-L1 to activate PI3K/AKT signaling pathway in HNSC, and targeting IFIT3 may be a novel strategy for the treatment of patients with HNSC.

LncRNA ROR promotes NLRP3-mediated cardiomyocyte pyroptosis by upregulating FOXP1 via interactions with PTBP1.

OBJECTIVE: The purpose of this design was to explore the specific role and related mechanism of long noncoding RNA (lncRNA) regulators of reprogramming (ROR) in viral myocarditis (VMC). METHODS: AC16 cells were infected with coxsackievirus B3 (CVB3) to establish a VMC cell model in vitro. The release of interleukin (IL)-1ß and IL-18 was evaluated by enzyme-linked immunosorbent assay (ELISA). Gene expression was calculated using quantitative real-time (qRT)-PCR. Cell pyroptosis was determined by flow cytometry and Western blot assays. Cell counting Kit-8 (CCK-8) detected cell viability. The molecular associations were verified by employing RNA immunoprecipitation (RIP), RNA pulldown and chromatin immunoprecipitation (ChIP) assays. RESULTS: The lncRNA ROR was more highly expressed in CVB3 virus-infected AC16 cells than in controls. Knockdown of ROR markedly rescued cell viability and reduced the release of IL-1ß and IL-18, cell pyroptosis and pyroptotic proteins such as NLRP3, ASC and cleaved caspase 1. Mechanistically, ROR destroyed the mRNA stability of Forkhead Box P Factor 1 (FOXP1) by binding polypyrimidine tract binding protein 1 (PTBP1). FOXP1 repressed the transcription of NLRP3 by directly interacting with its promoter. Importantly, coinhibition of FOXP1 impeded the protective role of ROR silencing in CVB3-infected AC16 cells. CONCLUSION: In conclusion, these findings elucidated that ROR knockdown inhibited CVB3-induced cardiomyocyte inflammation and NLRP3-mediated pyroptosis by regulating the PTBP1/FOXP1 axis, implying that ROR might be a new inducer in CVB3-infected VMC.

Nuclear Klf4 accumulation is associated with cetuximab drug-resistance and predicts poor prognosis of nasopharyngeal carcinoma.

BACKGROUND: The functions of the protein expressed in the nucleus and cytoplasm were different or opposite. The previous study found that oncogene Klf4 played a role of tumor suppressor in the nasopharyngeal cytoplasm. Cetuximab targeted epidermal growth factor receptor (EGFR) for the treatment of nasopharyngeal carcinoma. METHODS: A cohort of 231 cases of advanced nasopharyngeal carcinoma (7th AJCC III-IVa) samples was assessed by immunohistochemistry (IHC), of which, 63 cases were treated with basic treatment without cetuximab, the basic treatment include chemotherapy and radiotherapy, the regent of the chemotherapy include cisplatin and fluorouracil and 168 cases were treated with cetuximab in addition to the basic treatment. The expression of the KLF4 protein was detected in nucleus and cytoplasm, c-Met protein and nuclear EGFR protein (nEGFR) by IHC, and H-Ras and PI3K mutations by an arms-PCR method in vivo. KLF4 was found to specifically express in the cytoplasm by deleting the NES, while H-Ras and PI3K genes were mutated in the nasopharyngeal carcinoma 5-8F and HONE1cell line. The cetuximab resistance in differentially mutated 5-8F and HONE1 cells was analyzed. RESULTS: The expression of Klf4 in the nucleus was associated with prognosis in 168 patients with cetuximab-treated nasopharyngeal carcinoma, which was found by retrospective analysis. The KLF4 expression in the nucleus was not significantly correlated with the prognosis in 63 nasopharyngeal carcinoma patients treated with basic treatment (P = 0.261). The expression of Klf4 in the nucleus was correlated with mutations of H-Ras and PI3K in 168 cases of nasopharyngeal carcinoma with cetuximab treatment. In vitro experiments showed that Klf4 was specifically expressed in the nucleus of 5-8F and HONE1 cells as assessed by deleting nuclear export signal, which led to cetuximab resistance. H-Ras and PI3K mutations in 5-8F and HONE1 cells also led to the expression of Klf4 in the nucleus and resistance to cetuximab. In HONE1 cells, Klf4 was specifically localized in the cytoplasm by deleting the NES, and the H-Ras and PI3K mutations did not result in an increased expression of Klf4 in the nucleus and cetuximab resistance. CONCLUSION: The prognosis of nasopharyngeal carcinoma was not significantly improved by cetuximab treatment when the Klf4 was highly expressed in the nucleus of nasopharyngeal carcinoma tissues. The expression of Klf4 in the nucleus can be used as a biomarker for predicting the effects of cetuximab treatment in nasopharyngeal carcinoma, which might be attributed to the H-RAS and PI3K mutations, leading to cetuximab resistance.

Six Glycolysis-Related Genes as Prognostic Risk Markers Can Predict the Prognosis of Patients with Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is one of the worst-prognosis malignant tumors. This study used bioinformatic analysis of the transcriptome sequencing data of HNSCC and the patients' survival and clinical data to construct a prediction signature of glycolysis-related genes as the prognostic risk markers. METHODS: Gene expression profile data about HNSCC tissues (n = 498) and normal tissues in the head and neck (n = 44) were got from The Cancer Genome Atlas (TCGA), as well as patients' survival and clinical data. Then, we obtained core genes; their expression in head and neck squamous cell carcinoma tissues is significantly different from that in normal head and neck tissues. The predicted glycolysis-related genes are screened through univariate Cox regression analysis, and then, the prognostic risk markers were constructed through further correction of multivariate Cox regression analysis. The Kaplan-Meier curve and receiver operating characteristic curve are used to analyze the potential value of these risk markers in diagnosis and prognosis. We also evaluated that the glycolysis-related prognostic risk markers composed of 6 oncogenes are correlated with clinical features, such as age, gender, grade, and clinical stage of the tumor, by univariate and multivariate Cox regression analyses. RESULTS: Differentially expressed glycolytic genes in HNSCC tissues and normal head and neck tissues were screened from TCGA databases using the bioinformatic method. We confirmed a set of six glycolytic genes that were significantly associated with OS in the test series. According to our analysis, the prognostic risk markers composed of HPRT1, STC2, PLCB3, GPR87, PYGL, and SLC5A12 may be an independent risk factor for the prognosis of HNSCC. CONCLUSIONS: Through this analysis, we constructed new prognostic risk markers related to glycolysis as a prognostic risk marker for patients with HNSCC and provided new ideas and molecular targets for the research and individualized treatment of HNSCC.

Anticancer targets and mechanisms of calycosin to treat nasopharyngeal carcinoma.

Calycosin is a naturally occurring phytoestrogen, and it has the anti-nasopharyngeal carcinoma (NPC) action played by calycosin. However, the elaborate mechanisms of calycosin treating NPC remain to be unrevealed. In current report, a promising tool of network pharmacology method was used to uncover the anti-NPC targets and therapeutic mechanisms played by calycosin. Furthermore, were conducted to validate the bioinformatic findings in human and preclinical studies. As results, the bioinformatic findings showed the core anti-NPC targets played by calycosin included tumor protein p53 (TP53), mitogen-activated protein kinase 14 (MAPK14), caspase 8 (CASP8), mitogen-activated protein kinase 3 (MAPK3), caspase 3 (CASP3), receptor interacting protein kinase 1 (RIPK1), proto-oncogene c (JUN), and estrogen receptor 1 (ESR1). Concurrently, the top 20 biological processes and top 20 pharmacological pathways of calycosin treating NPC were identified and illustrated. In clinical data, NPC samples showed up-regulated expression of MAPK14, reduced TP53, and CASP8 expressions in comparison with those in non-NPC controls. As revealed in experimental data, calycosin-treated NPC cells resulted in reduced cell survival rate, increased cell apoptosis. In apoptosis-specific staining, calycosin-treated NPC cells exhibited elevated apoptotic cell number. Following the immunostaining assays, the results indicated increased TP53-, CASP8-positive cells, and reduced MAPK14-positive cells in calycosin-treated NPC cells and xenograft tumor sections. Altogether, the bioinformatic findings from network pharmacology reveal all core targets and mechanisms of calycosin treating NPC, and some of bioinformatic findings are identified using human and preclinical experiments. Notably, the screened biotargets may be potentially used to clinically treat NPC.

Clinical and biological significances of heat shock protein 90 (Hsp90) in human nasopharyngeal carcinoma cells and anti-cancer effects of Hsp90 inhibitor.

Nasopharyngeal carcinoma (NPC) is a malignant tumor in South China, characterized with high death rate. If untreated, NPC cells will be invasiveness and then spread to other tissues. In clinical practice, however, lack of early effective screening to prevent the NPC development. Therefore, candidate biomarker for detecting NPC is developing urgently. In current study, human NPC data and samples were collected for tests, followed by cell culture study. As results, Epstein-Barr virus (EBV)-based human NPC sections showed increased expressions of heat shock protein 90 (Hsp90), protein kinase B (AKT), and Hsp90 levels were positively expressed than those in cytokeratin 19 (CK19). The clinical data showed unchanged contents of blood cancer markers. In cell line study, Hsp90-treated cells (CNE1, 5-8 F) resulted in promoted cellular growth and proliferation. Additionally, proliferative proteins of cellular extracellular regulated protein kinase (Erk1/2), phospho-Erk1/2 (Thr202+Tyr204), B-cell lymphoma-2 (Bcl-2), AKT, phospho-AKT (Ser473), Ki-67 were up-regulated in Hsp90 treatments, while the apoptotic protease activating factor-1 (Apaf1) were down-regulated. Followed by treatment with Hsp90 inhibitor, the NPC cells exhibited inhibited cellular proliferation and growth, induced cell apoptosis, reduced proliferative proteins of Erk1/2, phospho-Erk1/2 (Thr202+Tyr204), AKT, phospho-AKT (Ser473), Bcl-2, Ki-67, and elevated Apaf1 expression. In conclusion, the current findings obtained from this study demonstrate that Hsp90 effectively promotes cell proliferation through activating carcinomatous ERK1/2, phospho-Erk1/2 (Thr202+Tyr204), AKT, phospho-AKT (Ser473) expressions in NPC cells. Briefly, Hsp90 may be a promising biomarker to screen NPC, including early stage.

SIRT6 overexpression induces apoptosis of nasopharyngeal carcinoma by inhibiting NF-κB signaling.

BACKGROUND: Previous reports show that SIRT6 serves as a critical modulator of the development of multiple malignancies as well as other disorders. However, its role in nasopharyngeal carcinoma (NPC) is unknown. Thus, we elucidated the effects of SIRT6 on the survival of NPC cells, and modulation of cell death. METHODS: We found that expression of SIRT6 is downregulated in ten human NPC specimens as well as in the human NPC cell lines, 5-8 F and CNE1, as compared with that in healthy tissues and normal nasopharyngeal NP69 cells. The MTT assay and colony formation assay revealed that upregulation of SIRT6 impaired the proliferation, as well as the survival of 5-8 F and CNE1 cells. The TUNEL assay, annexin V-FITC/propidium iodide, and flow cytometry were performed to detect apoptosis. The results revealed that the expression of SIRT6 resulted in increased apoptosis. RESULTS: Western blotting results showed that SIRT6 overexpression decreased anti-apoptotic Bcl-2 levels, whereas it promoted an increase in pro-apoptotic Bax and cleaved caspase-3 levels. Moreover, NF-κB levels were markedly reduced in cells expressing SIRT6, whereas they were increased in cells transfected with shRNA-SIRT6. Recovery of NF-κB expression was found to counter the suppressive influence of SIRT6 on NPC cell survival, whereas, NF-κB knockdown increased apoptosis of NPC cells. CONCLUSION: Thus, the findings of our study offer insight into the biological and molecular mechanisms underlying the development of NPC and may lead to the development of new and innovative strategies for the treatment of NPC.

[Expression of FOXC1 and its relationship with E-cadherin in nasopharyngeal carcinoma tissues].

OBJECTIVE: To investigate the significance and relationship between the expression of FOXC1 and clinicopathological features, and to explore its correlation with E-cadherin. METHOD: Immunohistochemical SP method was used to detected the expression of FOXC1 in nasopharyngeal carcinoma tissues and nasopharyngitis tissues. RESULT: (1) Immunoreaction to FOXC1 was mainly located in nucleus of nasopharyngeal carcinoma cells. The positive expression rate of FOXC1 in nasopharyngeal carcinoma tissues was 85.3% (81/95), which was significantly higher than that in nasopharyngitis tissues (59.4%) (P < 0.05). (2) The expression of FOXC1 was not related to patients' age and gender, clinical stage of cancer and lymph node metastasis (P > 0.05). (3) There was a correlation between the expression of FOXC1 and down-regulated expression of E-cadherin in nasopharyngeal carcinoma tissues (P < 0.05). CONCLUSION: FOXC1 may play an important role in generation and progression of nasopharyngeal carcinoma, there may be a correlation between the expression of FOXC1 and down-regulated expression of E-cadherin, also FOXC1 may play an important role in the process of EMT in nasopharyngeal carcinoma by regulating E-cadherin.

[Signifinace of cyclin D1 expression in CNE2 cells processed by EGCG].

OBJECTIVE: To study the expression of Cyclin D1 in nasopharyngeal carcinoma cells processed by epigallocatechin gallate(EGCG) and it's significance, and revealed the anti-tumor mechanism of EGCG against nasopharyngeal carcinoma. METHOD: CNE-2 cells were treated by EGCG at different concentrations, the morphological changes of CNE-2 cells were observed by inverted microscope; the inhibition ratio of cell proliferation was detected by MTT colorimetric method, flow cytometry was used to analyze the changes of cell cycle. The expression of Cyclin D1 mRNA was detected by RT-PCR. RESULT: After treated by EGCG, the CNE2 cells decreased in amount and density, some of which became roll and small; Floating and dead cells can be seen in the inverted microscopy; cell proliferation was significantly inhibited in a time and dose dependent (P < 0.05). CNE-2 cells were arrested at G1/G0 phase. The expression of Cyclin D1 mRNA was down-regulated by EGCG with concentration and action time dependent (P < 0.05). CONCLUSION: EGCG resisted nasopharyngeal carcinoma by inhibiting the cell proliferation, The down regulation of Cyclin D1 mRNA expression in a time and dose dependent may be the possible mechanisms.

[Effects of EGCG on the nasopharyngeal carcinoma cell line CNE-2 and the expression of related gene].

OBJECTIVE: To study the effects of epigallocatechin-3-gallate (EGCG) on proliferation and apoptosis of nasopharyngeal carcinoma CNE-2 cell line and analyze the expression of Bcl-2, Bax and Caspase-3 in the cell line which treated with EGCG. METHOD: MTT assay and flow cytometry were used to analyze cell proliferation and cell cycle. Hoechst33258 fluorescence staining was adopted to study cell apoptosis. RT-PCR was used to detect the expression of Bcl-2, Bax, Caspase-3. RESULT: EGCG could significantly inhibit proliferation of CNE-2 cell line and induce its apoptosis with dose-independent relationship. EGCG could suppress the expression of Bcl-2 and induce expression of Bax, Caspase-3. CONCLUSION: EGCG in vitro has efficacy of anti-nasopharyngeal carcinoma cells, which may be through regulating the expression of cell proliferation and apoptosis genes involved.