Pengkajian sekretom sel kanser payudara sasaran protein AGR2 menggunakan teknologi CRISPR/CAS9

Abstract

Anterior Gradient-2 (AGR2) is an overexpressed endoplasmic reticulum protein implicated in numerous epithelial-origin cancers, where it plays key roles in promoting tumour growth, metastasis, and cancer drug resistance. Recent studies revealed that AGR2 is secreted into the extracellular microenvironment, where extracellular AGR2 (eAGR2) exhibits pro-oncogenic properties. However, the precise mechanisms through which eAGR2 functions to modulate cancer progression remain poorly understood. Hence, this study aims to elucidate the role of eAGR2 within the cancer secretome—a subset of the proteome comprising extracellular signalling molecules that shape the tumour microenvironment. By expressing AGR2 structural variants, it is revealed that its secretion depends on specific structural elements, including the signal peptide, endoplasmic reticulum retention motif, and dimerization state. Characterization of CRISPR/Cas9-mediated AGR2 knockout (KO) in breast cancer cell lines (MCF-7, T47D, and 1833-BoM) revealed reduction in cell proliferation, migration, and invasion in 2D cultures, along with increased sensitivity to Doxorubicin and Tamoxifen. AGR2 depletion also activated unfolded protein response markers (UPR) IRE1α and ATF6α, underscoring its role in maintaining endoplasmic reticulum proteostasis. In 3D spheroid models, AGR2 KO cells formed significantly smaller and fewer spheroids, highlighting its role in anchorage-independent growth. Secretome analysis via mass spectrometry with data-independent acquisition and neural network (DIA-NN) analysis revealed dynamic shifts in secreted proteins. A total of 1041, 1626, and 1469 proteins were identified in the secretome of MCF-7, T47D, and 1833-BoM cells, respectively, with 40, 401, and 33 proteins differentially expressed upon AGR2 KO. These data reveal distinct, cell line-specific secretome alterations driven by AGR2, underscoring the heterogeneity of its modulation. Protein-protein interaction network analysis identified key AGR2-associated binding partners, with pathways related to cellular growth, metabolism, and immune regulation significantly enriched. Western blot analysis validated the differential expression of PKM, ENO1 and MAPK1 in MCF-7 and T47D, as well as PKM and IL-6 in 1833-BoM secretome. Collectively, this study provides novel insights into the mechanisms of eAGR2 secretion, its modulation of the tumour microenvironment, and its contribution to breast cancer progression. These findings reinforced AGR2 as a critical player in cancer biology and a potential therapeutic target