Matrix rigidity control of breast cancer cell malignancy

Abstract

The extracellular matrix (ECM) is a dynamic and essential scaffold that orchestrates tissue architecture and cellular behavior through a complex interplay of biochemical and biomechanical cues. In breast cancer, the ECM undergoes extensive remodeling, characterized by increased deposition and cross-linking of collagen fibers. This remodeling leads to a progressively stiffer microenvironment, a hallmark of many solid tumors, which disrupts normal tissue homeostasis and drives malignant transformation. Despite the recognized importance of ECM stiffness in tumor progression, the molecular mechanisms by which mechanical cues regulate breast cancer cell phenotypes remain incompletely understood.This thesis systematically investigates the role of ECM stiffness in modulating breast cancer cell behavior, employing an integrated multi-omic approach that combines quantitative proteomics, kinome profiling, and functional genetic screens. Utilizing the high-grade breast cancer cell line MCF10CA1a, we confirmed that an increase in matrix stiffness induces a phenotypic transition from a ductal carcinoma in situ (DCIS)-like state observed on compliant matrices, reflective of normal mammary gland architecture, to an invasive ductal carcinoma (IDC)-like phenotype on rigid matrices, recapitulating the mechanical properties of malignant breast tissue.In Study I, quantitative mass spectrometry revealed that matrix stiffening upregulates several enzymes of the mevalonate pathway, particularly Hydroxymethylglutaryl-CoA Synthase 1 (HMGCS1), at the protein level without corresponding changes in mRNA, indicating post-transcriptional regulation. Functional assays demonstrated that HMGCS1 is essential for the stiffness-induced malignant phenotype, and its synthesis is regulated by integrin and Rac1 signaling pathways. Notably, HMGCS1 expression was also elevated in human breast tumor tissues and correlated with collagen organization, underscoring the clinical relevance of these findings.Study II employed peptide chip arrays to profile kinase activity across stiffness-dependent cellular states, identifying the inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) and mitogen-activated protein kinase 8 (MAPK8) as key mediators of the stiffness-induced IDC phenotype. Both siRNA-mediated knockdown and pharmacological inhibition of IKBKE and MAPK8 successfully reverted the invasive phenotype to a DCIS-like state, suggesting that targeting these kinases could offer new therapeutic strategies for breast cancer.In Study III, integration of proteomic, kinomic, and transcriptomic data, followed by a targeted RNAi screen, identified several critical regulators of stiffness-induced malignancy, including FES proto-oncogene-tyrosine kinase (FES), Heat Shock Protein Family B (Small) Member 8 (HSPB8), Pyruvate dehydrogenase kinase 1 (PDK1), and Tensin-4 (TNS4). Of particular interest, TNS4 was consistently upregulated at the protein level in response to stiff ECM across multiple breast cancer cell lines. Importantly, at a stiffness resembling breast cancer, TNS4 predominantly localizes in hemidesmosomes (HDs) and is essential for the assembly of HDs. Moreover, disruption of TNS4 or the HD-linked keratin network abrogated the stiffness-induced malignant phenotype.Collectively, these findings provide novel insights into the molecular pathways by which the matrix stiffness regulates breast cancer cell behaviors and highlight potential targets for therapeutic intervention aimed at disrupting the biomechanical drivers of malignancy.List of scientific papersI. Sara Göransson, Helene Olofsson, Henrik J. Johansson, Feifei Yan, Christos Vogiatzakis, Shuo Liang, Hermano Martins Bellato, Laia Masvidal, Inci Aksoylu, Johan Hartman, Glaucia NM Hajj, Ola Larsson, Janne Lehtio, Staffan Stromblad. Mechanical control of breast cancer malignancy by promotion of mevalonate pathway enzyme synthesis. MATRIX BIOLOGY. 2025;140:1-15. https://doi.org/10.1016/j.matbio.2025.05.005II. Feifei Yan*, Sara Göransson*, Helene Olofsson, Christos Vogiatzakis, Anagha Acharekar, Staffan Stromblad. Matrix stiffness-induced IKBKE and MAPK8 signaling drives a phenotypic switch from DCIS to invasive breast cancer. CELL COMMUNICATION AND SIGNALING. 2025;23(1):269. https://doi.org/10.1186/s12964-025-02276-yIII. Feifei Yan, Sara Göransson, Jianjiang Hu, Staffan Strömblad. Matrix rigidity control of hemidesmosomes governs breast cancer cell malignancy. [Manuscript]*: Equal contribution</p