G0/G1 Switch 2 regulates radiation sensitivity of human head and neck cancer through a G1-lipid checkpoint

Patel, N., Kalen, A. L., Asha, W., Son, J., Schibler, J., Goswami, P. C., and Sarsour, E. H.

Background: Head and neck squamous cell carcinoma (HNSCC) resistance to radiotherapy has prompted a need to develop adaptive radiation therapy protocols to improve patient outcomes. Previous studies showed that HNSCC tumors with more G0+G1 phase cells (low proliferative index, LPI) are resistant to radiation versus HNSCC tumors with more S+G2 phase cells (high proliferative index, HPI). RNA-seq and bioinformatics have identified lipid metabolism as the major intrinsic pathway that differs between HPI and LPI HNSCC cultures. mRNA and protein levels of G0/G1 Switch 2 gene (G0S2), regulator of quiescence and lipid metabolism, have been found to be upregulated in LPI compared to HPI HNSCC cultures. G0S2 negatively regulates adipose triglyceride lipase (ATGL), resulting in less lipolytic activity. This study investigates the hypothesis that lipid metabolism regulates cell cycle phase specific radiation sensitivity of HNSCC cells.

Methods: In vitro cell culture methods were optimized within individual human tongue squamous cell carcinoma cell lines to obtain cultures with high proliferative index (HPI; >60% S+G2+M; higher RNA content) and cultures with low proliferative index (LPI; >70% G0+G1 cells; lower RNA content). Flow cytometry was utilised to measure DNA and RNA content. To override G0S2 action, we incubated LPI cultures with the fatty acid palmitate and examined cellular metabolic stress markers, such as reactive oxygen species and lipid peroxidation.

Results: LPI cultures were noted to be more radiation resistant compared to HPI cultures. Radiation sensitivity of HPI cultures was found to be associated with a significant increase in lipid peroxidation. Compared to controls, LPI cultures treated with palmitate showed increased reactive oxygen species levels, lipid peroxidation and oxygen consumption rate coupled with increased mitochondrial fission. Palmitate also resulted in significant radiation sensitization. Furthermore, using the fluorescent based cell cycle real-time imaging system, we showed that palmitate treatment sustained cell proliferation (higher S+G2) compared to controls (higher G1).

Conclusion: In summary, we demonstrated that G0S2 dependent lipid metabolism regulates cell cycle phase specific radiation sensitivity of HNSCC cells. G0S2 served as an effective inhibitor of lipolysis, which in turn reduced availability of free fatty acids and increased the radiation resistance of cancer cells. siG0S2 treatment of LPI cultures recruited cells into the proliferative cycle and exacerbated radiation sensitivity. We identified G0S2 and free fatty acids as novel targets for radiation therapy.