Protein expression and interaction of the kinases Src and Aurora A in esophageal carcinoma

Abstract: Esophageal carcinoma is the seventh most common cancer worldwide. The two main histotypes are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Overall, ESCC is more common, but the incidence of EAC is higher in Western countries.
The kinases SRC and AURKA are both involved in processes and signaling pathways that are crucial for normal cellular behavior. Several studies indicate an association of SRC and AURKA dysregulation and the development of ESCC and EAC. Nevertheless, the kinases’ detailed function in esophageal cancer cells in terms of cancer progression as well as a potential interaction of the kinases have not yet been elucidated.
Hence, the first objective of this experimental MD thesis focused on characterizing the protein expression patterns of SRC, AURKA and their phosphorylated forms in esophageal carcinoma. In situ, ESCC and EAC hardly differed at the level of mere SRC and AURKA protein expression. However, SRC activation by means of phosphorylation at Tyr419 was detected more frequently in EAC than in ESCC (55 % vs. 34 %). In contrast to in situ results, elevated SRC expression levels were found more often in EAC than in ESCC cell lines. Regarding SRC activation, in situ and in vitro data were consistent in that phosphorylation was more frequently detected in EAC. As expected, due to AURKA’s crucial role during mitosis, approximately 50 % of all tumors showed corresponding expression levels of AURKA and the mitotic marker H3S10P. Nevertheless, around 40 % of the tumors expressed higher levels of AURKA than of H3S10P, thus pointing to another, non-mitotic role of AURKA. In vitro expression levels of AURKA and P-AURKA exhibited great heterogeneity, likely attributable to cells not synchronized in cell cycle phases.
The second objective of this MD thesis was the development of a model system to induce and visualize the targeted overexpression of SRC and AURKA. For this, both proteins of interest were first labelled with fluorescent markers, SRC with mCherry and AURKA with eGFP. Next, a tetracycline-inducible expression system was successfully established in the non-neoplastic esophageal epithelial cell line Het-1A by lentiviral transduction and subsequent antibiotic selection. Then, monoclonal cell populations of the transduced cells were generated by limiting dilution cloning. Adequately inducible monoclonal cell populations were selected based on analyses by fluorescence microscopy, Western blot and flow cytometry, before being used for further analyses.
As protein-protein interactions are a central part in cell signaling systems, the third objective of this MD thesis was to analyze a possible interaction between SRC and AURKA using different methods. Immunofluorescence, co-immunoprecipitation and in situ proximity ligation assay were applied. Co-immunoprecipitation and proximity ligation assay results suggest a direct interaction of SRC and AURKA in the case of both aberrant and endogenous AURKA expression. Additionally, immunofluorescence staining and precipitation of SRC hint a nuclear localization of SRC in normal and adenocarcinoma cells of the esophagus.
This MD thesis demonstrates for the first time that the two main histotypes of esophageal carcinoma display similar expression patterns of SRC and AURKA and that, in situ and in vitro, activation of SRC is observed more frequently in EAC than in ESCC. As well, this thesis provides evidence that AURKA has a non-mitotic role in esophageal carcinoma, independent of histological subtype. Furthermore, this MD thesis suggests for the first time a direct interaction of SRC and AURKA in esophageal epithelial cells as well as nuclear localization of SRC in normal and adenocarcinoma cells of the esophagus.
The findings of this thesis pave the way for a better understanding of two central kinases in esophageal carcinoma for possible future use as relevant prognostic biomarkers and/or targets for therapeutic intervention

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2021

Classification and Topics

• Keyword: Expression ; Cancer

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2021
• Creator: Schäfer, Luisa
• Contributor: Laßmann, Silke ; Albert-Ludwigs-Universität Freiburg. Medizinische Fakultät

Further information

• DOI: 10.6094/UNIFR/193839
• URN: urn:nbn:de:bsz:25-freidok-1938395
• Rights: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 25.03.2025, 1:41 PM CET