Abstract 5884: 4EBP1 is a tumor suppressor gene unleashed by mTOR inhibition in head and neck squamous cell carcinoma
Authors: Wang Zhiyong , Martin Daniel , Vitale-Cross Lynn , Feng Xiaodong , Molinolo Alfredo A. , Allevato Michael M. , Wu Victoria H. , Gilardi Mara , Xu Hao , Chen Qianming , Gutkind J.Silvio 
Published: August 2018

Abstract
Persistent aberrant activation of the PI3K/AKT/mTOR signaling pathway occurs in over 80% of head and neck squamous cell carcinomas (HNSCC). In prior studies we have shown that mTOR regulates proliferation, angiogenesis and apoptosis, thus playing an essential role in HNSCC initiation and progression. The frequent over-reliance on this signaling circuit for HNSCC growth may in turn represent a cancer vulnerability that can be exploited therapeutically. Indeed, we have shown that mTOR inhibitors (mTORi) promote tumor regression in multiple genetically defined and chemically induced carcinogenesis animal models, and encouraging results have been recently obtained using mTORi in HNSCC in the clinic. However, the complexity of genomic alterations in HNSCC may facilitate the emergence of drug resistance, especially in cases with patients who may receive multiple rounds of chemotherapy and/or radiation. Understanding the precise molecular processes by which mTOR acts in HNSCC may help predict intrinsic resistance mechanisms and select patients who may benefit the most from the administration of mTORi. Here, we focused on EIF4EBP1 (4EBP1), a direct target of mTOR that serves as key effector for protein synthesis. Remarkably, we found a negative correlation between 4EBP1 gene copy number loss and disease-free survival (p&lt;0.001) and overall survival (p&lt;0.01) in the HNSCC TCGA database (n=181). Furthermore, IHC analysis of 4EBP1 protein expression revealed that 16.3% of HNSCC cases analyzed (n=49) lack 4EBP1, with progressive protein loss in advanced cases. Aligned with a tumor-suppressive role, 4ebp1-knockout mice formed larger and more HNSCC lesions (p&lt;0.001) in 4NQO oral carcinogenesis models. We engineered a mutant 4EBP1 that cannot be phosphorylated by mTOR (4EBP1-mut), resembling de-phospho-4EBP1, and generated HNSCC cells expressing this mutant in a tetracycline-inducible fashion. Protein-protein interactions and assembly of the translational complex were analyzed using 7-methyl GTP pull-down and co-IP assays. Expression of 4EBP1-mut disrupted the function of the translation initiation complex and was sufficient to prevent tumor growth in HNSCC xenograft models. Similarly, mTORi induced the accumulation of dephosphorylated endogenous wild-type 4EBP1 and resulted in HNSCC xenograft regression. Remarkably, CRISPR-CAS9-mediated knockout of 4EBP1 in HNSCC cells caused insensitivity to mTORi in vitro and in vivo. Overall, our findings suggest that 4EBP1 needs to be persistently repressed by mTOR phosphorylation in HNSCC and that mTORi may unleash 4EBP1's tumor-suppressive activity, thereby reducing the translation of growth-promoting and cancer cell stemness-related proteins. In turn, 4EBP1 gene and protein loss may underlie mTORi resistance, supporting that 4EBP1 expression and phosphorylation status may represent a mechanistic biomarker of mTORi sensitivity in HNSCC patients.
Citation Format: Zhiyong Wang, Daniel Martin, Lynn Vitale-Cross, Xiaodong Feng, Alfredo A. Molinolo, Michael M. Allevato, Victoria H. Wu, Mara Gilardi, Hao Xu, Qianming Chen, J.Silvio Gutkind. 4EBP1 is a tumor suppressor gene unleashed by mTOR inhibition in head and neck squamous cell carcinoma [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 5884.