Endoplasmic reticulum-localized TurboID-mediated labelling reveals distinct secretome profiles of MCF-10A and MDA-MB-231 cells

Abstract

Breast cancer is one of the most prevalent cancers and the leading causes of cancer-related deaths. The lack of reliable biomarkers for accurate subtyping and early diagnosis continues to hinder early detection and treatment. Secretome proteins represent an accessible and valuable source of biomarkers due to their roles in cell communication, signalling and shaping the extracellular microenvironment. In this study, secretome proteins from two cell lines, MCF-10A and MDA-MB-231, representing healthy and aggressive breast cells, respectively, were labelled with ER-localized TurboID-mediated enzymatic biotinylation approach at the endoplasmic reticulum. The biotinylated samples were enriched using streptavidin-coated magnetic beads and analysed by label-free quantitation using nHPLC LC-MS/MS. The regulated proteins were subjected to bioinformatics analyses using STRING and g:Profiler tools to identify candidate biomarkers. Proteomic analysis identified 206 proteins, with approximately 82% belonged to secretome proteins. Among them, 65 were differentially regulated which were associated with hydrolytic activity, cell adhesion, and lipid metabolism. CST1, APOC1, and POSTN had previously been associated with cancer, while TEX10, LZIC, and PSMA3 were implicated in breast cancer for the first time. Our findings demonstrates extensive secretome remodelling in invasive breast cancer cells, unveiling potential secreted biomarker candidates that may improve breast cancer diagnosis and treatment strategies.

Keywords

• Breast cancer
• secretome
• biomarkers
• biotinylation

Endoplazmik retikuluma lokalize edilen TurboID aracılı işaretleme ile MCF-10A ve MDA-MB-231 hücrelerine ait sekretom profillerinin belirlenmesi

Abstract

Meme kanseri, insidansı en yüksek olan kanser türlerinden biri olup, kanser kaynaklı ölümlerin başlıca nedenleri arasındadır. Alt tiplerin doğru sınıflandırılması ve erken tanı için güvenilir biyobelirteçlerin eksikliği, hastalığın erken teşhis ve tedavisini güçleştirmektedir. Hücre iletişimi, sinyal iletimi ve hücre dışı mikroçevrenin şekillenmesindeki rolleri nedeniyle sekretom proteinleri, biyobelirteç keşfi açısından erişilebilir ve değerli bir kaynaktır. Bu çalışmada, sağlıklı (MCF-10A) ve invaziv (MDA-MB-231) meme epitel hücre hatlarının sekretom proteinleri, endoplasmik retikuluma-lokalize edilen TurboID biyotin ligaz aracılı enzimatik biyotinilasyon yöntemiyle işaretlenmiştir. Etiketlenen proteinler streptavidin-kaplı manyetik boncuklar yardımı ile zenginleştirildikten sonra, etiketsiz miktar tayini için nHPLC LC-MS/MS yöntemi ile analiz edilmiştir. Anlamlı regülasyon gösteren proteinler STRING ve g:Profiler araçlarıyla biyoinformatik analizlere tabi tutulara biyobelirteç adayları belirlenmeye çalışılmıştır. Analizler sonucu toplam 206 protein tanımlanmış olup, bunların yaklaşık %82’si sekretom proteinleri içerine dahildir. Belirlenen bu proteinlerin 65 tanesinin gruplar arasında anlamlı farklılık gösterdiği ve bunların özellikle hidrolitik aktivite, hücre adezyonu ve lipid metabolizmasıyla ilişkili olduğu belirlenmiştir. Bu proteinlerden; CST1, APOC1 ve POSTN daha önce kanserle ilişkilendirilmişken, TEX10, LZIC ve PSMA3 ilk kez meme kanseriyle ilişkilendirilmiştir. Bulgularımız, invaziv meme kanseri hücrelerinde kapsamlı bir sekretom analizini ortaya koymuş ve meme kanseri tanı ve tedavi stratejilerini kullanılabilecek biyobelirteç adaylarının potansiyelleri sekretom düzeyinde değerlendirilmiştir.

Keywords

• Meme kanseri
• sekretom
• biyomarker
• biyotinilasyon

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