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Matrix Stiffness-Driven Cytoskeletal Remodeling and Tumor Progression in Anaplastic Thyroid Cancer via Integrin-Focal Adhesion Kinase Signaling

ABSTRACT: Anaplastic thyroid cancer (ATC) is a highly lethal malignancy characterized by rapid progression and therapeutic resistance. This study uncovers the pivotal role of extracellular matrix (ECM) stiffness in driving ATC aggressiveness through mechanotransduction mediated by the Integrin α6β4/Focal Adhesion Kinase (FAK) axis. By engineering collagen-coated polyacrylamide hydrogels with tunable rigidity, we demonstrated that high ECM stiffness (60 kPa) markedly enhanced ATC cell proliferation, clonogenicity, migration, and invasion. Mechanistically, stiff matrices induced cytoskeletal reorganization, activated RhoA/Rac1/Cdc42 signaling, and upregulated Integrin α6β4-FAK pathway components, as validated by transcriptomic, proteomic, and functional assays. Pharmacological inhibition of FAK reversed stiffness-dependent tumor-promoting effects in vitro. In vivo, mice implanted with high-stiffness ECM-mimicking hydrogels exhibited accelerated subcutaneous tumor growth and increased lung metastatic burden, which were significantly attenuated by FAK-targeted therapy. These findings establish ECM stiffness as a biomechanical determinant of ATC progression and metastasis, offering novel insights into microenvironment-driven malignancy and highlighting FAK as a promising therapeutic target to disrupt mechanosignaling in ATC.

ORGANISM(S): Homo Sapiens

SUBMITTER: Guang Chen

PROVIDER: PXD067033 | iProX | Thu Aug 07 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.

Project description:Background Colorectal cancer (CRC), a malignancy with high incidence and mortality rates, presents particularly severe prognoses at the stages of invasion and metastasis despite significant advancements in diagnostic and therapeutic technologies. The tumour microenvironment, especially the extracellular matrix (ECM) stiffness, has garnered considerable attention for its impact on CRC progression and metastasis. However, the precise relationship and molecular mechanisms between ECM stiffness and CRC prognosis remain unclear. Methods This study included 107 CRC patients. Tumor stiffness was assessed using magnetic resonance elastography (MRE), and collagen content was analysed with Masson staining. CRC cell lines SW480 and HCT116 were cultured on matrices of varying stiffness, followed by transcriptome sequencing to identify stiffness-related differential genes and investigate their functions and signalling pathways. Additionally, an HSF4 knockout CRC cell model was constructed to evaluate the effects of HSF4 on cell proliferation, migration, and invasion through both in vitro and in vivo experiments, exploring its role in tumour growth and metastasis. Results The study found that CRC tumour stiffness was significantly higher than normal tissue and positively correlated with collagen content and TNM staging. Transcriptome analysis revealed that high-stiffness matrices significantly regulated cell functions and signalling pathways. High expression of HSF4 was strongly associated with tumour stiffness and poor prognosis. HSF4 expression increased with higher TNM stages in CRC tissues, and its knockout significantly inhibited cell proliferation, migration, and invasion, particularly on high-stiffness matrices. In vivo experiments further confirmed that HSF4 promoted tumour growth and metastasis, independent of collagen protein increase. Conclusion This study reveals a close relationship between tumour stiffness and CRC prognosis, identifying HSF4 as a critical player in CRC progression. HSF4 promotes tumour proliferation and metastasis by regulating EMT-related signalling pathways, with its high expression closely linked to tumour stiffness. Although LOXL1 increased collagen content and a-SMA expression, HSF4's role in promoting tumour growth and metastasis was independent of collagen protein. HSF4 holds potential as a target for CRC prognosis evaluation and treatment. Future research should further explore the regulatory mechanisms of HSF4 and its potential for clinical application.

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Matrix Stiffness-Driven Cytoskeletal Remodeling and Tumor Progression in Anaplastic Thyroid Cancer via Integrin-Focal Adhesion Kinase Signaling

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