Abstract 4372: Genomic, RNAi screening, and functional validation reveal cross activation of classical and alternative NFκB pathways in head and neck squamous cell carcinoma of different HPV status

Abstract

Abstract Aberrant activation of transcription factor NF-κB promotes tumorigenesis of head and neck squamous cell carcinoma (HNSCC), but the genomic alterations and pathways affecting NF-κB signaling have not been fully dissected. As part of The Cancer Genome Atlas (TCGA) project for head and neck cancer, our laboratory recently uncovered that HNSCC harbored frequent alterations affecting gene expression of signal molecules in NF-κB and cell death pathways. We analyzed the transcriptome, integrated with the genetic alterations of 279 TCGA tumors with different HPV status in parallel with 15 HPV(-) and 11 HPV(+) HNSCC cell lines sequenced in our laboratory through whole exome and transcriptome sequencing. Using bioinformatics and pathway analyses, we identified 61 genes interacting with the genetically altered components of the NF-κB pathway. We found expression for 38 of these 61 genes were significantly higher in tumors than in mucosa tissues. Differential expression affecting more components of the canonical than the alternative NF-κB pathways were found in the HPV(-) group, while the alternative NF-κB pathway components were predominantly affected in the HPV(+) group. The distribution of genomic DNA copy number variation (CNV) between groups differing in HPV status support this observation. The mRNA levels are significantly correlated with their corresponding CNV for more than 2/3 of top 30 genes in both TCGA cohort and the cell lines. Supervised clustering analysis of mRNA expression of the TCGA and cell lines revealed distinct gene expression patterns related to their genetic features and HPV status. Functional genome-wide RNAi screening was performed using HNSCC cell lines stably transfected with the β-lactamase NF-κB reporter construct (Gene BLAzer). We identified 16 genes that significantly modulated TNF-α or Lymphotoxin β (LTβ) induced NF-κB activity and/or cell survival in both HPV(-) or HPV(+) HNSCC lines. Knocking down TNFR, LTβR, or downstream signaling components demonstrated cross-activation of classical and alternative NF-κB pathways in response to TNF-α or LTβ in HPV(-) or HPV(+) cells. Our study uncovers the prominent contribution of key molecules involved in canonical and alternative NF-κB activation, which modulate survival and migration of HNSCC. (Supported by NIDCD/NIH intramural projects ZIA-DC-000016, 73, and 74; and NCI/NIH, under contract number HHSN261200800001E). Citation Format: Xinping Yang, Hui Cheng, Ru Wang, Jianhong Chen, Han Si, Anthony Saleh, Carter Van Waes, Zhong Chen. Genomic, RNAi screening, and functional validation reveal cross activation of classical and alternative NFκB pathways in head and neck squamous cell carcinoma of different HPV status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2017-4372