Universität zu Köln

Ju, Feng (2024). Aldo-keto reductase 1C3 enhances radioresistance in esophageal adenocarcinoma via inhibiting ferroptosis. PhD thesis, Universität zu Köln.

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Abstract

Background: Esophageal cancer (EC) is the 6th highest mortality and 7th highest incidence worldwide. Overall, esophageal adenocarcinoma (EAC) is the most common subtype of EC in high-income countries. Aldo-keto reductase 1C 3 (AKR1C3) represents a promising therapeutic target to overcome radioresistance in many cancers, while the molecular mechanism of AKR1C3 in the radioresistance of EAC is still unclear. Methods: The radioresistant model of OE33 was established by long-term small-dose irradiation (IR). Colony formations were performed to validate the survival fraction of EAC cells. RNA-seq analysis was applied for the in vitro radioresistant model. AKR1C3 overexpressing cells (generated from OE33) and knockdown cells (modified from SKGT-4) were established for in vitro analysis. The IR-induced apoptosis levels were measured by flow cytometry with DAPI and Annexin V staining. DNA damage levels were measured by immunofluorescence with gamma-H2AX staining and by Comet assay. The mitochondrial morphology of EAC cells was photographed by transmission electron microscope. Seahorse XF cell mito stress tests and Seahorse XF glycolytic rate assays were performed to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. Tetramethyl rhodamine ethyl ester (TMRE) staining was used for measuring mitochondrial activities. SLC7A11 and GPX4 protein levels were measured by Western blot. Lipid peroxidation levels were measured by flow cytometry with C11-Bodipy staining. MTT assays were applied to measure cell viability after erastin-treatment in EAC cells. Furthermore, Kaplan-Meier survival analysis was performed to draw the survival curve from the TCGA survival data. Results: Colony formation assay validated the radioresistant model of EAC cell line. The RNA-seq analysis showed a higher mRNA level of AKR1C3, as well as some ferroptosis-related genes such as FSP1 and SLC7A11 in the radioresistant cell line than in the parental cell line. Western blot and qRT-PCR confirmed the expression of AKR1C3 in our radioresistant model. In addition, overexpression of AKR1C3 in EAC cells significantly improved the survival fraction and decreased the apoptotic cells after irradiation. Overexpression of AKR1C3 could also prevent EAC cells from DNA damage after irradiation. The reversed results were observed in AKR1C3 knockdown cells. Transmission electron microscope exhibited mitochondrial morphological changes after irradiation in EAC cells. AKR1C3 overexpressing cells showed more oxygen consumption rate changes after irradiation than the control cells. Higher TMRE levels were observed in AKR1C3 overexpressing cells after irradiation, while TMRE levels were lower in AKR1C3 knockdown cells. SLC7A11 and GPX4 expression levels showed a positive correlation with AKR1C3 by Western blot and GEPIA database analysis. Overexpression of AKR1C3 exhibited more resistant to erastin treatment, while knockdown of AKR1C3 sensitized cells to erastin treatment. Lipid peroxidation levels were lower in AKR1C3 overexpression cells and radioresistant cells while higher in AKR1C3 knockdown cells after irradiation or erastin treatment. MPA, a selective AKR1C3 inhibitor, could re-sensitize EAC cells to erastin treatment. The survival curve showed the median survival time was 23.1 months in the high AKR1C3 group and 27.1 months in the low AKR1C3 group in EAC cohort. Conclusions: In this study, we demonstrated AKR1C3 could regulate the radioresistance of EAC cells. AKR1C3 renders radioresistance through inhibition of ferroptosis via mediating SLC7A11/GSH/GPX4 axis. Targeting AKR1C3 may provide a potential approach to increase the treatment response in EAC patients for individualized therapy.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Ju, Feng juf908@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-736253
Date:	2024
Language:	English
Faculty:	Faculty of Medicine
Divisions:	Faculty of Medicine > Chirurgie
Subjects:	Medical sciences Medicine
Uncontrolled Keywords:	Keywords Language esophageal adenocarcinoma English Aldo-keto reductase 1C 3 (AKR1C3) English radioresistance English ferroptosis English lipid peroxidation English
Date of oral exam:	23 July 2024
Referee:	Name Academic Title Bruns, C. J. Univ.-Prof. Dr. med Kashkar, H. Univ.-Prof. Dr. rer. nat
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/73625