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.

Public Health Relevance

Epithelial-mesenchymal transition (EMT) is a fundamental biological process that has critical implications in development and wound healing, but that has also been linked to pathological conditions, including cancer and fibrosis. The current proposal will use innovative and collaborative approaches to explore a role for mitochondrial turnover (via mitochondrial-targeted autophagy) in promoting esophageal cell plasticity via EMT. Results from these studies will lead to a more precise understanding of the biology that underlies epithelial cell plasticity and will provide the basis for future studies with translational applications in therapy for various esophageal diseases.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Scientist Development Award - Research & Training (K01)
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Special Emphasis Panel (ZDK1)
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Saslowsky, David E
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Temple University
Internal Medicine/Medicine
Schools of Medicine
United States
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Kasagi, Yuta; Chandramouleeswaran, Prasanna M; Whelan, Kelly A et al. (2018) The Esophageal Organoid System Reveals Functional Interplay Between Notch and Cytokines in Reactive Epithelial Changes. Cell Mol Gastroenterol Hepatol 5:333-352
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