BACKGROUND: Despite the known growth-promoting role of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC), EGFR tyrosine kinase inhibitors (TKIs) have shown low efficacy in this disease. The insulin-like growth factor-1 receptor (IGF-1R) has been shown to induce resistance to EGFR TKIs in HNSCC predominantly through anti-apoptotic pathways. PRAS40 is an inhibitor of mTOR that ceases inhibition upon phosphorylation by Akt. Phosphorylated PRAS40 in turn inhibits FOXO3a, contributing to an overall pro-survival state. This study evaluates the role of PRAS40 in IGF-1R mediated resistance to EGFR TKIs.

METHODS: In vitro experiments using alamarBlue, CyQuant, reverse phase protein microarray (RPPA), and immunoblot techniques to evaluate protein expression/phosphorylation and correlate with cell physiologic behavior.

RESULTS: Proliferation assays were used to separate 6 HNSCC cell lines into 2 groups: those rescued from EGFR inhibition by IGF-1R activation and those not rescued. RPPA analysis identified a correlation between PRAS40 phosphorylation and rescue status. Immunoblot analysis confirmed the RPPA findings: in rescued cell lines, PRAS40 phosphorylation decreases with EGFR inhibition, but phosphorylation is restored by IGF-1R activation. However, these treatments have little effect on PRAS40 phosphorylation in non-rescued cell lines. In a representative cell line from each group, p70S6K phosphorylation was found to follow this pattern as well, suggesting possible involvement of mTOR in the rescue mechanism. However the addition of temsirolimus, an mTORC1 inhibitor, to treatment with an EGFR TKI was not sufficient to overcome IGF-induced rescue.

CONCLUSIONS: PRAS40 phosphorylation is tightly correlated with IGF1R activation in HNSCC cells that exhibit IGF1R-induced rescue from EGFR TKI treatment. This phenomenon is absent in non-rescued cells, suggesting a potential role for pPRAS40 in IGF1R-based therapeutic resistance. While PRAS40 phosphorylation results in mTOR activation, the inability of mTOR inhibition to overcome IGF-induced rescue from EGFR antagonism suggests an important alternative downstream pathway. One possible mechanism is through inhibition of FOXO3a, a function of pPRAS40 that has been previously reported in other cell types.

Citation Format: Michael I. Dougherty, Christine E. Lehman, Rolando E. Mendez, Linnea E. Taniguchi, Julie Wulfkuhle, Emanuel F. Petricoin, Daniel G. Gioeli, Mark J. Jameson. PRAS40 as a mediator of insulin-like growth factor-1 receptor-induced resistance to epidermal growth factor receptor inhibition in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1829.