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International Journal of Molecular... Nov 2023Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in...
Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in various cancer types, with tyrosine phosphorylation shown to play a critical role in driving cancer cell migration. However, the specific impact of the distinct tyrosine phosphorylation events of paxillin in the progression of breast cancer remains to be fully elucidated. Here, we found that paxillin overexpression in breast cancer tissue is associated with a patient's poor prognosis. Paxillin knockdown inhibited the migration and invasion of breast cancer cells. Furthermore, the phosphorylation of paxillin tyrosine residue 31 (Tyr31) was significantly increased upon the TGF-β1-induced migration and invasion of breast cancer cells. Inhibiting Fyn activity or silencing Fyn decreases paxillin Tyr31 phosphorylation. The wild-type and constitutively active Fyn directly phosphorylate paxillin Tyr31 in an in vitro system, indicating that Fyn directly phosphorylates paxillin Tyr31. Additionally, the non-phosphorylatable mutant of paxillin at Tyr31 reduces actin stress fiber formation, migration, and invasion of breast cancer cells. Taken together, our results provide direct evidence that Fyn-mediated paxillin Tyr31 phosphorylation is required for breast cancer migration and invasion, suggesting that targeting paxillin Tyr31 phosphorylation could be a potential therapeutic strategy for mitigating breast cancer metastasis.
Topics: Female; Humans; Breast Neoplasms; Cell Movement; Paxillin; Phosphorylation; Tyrosine
PubMed: 37958964
DOI: 10.3390/ijms242115980 -
Fish & Shellfish Immunology Nov 2023Cell migration is an essential process in immunity and wound healing. The in vitro scratch assay was optimized for the SAF-1 cell line, obtained from gilthead seabream...
Cell migration is an essential process in immunity and wound healing. The in vitro scratch assay was optimized for the SAF-1 cell line, obtained from gilthead seabream (Sparus aurata) fin. In addition, selected cells from the cell front were tracked for detailed individual cell movement and morphological analysis. Modulation of migration and cell tracking of the SAF-1 cell line by probiotics was evaluated. Cells were cultured and incubated for 24 h with three species of extremophilic yeasts [Yarrowia lipolytica (D1 and N6) and Debaryomyces hansenii (CBS004)] and the bacterium Shewanella putrefaciens (known as SpPdp11) and then scratch and cell tracking assays were performed. The results indicated that the forward velocity was significantly (p < 0.05) increased in SAF-1 cells incubated with CBS004 or SpPdp11. However, cell velocity, cumulative distance and Euclidean distance were only significantly increased in SAF-1 cells incubated with SpPdp11. Furthermore, to increase our understanding of the genes involved in cell movement, the expression profile of ten structural proteins (α-1β tubulin, vinculin, focal adhesion kinase type, alpha-2 integrin, tetraspanin, integrin-linked kinase 1, tensin 3, tensin 4, paxillin, and light chain 2) was studied by real time-PCR. The expression of these genes was modulated as a function of the probiotic tested and the results indicate that CBS004 and SpPdp11 increase the movement of SAF-1 cells.
Topics: Animals; Sea Bream; Cell Tracking; Tensins; Cell Movement; Probiotics
PubMed: 37858786
DOI: 10.1016/j.fsi.2023.109149 -
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 -
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 -
Cancers Sep 2023Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is...
Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody-drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients' data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies.
PubMed: 37686651
DOI: 10.3390/cancers15174374 -
Journal of Cell Science Sep 2023Liver injury leads to fibrosis and cirrhosis. The primary mechanism underlying the fibrogenic response is the activation of hepatic stellate cells (HSCs), which are...
Liver injury leads to fibrosis and cirrhosis. The primary mechanism underlying the fibrogenic response is the activation of hepatic stellate cells (HSCs), which are 'quiescent' in normal liver but become 'activated' after injury by transdifferentiating into extracellular matrix (ECM)-secreting myofibroblasts. Given that integrins are important in HSC activation and fibrogenesis, we hypothesized that paxillin, a key downstream effector in integrin signaling, might be critical in the fibrosis pathway. Using a cell-culture-based model of HSC activation and in vivo models of liver injury, we found that paxillin is upregulated in activated HSCs and fibrotic livers. Overexpression of paxillin (both in vitro and in vivo) led to increased ECM protein expression, and depletion of paxillin in a novel conditional mouse injury model reduced fibrosis. The mechanism by which paxillin mediated this effect appeared to be through the actin cytoskeleton, which signals to the ERK pathway and induces ECM protein production. These data highlight a novel role for paxillin in HSC biology and fibrosis.
Topics: Mice; Animals; Paxillin; Actins; Hepatic Stellate Cells; Polymerization; Liver Cirrhosis; Liver; Fibrosis; Disease Models, Animal
PubMed: 37667902
DOI: 10.1242/jcs.261122 -
EMBO Molecular Medicine Oct 2023Periventricular neuronal heterotopia (PH) is one of the most common forms of cortical malformation in the human cortex. We show that human neuronal progenitor cells...
Periventricular neuronal heterotopia (PH) is one of the most common forms of cortical malformation in the human cortex. We show that human neuronal progenitor cells (hNPCs) derived from PH patients with a DCHS1 or FAT4 mutation as well as isogenic lines had altered migratory dynamics when grafted in the mouse brain. The affected migration was linked to altered autophagy as observed in vivo with an electron microscopic analysis of grafted hNPCs, a Western blot analysis of cortical organoids, and time-lapse imaging of hNPCs in the presence of bafilomycin A1. We further show that deficits in autophagy resulted in the accumulation of paxillin, a focal adhesion protein involved in cell migration. Strikingly, a single-cell RNA-seq analysis of hNPCs revealed similar expression levels of autophagy-related genes. Bolstering AMPK-dependent autophagy by metformin, an FDA-approved drug, promoted migration of PH patients-derived hNPCs. Our data indicate that transcription-independent homeostatic modifications in autophagy contributed to the defective migratory behavior of hNPCs in vivo and suggest that modulating autophagy in hNPCs might rescue neuronal migration deficits in some forms of PH.
PubMed: 37609821
DOI: 10.15252/emmm.202216908 -
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 -
Diagnostics (Basel, Switzerland) Jul 2023Paxillin is a cytoskeletal protein involved in the pathogenesis of several types of cancers. However, the roles of paxillin in epithelial dysplasia (ED), oral squamous...
Evaluation of Paxillin Expression in Epithelial Dysplasia, Oral Squamous Cell Carcinoma, Lichen Planus with and without Dysplasia, and Hyperkeratosis: A Retrospective Cross-Sectional Study.
BACKGROUND
Paxillin is a cytoskeletal protein involved in the pathogenesis of several types of cancers. However, the roles of paxillin in epithelial dysplasia (ED), oral squamous cell carcinoma (OSCC), oral lichen planus with dysplasia (OLPD), hyperkeratosis (HK), and oral lichen planus (OLP) have remained unnoticed in the literature. This study aimed to evaluate its attainable functions in the pathogenesis and malignant transformation of potentially malignant oral epithelium and benign lesions.
METHODS
In this retrospective cross-sectional study, paxillin expression was investigated in 99 tissue samples, including 18 cases of OSCC, 21 ED, 23 OLP, 21 OLPD, and 16 cases of HK. The tissue sections also underwent immunohistochemical paxillin staining using 3,3-diaminobenzidine (DAB) chromogen. The intensity, location, and percentage of staining were examined across all groups. Data were analyzed using the Shapiro-Wilk test, ANOVA, Pearson chi-square, Kruskal-Wallis, and Dunn's post hoc test.
RESULTS
The cytoplasmic percentage and intensity staining of Paxillin expression were evident in the central/suprabasal and basal/peripheral layers of all the obtained samples. The final staining score was significantly higher in OSCC and dysplasia compared to HK and OLP ( = 0.004). It was found that paxillin expression is associated with the grade of dysplastic samples ( < 0.001).
CONCLUSION
The present study provides evidence that paxillin may be involved in the pathogenesis of OSCC and the development and progression of dysplastic tissue, since the paxillin expression was higher than that of HK and OLP.
PubMed: 37568839
DOI: 10.3390/diagnostics13152476 -
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