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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
1K01DK103953-01A1
Application #
8967804
Study Section
Special Emphasis Panel (ZDK1)
Program Officer
Saslowsky, David E
Project Start
2015-07-01
Project End
2020-06-30
Budget Start
2015-07-01
Budget End
2016-06-30
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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
Hall, Timothy M; Tétreault, Marie-Pier; Hamilton, Kathryn E et al. (2018) Autophagy as a cytoprotective mechanism in esophageal squamous cell carcinoma. Curr Opin Pharmacol 41:12-19
Whelan, Kelly A; Muir, Amanda B; Nakagawa, Hiroshi (2018) Esophageal 3D Culture Systems as Modeling Tools in Esophageal Epithelial Pathobiology and Personalized Medicine. Cell Mol Gastroenterol Hepatol 5:461-478
Giroux, Véronique; Lento, Ashley A; Islam, Mirazul et al. (2017) Long-lived keratin 15+ esophageal progenitor cells contribute to homeostasis and regeneration. J Clin Invest 127:2378-2391
Maehara, Osamu; Suda, Goki; Natsuizaka, Mitsuteru et al. (2017) Fibroblast growth factor-2-mediated FGFR/Erk signaling supports maintenance of cancer stem-like cells in esophageal squamous cell carcinoma. Carcinogenesis 38:1073-1083
Whelan, Kelly A; Merves, Jamie F; Giroux, Veronique et al. (2017) Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis. Gut 66:1197-1207
Whelan, K A; Chandramouleeswaran, P M; Tanaka, K et al. (2017) Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance. Oncogene 36:4843-4858
Natsuizaka, Mitsuteru; Whelan, Kelly A; Kagawa, Shingo et al. (2017) Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma. Nat Commun 8:1758
Merves, Jamie F; Whelan, Kelly A; Benitez, Alain J et al. (2016) ATG7 Gene Expression as a Novel Tissue Biomarker in Eosinophilic Esophagitis. Am J Gastroenterol 111:151-3
Muir, Amanda B; Dods, Kara; Henry, Steven J et al. (2016) Eosinophilic Esophagitis-Associated Chemical and Mechanical Microenvironment Shapes Esophageal Fibroblast Behavior. J Pediatr Gastroenterol Nutr 63:200-9

Showing the most recent 10 out of 15 publications