Functional and molecular analysis of a NF-kB pathway inhibitor, CYLD, in nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma of the head and neck area with a distinct gender, geographical and racial distribution across the world. It ranked as the tenth in cancer incidence with a high risk of mortality in Hong Kong. The pathogenesis of NPC is attributed to three main etiological factors: EBV infection, genetic factors and environmental factors. NF-kB is an inducible transcriptional factor, regulating inflammation, angiogenesis, cell proliferation and metastasis in cancer. Its activation plays critical roles in NPC development. By our previous whole-exome sequencing and targeted sequencing of NPC patients, CYLD, a NF-kB inhibitor, was screened out as one of the top mutated candidate genes. As CYLD was downregulated in NPC clinical samples and NPC cell lines, it was expected to play an inhibitory role in NPC development. In order to investigate the potential functional effects of CYLD, the CRISPR-Cas9 system was used to knock out CYLD in NPC cell lines. Subsequent in vitro functional assays revealed CYLD inhibits NPC cell proliferation, survival and migration. More importantly, the in vivo assays showed that CYLD knockout contributed to enhanced NPC tumorigenicity and metastasis abilities. Staining of the xenografts showed an increased number of cells undergoing mitosis and microvessel formation after CYLD knockout. Mechanistic study also confirmed that the CYLD knockout increases p65 nuclear translocation, DNA binding activity, and NF-kB downstream target genes expression in vitro and in vivo. As NF-kB mediates a crosstalk between inflammation and cancer in multiple aspects, hyperactive NF-kB is able to regulate the host immune response to recruit more stromal cells to support the tumor growth. By the method of single cell RNA-Seq analysis of NPC xenografts, CYLD was found to inhibit fibroblasts and endothelial stromal cells recruitment in the tumor microenvironment (TME). Therefore, CYLD can regulate NPC via inhibition of three cancer hallmarks, cell proliferation, angiogenesis and metastasis and regulate TME via inhibition of fibroblast and endothelial stromal cells recruitment, by suppressing the NF-kB pathway.

Two truncation mutations (S323X and S371X) and one missense mutation (758Q) identified from our previous WES were further studied to enhance our understanding of how mutated CYLD contributes to cancer using a lentiviral transduction system to re-express these mutants in NPC cell lines. The in vitro assays showed the truncation mutations are associated with loss of the CYLD inhibitory roles in suppressing cell migration and NF-kB DNA binding activity. Further assays were applied to study the missense mutation, R758Q. It fails to inhibit cell proliferation and migration, NF-kB nucleus translocation, DNA binding activity and NF-kB downstream targets expression in vitro. While the in vivo assays for S323X, 371X and 758Q failed to significantly differ from the CYLD WT, further assays need to be performed in the future to have a better understanding of these mutations.