Functional Genomics Screen with Pooled shRNA Library and Gene Expression Profiling with Extracts of Azadirachta indica Identify Potential Pathways for Therapeutic Targets in Head and Neck Squamous Cell Carcinoma

We investigate the molecular targets and pathways that the neem extracts and the associated compounds act through, to bring about tumor suppression by using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell line treated with crude neem leaf extracts. We analyzed differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing. We further analyzed differences in gene expression in HSC-4 cells, upon treatment with neem leaf extract in a time-dependent manner, followed by a time-dependent rescue. Our results indicate that neem extract simultaneously affects various important molecular signaling pathways in head and neck cancer cells, some (TGF-β/HSF-1) of which may be therapeutic targets for this devastating tumor. RNA was extracted from HSC-4 cells, seeded and treated with neem leaf extract (200μg/ml) at nine pre-defined time points (5min, 10min, 15min, 30min, 1hr, 3hr, 6hr, 10hr, and 24hr) and rescued with fresh complete medium post 48-hour treatment at these time-points. The samples were assayed for gene expression using Illumina whole-genome HumanHT-12 v4 expression BeadChip (Illumina, San Diego, CA), following the manufacturer’s specifications.