Abstract 3442: Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma
Authors: Mingdan Deng, Wei Dai, Maria Lung
Journal: Cancer Research (2019)

Abstract Background: NF-κB is an important signaling pathway for regulating multiple signaling cascades involved in cancer development such as inflammation, immune response, cell survival, and cell proliferation. Constitutive activation of the NF-κB pathway plays a key role in the development of NPC. Our previous whole-exome sequencing (WES) study in NPC biopsies has identified five mutations in CYLD, including R758Q. As R758X is a mutational hotspot in CYLD, it has been reported in multiple familial trichoepithelioma type 1, Brooke-Spiegler syndrome and familial cylindromatosis. Thus, we attempted to evaluate the functional effect of CYLD WT and R758Q in regulating NF-κB activity in NPC, which is crucial in NPC progression and development. Methods: C17, C666-1 and NPC43 EBV-positive cell lines and HK1 were co-infected by CYLD wild-type (WT) and mutant constructs with a NF-κB specific dual luciferase promoter plasmid. CRISPR-CAS9 system was used to knock out CYLD. The functional role of CYLD was determined by MTT, CFA and wound healing assays. NF-κB downstream targets expression was evaluated by the method of RT-QPCR. Co-immunoprecipitation (Co-IP) experiments were performed with the Sigma Anti-FLAG M2 magnetic beads and the results were confirmed by Western blot analysis. Female athymic BALB/cAnN (nude) mice were utilized to investigate tumorigenicity. NPC cell lines were treated with cisplatin to check cell sensitivity. Results: The reporter assay showed that CYLD can downregulate NF-κB binding activities in comparison to the vector-alone (VA), but R758Q failed to inhibit the NF-κB binding activities. Over-expression of CYLD can suppress NF-κB downstream target genes expression. Knocking out of CYLD results in higher NF-κB downstream genes expression compared with VA, which confirmed that CYLD can suppress NF-κB activity. R758Q increases these genes expression compared with WT. CYLD can suppress cell growth rate, while R758Q promoted cell proliferation and migration compared to WT. Furthermore, WT CYLD can significantly suppress tumor growth rate, whereas R758Q has the same growth rate with VA, consistent with R758Q loss of WT function, causing tumor suppression. Moreover, CYLD can enhance cell sensitivity to cisplatin treatment compared to the VA. Conclusions: These results suggest that CYLD can suppress NF-κB activity compared to VA, while R758Q lost WT function in NPC. CYLD can inhibit tumorigenesis compared to VA, whereas R758Q induces tumor growth compared to WT. The NPC cell line with CYLD WT is more sensitive to cisplatin treatment. Acknowledgement: Research Grants Council Area of Excellence grant AoE-M06/08 to MLL. Citation Format: Mingdan Deng, Wei Dai, Maria Lung. Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3442.