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ELife Jul 2023Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal...
Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal adhesion assembly is unclear. We provide evidence that the Src-family kinase (SFK) substrate Cas (Crk-associated substrate, p130Cas, BCAR1) is phosphorylated and associated with its Crk/CrkL effectors in clusters that are precursors of focal adhesions. The initial phospho-Cas clusters contain integrin β1 in its inactive, bent closed, conformation. Later, phospho-Cas and total Cas levels decrease as integrin β1 is activated and core focal adhesion proteins including vinculin, talin, kindlin, and paxillin are recruited. Cas is required for cell spreading and focal adhesion assembly in epithelial and fibroblast cells on collagen and fibronectin. Cas cluster formation requires Cas, Crk/CrkL, SFKs, and Rac1 but not vinculin. Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest a two-step model for focal adhesion assembly in which clusters of phospho-Cas, effectors and inactive integrin β1 grow through positive feedback prior to integrin activation and recruitment of core focal adhesion proteins.
Topics: Phosphorylation; Focal Adhesions; Phosphoproteins; Integrin beta1; Crk-Associated Substrate Protein; Protein-Tyrosine Kinases; Integrins; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesion Kinase 1
PubMed: 37489578
DOI: 10.7554/eLife.90234 -
The Journal of Clinical Investigation Nov 2023Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is...
Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe's largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α-expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.
Topics: Humans; Animals; Mice; Transforming Growth Factor alpha; Paxillin; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Phenotype; Cell Line, Tumor
PubMed: 37607005
DOI: 10.1172/JCI166333 -
Frontiers in Cell and Developmental... 2023Cancer progression and metastasis are processes heavily controlled by the integrin receptor family. Integrins are cell adhesion molecules that constitute the central... (Review)
Review
Cancer progression and metastasis are processes heavily controlled by the integrin receptor family. Integrins are cell adhesion molecules that constitute the central components of mechanosensing complexes called focal adhesions, which connect the extracellular environment with the cell interior. Focal adhesions act as key players in cancer progression by regulating biological processes, such as cell migration, invasion, proliferation, and survival. Src family kinases (SFKs) can interplay with integrins and their downstream effectors. SFKs also integrate extracellular cues sensed by integrins and growth factor receptors (GFR), transducing them to coordinate metastasis and cell survival in cancer. The non-receptor tyrosine kinase CSK is a well-known SFK member that suppresses SFK activity by phosphorylating its specific negative regulatory loop (C-terminal Y residue). Consequently, CSK may play a pivotal role in tumour progression and suppression by inhibiting SFK oncogenic effects in several cancer types. Remarkably, CSK can localise near focal adhesions when SFKs are activated and even interact with focal adhesion components, such as phosphorylated FAK and Paxillin, among others, suggesting that CSK may regulate focal adhesion dynamics and structure. Even though SFK oncogenic signalling has been extensively described before, the specific role of CSK and its crosstalk with integrins in cancer progression, for example, in mechanosensing, remain veiled. Here, we review how CSK, by regulating SFKs, can regulate integrin signalling, and focus on recent discoveries of mechanotransduction. We additionally examine the cross talk of integrins and GFR as well as the membrane availability of these receptors in cancer. We also explore new pharmaceutical approaches to these signalling pathways and analyse them as future therapeutic targets.
PubMed: 37519303
DOI: 10.3389/fcell.2023.1214787 -
Journal of Oral and Maxillofacial... 2023Cell adhesion molecules (CAMs) are found on the surface of all cells, where they allow dynamic processes to take place. These include cadherins, integrins, selectins and... (Review)
Review
BACKGROUND
Cell adhesion molecules (CAMs) are found on the surface of all cells, where they allow dynamic processes to take place. These include cadherins, integrins, selectins and Immunoglobulin superfamily. Directly associated with β-integrin tails is a multidomain protein known as paxillin. However, CAMs participate in cell-cell and extracellular matrix-cell interactions during histomorphogenesis in the various phases of odontogenesis. Some tumours or cysts like ameloblastoma (AB) or odontogenic keratocyst (OKC) having odontogenic origin show disturbance in the interaction of these CAMs. Hence, the assessment of paxillin expression in AB and OKC was carried out.
MATERIALS AND METHODS
The present observational study comprised 30 clinically and histologically confirmed cases of AB and OKC. All the slides were stained immunohistochemically using a paxillin antibody.
RESULTS
Upon comparison of staining intensity of paxillin among AB and OKC showed statistically significant result, whereas quantitative staining and final summation showed non-significant result. Gender-wise comparison of paxillin staining intensity, quantitative staining and final summation among OKC showed significant result; however, in AB, staining intensity showed non-significant result, whereas quantitative staining and final summation showed significant result.
CONCLUSION
Paxillin has the greatest influence on tissue morphogenesis and development. The regulation of cell mobility is aided by the multiple roles that paxillin plays in a range of cells and tissues. However, further studies using a large sample size, along with other molecular analytical methods, may be essential to draw a definite conclusion about the association of paxillin and its exact function in OKC and AB.
PubMed: 38304525
DOI: 10.4103/jomfp.jomfp_312_23 -
EMBO Reports Nov 2023The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of...
The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well-characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN-dependent mechanotransduction.
Topics: Humans; Female; Paxillin; Breast Neoplasms; Mechanotransduction, Cellular; Phosphorylation; Cell Movement; Serine; Autophagy-Related Protein-1 Homolog; Intracellular Signaling Peptides and Proteins
PubMed: 37846507
DOI: 10.15252/embr.202356850 -
HGF/c-Met/β1-integrin signalling axis induces tunneling nanotubes in A549 lung adenocarcinoma cells.Life Science Alliance Oct 2023Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and...
Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and signalling molecules. In cancer cells, TNT formation contributes to cell survival, chemoresistance, and malignancy. However, the molecular mechanisms underlying TNT formation are not well defined, especially in different cancers. TNTs are present in non-small cell lung cancer (NSCLC) patients with adenocarcinoma. In NSCLC, hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated, causing increased cancer cell growth, survival, and invasion. This study identifies c-Met, β1-integrin, and paxillin as novel components of TNTs in A549 lung adenocarcinoma cells, with paxillin localised at the protrusion site of TNTs. The HGF-induced TNTs in our study demonstrate the ability to transport lipid vesicles and mitochondria. HGF-induced TNT formation is mediated by c-Met and β1-integrin in conjunction with paxillin, followed by downstream activation of MAPK and PI3K pathways and the Arp2/3 complex. These findings demonstrate a potential novel approach to inhibit TNT formation through targeting HGF/c-Met receptor and β1-integrin signalling interactions, which has implications for multi-drug targeting in NSCLC.
Topics: Humans; Paxillin; Carcinoma, Non-Small-Cell Lung; Phosphatidylinositol 3-Kinases; Lung Neoplasms; Adenocarcinoma of Lung; Integrins; Hepatocyte Growth Factor
PubMed: 37550007
DOI: 10.26508/lsa.202301953 -
Cancers Dec 2023The aberrant transformation of normal cells into cancer cells, known as carcinogenesis, is a complex process involving numerous genetic and molecular alterations in... (Review)
Review
The aberrant transformation of normal cells into cancer cells, known as carcinogenesis, is a complex process involving numerous genetic and molecular alterations in response to innate and environmental stimuli. The Src family kinases (SFK) are key components of signaling pathways implicated in carcinogenesis, with c-Src and its oncogenic counterpart v-Src often playing a significant role. The discovery of c-Src represents a compelling narrative highlighting groundbreaking discoveries and valuable insights into the molecular mechanisms underlying carcinogenesis. Upon oncogenic activation, c-Src activates multiple downstream signaling pathways, including the PI3K-AKT pathway, the Ras-MAPK pathway, the JAK-STAT3 pathway, and the FAK/Paxillin pathway, which are important for cell proliferation, survival, migration, invasion, metastasis, and drug resistance. In this review, we delve into the discovery of c-Src and v-Src, the structure of c-Src, and the molecular mechanisms that activate c-Src. We also focus on the various signaling pathways that c-Src employs to promote oncogenesis and resistance to chemotherapy drugs as well as molecularly targeted agents.
PubMed: 38201459
DOI: 10.3390/cancers16010032 -
ACS Applied Materials & Interfaces Feb 2024The complex interplay between cells and materials is a key focus of this research, aiming to develop optimal scaffolds for regenerative medicine. The need for tissue...
The complex interplay between cells and materials is a key focus of this research, aiming to develop optimal scaffolds for regenerative medicine. The need for tissue regeneration underscores understanding cellular behavior on scaffolds, especially cell adhesion to polymer fibers forming focal adhesions. Key proteins, paxillin and vinculin, regulate cell signaling, migration, and mechanotransduction in response to the extracellular environment. This study utilizes advanced microscopy, specifically the AiryScan technique, along with advanced image analysis employing the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) cluster algorithm, to investigate protein distribution during osteoblast cell adhesion to polymer fibers and glass substrates. During cell attachment to both glass and polymer fibers, a noticeable shift in the local maxima of paxillin and vinculin signals is observed at the adhesion sites. The focal adhesion sites on polymer fibers are smaller and elliptical but exhibit higher protein density than on the typical glass surface. The characteristics of focal adhesions, influenced by paxillin and vinculin, such as size and density, can potentially reflect the strength and stability of cell adhesion. Efficient adhesion correlates with well-organized, larger focal adhesions characterized by increased accumulation of paxillin and vinculin. These findings offer promising implications for enhancing scaffold design, evaluating adhesion to various substrates, and refining cellular interactions in biomedical applications.
Topics: Paxillin; Vinculin; Focal Adhesions; Mechanotransduction, Cellular; Cell Adhesion; Polymers; Phosphoproteins; Focal Adhesion Protein-Tyrosine Kinases
PubMed: 38354103
DOI: 10.1021/acsami.3c19035 -
Hepatology Communications Jul 2023HCC characterizes malignant metastasis with high incidence and recurrence. Thus, it is pivotal to discover the mechanisms of HCC metastasis. TATA-box-binding protein...
BACKGROUND
HCC characterizes malignant metastasis with high incidence and recurrence. Thus, it is pivotal to discover the mechanisms of HCC metastasis. TATA-box-binding protein (TBP), a general transcriptional factor (TF), couples with activators and chromatin remodelers to sustain the transcriptional activity of target genes. Here, we investigate the key role of TBP in HCC metastasis.
METHODS
TBP expression was measured by PCR, western blot, and immunohistochemistry. RNA-sequencing was performed to identify downstream proteins. Functional assays of TBP and downstream targets were identified in HCC cell lines and xenograft models. Luciferase reporter and chromatin immunoprecipitation assays were used to demonstrate the mechanism mediated by TBP.
RESULTS
HCC patients showed high expression of TBP, which correlated with poor prognosis. Upregulation of TBP increased HCC metastasis in vivo and in vitro, and muscleblind-like-3 (MBNL3) was the effective factor of TBP, positively related to TBP expression. Mechanically, TBP transactivated and enhanced MBNL3 expression to stimulate exon inclusion of lncRNA-paxillin (PXN)-alternative splicing (AS1) and, thus, activated epithelial-mesenchymal transition for HCC progression through upregulation of PXN.
CONCLUSIONS
Our data revealed that TBP upregulation is an HCC enhancer mechanism that increases PXN expression to drive epithelial-mesenchymal transition.
Topics: Humans; Biological Assay; Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Liver Neoplasms; RNA-Binding Proteins; TATA-Box Binding Protein; Animals
PubMed: 37314767
DOI: 10.1097/HC9.0000000000000155 -
Biochemical and Biophysical Research... Nov 2023Self-renewal and differentiation of mouse embryonic stem cells (mESCs) are greatly affected by the extracellular matrix (ECM) environment; the composition and stiffness...
Self-renewal and differentiation of mouse embryonic stem cells (mESCs) are greatly affected by the extracellular matrix (ECM) environment; the composition and stiffness of which are sensed by the cells via integrin-associated focal adhesions (FAs) which link the cells to the ECM. Although FAs have been studied extensively in differentiated cells, their composition and function in mESCs are not as well elucidated. To gain more detailed knowledge of the molecular compositions of FAs in mESCs, we adopted the proximity-dependent biotinylation (BioID) proteomics approach. Paxillin, a known FA protein (FAP), is fused to the promiscuous biotin ligase TurboID as bait. We employed both SILAC- and label-free (LF)-based quantitative proteomics to strengthen as well as complement individual approach. The mass spectrometry data derived from SILAC and LF identified 38 and 443 proteins, respectively, with 35 overlapping candidates. Fifteen of these shared proteins are known FAPs based on literature-curated adhesome and 7 others are among the reported "meta-adhesome", suggesting the components of FAs are largely conserved between mESCs and differentiated cells. Furthermore, the LF data set contained an additional 18 literature-curated FAPs. Notably, the overlapped proteomics data failed to detect LIM-domain proteins such as zyxin family proteins, which suggests that FAs in mESCs are less mature than differentiated cells. Using the LF approach, we are able to identify PDLIM7, a LIM-domain protein, as a FAP in mESCs. This study illustrates the effectiveness of TurboID in mESCs. Importantly, we found that application of both SILAC and LF methods in combination allowed us to analyze the TurboID proteomics data in an unbiased, stringent and yet comprehensive manner.
PubMed: 37725837
DOI: 10.1016/j.bbrc.2023.09.017