Interrogation of structural variations of pro-oncogenic AGR2 protein in supporting esophageal cancer progression / Nurul Hanani Baderol Hisham

Nurul Hanani , Baderol Hisham (2022) Interrogation of structural variations of pro-oncogenic AGR2 protein in supporting esophageal cancer progression / Nurul Hanani Baderol Hisham. Masters thesis, Universiti Malaya.

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Abstract

A non-hormone-dependent cancer, esophageal adenocarcinoma was classified as the sixth deadliest cancer with an elevated mortality rate resulting from failure detection at an onset of the disease hence causing the tumor to be at either an advanced or metastatic stage upon diagnosis. Such cancer progression initially reported to be due to many factors including the emergence of pro-oncogenic protein such as the AGR2 protein, known to play roles mainly in the folding of proteins as well as maintaining proteostasis in the ER. The poor understanding on the structural functions of the AGR2 protein in supporting esophageal cancer progression has led to the design of this research which involved constructing four AGR2 protein variants comprised of the AGR2 – WT variant as to compare the basal expression to the other mutated variants, the AGR – KDEL variant to enhance localization of the protein in the ER, the mAGR2 – ΔKTEL variant to enhance secretion of the protein into the extracellular environment and the ΔNterm AGR2 – KDEL variant to enhance the dimerization of the protein. All constructs of AGR2 protein which were AGR – WT, AGR2 – KDEL, mAGR2 – ΔKTEL and ΔNterm AGR2 – KDEL were cloned into the pSF-CMV vector before basal expression of the AGR2 protein variants were determined by transiently transfecting them into the FLO-1 esophageal adenocarcinoma cell line and conduction of immunoblotting and immunofluorescence. The intracellular expressions of the FLO-1 showed the highest expression observed by the AGR2 – WT variant followed by AGR – KDEL. Such expressions were further validated by immunofluorescence. Reduced extracellular expressions of the AGR2 variants in FLO-1 were observed only in two variants, the AGR2 – WT and the AGR – KDEL, while no extracellular expression was observed for the mAGR2 – ΔKTEL variant. The expressions of the ΔNterm AGR2 – KDEL variant either intracellularly or extracellularly could not be validated either by Western blot or immunofluorescence due to the lack of transfection efficiency. The accession on the influence of the AGR2 protein variants towards proliferation and migration of the FLO-1 observed from wound healing assay revealed significant elevations for FLO-1 proliferation and migration in AGR2 – WT, AGR2 – KDEL and mAGR2 – ΔKTEL (p<0.05). The viability of the FLO-1 transfected to AGR2 – WT, AGR2 – KDEL and mAGR2 – ΔKTEL through conduction of MTT assay revealed a significant result (p<0.05) supported by an increase in the absorbances of the cells with respect to time in contrary to Alamar Blue assay conducted which revealed an insignificant result (p>0.05) suggesting that the transfected AGR2 protein variants were independent towards the viability of the FLO-1.

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