Kirsten H Limesand

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• Limesand, K. H., Nguyen, T., Wong, A., & Chibly, A. (2016). Sensitivity of the salivary glands to radiation: Understanding the inefficient regenerative response and potential therapeutic interventions.. In Salivary Gland Regeneration.

Journals/Publications

• Buss, L. G., Rheinheimer, B. A., & Limesand, K. H. (2024). Radiation-induced changes in energy metabolism result in mitochondrial dysfunction in salivary glands. Scientific reports, 14(1), 845.
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  Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.
• Buss, L. G., De Oliveira Pessoa, D., Snider, J. M., Padi, M., Martinez, J. A., & Limesand, K. H. (2023). Metabolomics analysis of pathways underlying radiation-induced salivary gland dysfunction stages. PloS one, 18(11), e0294355.
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  Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.
• Meyer, R. K., Gilman, K. E., Rheinheimer, B. A., Meeks, L., & Limesand, K. H. (2023). AMPK Activation Restores Salivary Function Following Radiation Treatment. Journal of dental research, 102(5), 546-554.
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  Head and neck cancers represent a significant portion of cancer diagnoses, with head and neck cancer incidence increasing in some parts of the world. Typical treatment of early-stage head and neck cancers includes either surgery or radiotherapy; however, advanced cases often require surgery followed by radiation and chemotherapy. Salivary gland damage following radiotherapy leads to severe and chronic hypofunction with decreased salivary output, xerostomia, impaired ability to chew and swallow, increased risk of developing oral mucositis, and malnutrition. There is currently no standard of care for radiation-induced salivary gland dysfunction, and treatment is often limited to palliative treatment that provides only temporary relief. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme that activates catabolic processes and has been shown to influence the cell cycle, proliferation, and autophagy. In the present study, we found that radiation (IR) treatment decreases tissue levels of phosphorylated AMPK following radiation and decreases intracellular NAD and AMP while increasing intracellular adenosine triphosphate. Furthermore, expression of sirtuin 1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) was lower 5 d following IR. Treatment with AMPK activators, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, attenuated compensatory proliferation (days 6, 7, and 30) following IR and reversed chronic (day 30) salivary gland dysfunction post-IR. In addition, treatment with metformin or AICAR increased markers of apical/basolateral polarity (phosphorylated aPKCζ-positive area) and differentiation (amylase-positive area) within irradiated parotid glands to levels similar to untreated controls. Taken together, these data suggest that AMPK may be a novel therapeutic target for treatment of radiation-induced salivary damage.
• Gilman, K. E., Camden, J. M., Woods, L. T., Weisman, G. A., & Limesand, K. H. (2021). Indomethacin Treatment Post-irradiation Improves Mouse Parotid Salivary Gland Function via Modulation of Prostaglandin E Signaling. Frontiers in bioengineering and biotechnology, 9, 697671.
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  Annually, >600,000 new cases of head and neck cancer (HNC) are diagnosed worldwide with primary treatment being surgery and radiotherapy. During ionizing radiation (IR) treatment of HNC, healthy salivary glands are collaterally damaged, leading to loss of function that severely diminishes the quality of life for patients due to increased health complications, including oral infections and sores, cavities, and malnutrition, among others. Therapies for salivary hypofunction are ineffective and largely palliative, indicating a need for further research to uncover effective approaches to prevent or restore loss of salivary gland function following radiotherapy. Previous work in our lab implicated prostaglandin E (PGE) as an inflammatory mediator whose release from radiation-exposed cells promotes salivary gland damage and loss of function. Deletion of the P2X7 purinergic receptor for extracellular ATP reduces PGE secretion in irradiated primary parotid gland cells, and salivary gland function is enhanced in irradiated P2X7R mice compared to wild-type mice. However, the role of PGE signaling in irradiated salivary glands is unclear and understanding the mechanism of PGE action is a goal of this study. Results show that treatment of irradiated mice with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, which reduces PGE production via inhibition of cyclooxygenase-1 (COX-1), improves salivary gland function compared to irradiated vehicle-treated mice. To define the signaling pathway whereby PGE induces salivary gland dysfunction, primary parotid gland cells treated with PGE have increased c-Jun N-terminal Kinase (JNK) activation and cell proliferation and reduced amylase levels and store-operated calcium entry (SOCE). The effects of blocking PGE production were also examined and irradiated mice receiving indomethacin injections have reduced JNK activity at 8 days post-irradiation and reduced proliferation and increased amylase levels at day 30, as compared to irradiated mice without indomethacin. Combined, these data suggest a mechanism whereby irradiation-induced PGE signaling to JNK blocks critical steps in saliva secretion manifested by a decrease in the quality (diminished amylase) and quantity (loss of calcium channel activity) of saliva, that can be restored with indomethacin. These findings encourage further attempts evaluating indomethacin as a viable therapeutic option to prevent damage to salivary glands caused by irradiation of HNC in humans.
• Meeks, L., De Oliveira Pessoa, D., Martinez, J. A., Limesand, K. H., & Padi, M. (2021). Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction. Physiological genomics, 53(3), 85-98.
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  Radiation therapy for head and neck cancer causes damage to the surrounding salivary glands, resulting in salivary gland hypofunction and xerostomia. Current treatments do not provide lasting restoration of salivary gland function following radiation; therefore, a new mechanistic understanding of the radiation-induced damage response is necessary for identifying therapeutic targets. The purpose of the present study was to investigate the metabolic phenotype of radiation-induced damage in parotid salivary glands by integrating transcriptomic and metabolomic data. Integrated data were then analyzed to identify significant gene-metabolite interactions. Mice received a single 5 Gy dose of targeted head and neck radiation. Parotid tissue samples were collected 5 days following treatment for RNA sequencing and metabolomics analysis. Altered metabolites and transcripts significantly converged on a specific region in the metabolic reaction network. 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 salivary glands, such as reduced glutathione metabolism, that may be driving salivary gland dysfunction.
• Gilman, K. E., & Limesand, K. H. (2020). The complex role of Prostaglandin E-EP receptor signaling in wound healing. American journal of physiology. Regulatory, integrative and comparative physiology.
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  Prostaglandins are critical lipid mediators involved in the wound healing response, with prostaglandin E (PGE) being the most complex and exhibiting the most diverse physiological outputs. PGE signals via four G-protein coupled receptors, termed EP-receptors 1-4, that induce distinct signaling pathways upon activation and lead to an array of different outputs. Recent studies examining the role of PGE and EP receptor signaling in wound healing following various forms of tissue damage are discussed in this review.
• Jasmer, K. J., Gilman, K. E., Muñoz Forti, K., Weisman, G. A., & Limesand, K. H. (2020). Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions. Journal of clinical medicine, 9(12).
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  Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell-cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.
• Khalafalla, M. G., Woods, L. T., Jasmer, K. J., Forti, K. M., Camden, J. M., Jensen, J. L., Limesand, K. H., Galtung, H. K., & Weisman, G. A. (2020). P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction. Frontiers in pharmacology, 11, 222.
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  Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren's syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.
• Mulic, A., Tveit, A., Vieira, N., Limesand, K. H., & Vieira, A. (2020). Protein Profiles of Individuals with Erosive Tooth Wear. Pesquisa Brasileira em Odontopediatria e Clínica Integrada, 20, e0026. doi:https://doi.org/10.1590/pboci.2020.133
• Vieira, A., Vieira, N., Limesand, K. H., & Modesto, A. (2020). Differences in Proteomic Profiles Between Caries Free and Caries Affected Children. Pesquisa Brasileira em Odontopediatria e Clínica Integrada;, 20, e5592. doi:https://doi.org/10.1590/pboci.2020.131
• Winslow, J. W., Limesand, K. H., & Zhao, N. (2020). The Functions of ZIP8, ZIP14, and ZnT10 in the Regulation of Systemic Manganese Homeostasis. International journal of molecular sciences, 21(9).
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  As an essential nutrient, manganese is required for the regulation of numerous cellular processes, including cell growth, neuronal health, immune cell function, and antioxidant defense. However, excess manganese in the body is toxic and produces symptoms of neurological and behavioral defects, clinically known as manganism. Therefore, manganese balance needs to be tightly controlled. In the past eight years, mutations of genes encoding metal transporters (), (), and () have been identified to cause dysregulated manganese homeostasis in humans, highlighting the critical roles of these genes in manganese metabolism. This review focuses on the most recent advances in the understanding of physiological functions of these three identified manganese transporters and summarizes the molecular mechanisms underlying how the loss of functions in these genes leads to impaired manganese homeostasis and human diseases.
• Wong, W. Y., Gilman, K., & Limesand, K. H. (2020). Yap activation in irradiated parotid salivary glands is regulated by ROCK activity. PloS one, 15(11), e0232921.
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  Radiotherapy plays a major role in the curative treatment of head and neck cancer, either as a single modality therapy, or in combination with surgery or chemotherapy, or both. Despite advances to limit radiation-induced side-effects, the major salivary glands are often affected. This frequently leads to hyposalivation which causes an increased risk for xerostomia, dental caries, mucositis, and malnutrition culminating in a significant impact on patients' quality of life. Previous research demonstrated that loss of salivary function is associated with a decrease in polarity regulators and an increase in nuclear Yap localization in a putative stem and progenitor cell (SPC) population. Yap activation has been shown to be essential for regeneration in intestinal injury models; however, the highest levels of nuclear Yap are observed in irradiated salivary SPCs that do not regenerate the gland. Thus, elucidating the inputs that regulate nuclear Yap localization and determining the role that Yap plays within the entire tissue following radiation damage and during regeneration is critical. In this study, we demonstrate that radiation treatment increases nuclear Yap localization in acinar cells and Yap-regulated genes in parotid salivary tissues. Conversely, administration of insulin-like growth factor 1 (IGF1), known to restore salivary function in mouse models, reduces nuclear Yap localization and Yap transcriptional targets to levels similar to untreated tissues. Activation of Rho-associated protein kinase (ROCK) using calpeptin results in increased Yap-regulated genes in primary acinar cells while inhibition of ROCK activity (Y-27632) leads to decreased Yap transcriptional targets. These results suggest that Yap activity is dependent on ROCK activity and provides new mechanistic insights into the regulation of radiation-induced hyposalivation.
• Gilman, K. E., Camden, J. M., Klein, R. R., Zhang, Q., Weisman, G. A., & Limesand, K. H. (2019). P2X7 receptor deletion suppresses γ-radiation-induced hyposalivation. American journal of physiology. Regulatory, integrative and comparative physiology, 316(5), R687-R696.
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  Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E (PGE) release from primary parotid cells of wild-type but not P2X7R mice. To attempt to uncover the mechanism underlying differential PGE release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE release in P2X7R mice. Irradiated P2X7R mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.
• Jensen, S. B., Vissink, A., Limesand, K. H., & Reyland, M. E. (2019). Salivary Gland Hypofunction and Xerostomia in Head and Neck Radiation Patients. Journal of the National Cancer Institute. Monographs, 2019(53).
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  The most manifest long-term consequences of radiation therapy in the head and neck cancer patient are salivary gland hypofunction and a sensation of oral dryness (xerostomia).
• Meyer, R., Wong, W. Y., Guzman, R., Burd, R., & Limesand, K. (2019). Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics. Journal of visualized experiments : JoVE.
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  Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.
• Wong, W. Y., Allie, S., & Limesand, K. H. (2019). PKCζ and JNK signaling regulate radiation-induced compensatory proliferation in parotid salivary glands. PloS one, 14(7), e0219572.
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  Radiotherapy is a common treatment option for head and neck cancer patients; however, the surrounding healthy salivary glands are often incidentally irradiated during the process. As a result, patients often experience persistent xerostomia and hyposalivation, which deceases their quality of life. Clinically, there is currently no standard of care available to restore salivary function. Repair of epithelial wounds involves cellular proliferation and establishment of polarity in order to regenerate the tissue. This process is partially mediated by protein kinase C zeta (PKCζ), an apical polarity regulator; however, its role following radiation damage is not completely understood. Using an in vivo radiation model, we show a significant decrease in active PKCζ in irradiated murine parotid glands, which correlates with increased proliferation that is sustained through 30 days post-irradiation. Additionally, salivary glands in PKCζ null mice show increased basal proliferation which radiation treatment did not further potentiate. Radiation damage also activates Jun N-terminal kinase (JNK), a proliferation-inducing mitogen-activated protein kinase normally inhibited by PKCζ. In both a PKCζ null mouse model and in primary salivary gland cell cultures treated with a PKCζ inhibitor, there was increased JNK activity and production of downstream proliferative transcripts. Collectively, these findings provide a potential molecular link by which PKCζ suppression following radiation damage promotes JNK activation and radiation-induced compensatory proliferation in the salivary gland.
• Chibly, A. M., Wong, W. Y., Pier, M., Cheng, H., Mu, Y., Chen, J., Ghosh, S., & Limesand, K. H. (2018). aPKCζ-dependent Repression of Yap is Necessary for Functional Restoration of Irradiated Salivary Glands with IGF-1. Scientific reports, 8(1), 6347.
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  Xerostomia and salivary hypofunction often result as a consequence of radiation therapy for head and neck cancers, which are diagnosed in roughly 60,000 individuals every year in the U.S. Due to the lack of effective treatments for radiation-induced salivary hypofunction, stem cell-based therapies have been suggested to regenerate the irradiated salivary glands. Pharmacologically, restoration of salivary gland function has been accomplished in mice by administering IGF-1 shortly after radiation treatment, but it is not known if salivary stem and progenitor cells play a role. We show that radiation inactivates aPKCζ and promotes nuclear redistribution of Yap in a population of label-retaining cells in the acinar compartment of the parotid gland (PG)- which comprises a heterogeneous pool of salivary progenitors. Administration of IGF-1 post-radiation maintains activation of aPKCζ and partially rescues Yap's cellular localization in label retaining cells, while restoring salivary function. Finally, IGF-1 fails to restore saliva production in mice lacking aPKCζ, demonstrating the importance of the kinase as a potential therapeutic target.
• Lin, H. H., Chung, Y., Cheng, C. T., Ouyang, C., Fu, Y., Kuo, C. Y., Chi, K. K., Sadeghi, M., Chu, P., Kung, H. J., Li, C. F., Limesand, K. H., & Ann, D. K. (2018). Autophagic reliance promotes metabolic reprogramming in oncogenic KRAS-driven tumorigenesis. Autophagy, 14(9), 1481-1498.
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  Defects in basal autophagy limit the nutrient supply from recycling of intracellular constituents. Despite our understanding of the prosurvival role of macroautophagy/autophagy, how nutrient deprivation, caused by compromised autophagy, affects oncogenic KRAS-driven tumor progression is poorly understood. Here, we demonstrate that conditional impairment of the autophagy gene Atg5 (atg5-KO) extends the survival of KRAS-driven tumor-bearing mice by 38%. atg5-KO tumors spread more slowly during late tumorigenesis, despite a faster onset. atg5-KO tumor cells displayed reduced mitochondrial function and increased mitochondrial fragmentation. Metabolite profiles indicated a deficiency in the nonessential amino acid asparagine despite a compensatory overexpression of ASNS (asparagine synthetase), key enzyme for de novo asparagine synthesis. Inhibition of either autophagy or ASNS reduced KRAS-driven tumor cell proliferation, migration, and invasion, which was rescued by asparagine supplementation or knockdown of MFF (mitochondrial fission factor). Finally, these observations were reflected in human cancer-derived data, linking ASNS overexpression with poor clinical outcome in multiple cancers. Together, our data document a widespread yet specific asparagine homeostasis control by autophagy and ASNS, highlighting the previously unrecognized role of autophagy in suppressing the metabolic barriers of low asparagine and excessive mitochondrial fragmentation to permit malignant KRAS-driven tumor progression.
• Lin, H. H., Limesand, K. H., & Ann, D. K. (2018). Current State of Knowledge on Salivary Gland Cancers. Critical reviews in oncogenesis, 23(3-4), 139-151.
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  Salivary gland cancers (SGCs), categorized as head and neck cancers (HNCs), constitute about 6% of head and neck cancer diagnoses based on estimate by American Head and Neck Society. Salivary gland tumors originate from different glandular cell types and are thus morphologically diverse. These tumors arise from any of the three major and various minor salivary glands. The incidence of SGCs has slowly increased during the last four decades. The etiology of SGCs is mostly unknown; however, specific gene mutations are associated with certain types of salivary tumors. Treatment options include surgical resection, radiation therapy (RT), chemotherapy, and multimodality therapy. HNC patients treated with RT often develop xerostomia and salivary hypofunction due to damaged salivary glands. In this review, we discuss etiology of SGCs, present findings on the role of autophagy in salivary tumorigenesis, review adverse effects of radiation treatment, and examine remedies for restoration of salivary function.
• Nguyen, V. T., Dawson, P., Zhang, Q., Harris, Z., & Limesand, K. H. (2018). Administration of growth factors promotes salisphere formation from irradiated parotid salivary glands. PloS one, 13(3), e0193942.
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  Worldwide, 500,000 cases of head and neck cancer (HNC) are reported each year and the primary treatment for HNC is radiotherapy. Although the goal of radiotherapy is to target the tumor, secondary exposure occurs in surrounding normal tissues, such as the salivary glands. As a result, despite successful treatment of the cancer, patients are left with long-term side effects due to direct damage to the salivary glands. The effect is chronic and currently there is no treatment. Stem cells are an attractive therapeutic option for treatment of radiation-induced glandular dysfunction because of the potential to regenerate damaged cell populations and restore salivary gland function. However, limited knowledge about the endogenous stem cell population post irradiation hinders the development for stem cell-based therapies. In this study, an ex vivo sphere formation cell culture system was utilized to assess the self-renewal capacity of cells derived from parotid salivary glands at a chronic time point following radiation. Salivary glands from irradiated mice generate significantly fewer salispheres, but can be stimulated with fetal bovine serum (FBS) to generate an equivalent number of salispheres as unirradiated salivary glands. Interestingly, the number and size of salispheres formed is dependent on the concentration of FBS supplemented into the media. Salispheres derived from irradiated glands and cultured in FBS media were found to contain cells that proliferate and express progenitor and acinar cell markers such as Keratin 5, Keratin 14, Aquaporin 5, and NKCC1. Utilization of insulin-like growth factor (IGF1) injections following radiation treatment restores salivary gland function and improves salisphere generation. These findings indicate that stimulation of these cellular populations may provide a promising avenue for the development of cell-based therapies for radiation-induced salivary gland damage.
• Wong, W. Y., Pier, M., & Limesand, K. H. (2018). Persistent disruption of lateral junctional complexes and actin cytoskeleton in parotid salivary glands following radiation treatment. American journal of physiology. Regulatory, integrative and comparative physiology, 315(4), R656-R667.
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  Xerostomia and hyposalivation are debilitating side effects for patients treated with ionizing radiation for head and neck cancer. Despite technological advances, collateral damage to the salivary glands remains a significant problem for patients and severely diminishes their quality of life. During the wound healing process, restoration of junctional contacts is necessary to maintain polarity, structural integrity, and orientation cues for secretion. However, little is known about whether these structural molecules are impacted following radiation damage and more importantly, during tissue restoration. We evaluated changes in adherens junctions and cytoskeletal regulators in an injury model where mice were irradiated with 5 Gy and a restoration model where mice injected postradiation with insulin-like growth factor 1 (IGF1) are capable of restoring salivary function. Using coimmunoprecipitation, there is a decrease in epithelial (E)-cadherin bound to β-catenin following damage that is restored to untreated levels with IGF1. Via its adaptor proteins, β-catenin links the cadherins to the cytoskeleton and part of this regulation is mediated through Rho-associated coiled-coil containing kinase (ROCK) signaling. In our radiation model, filamentous (F)-actin organization is fragmented, and there is an induction of ROCK activity. However, a ROCK inhibitor, Y-27632, prevents E-cadherin/β-catenin dissociation following radiation treatment. These findings illustrate that radiation induces a ROCK-dependent disruption of the cadherin-catenin complex and alters F-actin organization at stages of damage when hyposalivation is observed. Understanding the regulation of these components will be critical in the discovery of therapeutics that have the potential to restore function in polarized epithelium.
• Khalafalla, M. G., Woods, L. T., Camden, J. M., Khan, A. A., Limesand, K. H., Petris, M. J., Erb, L., & Weisman, G. A. (2017). P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy. The Journal of biological chemistry, 292(40), 16626-16637.
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  Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1β and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1β, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1β release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1β release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.
• Meyer, S., Chibly, A. M., Burd, R., & Limesand, K. H. (2017). Insulin-Like Growth Factor-1-Mediated DNA Repair in Irradiated Salivary Glands Is Sirtuin-1 Dependent. Journal of dental research, 96(2), 225-232.
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  Ionizing radiation is one of the most common cancer treatments; however, the treatment leads to a wide range of debilitating side effects. In patients with head and neck cancer (HNC), the surrounding normal salivary gland is extremely sensitive to therapeutic radiation, and damage to this tissue results in various oral complications and decreased quality of life (QOL). In the current study, mice treated with targeted head and neck radiation showed a significant increase in double-stranded breaks (DSB) in the DNA of parotid salivary gland cells immediately after treatment, and this remained elevated 3 h posttreatment. In contrast, mice pretreated with insulin-like growth factor-1 (IGF-1) showed resolution of the same amount of initial DNA damage by 3 h posttreatment. At acute time points (30 min to 2 h), irradiated parotid glands had significantly decreased levels of the histone deactylase Sirtuin-1 (SirT-1) which has been previously shown to function in DNA repair. Pretreatment with IGF-1 increased SirT-1 protein levels and increased deacetylation of SirT-1 targets involved in DNA repair. Pharmacological inhibition of SirT-1 activity decreased the IGF-1-mediated resolution of DSB. These data suggest that IGF-1 promotes DNA repair in irradiated parotid glands through the maintenance and activation of SirT-1.
• Goldberg, E. L., Romero-Aleshire, M. J., Renkema, K. R., Ventevogel, M. S., Chew, W. M., Uhrlaub, J. L., Smithey, M. J., Limesand, K. H., Sempowski, G. D., Brooks, H. L., & Nikolich-Žugich, J. (2015). Lifespan-extending caloric restriction or mTOR inhibition impair adaptive immunity of old mice by distinct mechanisms. Aging cell, 14(1), 130-8.
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  Aging of the world population and a concomitant increase in age-related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age-related decline of the immune system, infectious diseases remain among the top 5-10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T-cell development in the thymus, peripheral T-cell maintenance, T-cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.
• Morgan-Bathke, M., Lin, H. H., Ann, D. K., & Limesand, K. H. (2015). The Role of Autophagy in Salivary Gland Homeostasis and Stress Responses. Journal of dental research, 94(8), 1035-40.
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  Autophagy is a catabolic process that has been shown to have a role in many cellular processes including the removal of excessive or damaged proteins and protein aggregates. The salivary glands play a critical role in oral health, and their secretory capacity may be critically intertwined with the autophagic process. This review describes the role of autophagy activation in normal salivary gland homeostasis and during the glandular stress responses of therapeutic radiation, ductal ligation, autoimmunity, and salivary gland adenoid cystic carcinoma.
• Hill, G., Headon, D., Harris, Z. I., Huttner, K., & Limesand, K. H. (2014). Pharmacological activation of the EDA/EDAR signaling pathway restores salivary gland function following radiation-induced damage. PloS one, 9(11), e112840.
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  Radiotherapy of head and neck cancers often results in collateral damage to adjacent salivary glands associated with clinically significant hyposalivation and xerostomia. Due to the reduced capacity of salivary glands to regenerate, hyposalivation is treated by substitution with artificial saliva, rather than through functional restoration of the glands. During embryogenesis, the ectodysplasin/ectodysplasin receptor (EDA/EDAR) signaling pathway is a critical element in the development and growth of salivary glands. We have assessed the effects of pharmacological activation of this pathway in a mouse model of radiation-induced salivary gland dysfunction. We report that post-irradiation administration of an EDAR-agonist monoclonal antibody (mAbEDAR1) normalizes function of radiation damaged adult salivary glands as determined by stimulated salivary flow rates. In addition, salivary gland structure and homeostasis is restored to pre-irradiation levels. These results suggest that transient activation of pathways involved in salivary gland development could facilitate regeneration and restoration of function following damage.
• Lin, H. H., Lin, S. M., Chung, Y., Vonderfecht, S., Camden, J. M., Flodby, P., Borok, Z., Limesand, K. H., Mizushima, N., & Ann, D. K. (2014). Dynamic involvement of ATG5 in cellular stress responses. Cell death & disease, 5, e1478.
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  Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5(f/-);Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5(f/-);Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated β-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.
• Morgan-Bathke, M., Harris, Z. I., Arnett, D. G., Klein, R. R., Burd, R., Ann, D. K., & Limesand, K. H. (2014). The Rapalogue, CCI-779, improves salivary gland function following radiation. PloS one, 9(12), e113183.
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  The standard of care for head and neck cancer typically includes surgical resection of the tumor followed by targeted head and neck radiation. However depending on tumor location and stage, some cases may not require surgical resection while others may be treated with chemoradiation. Unfortunately, these radiation treatments cause chronic negative side effects for patients. These side effects are associated with damage to surrounding normal salivary gland tissue and include xerostomia, changes in taste and malnutrition. The underlying mechanisms of chronic radiation-induced salivary gland dysfunction are unknown, however, in rodent models persistently elevated proliferation is correlated with reduced stimulated salivary flow. The rapalogue, CCI-779, has been used in other cell systems to induce autophagy and reduce proliferation, therefore the aim of this study was to determine if CCI-779 could be utilized to ameliorate chronic radiation-induced salivary gland dysfunction. Four to six week old Atg5f/f; Aqp5-Cre, Atg5+/+; Aqp5-Cre and FVB mice were treated with targeted head and neck radiation. FVB mice were treated with CCI-779, chloroquine, or DMSO post-radiation. Stimulated salivary flow rates were determined and parotid and submandibular salivary gland tissues were collected for analyses. Mice with a defect in autophagy, via a conditional knockout of Atg5 in the salivary glands, display increased compensatory proliferation in the acinar cell compartment and hypertrophy at 24-72 hours following radiation. FVB mice treated with post-therapy CCI-779 have significant improvements in salivary gland physiology as determined by stimulated salivary flow rates, proliferation indices and amylase production and secretion. Consequently, post-radiation use of CCI-779 allows for improvement of salivary gland function and reestablishment of glandular homeostasis. As CCI-779 is already FDA approved for other uses, it could have a secondary use to alleviate the chronic side effects in head and neck cancer patients who have completed anti-tumor therapy.

Presentations

• Limesand, K. H. (2021). Wound healing as a framework for regeneration. International Association for Dental Research.
• Limesand, K. H. (2021). Wound healing as a framework for regeneration. NIH Salivary Gland Interest Group Meeting. virtual.
• Limesand, K. H. (2017, February). Signaling Mechanisms involved in Radiation-induced Chronic Loss of Function. Gordon Research Conference on Salivary Gland and Exocrine Research.

Poster Presentations

• Meeks, L., De Oliveira Pessoa, D., Martinez, J. A., Limesand, K. H., & Padi, M. (2021). Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction.. Cold Springs Harbor Network Biology conference. virtual.
• Gilman, K., Camden, J., Weisman, G., & Limesand, K. H. (2019, February). P2X7 receptor deletion suppresses γ-radiation induced hyposalivation. Gordon Research Conference on Salivary Gland and Exocrine Research. Galveston, TX.
• Meyer, R., & Limesand, K. H. (2019, February). AMP-activated Protein Kinase Activation Impacts Acinar Cell Proliferation and Salivary Flow Rates following Radiation Therapy. Gordon Research Conference on Salivary Gland and Exocrine Research. Galveston, TX.
• Wong, W. Y., Allie, S., & Limesand, K. H. (2019, February). Radiation-induced compensatory proliferation in parotid salivary glands is PKCζ-dependent and regulated by JNK signaling. Gordon Research Conference on Salivary Gland and Exocrine Research. Galveston, TX.
• Chibly, A., Ghosh, S., & Limesand, K. H. (2017, February). aPKCz Inhibits Nuclear Yap in Salivary Progenitors During Gland Regeneration. Gordon Research Conference on Salivary Gland and Exocrine Research.
• Nguyen, V. T., & Limesand, K. H. (2017, February). Radiation treatment limits salisphere potential of parotid progenitor cells. Gordon Research Conference on Salivary Gland and Exocrine Research.
• Wong, W. Y., & Limesand, K. H. (2017, February). Regulation of E-cadherin/β-catenin Interactions in Irradiated Salivary Glands. Gordon Research Conference on Salivary Gland and Exocrine Research.
• Limesand, K. H., & Chibly, A. (2015, Feb 14-18). Loss of Salivary Gland Cell Polarity due to Radiation. Gordon Research Conference on Salivary Gland and Exocrine Biology.
• Limesand, K. H., Nguyen, T., Chibly, A., & Harris, Z. (2015, Feb 14-18). Potential Role of Keratin 5 and 14 in Salisphere-Forming Ability of Adult Murine Parotid Glands after Radiation. Gordon Research Conference on Salivary Gland and Exocrine Biology.

Reviews

• Harris, Z., Donovan, M. G., Branco, G. M., Limesand, K. H., & Burd, R. (2016. Quercetin as an Emerging Anti-Melanoma Agent: A Four-Focus Area Therapeutic Development Strategy(p. 48).
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  Replacing current refractory treatments for melanoma with new prevention and therapeutic approaches is crucial in order to successfully treat this aggressive cancer form. Melanoma develops from neural crest cells, which express tyrosinase - a key enzyme in the pigmentation pathway. The tyrosinase enzyme is highly active in melanoma cells and metabolizes polyphenolic compounds; tyrosinase expression thus makes feasible a target for polyphenol-based therapies. For example, quercetin (3,3',4',5,7-pentahydroxyflavone) is a highly ubiquitous and well-classified dietary polyphenol found in various fruits, vegetables, and other plant products including onions, broccoli, kale, oranges, blueberries, apples, and tea. Quercetin has demonstrated antiproliferative and proapoptotic activity in various cancer cell types. Quercetin is readily metabolized by tyrosinase into various compounds that promote anticancer activity; additionally, given that tyrosinase expression increases during tumorigenesis, and its activity is associated with pigmentation changes in both early- and late-stage melanocytic lesions, it suggests that quercetin can be used to target melanoma. In this review, we explore the potential of quercetin as an anti-melanoma agent utilizing and extrapolating on evidence from previous in vitro studies in various human malignant cell lines and propose a "four-focus area strategy" to develop quercetin as a targeted anti-melanoma compound for use as either a preventative or therapeutic agent. The four areas of focus include utilizing quercetin to (i) modulate cellular bioreduction potential and associated signaling cascades, (ii) affect transcription of relevant genes, (iii) regulate epigenetic processes, and (iv) develop effective combination therapies and delivery modalities/protocols. In general, quercetin could be used to exploit tyrosinase activity to prevent, and/or treat, melanoma with minimal additional side effects.