P1 Downregulation of hypoxia-inducible factor 1 α expression inhibits growth and enhances IGF1R inhibitor OSI906 sensitivity in head and neck squamous cell carcinoma cells
Ashraf Khalil1,2, Mark Jameson1
1Department of Clinical Biochemistry and Molecular Diagnostics, National Liver Institute, Menoufia University, Shebin ELkom, Egypt; 2Department of Otolaryngology – Head and Neck Surgery. University of Virginia, Charlottesville, Virginia, USA
Correspondence: Ashraf Khalil (ashkalil2010@gmail.com)
Background
Hypoxia-inducible factor 1 α (HIF‑1α) is a central regulator for cells to adapt to low cellular oxygen levels, is also often overexpressed and activated in many human cancers. HIF‑1α mediates the primary transcriptional response of a wide range of genes in response to hypoxia. Insulin‑like growth factor‑1 receptor (IGF‑1R) is a cell membrane receptor involved in cell proliferation is expressed in the head and squamous cell carcinoma.
Aim
To detect the effect of HIF‑1α downregulation on the sensitivity of squamous cell carcinoma (SCC) to small molecule IGF1R inhibitor OSI-906.
Methods
A lentivirus-mediated doxycycline-inducible pTRIPZ short hairpin RNA micro (shRNAmir) plasmid targeting HIF‑1α was transfected into two head and neck squamous cell carcinoma (HNSCC) cell lines to silence HIF‑1α expression and to assess the effect of its downregulation on cell proliferation and sensitivity to IGF1R inhibitor. A nude mice animal model was developed by the transfection of a luciferase gene into the inducible shRNA HIF‑1α cells and growing tumors by direct inoculation of the cells in the tongue. Tumor growth was determined by detecting the bioluminescence signal by the advanced IVIS 200 technology (Figure 2d).
Result
Downregulation of the expression of HIF‑1α (Figure1 a-b) led to an increase in the sensitivity of head and neck squamous cell carcinoma to the OSI-906 IGF1R small molecule inhibitor (Figure 2a-b) by a mechanism involving the increase in apoptosis (Figure 2c).
Conclusion
The preliminary findings suggest that co-targeting of IGF‑1R and HIF‑1α may represent a novel approach for resistant head and neck tumors.
This study received supported from the National Institutes of Health (NIH) (K08 grant DE019477) (M.J.J.) and the University of Virginia (UVA) Cancer Center/UVA Department of Otolaryngology–Head and Neck Surgery Pilot Project Grant (M.J.J./D.G.G.). The authors have no other funding, financial relationships, or conflicts of interest to disclose.
Lentivirus-mediated shRNA efficiently inhibited the expression of HIF‑1α in head and neck squamous carcinoma Cal27 and SCC2 cells. A. Fluorescence microscopic picture of the expressed reporter Turbo-RFP protein as a marker of successful transfection and doxycycline indelibility. Lentivirus doxycycline-inducible pTRIPZ-shRNAmir constructs directed against HIF‑1α used to transfect Cal27 and SCC25 HNSCC cells. The construct also contains a doxycycline-inducible TurboRFP as a reporter gene co-expressed with the shRNA sequence. B. Immunoblot with anti- HIF‑1α showed that doxycycline-induced HIF-shRNAmir reduced the HIF‑1α protein level by 90% relative to the untreated cells. Cobalt chloride at 2mM was added for 16h to induce hypoxia in the cell and induce HIF‑1α.
Downregulation of HIF‑1α suppress cell growth, colony formation, increased apoptosis, and the sensitivity of cells to IGF1R inhibition. A. Cells transfected with the silencing shRNAmir HIF‑1α were treated with 2 μg/ml doxycycline for three days, then with OSI901 as indicated, and cell proliferation was determined at 72 h using alamarBlue. B. Colony formation assay at 14 days. C. Apoptosis assay 72h by Annexin V and PI by flow cytometry. D. Tumor detection and quantification by Bioluminescence imaging.
