Proximity Mapping of the ER Proteome Reveals Metastasis-Specific Candidate Biomarkers in Breast Cancer Cell lines

Breast cancer is the most frequently diagnosed cancer among women and a leading cause of cancer-related mortality worldwide. Biomarkers play a pivotal role in early detection, prognosis and therapeutic targeting; however, current biomarkers lack sufficient specificity and sensitivity, highlighting the need for novel, robust candidates. The endoplasmic reticulum (ER) plays a central role in protein synthesis, folding, post-translational modification and lipid metabolism. Alterations in the ER proteome are closely associated with tumor progression, invasion and metastasis. We performed a comparative analysis of the ER proteome in non-invasive MCF-7 and invasive, metastatic MDA-MB-231 breast cancer cell lines using an ER-targeted TurboID proximity-labelling approach. This strategy enabled efficient and specific biotinylation of ER proteins, overcoming limitations of conventional methods. LC–MS/MS analyses identified a total of 2079 proteins, including 1378 ER-associated proteins. To ensure reliability, proteins with at least two unique peptides were considered, yielding 403 ER-resident and 771 ER-associated proteins. Comparative analysis revealed 185 ER-resident proteins that were upregulated and 28 that were downregulated in invasive MDA-MB-231 cells, of which 116 upregulated and 12 downregulated proteins were associated with invasion and metastasis, highlighting key molecular differences underlying the aggressive phenotype. Among more than four hundred regulated proteins, upregulated ones were associated with cellular localization, protein folding, ER stress, vesicle transport, migration, adhesion, hydrolase activity and extracellular matrix organization, reflecting aggressive tumor behaviour, whereas downregulated proteins were primarily involved in aerobic respiration, indicating altered metabolic activity. Additionally, with this study, many candidate biomarkers for breast cancer through transcriptomic and bioinformatic analyses were examined at the protein level and demonstrating their relevance to breast cancer Notably, 36 ER proteins were identified as novel candidate proteins that had not previously been linked to breast cancer. These findings provide the first protein-level evidence of ER proteome remodelling during breast cancer invasion and metastasis. Overall, ER-targeted proximity labelling offers a powerful strategy to uncover functionally relevant proteomic alterations and identify novel biomarkers with clinical potential.