Targeted combinatorial therapy with XRT is necessary to overcome adaptive radioresistance. RPPA is a powerful proteomic platform, suggesting alterations in energy metabolism following XRT that are targetable by inhibition of the enzyme glutaminase. Further in vivo experiments with glutaminase inhibition and xenograft models to assess combinatorial efficacy with radiation are warranted.
Vinita Takiar, MD, PhD, Jennifer B. Dennison, PhD, Shreya Mitra, PhD, Dominic Ma, BS, Jack Phan, MD, PhD, Gordon B. Mills, MD, PhD; UT MD Anderson Cancer Center
OBJECTIVE: Locoregional recurrence after definitive radiation therapy (XRT) for head and neck (H&N) cancer is a significant clinical challenge, with many patients failing after combined doses > 100 Gy. Although multimodality treatment is prescribed for many cancers, such treatment algorithms are generally empiric, with no identified molecular basis. The efficacy of XRT is limited by intrinsic and acquired radioresistance. We hypothesize that resistance to XRT is, at least in part, mediated by adaptive signaling events induced by the XRT itself. Using signal transduction to better understand these adaptive responses is therefore crucial to developing rational treatment combinations that increase the therapeutic efficacy of XRT.
MATERIALS AND METHODS: Two representative H&N cell lines (FaDu and HN5) were grown in both two dimensions (2D) and three dimensions (3D) (spheroids suspended in Matrigel). Based on cell survival assays conducted in 2D culture, a dose of 4 Gy (~LD50) was chosen for subsequent experiments. Cells in both 2D and 3D were exposed to either 0 Gy or 4 Gy with a cobalt-60 source and then subjected to high-throughput reverse phase protein analysis (RPPA) to screen for differential protein expression in cells able to survive radiation. As the expression of multiple energy-related metabolites was increased, oxygen consumption rate (OCR), which is a readout of aerobic respiration, was subsequently analyzed using an extracellular flux analyzer.
RESULTS: Both HN5 and FaDu cells formed intact monolayers in 2D and spheroids in 3D. Radiation of 4 Gy in one fraction resulted in differential expression of multiple signaling pathways in both cell lines, with significant upregulation of multiple components of the mammalian target of rapamycin (mTOR) pathway (P < .05), including pACC, pAMPK, pAkt, and p70S6K-collectively suggesting altered energy metabolism. Glutamine metabolism, regulated by the mTOR pathway, serves as an anaplerotic and limiting reagent for citric acid cycle progression and thus serves as a proof-of-concept pathway. Using a glutaminase-specific inhibitor that prevents the conversion of glutamine to glutamate, we report that the cellular OCR was significantly reduced (P < .001) in the presence of glutamine. However, the OCR was unchanged in the presence of glucose alone, suggesting that glutamine metabolism is indeed the driver of aerobic respiration in these cells.
CONCLUSIONS: Targeted combinatorial therapy with XRT is necessary to overcome adaptive radioresistance. RPPA is a powerful proteomic platform, suggesting alterations in energy metabolism following XRT that are targetable by inhibition of the enzyme glutaminase. Further in vivo experiments with glutaminase inhibition and xenograft models to assess combinatorial efficacy with radiation are warranted.
Proceedings of the 97th Annual Meeting of the American Radium Society- americanradiumsociety.org