Metabolic Changes in the Salivary Gland Following Radiation Treatment

Abstract

Over 54,000 new cases of head and neck cancer (HNC) are estimated for 2022 in the Unites States and the current treatment involves surgery and radiotherapy. Due to the proximity of the salivary glands to HNC tumors, they are indirectly damaged by radiotherapy and lose secretory function due to their high level of radiation sensitivity. Salivary gland hypofunction leads to xerostomia and other deleterious conditions, such as malnutrition, dental caries, and periodontal disease. Salivary hypofunction treatments include topical agents and saliva stimulants, which temporarily treat the symptoms without regenerating the tissue to restore function for HNC patients who experience chronic xerostomia following radiotherapy. Progress has been made in identifying the mechanisms driving the radiation-damage response, but the metabolic reprogramming that facilitates these mechanisms in the salivary gland is not understood.Metabolomics analysis has been useful for identifying biomarkers of radiation damage in tissues using animal models and in urine and serum of humans. By incorporating additional “omics” data with metabolomics data, more accurate metabolic reactions can be mapped to identify biological networks affected by radiation. To investigate the metabolic phenotype of radiation-induced damage in the salivary gland, we integrated transcriptomic and metabolomic data analysis to identify significant gene-metabolite interactions (Chapter II). Altered metabolites and transcripts significantly converged on a specific region in the metabolic reaction network in parotid tissue glands of mice collected 5 days following radiation treatment. Both integrative pathway enrichment using rank-based statistics and network analysis highlighted significantly coordinated changes in glutathione metabolism, energy metabolism (TCA cycle and thermogenesis), peroxisomal lipid metabolism, and bile acid production with radiation. Integrated changes observed in energy metabolism suggest that radiation induces a mitochondrial dysfunction phenotype. These findings validated previous pathways involved in the radiation-damage response, such as altered energy metabolism, and identified robust signatures in the salivary gland, such as reduced glutathione metabolism, that may be driving salivary gland dysfunction. The aforementioned study identified metabolic changes at 5 days after radiation treatment in the salivary gland, which is the time point when compensatory proliferation begins in the gland in an attempt to heal the damaged tissue. However, the cells are not differentiating properly and becoming fully functional as assessed by decreased amylase levels in the cells. Additionally, decreases in apical and basolateral polarity markers begin at 5 days post-radiation, contributing to the loss of function in the gland. Day 5 denotes the transition between the acute phase of the radiation-damage response marked by increased DNA damage, apoptosis, and reactive oxygen species (ROS) production leading to loss of salivary gland function at 3 days post-radiation, to the chronic phase where increased ROS production continues as well as increased compensatory proliferation and decreased differentiation markers. Due to the complexity of the radiation-damage response over time, we sought to delineate metabolic changes at the acute, intermediate, and chronic radiation-damage response stages in the salivary glands of mice following a single 5 Gy dose (Chapter III). Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation treatment. Pathway enrichment analysis, network analysis based on metabolite structural information, and weighted-gene correlation network analysis were used to incorporate both metabolite levels and structural annotation into the analysis for robust results. The greatest number of enriched pathways was observed at 3 days following radiation and the lowest at 30 days following radiation, and various amino acid metabolism pathways were significantly enriched at all three radiation time points as well as glutathione metabolism and central carbon metabolism in cancer. This study identified the temporal changes in pathways underlying the radiation damage response in the salivary gland to aid in the development of precision therapy for different stages of radiation-induced salivary gland dysfunction. Due to the changes observed in energy metabolism by integrating transcripts and metabolites at day 5 following radiation in the salivary gland, we decided to pursue a deeper investigation of the effects of radiation on energy metabolism in the salivary gland (Chapter IV). The reprogramming of energy metabolism has been observed in cancer and wound healing models to provide the necessary fuel for cell proliferation, but the reprogramming of energy metabolism in the salivary gland in response to radiation has not been investigated. We hypothesized that glycolytic flux increases to fuel compensatory proliferation while oxidative phosphorylation decreases due to mitochondrial dysfunction following radiation. We measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of irradiated primary acinar cells to assess glycolytic flux and oxidative phosphorylation and ATP production produced from each pathway. We measured protein levels and enzymatic activity of the rate-limiting enzymes in glycolysis and measured lactate concentrations in irradiated parotid gland tissue. To assess mitochondrial function, we measured protein levels of complex I and III subunits of the electron transport chain and measured mitochondrial DNA (mtDNA) copy number and spare respiratory capacity. Lastly, we tested fuel dependency and flexibility of irradiated acinar cells. Our results showed an increase in OCR, ECAR, and ATP production rate at 24 hours and 5 days post-radiation with a subsequent decrease at chronic time points. Hexokinase protein levels and activity increased at 3 days post-IR and lactate concentration increased at days 5 and 14 post-radiation. Complex I and III subunit protein levels decreased at days 3 and 5 post-radiation while mitochondrial DNA copy number increased chronically post-radiation. Primary acinar cells became more dependent on long-chain fatty acids for fuel at day 5 post-radiation. These results elucidated the reprogramming of energy metabolism in the salivary gland in response to radiation over time, which may contribute to the radiosensitivity of the gland and aid in identifying therapeutic targets for restoring function.