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

PeerJ. 2019 Mar 1;7:e6464. doi: 10.7717/peerj.6464. eCollection 2019.


Tumor suppression by the extracts of Azadirachta indica (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that neem extracts and their associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell lines treated with crude neem leaf extracts, known for their anti-tumorigenic activity. 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 found 225 genes affected the cancer cell growth in the shRNA-infected cells treated with neem extract. Pathway enrichment analyses of whole-genome gene expression data from cells temporally treated with neem extract revealed important roles played by the TGF-β pathway and HSF-1-related gene network. Our results indicate that neem extract affects various important molecular signaling pathways in head and neck cancer cells, some of which may be therapeutic targets for this devastating tumor.

Keywords: Anti-tumorogenic; Azadirachta indica; Drug target; Gene expression; HSC-4; HSF-1; Head and neck squamous cell carcinoma; Neem; Pooled shRNA screen; TGF-β.

Grant support

The research is funded under the India-Japan Cooperative Science Programme by the Department of Science and Technology (DST), India to Binay Panda (Ref No. DST/INT/JSPS/P-181/2014), by the Japanese Society of Promotion of Science (JSPS) to Shumpei Ishikawa, by the Medical Research Institute, Tokyo Medical and Dental University for International Joint Research Project 2017, and by PRESTO, JST to Hiroto Katoh. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.