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Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

ABSTRACT: Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.

SUBMITTER: Mekhdjian AH

PROVIDER: S-EPMC5449147 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Mekhdjian Armen H AH Kai FuiBoon F Rubashkin Matthew G MG Prahl Louis S LS Przybyla Laralynne M LM McGregor Alexandra L AL Bell Emily S ES Barnes J Matthew JM DuFort Christopher C CC Ou Guanqing G Chang Alice C AC Cassereau Luke L Tan Steven J SJ Pickup Michael W MW Lakins Jonathan N JN Ye Xin X Davidson Michael W MW Lammerding Jan J Odde David J DJ Dunn Alexander R AR Weaver Valerie M VM

Molecular biology of the cell 20170405 11

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and ...[more]

PMID: 28381423

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Project description:Keratin intermediate filaments are dynamic cytoskeletal components that are responsible for tuning the mechanical properties of epithelial tissues. Although it is known that keratin filaments (KFs) are able to sense and respond to changes in the physicochemical properties of the local niche, a direct correlation of the dynamic three-dimensional network structure at the single filament level with the microenvironment has not been possible. Using conventional approaches, we find that keratin flow rates are dependent on extracellular matrix (ECM) composition but are unable to resolve KF network organization at the single filament level in relation to force patterns. We therefore developed a novel method that combines a machine learning-based image restoration technique and traction force microscopy to decipher the fine details of KF network properties in living cells grown on defined ECM patterns. Our approach utilizes Content-Aware Image Restoration (CARE) to enhance the temporal resolution of confocal fluorescence microscopy by at least five fold while preserving the spatial resolution required for accurate extraction of KF network structure at the single KF/KF bundle level. The restored images are used to segment the KF network, allowing numerical analyses of its local properties. We show that these tools can be used to study the impact of ECM composition and local mechanical perturbations on KF network properties and corresponding traction force patterns in size-controlled keratinocyte assemblies. We were thus able to detect increased curvature but not length of KFs on laminin-322 versus fibronectin. Photoablation of single cells in microprinted circular quadruplets revealed surprisingly little but still significant changes in KF segment length and curvature that were paralleled by an overall reduction in traction forces without affecting global network orientation in the modified cell groups irrespective of the ECM coating. Single cell analyses furthermore revealed differential responses to the photoablation that were less pronounced on laminin-332 than on fibronectin. The obtained results illustrate the feasibility of combining multiple techniques for multimodal monitoring and thereby provide, for the first time, a direct comparison between the changes in KF network organization at the single filament level and local force distribution in defined paradigms.

Project description:BackgroundMetastatic melanoma is an aggressive malignancy that is resistant to therapy and has a poor prognosis. The progression of primary melanoma to metastatic disease is a multi-step process that requires dynamic regulation of gene expression through currently uncharacterized epigenetic mechanisms. Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes. Understanding the mechanisms involved in the regulation of gene expression during metastasis is important for developing an effective strategy to treat metastatic melanoma. SWI/SNF enzymes are multisubunit complexes that contain either BRG1 or BRM as the catalytic subunit. We previously demonstrated that heterogeneous SWI/SNF complexes containing either BRG1 or BRM are epigenetic modulators that regulate important aspects of the melanoma phenotype and are required for melanoma tumorigenicity in vitro.ResultsTo characterize BRG1 expression during melanoma progression, we assayed expression of BRG1 in patient derived normal skin and in melanoma specimen. BRG1 mRNA levels were significantly higher in stage IV melanomas compared to stage III tumors and to normal skin. To determine the role of BRG1 in regulating the expression of genes involved in melanoma metastasis, we expressed BRG1 in a melanoma cell line that lacks BRG1 expression and examined changes in extracellular matrix and adhesion molecule expression. We found that BRG1 modulated the expression of a subset of extracellular matrix remodeling enzymes and adhesion proteins. Furthermore, BRG1 altered melanoma adhesion to different extracellular matrix components. Expression of BRG1 in melanoma cells that lack BRG1 increased invasive ability while down-regulation of BRG1 inhibited invasive ability in vitro. Activation of metalloproteinase (MMP) 2 expression greatly contributed to the BRG1 induced increase in melanoma invasiveness. We found that BRG1 is recruited to the MMP2 promoter and directly activates expression of this metastasis associated gene.ConclusionsWe provide evidence that BRG1 expression increases during melanoma progression. Our study has identified BRG1 target genes that play an important role in melanoma metastasis and we show that BRG1 promotes melanoma invasive ability in vitro. These results suggest that increased BRG1 levels promote the epigenetic changes in gene expression required for melanoma metastasis to proceed.

Dataset Information

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Publications

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

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