Kirsten H. Limesand - US grants

DESCRIPTION: (provided by applicant) The induction of apoptosis is an important event in mammalian development; however, apoptosis of salivary gland cells resulting from head and neck irradiation, chemotherapeutic drugs, or autoimmune diseases ultimately decreases the quality of life for these patients. The goal of this project is to define the signaling pathways activated in the salivary glands by various apoptotic stimuli and determine if these events can be suppressed. Specific aim 1 identifies the activation of the anti-apoptotic protein kinase Akt by growth factors in C5 parotid and C6 submandibular cell lines and the protection activated Akt confers after an apoptotic insult (etoposide, taxol, irradiation, and FasL). Apoptosis will be quantitated after each stimulus by flow cytometry to measure Sub G1 fraction, caspase 3 activation, TUNEL assay, and immunoblotting for Akt substrates. Transgenic mice have been generated to express a constitutively active form of the pro-survival molecule Akt (Myr-Akt) in the salivary glands. Specific aim 2 will assess the ability of the primary murine salivary gland acinar cells from the Myr-Akt mice to suppress apoptosis. Specific aim 3 examines the apoptotic response of the transgenic mice to various stimuli including: isoproterenol, irradiation, and FasL. After each stimulus, salivary gland flow rate will be measured and salivary gland tissues will be analyzed by standard histology, TUNEL staining, and immunoblotting for Akt substrates and changes in salivary protein concentrations. These studies will provide greater insight to the understanding of this tissue and potentially provide clinical treatment of cancer-induced apoptosis in salivary glands.

DESCRIPTION (provided by applicant): Apoptosis plays a crucial role in mammalian development and complex control mechanisms exist to regulate these signaling pathways. Aberrant apoptosis of the salivary glands is induced by secondary side effects of head and neck irradiation, chemotherapeutics, or Sjogren's syndrome. p53 is an important regulator of apoptosis induced by DNA damage. The identification of two p53 homologues, p73 and p63, along with the selective activation of apoptotic target genes by these different homologues have added additional complexity in the understanding of how the decision to undergo apoptosis is executed. Increased expression of the p53 responsive genes bax and Fas has been shown in the lacrimal and salivary glands of Sjogren's syndrome patients and p53 is hypothesized to be associated in the pathogenesis of autoimmune disorders. The general goal of this proposal is to understand the induction of the p53 responsive genes in salivary acinar cells and regulation of this activity by the pro-survival serine/threonine protein kinase Akt. Specifically, the role Akt serves in suppressing apoptosis following DNA damage and the interaction with the p53 family of proteins will be examined. We hypothesize that Akt suppresses DNA damage induced apoptosis through a modulation of the p53 family of transcription factors. Specific Aim 1 will define the in vivo mechanisms by which Akt suppresses gamma-irradiation-induced apoptosis in the salivary gland. Specific Aim 2 will identify the requirement for p53 transcriptional activation in gamma-irradiation-induced salivary gland apoptosis. Specific Aim 3 will examine the induction of p73 activity in salivary acinar cells and the effect of its suppression by activated Akt. Specific Aim 4 will investigate the functions of p63 in salivary acinar cells and the ability of Akt to regulate these functions. Transiently cultured primary submandibular or parotid acinar cells isolated from mice provide a unique environment to examine the regulation of apoptosis. In addition, a transgenic mouse strain that expresses activated Akt has been used as a novel reagent in understanding the suppression of apoptosis induced by a variety of stimuli in salivary acinar cells. New understanding of the fundamental biological process of p53 regulation could have a powerful impact on clinical therapeutics for salivary glands.

[unreadable] DESCRIPTION (provided by applicant): Salivary glands are the site of aberrant apoptosis following head and neck irradiation resulting in severe glandular dysfunction. During therapy, these patients are exposed to multiple rounds of radiation treatment which takes a significant toll on the normal salivary gland. Transgenic mice have been created expressing a constitutively activated Akt1 (myr-Akt1) which significantly reduces the level of apoptosis in the salivary glands following exposure to targeted head and neck radiation. Preliminary results presented in this proposal have indicated the myr-Akt1 transgenic mice also rescue salivary flow rates following a single therapeutic dose of ionizing radiation providing a correlation between apoptosis and salivary gland dysfunction. While these results are very promising, patients undergoing therapeutic radiation for head and neck cancer receive multiple doses of radiation. The general goal of this proposal is to define the ability of Akt to maintain salivary gland function and homeostasis following fractionated doses of radiation. As a means to translate these studies, we have previously shown that IGF1 induces robust Akt activation in salivary acinar cells when compared to other growth factors (1). The long-term goal of this proposal is to evaluate whether IGF1 treatment of salivary glands prior to head and neck irradiation could activate Akt in situ and impact clinical therapeutics for salivary gland dysfunction and xeriostomia. We hypothesize that Akt plays a pivotal role in improved salivary function following therapeutic radiation by enhancement of DNA repair pathways and suppression of apoptotic pathways. Specific Aim 1 will evaluate the ability of Akt to maintain salivary gland function following fractionated doses of head and neck radiation. Specific Aim 2 will investigate the role of Akt in regulating DNA repair in ?-irradiation-induced salivary gland dysfunction. A unique and innovative strength of these studies is the use of transgenic mice that express a constitutively active mutant of Akt1. This mouse model and primary salivary acinar cell cultures from these mice allow the direct comparison of transgenic mice with control mice in targeted head and neck animal irradiation and the ability to relate in vitro preliminary results from primary cultures with in vivo examination of histological or biochemical changes. This proposal also focuses on a mechanistic analysis of salivary gland dysfunction and sensitivity to therapeutic radiation in order to apply these results towards translation application studies. These studies will significantly improve our understanding of salivary gland biology in stressed environments and the unique ability to cannulate salivary gland ducts in patients provides the optimism for clinical application. [unreadable] [unreadable] Radiation is a common treatment in most head and neck cancer cases and results in the loss of saliva in most patients. The resulting lack of salivary gland activity results in significant side effects, which diminish the effectiveness of anti-cancer therapies, and decreases the quality of life for these patients. The general goal of this proposal is to define the role cell death serves in loss of salivary gland function following exposure to radiation and evaluate whether substances that prevent cell death could be used prior to head and neck irradiation in order to prevent salivary gland dysfunction and xerostomia. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Radiation therapy for head and neck cancer causes significant secondary side effects in the normal salivary gland resulting in diminished quality of life for these patients. Classically radio- sensitive tissues have high rates of proliferation coupled with a low level of differentiation. However, salivary glands convey the opposite characteristics with a low level of proliferation and high level of differentiation suggesting that they should not be sensitive to radiation. Preliminary results have indicated that apoptotic pathology induced early after radiation exposure correlates with salivary gland hypofunction. Transgenic mice expressing a constitutively activated Akt (myr-Akt1) rescues salivary flow rates following a therapeutic dose of ionizing radiation. As a means to translate these studies, we have previously shown that insulin-like growth factor (IGF1) induces robust Akt activation in salivary acinar cells when compared to other growth factors (59). Intravenous injections of recombinant IGF1 prior to radiation exposure completely rescues salivary flow rates 30 days after treatment. The general goal of this proposal is to identify the mechanisms of IGF1 preservation of salivary gland function by investigating cell cycle arrest and proliferation following radiation therapy. We hypothesize that the exquisite sensitivity of the salivary glands to radiation may be due to immediate activation of p53-mediated apoptotic pathology without cell cycle arrest. Appropriate activation and release of cell cycle checkpoints may be one mechanism to improve salivary gland function. Specific Aim 1 will evaluate the ability of IGF1 to prevent radiation-induced apoptotic pathology in the salivary glands of mice. Specific Aim 2 will investigate cell cycle arrest activation following treatment with radiation. Specific Aim 3 will determine the ability of delayed IGF1 administration to restore proliferation and function in mice with radiation-induced salivary gland dysfunction. A unique and innovative strength of these studies is the translational promise of using IGF1 to counter radiation-induced salivary gland dysfunction and transgenic mice that express a constitutively active mutant of Akt to decipher potential mechanisms of IGF1. The long-term goal of this proposal is to evaluate whether IGF1 treatment of salivary glands prior to head and neck irradiation could improve clinical therapeutics for salivary gland dysfunction and xerostomia. These studies will significantly improve our understanding of salivary gland biology in stressed environments and the unique ability to cannulate salivary gland ducts in patients provides optimism for clinical application. PUBLIC HEALTH RELEVANCE: Radiation is a common treatment in most head and neck cancer cases and results in the loss of saliva in most patients. The resulting lack of salivary gland activity results in significant adverse side effects, which diminish the effectiveness of anti-cancer therapies, and decreases the quality of life for these patients. The general goal of this proposal is to identify mechanisms to preserve salivary gland function following exposure to radiation by evaluating cellular repair mechanisms and cell growth. The studies could have the potential to prevent or restore salivary gland function to head and neck cancer patients.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-DE-104 Functional Restoration of Salivary Glands. Proper salivary gland function is critical for oral health. Radiation therapy for head and neck cancer often causes significant secondary side effects that impact normal salivary gland function, most commonly xerostomia. The ensuing salivary gland hypofunction results in significant morbidity, diminishes the effectiveness of anti-cancer therapies and decreases the quality of life for these patients. Current therapies are unable to permanently restore salivary function, which remains a major therapeutic challenge. The primary goal of our research is to elucidate the role of radiation-induced autophagy in salivary glands in response to irradiation. Secondarily, through the use of small molecule therapeutic approaches, we wish to investigate whether activation of autophagy leads to cytoprotection or exacerbation of radiation-induced injury to salivary tissues. Autophagy is a constitutive cellular catabolic degradation process whereby cellular proteins and organelles are engulfed, digested through the lysosomal machinery and recycled. Depending on the cellular context, autophagy can affect pro-survival or pro-death outcomes. The autophagy-related 5 gene, Atg5, has been established as an indispensable player in autophagy. We have generated Atg5f/f;Aqp5-Cre transgenic mice, in which the Aquaporin-5 (Aqp5)-driven Cre recombinase is targeted to impair autophagy by excising the floxed exon 3 of Atg5 specifically in salivary acinar cells. We propose to investigate the role of autophagy in influencing the fate of salivary acinar cells following radiation and to utilize the FDA approved autophagy activator rapamycin or autophagy inhibitor chloroquine in a restoration of salivary function model. Our central hypotheses are: 1) Appropriate activation of autophagy is important in the removal of damaged organelles/proteins within damaged acinar and ductal cells as well as whole cell corpses following irradiation, and 2) Timely and efficient removal of cell corpses following irradiation by autophagy will accelerate restoration of salivary function. The expected outcomes from these experiments will contribute substantially to our understanding on the role of autophagy in protecting salivary gland function against radiation-induced damage. In addition, they will provide a unique opportunity to evaluate the feasibility of autophagy-targeted therapies to restore salivary gland function in thousands of head and neck cancer patients that have completed anti-cancer therapies yet continue to suffer from the side effect of salivary gland hypofunction. This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-DE-104 Functional Restoration of Salivary Glands. Proper salivary gland function is critical for oral health. Radiation therapy for head and neck cancer often causes significant secondary side effects that impact normal salivary gland function. This proposal will provide a unique opportunity to evaluate the feasibility of autophagy-targeted therapies to restore salivary gland function in thousands of head and neck cancer patients that have completed anti-cancer therapies yet continue to suffer from the side effect of salivary gland hypofunction.

DESCRIPTION (provided by applicant): The field of salivary gland biology and regeneration is severely hampered by a lack of knowledge regarding the identification of stem or progenitor cells and the fate of this population following salivary gland damage. The cell label retaining approach can objectively identify long living and non-proliferative cells that are hypothesized to be stem and/or progenitor cells. This proposal will utilize the label retaining cell (LRC) techniqu within two established models of salivary gland restoration, post-radiation injections of insulin-like growth factor (IGF1) [3] and ductal ligation/deligation [4], in order to create a more universl understanding of salivary gland regeneration following injury. The central hypothesis of this proposal is label retaining cells play an important role in orchestrating salivary gland regeneration through reestablishment of apical- basal polarity. Specific Aim 1 will demonstrate the regenerative potential of salivary BrdU label retaining cells (LRC). Specific Aim 2 will evaluate the intrinsic role of apical-basal polarity within label retaining cells in restoration of cellular differentiation. Specific Aim 3 will determine the non-cell autonomous effect of loss of polarity on the compensatory proliferation response of label retaining cells. The general goal of this proposal is to identify the capacity of label retaining cells to regenerate differentiated salivary gland structures, and to identify the role of polarity signaling mechanism in promoting differentiation and functional reconstitution. The long-term goal of this proposal is to evaluate whether novel therapies that promote apical-basal polarity within salivary stem/progenitor cells and their respective niches could improve clinical therapeutics for chronic salivary gland dysfunction and xerostomia following radiation therapy for head and neck cancer.

Abstract: More than 73% of head and neck cancer patients continue to suffer from the chronic consequences of xerostomia months to years after the completion of radiotherapy making this one of the most compelling issues in salivary gland biology. Despite technological advancements in cancer therapies, collateral damage to salivary glands remains a significant problem for these patients and severely diminishes their quality of life. The field of radiation-induced salivary gland damage is severely hampered by the lack of a comprehensive model detailing the molecular stages of damage. The overall vision is to restore salivary gland function in patients following radiotherapy by identifying healing stages in salivary glands that lead to the stratification and administration of precise therapeutics for their stage. This proposal will use the sequential phases of wound healing involving inflammation to its resolution and reconstitution of tissue through proliferation and differentiation of epithelial tissue as steps to accomplish this vision. We hypothesize that irradiated salivary glands fail to efficiently progress through the wound healing phases leading to prolonged dysfunction. Our prior work has demonstrated that radiation-induced proliferation in salivary glands is due in part to disruption of the PKC? apical polarity complex leading to enhanced nuclear localization of Yap, while models that restore salivary function have repaired apical polarity and reduced nuclear localization of Yap. We propose to develop a model that integrates each phase of wound healing detailing the interactions between phases and the impact of nuclear Yap on the progression through these phases. The outcomes from this work include: 1) inputs regulating sustained Yap nuclear translocation, 2) ability of chronic nuclear Yap to prevent re-differentiation after IR, 3) when/if Yap is necessary for restoration of salivary gland function, 4) uncovering the interplay between wound healing phases that prevent restoration of salivary gland function. Understanding this process would have a positive impact by revealing intervention points that promote restoration of salivary gland function.

Abstract: More than 73% of head and neck cancer patients continue to suffer from the chronic consequences of xerostomia months to years after the completion of radiotherapy making this one of the most compelling issues in salivary gland biology. Despite technological advancements in cancer therapies, collateral damage to salivary glands remains a significant problem for these patients and severely diminishes their quality of life. The field of radiation- induced salivary gland damage is severely hampered by the lack of a comprehensive model detailing the molecular stages of damage. The overall vision (long-term goal) is to restore salivary gland function in patients following radiotherapy by identifying healing stages in salivary glands that lead to the stratification and administration of precise therapeutics for their stage. This proposal will integrate metabolic changes at three time points of radiation-induced dysfunction that build a mechanistic foundation to bridge to clinical therapeutic interventions. We hypothesize that changes in salivary gland metabolism enable the loss of function phenotype following radiation treatment of salivary glands. The outcomes from this work include: 1) Metabolic networks that are kinetically altered following IR, 2) Identification of novel regulators of the metabolic phenotype following IR, 3) Linkage of changes in metabolic networks with cellular mechanisms that are involved in the response of salivary glands to IR damage. Understanding this process would have a positive impact by revealing intervention points that promote restoration of salivary gland function.