Abstract 81: RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines

Abstract

Abstract We previously observed aberrant activation of NF-kB in head and neck squamous cell carcinoma (HNSCC), however, the signaling constitutively activating NF-kB in solid cancers has not been well defined. Recently, The Cancer Genome Atlas (TCGA) project has investigated 279 HNSCC tissue specimens, and uncovered significant genomic alterations of key molecules involved in inflammation, NF-kB, and death pathways. These genetic alterations include amplification of FADD and BIRC2/3, mutations of caspase 8 and RIPKs in HPV(-), or deletion of TRAF3 in HPV(+) HNSCC tissues. Using bioinformatics analyses and protein structural and interactive tools, we identified ∼60 proteins that potentially interact with these genetically altered molecules. More than 90% HNSCC tissues studied in TCGA exhibited expression or genetic alterations of these genes, of which FADD amplification and/or overexpression ranked first with 37%. We searched this group of genes among more than 20 major cancer types investigated by TCGA, among which HNSCC ranked the highest for these alterations. To further investigate and identify experimental models for functional studies of these genetic and phenotypic alterations, we have performed RNA-seq and exome DNA-seq in 15 HPV(-) and 11 HPV(+) HNSCC lines, and compared them with three normal human oral mucosa lines and 8 matched blood samples. Among the top 30 genes identified from TCGA with the alteration rate greater than 8% in cancer tissues, we found consistent expression patterns for ∼57% molecules in our cell lines. To further test the function of these molecules, we established HNSCC cell lines stably transfected with a vector that contains NF-kB transcription factor response elements upstream of the α-lactamase reporter gene (Gene BLAzer). Using these NF-kB reporter cell lines, genome-wide RNAi screening assays have been performed and the regulatory and signaling molecules involved in NF-κB and death pathways in responding to TNF-α have been identified. We further validated the screening results by knockdown of 35 genes individually using two siRNAs for each genes, and found that knockdown of 16 genes significantly modulated TNF-α and Lymphotoxin β (LTβ) induced NF-κB activity and/or cell survival. We observed that TNF-α and LTβ cross-activate classical and alternative NF-κB pathways and regulate key molecules that serve differential roles in proliferation, survival and migration in HNSCC. Our data help define key molecules modulating aberrant classical and alternative NF-kB activation, as candidate molecular biomarkers for diagnosis and prognosis and targets for further preclinical and clinical investigation in HNSCC. (Supported by NIDCD/NIH intramural projects ZIA-DC-000016, 73, 74; and NCI/NIH, under contract number HHSN261200800001E). Citation Format: Hui Cheng, Xinping Yang, Anthony Saleh, Shaleeka Cornelius, Carter Van Waes, Zhong Chen, Emine Guven-Maiorov, Ozlem Keskin, Attila Gursoy, Ruth Nussinov. RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 81.