Linkage Of Nf-Kappa B Pathway And Mitosis-Related Molecules Using Integrated Rnai Screening And Transcriptomic Analysis Of Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with five-year overall survival rate of about 50%. Analysis of The Cancer Genome Atlas (TCGA) dataset showed that one or more molecules linked to the tumor necrosis factor receptor (TNF)-NF-κB/REL signal pathway are genetically altered in most HNSCC, and experimental studies show this pathway drives expression of pro-proliferative, inflammatory, angiogenic, and therapeutic resistance genes. Such prevalence and effect across diverse genes suggest that the TNF-NF-κB/REL pathway interacts with molecules that are involved in multiple pathways, but their identity and mechanistic relationship to increased NF-κB signaling have not been studied in detail. Towards this hypothesis, we established a stable NF-κB β−lactamase reporter cell line and performed a genome-wide RNAi screening to identify key regulators of NF-κB oncogenic signaling and proliferation. As expected, the screening and validation data confirmed most components of the TNF receptor complex and downstream NF-κB pathway genes, such as IKKs, contribute to TNFα induced NF-κB activation and cell viability. We further identified and validated a novel sets of genes, which unexpectedly co-modulate NF-κB activity, including those related to the G2M cell cycle checkpoint, such as WEE1, PLK1, AURKA, TTK, CDC2, CDC7 and CALM2, as well as components related to kinetochore such as TPR, NDC80, and NUF2. After confirming sustained knockdown of selected mRNAs and proteins using siRNA by 48-72-hours post transfection, their effects on UM-SCC 1 were evaluated by real-time cell impedance assay. Cell index was significantly decreased by NDC80, NUF2, PLK1, or AURKA knockdown as compared to control siRNA knockdown. In addition, TNF-α treatment further decreased cellular density after NUF2, TPR, PLK1, and AURKA knockdown. The targeted drugs such as volasertib (PLK1), alisertib (AURKA), and adavosertib (WEE1) decreased cell density in a dose-dependent manner, while adavosertib and volasertib also attenuated NF-κB signaling. Thus, we demonstrate a novel link between NF-κB signaling with G2M kinases and kinetochore-related molecules to co-modulate tumor cell growth and survival. Integrated genomics and mechanistic studies are currently underway to understand how G2M and kinetochore-related molecules modulate NF-κB activation and their potential for cancer therapeutics. Supported by NIDCD projects ZIA-DC-000016, -73, -74, and the Medical Research Scholars Program (JWJ). Citation Format: Jun W. Jeon, Anthony D. Saleh, Shaleeka Cornelius, Sophie Carlson, Scott Martin, Pinar Ormanoglu, Hui Cheng, Xinping Yang, Chris Silvin, Zhong Chen, Carter Van Waes. Linkage of NF-κB pathway and mitosis-related molecules using integrated RNAi screening and transcriptomic analysis of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 605.