Kelly A. Whelan, Ph.D. - US grants

DESCRIPTION (provided by applicant): Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human squamous cell carcinoma (SCC). ESCC patient prognosis remains poor due to late diagnosis and limited response to chemoradiotherapy, highlighting the need for novel therapeutic strategies in the treatmen of this disease. Tumor-initiating cells (TICs) are subsets of tumor cells possessing self-renewal and differentiation capabilities as well as enhanced chemoresistance, properties that have contributed to their emergence as cancer therapy targets. TICs, defined by high expression of CD44, the receptor for hyaluronic acid, have been identified in ESCC~ however, mechanisms regulating these subpopulations have yet to be elucidated. We have isolated CD44 High (CD44H) populations displaying characteristics of TICs from ESCC cells and demonstrated that transforming growth factor (TGF)-beta, promotes expansion of CD44H subpopulations concomitant with activation of Notch signaling. The Notch pathway is a context-dependent regulator of cell fate that is dysregulated in diverse cancer types~ however, the role of this pathway in ESCC is not yet known. In the current proposal, we hypothesize that Notch (N1) activation in the invasive front of ESCC acts in concert with TGF-beta to facilitate expansion of CD44H cell populations that are associated with the malignant properties of ESCC. We will test this through three interrelated specific aims. Aim 1 is to elucidate the role of N1 in expansion of CD44H ESCC cell populations. This will be achieved using inducible lentiviral overexpression and silencing systems to determine the requirement for N1 signaling in generation of CD44H cells in monolayer and 3-dimensional culture. These expression systems will also be used in concert with assays of migration, invasion, colony formation and xenograft tumor formation to assess the role of N1 in CD44H cell- mediated tumorigenicity. Aim 2 is to characterize the molecular mechanisms through which N1 and TGF- beta cooperate to promote CD44H cell expansion. Based on published findings and preliminary data, we hypothesize that TGF-beta- and N1-mediated signaling pathways may crosstalk to initiate a cell fate switch involving suppression of the N1 target gene Notch 3 (N3). We will test this by examining the influence of TGF-beta on N1-mediated transcriptional regulation of N3 using immunoprecipitation (IP), promoter activity assays and chromatin IP. We will also use inducible lentiviral-mediated overexpression and silencing systems to investigate the role o N3 signaling in generation of CD44H subpopulations. Aim 3 is to characterize the role of N1 in tumor growth and TIC expansion in vivo using genetically engineered mouse models. We will evaluate the role of N1 in ESCC progression by inactivating N1 in established ESCC lesions using inducible K5-ERT-Cre~Notch1floxf/lox mice and assessing effects on tumor progression and TIC content. Collectively, these studies will reveal mechanisms through which N1 supports ESCC progression while building a platform for new avenues toward translational applications for therapy of ESCC, which remains resistant to existing therapies.

? DESCRIPTION (provided by applicant): The squamous epithelium of the esophagus exhibits an exquisite differentiation gradient that is disrupted during malignant transformation. Epithelial mesenchymal transition (EMT), a process through which epithelial cells revert to a dedifferentiated mesenchymal phenotype, is a developmental feature that is activated in adult tissues during carcinogenesis. In the esophagus, EMT is linked to the pathogenesis of esophageal squamous cell carcinoma (ESCC), an aggressive form of cancer characterized by invasion, metastasis and treatment resistance. Thus, understanding mechanisms that regulate EMT may provide novel therapeutic targets in the treatment of ESCC. As a phenotypic switch, EMT exerts a high cellular energy demand upon cells. The primary source of cellular energy production is mitochondria. Mitochondria are also a source of cellular reactive oxygen species (ROS) that promote EMT in response to physiological stimuli; however, ROS level must be tightly regulated to prevent damage to cellular components, including mitochondria. Damaged mitochondria are targeted for removal from cells by mitochondrial-targeted autophagy (i.e. mitophagy). Our preliminary data indicate that mitophagy is activated in transformed esophageal keratinocytes undergoing EMT, a novel finding, and that this activation is concurrent with alterations in mitochondrial membrane potential and transient ROS accumulation, suggesting potential interplay between mitochondrial activity, ROS and mitophagy. We hypothesize that mitophagy is a critical EMT mediator in esophageal keratinocytes during malignant transformation. This hypothesis will be tested by pursuing the following three interrelated specific aims: Aim 1: Examine regulation of mitophagy during EMT. Keratinocytes will be stimulated to undergo EMT then we will assess the spatiotemporal dynamics of mitophagy as well as the relationship between mitochondrial activity and mitophagic initiation. Aim 2: Characterize the functional role of mitophagy in EMT. This will be achieved by depleting expression of Parkin, a critical mediator of mitophagy, in esophageal keratinocytes then examining effects upon EMT and oxidative stress. Aim 3: Elucidate the role of mitophagy in esophageal biology in vivo. To evaluate the effects of mitophagy deficiency upon esophageal homeostasis and malignant transformation in vivo, we will utilize Parkin knockout mice coupled with an innovative epithelial lineage-tracing model. EMT will be stimulated in vivo with the oral-esophageal carcinogen 4-nitroquinoline 1-oxide (4NQO). Effects of Parkin deficiency upon tissue morphology, EMT, mitochondria and oxidative stress will be assessed in the presence and absence of 4NQO. Our lineage-tracing model in which a fluorescent reporter is targeted to squamous epithelium of mice using the Keratin 5 promoter will provide definitive evidence of EMT and mitophagy in esophageal epithelium in vivo. Overall, these studies will provide mechanistic insight into the role of mitophagy in esophageal epithelial biology and EMT-mediated plasticity, which may build new therapeutic platforms in for pathological conditions in which EMT has been implicated, including ESCC.

Project Summary Esophageal squamous epithelium comprises a basal layer of proliferative cells that undergoes differentiation in the suprabasal layer and luminal desquamation, facilitating epithelial renewal. Although stem cells responsible for and Through work supported by my K01 grant, we have recently demonstrated that autophagy-mediated modulation of oxidative stress and mitochondrial function supports expansion of cancer stem cells in the tumor microenvironment; however, what role, if any, autophagy plays in normal esophageal stem cell biology remains to be determined. Our published and preliminary data demonstrate evidence of autophagy in a subset of basal cells in murine and human esophageal epithelium in situ. Additionally, pharmacologic or genetic autophagy impairment enhances 3D esophageal organoid formation ex vivo. The goal of the current R03 proposal is to investigate the role of autophagy in regulating basal cell dynamics, namely the balance between actively proliferating and quiescent stem cells. We hypothesize that autophagy limits basal cell proliferation to maintain a pool of slow-cycling esophageal stem/progenitor cells. To test this hypothesis we will pursue the following Specific Aims: Aim 1: To determine the relation relationship between autophagy level and the basal cell stemness/proliferation axis in esophageal epithelium. Aim 2: To define the functional consequences of genetic autophagy impairment upon esophageal basal cell dynamics. These innovative studies utilize functional evaluation of human endoscopic tissue biopsies, 3D esophageal organoids, RNA-sequencing, and a genetically engineered murine model with lineage tracing capacity to investigate autophagy as a novel regulator of esophageal basal cell dynamics, building a platform for future investigations into the specific molecular mechanisms underlying autophagy-mediated esophageal cell fate determination under conditions of health and disease. This R03 proposal represents a logical progression from my K01 grant seeking to establish mitophagy as a critical mediator of epithelial-mesenchymal transition under conditions of homeostasis and carcinogenesis and will facilitate expansion of my research program into the exciting field of stem cell biology. These studies will lay the foundation for an NIH R01 proposal, thereby supporting my transition to a fully independent investigator. the maintenance of esophageal epithelium characterization of these cells remains are thought to reside in the basal cell compartment, identification elusive.

Project Summary Esophageal dysfunction and pathology represent significant health burdens in the United States and worldwide. While patient age is an established risk factor for dysphagia, esophageal cancer and Eosinophilic Esophagitis (EoE)-associated subepithelial fibrosis, our understanding of the biology of aging in the esophagus remains elusive. Under homeostatic conditions, esophageal squamous epithelium comprises a basal compartment of proliferative cells that undergo differentiation in suprabasal layers and luminal desquamation, facilitating tissue renewal. Perturbation of this defined proliferation/differentiation gradient in esophageal epithelium is a feature of esophageal pathologies, including EoE. While the prevalence of BCH is nearly identical in pediatric and adult patients with active EoE, preliminary data indicate that BCH is present in ~20% of normal esophageal epithelial specimens from adults while remaining undetectable in normal pediatric specimens. We have recently demonstrated that autophagy (?self-eating?) is activated in esophageal epithelium in response to EoE inflammation, serving to limit BCH and eosinophilia. Preliminary data indicate that autophagy flux is stalled in aged esophageal epithelium under normal conditions. Moreover, EoE induction in aged mice results in diminished eosinophilia and subepithelial fibrosis. The overarching hypothesis is that age-associated decline in esophageal epithelial autophagy flux impairs tissue homeostasis and contributes to age-associated alterations in EoE phenotype. To test this hypothesis, we will define the functional relationship between mTORC1/autophagy signaling and age-associated esophageal basal cell hyperplasia (Aim 1); elucidate the functional role of epithelial autophagy in age-associated EoE fibrosis (Aim 2); and investigate the role of epithelial autophagy in the EoE inflammatory response in the context of aging (Am 3). The biology of aging in the esophagus represents a significant knowledge gap as understanding mechanisms of tissue aging has the potential to improve strategies for diagnosis, monitoring and therapy of widely prevalent esophageal diseases, including EoE and cancer. Here, we investigate epithelial autophagy as a novel regulator of aging in the esophagus under conditions of homeostasis and EoE inflammation using an innovative approach coupling functional evaluation of human endoscopic biopsies, 3D esophageal organoids and a murine model of EoE featuring age-associated fibrosis. These studies have great potential to provide novel insight into age-relevant mechanisms/cellular phenotypes in the esophagus and unveil new biomarkers and therapeutic targets for EoE and other age-associated disorders affecting the esophagus.

Project Summary Eosinophilic Esophagitis (EoE) is a chronic, food antigen-mediated disease characterized by eosinophil-rich esophageal inflammation and symptoms such as abdominal pain, dysphagia and food impaction. A recent study identified damaging variants in the gene encoding the mitochondrial protein DHTKD1 in EoE patients. Although this indicates a potential role for mitochondria in EoE pathobiology, it remains unclear whether alterations in mitochondrial DNA are present in EoE and may be leveraged to improve clinical care of EoE patients. Indeed, mitochondria house reactive oxygen species (ROS)-producing electron transport chain components and have less sophisticated DNA repair mechanisms as compared to nuclear DNA, making mitochondria DNA highly susceptible to ROS-induced genetic alterations. This information in concert with our preliminary findings indicating (i) that there is an increase in mitochondria in esophageal epithelium of patients with active EoE that fails to normalize in inactive EoE patients; and (ii) mitochondrial dysfunction is induced in esophageal keratinocytes upon exposure to the EoE inflammatory milieu, led us to hypothesize that alterations in mitochondrial DNA may be acquired in esophageal mucosa of patients with Active EoE and maintained in a subset of Inactive patients in clinical disease remission. To test this hypothesis, we will pursue the following specific aims: Aim 1: Perform mitochondrial DNA sequencing in biopsy specimens from a cross-sectional cohort of human subjects comprising Normal controls as well as patients with Active and Inactive EoE. Gastroesophageal reflux disease patients will be evaluated to determine whether identified mitochondrial DNA alterations are specific to EoE. Aim 2: Perform mitochondrial DNA sequencing in biopsy specimens from a longitudinal cohort of human subjects comprising patients with tissues available at the time of both Active EoE and Inactive EoE and classified as therapeutic responders or non-responders. This study will (i) determine if mitochondrial DNA damage is a feature of EoE, both Active and Inactive; (ii) assess the relationship between mitochondrial DNA mutational burden and therapeutic response in EoE; and (iii) identify recurrent mitochondrial mutations that may be targeted therapeutically in EoE. Examples of potential mitochondria- targeted therapies for EoE include, delivery of antioxidant compounds (e.g. Coenzyme Q10) or micronutrients (e.g. Zinc, Biotin) to improve mitochondrial function, enzyme replacement therapy to restore the function of faulty enzymes encoded by mitochondrial DNA, and Adenoviral or CRISPR-mediated editing of the mitochondrial genome. Data generated in this study will provide the foundation for a future NIH R01 proposal that will delineate the functional significance of mitochondrial DNA alterations in EoE pathobiology and also evaluate therapeutic strategies related to mitochondrial dysfunction using preclinical models, including primary epithelial cultures, patient-derived esophageal organoids, and murine EoE models.

Project Summary Esophageal cancer is the eighth most common cancer and sixth leading cause of cancer mortality worldwide. Chronic reflux-associated esophagitis, termed gastroesophageal reflux disease (GERD), is a primary risk factor for development of esophageal adenocarcinoma. Eosinophilic esophagitis (EoE) represents food allergen-mediated esophagitis characterized by esophageal eosinophilia. In contrast to patients with reflux esophagitis, epidemiological data indicates that EoE patients fail to develop esophageal cancer despite the presence of chronic esophageal inflammation. A negative correlation between allergic inflammation and cancer risk has been identified in a variety of organs via population-based studies; however, functional investigations are necessary to define the relationship between allergy and cancer as well as to determine the feasibility of approaches for leveraging allergic inflammation to improve clinical outcomes in cancer patients. To examine the relationship between EoE and cancer, we paired murine models of the two conditions. Our robust preliminary data indicate that exposure to EoE inflammation limits esophageal carcinogenesis in vivo. We hypothesize that EoE inflammation limits esophageal carcinogenesis by activating anti-tumor responses in the immune and epithelial cell compartments. We will test this hypothesis by pursuing the following Specific Aims: Aim 1: Identify the immune-mediated mechanisms responsible for tumor cell apoptosis induced by EoE inflammation. Aim 2: Delineate the impact of EoE inflammation upon esophageal epithelial cells in the context of carcinogenesis. These studies provide the first functional investigation of the relationship between EoE and esophageal cancer with the potential to unveil novel mechanisms for targeting the allergic immune response and/or allergy-mediated esophageal epithelial fate decisions to improve clinical care for cancer patients. These developmental R21 studies will identify the direct cellular/molecular mechanisms through which the EoE influences the epithelial and immune cell compartments to limit esophageal carcinogenesis. A future R01 proposal will aggressively pursue identified mechanisms in preclinical and clinical models to meet our long- term goal of defining novel strategies for improving esophageal cancer prevention, diagnosis, monitoring, and therapy. As a negative association between allergic inflammation and cancer has been identified in various organs, findings from this study may have broad implications for cancer prevention and therapy.