miR-31 is consistently inactivated in EBV-associated nasopharyngeal carcinoma and contributes to its tumorigenesis.

BACKGROUND: As a distinctive type of head and neck cancers, nasopharyngeal carcinoma (NPC) is genesis from the clonal Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cells accumulated with multiple genetic lesions. Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC. In addition to the reported CDKN2A/p16, herein, we elucidated the role of a miRNA, miR-31 within this 9p21.3 region as NPC-associated tumor suppressor. METHODS: The expression and promoter methylation of miR-31 were assessed in a panel of NPC tumor lines and primary tumors. Its in vitro and in vivo tumor suppression function was investigated through the ectopic expression of miR-31 in NPC cells. We also determined the miR-31 targeted genes and its involvement in the growth in NPC. RESULTS: Downregulation of miR-31 expression was detected in almost all NPC cell line, patient-derived xenografts (PDXs) and primary tumors. Both homozygous deletion and promoter hypermethylation were shown to be major mechanisms for miR-31 silencing in this cancer. Strikingly, loss of miR-31 was also obviously observed in the dysplastic lesions of nasopharynx. Restoration of miR-31 in C666-1 cells inhibited the cell proliferation, colony-forming and migratory capacities. Dramatic reduction of in vitro anchorage-independent growth and in vivo tumorigenic potential were demonstrated in the stable clones expressing miR-31. Furthermore, we proved that miR-31 suppressed the NPC cell growth via targeting FIH1 and MCM2. CONCLUSIONS: The findings provide strong evidence to support miR-31 as a new NPC-associated tumor suppressor on 9p21.3 region. The inactivation of miR-31 may contribute to the early development of NPC.

CD38 triggers inflammasome-mediated pyroptotic cell death in head and neck squamous cell carcinoma.

BACKGROUND: Pyroptosis is a form of inflammatory cell death. Although it is recognized that NLRP3 (nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3) inflammasome is involved in pyroptosis activation, the mechanism by which head and neck squamous cell carcinoma (HNSCC) inhibits pyroptotic cell death remains undefined. This study aims to delineate the role of calcium regulator CD38 in NLRP3 inflammasome-dependent pyroptosis in HNSCC. METHODS: CD38 overexpressing HNSCC cell lines (SAS, CAL27, SNU899) were generated using lentiviral vectors. NLRP3 and gasdermin D (GSDMD) quantity were detected using Western blot. Caspase-1 activity changes were measured using the Caspase-Glo® 1 inflammasome assay. Cell death proportion was determined by flow cytometry analysis. Proliferation assay was performed using xCELLigence RTCA system. Mouse xenotransplantation was performed to evaluate the potential oncogenic or tumor-suppressive function of CD38. ChIP assay was conducted to verify whether transcription factor NFAT1-mediated NLRP3 expression. RESULTS: Exogenous calcium treatment can lead to a significant increase in caspase-1 activity in HNSCC. This feature was also observed in HNSCC cells with stable CD38 overexpression. CD38-overexpressing cell lines showed a significant reduction in proliferation. Further, expression of NLRP3 protein level was significantly increased in CD38-overexpressing cell lines. The N-terminal effector domain of GSDMD was remarkably increased in the CD38-overexpressing HNSCC. ChIP assay indicated that calcium-sensitive transcription factor NFAT1 was possibly involved in the transcriptional upregulation of NLRP3 observed in CD38-overexpressing HNSCC. The pre-clinical xenograft model revealed that CD38 expression had an inhibiting function on HNSCC progression. CONCLUSION: In conclusion, our results suggested that activation of pyroptosis in HNSCC is a calcium-dependent process. Reduced expression of calcium ion regulator CD38 functions could prevent inflammasome-induced pyroptosis in HNSCC. CD38 may function as a tumor suppressor in HNSCC progression.

NADPH oxidase 5α promotes the formation of CD271 tumor-initiating cells in oral cancer.

Oral tongue squamous cell carcinoma (OTSCC) has a distinctive cell sub-population known as tumor-initiating cells (TICs). CD271 is a functional TIC receptor in head and neck cancers. The molecular mechanisms governing CD271 up-regulation remains unclear. Oxidative stress is a contributing factor in TIC development. Here, we explored the potential role of NADPH oxidase 5 (NOX5) and its regulatory mechanism on the development of CD271-expressing OTSCC. Our results showed that the splice variant NOX5α is the most prevalent form expressed in head and neck cancers. NOX5α enhanced OTSCC proliferation, migration, and invasion. Overexpression of NOX5α increased the size of OTSCC xenograft significantly in vivo. The tumor-promoting functions of NOX5α were mediated through the reactive oxygen species (ROS)-generating property. NOX5α activated ERK singling and increased CD271 expression at the transcription level. Also, NOX5α reduces the sensitivity of OTSCC to cisplatin and natural killer cells. The findings indicate that NOX5α plays an important part in the development of TIC in OTSCC.