Proteomics Analysis of Cell Surface Protein in AGR2-Targeted Breast Cancer Cell

Abstract

The anterior gradient-2 (AGR2) belongs to the protein disulfide isomerase (PDI) family that localized in the endoplasmic reticulum (ER) where it regulates the formation of disulfide bonds and maintaining protein homeostasis. Its overexpression in numerous cancer types is strongly associated with cancer progression and metastasis. Although predominantly ER-resident, AGR2 has been detected in other cellular compartments, with its functions beyond the ER largely elusive. Recent in-silico and in-vitro interatomics studies have revealed significant interactions between AGR2 and cell surface proteins. This prompts the hypothesis that AGR2 expression plays a role in the synthesis and maturation of cell surface proteins in cancer. To explore this hypothesis, CRISPR-Cas9 gene editing technology was employed to generate AGR2 knockout (AGR2-KO) in MCF-7 breast cancer cells. Two AGR2-KO cell lines were successfully established, along with a non-targeting control (NTC) cell line, confirmed through Western blotting, DNA sequencing and immunofluorescence. Notably, the findings indicated that the AGR2-KO cells exhibited decreased migratory, proliferative, and adhesive capacity compared to the NTC cells indicating a potential role for AGR2 in maintaining cancer cell fitness. Subsequently, the cell surface sub-proteome of these two isogenic cells were enriched and analyzed using mass spectrometry. Several unique proteins were identified in both AGR2-KO and NTC breast cancer cell populations suggesting that loss of AGR2 can reconfigure the cell surface protein landscape. Functional enrichment analysis revealed the involvement of these proteins in key biological processes such as signal transduction, cell communication, and cell growth. These data collectively suggest that AGR2 is integral in driving the synthesis and maturation of pro-oncogenic cell surface proteins in cancer