Elucidating the Mechanism of Secretion for Human Pro-Metastatic AGR2 Protein

Abstract

Anterior Gradient Protein-2 (AGR2) is a protein that belongs to the protein disulphide isomerase (PDI) family. As a member of PDI, AGR2 normally resides in the endoplasmic reticulum (ER) to assist with protein folding and maturation of client proteins entering the secretory pathway (SP). Multi-omics studies identified that AGR2 was overexpressed in several types of cancer and its expression enhanced tumour development and metastasis. Interestingly, this supposedly ER-resident protein can also be secreted into the extracellular milieu and confer tumourigenic properties. However, the mechanism of how AGR2 is secreted extracellularly is not fully understood. This study aims to assess the intracellular and extracellular AGR2 expression and to dissect the mechanism of AGR2 secretion in breast cancer cell model. Here, AGR2 was shown to be expressed intracellularly and extracellularly (cell culture media) in a panel of breast cancer cells namely 1833-BoM, MCF-7 and T47D cells. Using the triplenegative breast cancer (TNBC) cell line, 1833-BoM as a model, AGR2 could bypass ER-Golgi and follows the unconventional protein secretion pathway (UPS) and this event is more evident during ER stress condition. It was also found that extracellular AGR2 was not post-translationally modified with O- or N-glycosylation. Next, to identify constituents that are responsible for AGR2 secretion, AGR2-knockout (AGR2- KO) and non-targeting control (NTC) were generated in 1833-BoM cell pools using CRISPR-Cas9 genome editing technology and the proteome of these cells were analysed using quantitative mass spectrometry. Proteome profiling has identified several differentially expressed proteins exclusive to the AGR2-KO cells population that are potentially involved in trafficking/secreting AGR2 extracellularly. Functional enrichment showed that these proteins were largely involved in cell-cell adhesion, protein folding, and extracellular exosome formation. In conclusion, the data suggest an underlying mechanism of the AGR2 secretion process and provide potential client proteins crucial for AGR2 signalling.