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International Journal of Oral Science Aug 2023Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular...
Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling.
Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.
Topics: Humans; Paxillin; Protein-Lysine 6-Oxidase; Carcinoma, Squamous Cell; Epithelial-Mesenchymal Transition; Integrin alpha2beta1; Mouth Neoplasms; Collagen; Fibroblasts; Extracellular Vesicles; Cell Line, Tumor; Tumor Microenvironment
PubMed: 37532712
DOI: 10.1038/s41368-023-00236-1 -
Toxicology Mechanisms and Methods Feb 2016Formaldehyde (FA) is an environmental pollutant and an endogenous product believed to be involved in tumorigenesis. However, the underlying mechanism of observed FA...
Formaldehyde (FA) is an environmental pollutant and an endogenous product believed to be involved in tumorigenesis. However, the underlying mechanism of observed FA effects has not been clearly defined. Paxillin is a focal adhesion protein that may play an important role in several signaling pathways. Many paxillin-interacting proteins are involved in the regulation of actin cytoskeleton organization, which is necessary for cell motility events associated with diverse biological responses, such as embryonic development, wound repair and tumor metastasis. P53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. In this study, we investigated the effects of FA on paxillin-tyrosine phosphorylation and P53 expression in Hela cells by Western blot and immunofluorescence. Western blot analysis revealed that nonlethal concentrations of FA (0.5, 1.0 and 2.0 mM, with the exposure time for 0.5, 1.0 and 2.0 h, respectively) had downregulated paxillin and wild-type p53 genes expression while upregulated paxillin-tyrosine phosphorylation significantly. At the same time, phosphotyrosine at the focal adhesion sites detected by immunofluorescence assay obviously increased in Hela cells incubated with 2.0 mM FA for 2 h. The results suggested that paxillin and p53 genes expression may be involved in FA-related adverse effects and the mechanism may be involved in paxillin-tyrosine phosphorylation.
Topics: Blotting, Western; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Environmental Pollutants; Formaldehyde; HeLa Cells; Humans; Paxillin; Phosphorylation; Tumor Suppressor Protein p53; Tyrosine
PubMed: 26400731
DOI: 10.3109/15376516.2015.1082001 -
Molecular Biology of the Cell Dec 2019Recent studies indicate that adherent cells are keenly sensitive to external physical environment, such as substrate rigidity and topography, and internal physical...
Recent studies indicate that adherent cells are keenly sensitive to external physical environment, such as substrate rigidity and topography, and internal physical states, such as cell shape and spreading area. Many of these responses are believed to involve coupled output and input of mechanical forces, which may constitute the key sensing mechanism to generate downstream regulatory signals for cell growth and differentiation. Here, we show that the state of cell migration also plays a regulatory role. Compared with migrating cells, stationary cells generate stronger, less dynamic, and more peripherally localized traction forces. These changes are coupled to reduced focal adhesion turnover and enhanced paxillin phosphorylation. Further, using cells migrating along checkerboard micropatterns, we show that the appearance of new focal adhesions directly in front of existing focal adhesions is associated with the down-regulation of existing focal adhesions and associated traction forces. Together, our results imply a mechanism where cell migration regulates traction forces by promoting dynamic turnover of focal adhesions, which may then regulate processes such as wound healing and embryogenesis where cell differentiation must coordinate with migration state and proper localization.
Topics: 3T3 Cells; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Line; Cell Movement; Cell Shape; Focal Adhesions; Mice; Paxillin; Phosphorylation
PubMed: 31693433
DOI: 10.1091/mbc.E19-02-0099 -
Cellular Signalling Aug 2023The molecular mechanisms whereby angiopoietin-like 4 (ANGPTL4), a pluripotent protein implicated in cancer development, contributes to head and neck squamous cell...
OBJECTIVES
The molecular mechanisms whereby angiopoietin-like 4 (ANGPTL4), a pluripotent protein implicated in cancer development, contributes to head and neck squamous cell carcinoma (HNSCC) growth and dissemination are unclear.
MATERIALS AND METHODS
We investigated ANGPTL4 expression in human normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), oral leukoplakia cells (LEUK1), and HNSCC cell lines, as well as in tissue biopsies from patients with oral dysplasia, and primary and metastatic HNSCC. We further examined the contribution of ANGPTL4 cancer progression in an HNSCC orthotopic floor-of mouth tumor model and the signaling pathways linking ANGPTL4 to cancer cell migration.
RESULTS
ANGPTL4 expression was upregulated in premalignant DOKs and HNSCC cell lines compared to NOKs and was increased in tissue biopsies from patients with oral dysplasia, as well as in primary and metastatic HNSCC. We also observed that downregulation of ANGPTL4 expression inhibited primary and metastatic cancer growth in an HNSCC orthotopic tumor model. Interestingly, ANGPTL4 binding to the neuropilin1 (NRP1) receptor led to phosphorylation of the focal adhesion protein, paxillin (PXN), and tumor cell migration; this was dependent on the tyrosine kinase ABL1. Treatment with the ABL1 inhibitor, dasatinib and small interfering RNA silencing of NRP1 or ABL1 expression blocked PXN phosphorylation and tumor cell migration.
CONCLUSION
Our findings suggest an early, sustained, and angiogenesis-independent autocrine role for ANGPTL4 in HNSCC progression and expose ANGPTL4/NRP1/ABL1/PXN as an early molecular marker and vulnerable target for the prevention of HNSCC growth and metastasis.
Topics: Humans; Angiopoietins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Head and Neck Neoplasms; Neuropilin-1; Paxillin; Squamous Cell Carcinoma of Head and Neck
PubMed: 37169211
DOI: 10.1016/j.cellsig.2023.110697 -
Scientific Reports Aug 2018Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound...
Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12. Cell-free interaction assay with immobilized paxillin shows that TG-2 bind to paxillin directly. JNK was the strongest kinase for paxillin phosphorylation in the in-vitro kinase screen, but TG-2 could not phosphorylate paxillin directly. Increasing TG-2 concentrations did not increase the amount of JNK in the TG-2/paxillin complex. Immunofluoresent staining shows that TG-2 colocalises with vinculin and paxillin in FA of migrating cells. TG-2 binds to paxillin and JNK-containing FA but does not recruit JNK directly. Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate paxillin, these processes critically regulate corneal epithelial adhesion and migration.
Topics: Blotting, Western; Cell Line; Cell Movement; Focal Adhesions; GTP-Binding Proteins; Humans; Immunoprecipitation; MAP Kinase Signaling System; Mass Spectrometry; Paxillin; Phosphorylation; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; RNA, Small Interfering; Transglutaminases
PubMed: 30120307
DOI: 10.1038/s41598-018-30172-8 -
Acta Biochimica Polonica Apr 2021Globally, the tenth most common cancer is the oral squamous cell carcinoma (OSCC) and the treatment strategy for improving of OSCC patients survival rate still remains a...
Globally, the tenth most common cancer is the oral squamous cell carcinoma (OSCC) and the treatment strategy for improving of OSCC patients survival rate still remains a challenging one. Aberrant regulation of cell to extracellular matrix protein interactions leads to progression of human cancers. The focal adhesion kinase (FAK) and its downstream target paxillin have been implicated in cancer growth, migration, invasion and metastasis of different cancers. However, the clinical significance of FAK and paxillin in OSCC is not well characterized so far. In the present work, we showed that relative mRNA and protein expressions of FAK and paxillin are significantly higher in side population (SP) cells of OSCC cell line SCC-55. Concomitantly, the matrix metalloproteinase-11 (MMP-11) level is also significantly elevated in SP cells. The enhanced expression of paxillin is strongly correlated with increased chemoresistance, proliferation rate, migration and invasion potential of SP cells. In addition, inhibition of paxillin expression by RNAi makes SP cells more sensitive to chemotherapy drugs. Therefore, our results suggest that paxillin over expression might play a significant role in cancer progression, invasion and chemoresistance of OSCC.
Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Focal Adhesion Protein-Tyrosine Kinases; Humans; Matrix Metalloproteinase 11; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Paxillin; RNA Interference; Side-Population Cells
PubMed: 33860659
DOI: 10.18388/abp.2020_5583 -
Structure (London, England : 1993) Nov 2019Transmembrane integrin bridges the extracellular and intracellular environments and is activated by focal adhesion proteins, talin and kindlin. Activated integrin...
Transmembrane integrin bridges the extracellular and intracellular environments and is activated by focal adhesion proteins, talin and kindlin. Activated integrin engages ligands from the extracellular matrix and controls intracellular responses. In this issue of Structure, Zhu et al. (2019) describe an initial step involving recruitment of paxillin by ubiquitin-like kindlin-2 domain.
Topics: Cell Adhesion; Membrane Proteins; Neoplasm Proteins; Paxillin; Talin
PubMed: 31693910
DOI: 10.1016/j.str.2019.10.010 -
Tissue Engineering. Part A Mar 2022Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues...
Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue, resulting in junction-specific features. The ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue-specific pre-hydrogel digests. The ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities that were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissue-specific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. The ECM conditioned media had only a minor effect on the upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with a varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, another junction marker, type XXII collagen, had similar expression patterns as paxillin, with smaller effect sizes. Using tissue-specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscle-tendon junctions to study the expression of myotendinous junction-specific proteins. Impact statement The muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, the junction is often overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction-specific features and markers that should be included in tissue models of the muscle-tendon unit, while gaining a deeper understanding of the natural junction. This research aims at informing future methods to engineer a more relevant multi-tissue platform to study the muscle-tendon unit.
Topics: Collagen; Collagen Type I; Culture Media, Conditioned; Extracellular Matrix; Hydrogels; Muscles; Paxillin; Tendons
PubMed: 34375125
DOI: 10.1089/ten.TEA.2021.0070 -
Molecular Biology of the Cell Jun 2023Rab GTPase-mediated vesicle trafficking of cell surface proteins, including integrins, through endocytic and recycling pathways is important in controlling...
Rab GTPase-mediated vesicle trafficking of cell surface proteins, including integrins, through endocytic and recycling pathways is important in controlling cell-extracellular matrix interactions during cell migration. The focal adhesion adaptor protein, paxillin, plays a central role in regulating adhesion dynamics and was previously shown to promote anterograde vesicle trafficking through modulation of microtubule acetylation via its inhibition of the deacetylase HDAC6. The role of paxillin in retrograde trafficking is unknown. Herein, we identified a role for paxillin in the modulation of the Rab5 GTPase, which is necessary for regulating early endosome dynamics and focal adhesion turnover. Using MDA-MB-231 breast cancer cells and paxillin (-/-) fibroblasts, paxillin was shown to impact Rab5-associated vesicle size and distribution, as well as Rab5 GTPase activity, through its modulation of HDAC6. Using a combination of real-time imaging and particle tracking analysis, paxillin was shown to promote Rab5-associated vesicle motility through inhibition of HDAC6-mediated micro-tubule deacetylation, along with the localization of active integrin to focal adhesions.
Topics: Humans; Paxillin; Acetylation; Protein Processing, Post-Translational; Cell Movement; Focal Adhesions; Integrins; Microtubules; rab GTP-Binding Proteins; Cell Adhesion
PubMed: 37043310
DOI: 10.1091/mbc.E22-10-0455 -
International Journal of Molecular... Nov 2022Integrins allow cells to adhere to the extracellular matrix and promote the recruitment of other integrins, resulting in the formation of focal adhesion sites at the... (Review)
Review
Integrins allow cells to adhere to the extracellular matrix and promote the recruitment of other integrins, resulting in the formation of focal adhesion sites at the binding sites. Focal adhesion sites play essential roles in the assembly of the cytoskeleton and are vital in shaping the structure of cells. They also play other regulatory roles by influencing numerous biological functions, such as cell proliferation and apoptosis. Hydrogen peroxide‑inducible clone 5 (Hic‑5) is a member of the Paxillin family of proteins and is an adhesive plaque scaffolding protein. Its expression can be detected in both vascular and smooth muscle cells. Thus, it plays an essential role in vascular remodeling, as well as in fibrotic diseases. Hic‑5 functions as a coactivator of steroid receptors, thus playing a role in steroid hormone‑dependent diseases. It also plays a vital role in the invasive metastasis of various types of cancer. Moreover, several studies have demonstrated that Hic‑5 plays a critical role in transcriptional regulation, as well as in numerous signaling pathways. Therefore, the inhibition of the functions of Hic‑5 may prevent the development or halt the progression of several diseases. Its use as a therapeutic target in future investigations may thus aid in the treatment of several diseases, including various types of cancer. The present review article focused on the expression and functions of Hic‑5 in different organs, with the aim of highlighting novel possibilities for future research.
Topics: Cell Adhesion; Focal Adhesion Protein-Tyrosine Kinases; Hormones; Hydrogen Peroxide; Integrins; Paxillin; Phosphorylation
PubMed: 36222304
DOI: 10.3892/ijmm.2022.5194