Cell-matrix Adhesion Proteins Research Articles

Salivary Molecular Spectroscopy with Machine Learning Algorithms for a Diagnostic Triage for Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a genetic disease characterized by poor formation of tooth enamel. AI occurs due to mutations, especially in AMEL, ENAM, KLK4, MMP20, and FAM83H, associated with changes in matrix proteins, matrix proteases, cell-matrix adhesion proteins, and transport proteins of enamel. Due to the wide variety of phenotypes, the diagnosis of AI is complex, requiring a genetic test to characterize it better. Thus, there is a demand for developing low-cost, noninvasive, and accurate platforms for AI diagnostics. This case-control pilot study aimed to test salivary vibrational modes obtained in attenuated total reflection fourier-transformed infrared (ATR-FTIR) together with machine learning algorithms: linear discriminant analysis (LDA), random forest, and support vector machine (SVM) could be used to discriminate AI from control subjects due to changes in salivary components. The best-performing SVM algorithm discriminates AI better than matched-control subjects with a sensitivity of 100%, specificity of 79%, and accuracy of 88%. The five main vibrational modes with higher feature importance in the Shapley Additive Explanations (SHAP) were 1010 cm-1, 1013 cm-1, 1002 cm-1, 1004 cm-1, and 1011 cm-1 in these best-performing SVM algorithms, suggesting these vibrational modes as a pre-validated salivary infrared spectral area as a potential biomarker for AI screening. In summary, ATR-FTIR spectroscopy and machine learning algorithms can be used on saliva samples to discriminate AI and are further explored as a screening tool.

NANOPARTICLE ENCAPSULATION OF THE SPECIALIZED PRO-RESOLVING MEDIATOR MARESIN-2: A NOVEL APPROACH FOR PROMOTING MUCOSAL REPAIR IN THE INTESTINE

Abstract Epithelial cells form protective barriers at mucosal surfaces and play a critical role in maintaining tissue homeostasis. Active inflammation in inflammatory bowel disease (IBD) is associated with epithelial barrier disruption and mucosal erosions/wounds that contribute to the inflammatory response and disease symptoms. Therefore, efficient repair of epithelial erosions/wounds is crucial for restoring intestinal barrier function and resolving mucosal inflammation. In response to injury, epithelial cells proliferate and migrate to cover denuded surfaces and restore critical barrier properties. Resident and recruited immune cells at sites of injury release factors that play an important role in orchestrating mucosal repair. Bioactive lipid mediators released by leukocytes, including leukotrienes and specialized pro-resolving mediators (SPMs), regulate epithelial signaling pathways to influence mucosal repair processes. Liquid chromatography-mass spectrometry (LCMS) analyses of healing colonic mucosal wounds performed to detect SPMs identified high levels of MaR2 in healing colonic wounds. In vitro studies using primary colonic epithelial cells revealed MaR2-driven increases in epithelial wound repair that were potentiated by the pro-inflammatory cytokines TNFα and IFNγ. While MaR2 increased epithelial migration by activating cell matrix adhesion proteins, including Src-paxillin and focal adhesion kinase, we did not observe any influence of MaR2 on the activation of cyclin D1/D2 or on epithelial proliferation. Importantly, in vivo studies demonstrated enhanced colonic mucosal wound repair and recovery from dextran sulfate-induced colitis in mice administered MaR2. Given the thermolabile nature of MaR2 (and associated ultracold storage requirements), thermostable polylactic acid (PLA) nanoparticles (NP) containing MaR2 (PLA MaR2) were generated. PLA MaR2 nanoparticles retained bioactivity following extended storage at room temperature and exhibited potent effects on in vivo colonic biopsy induced wound repair. Taken together these data reveal that thermostable MaR2 containing nanoparticles represent a promising new therapeutic approach for regenerating epithelial barrier function and restoring mucosal homeostasis in individuals with IBD.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell–matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Methimazole, an Effective Neutralizing Agent of the Sulfur Mustard Derivative 2-Chloroethyl Ethyl Sulfide.

Sulfur mustard (SM), designated by the military as HD, is a highly toxic and dangerous vesicant that has been utilized as a chemical warfare agent since World War I. Despite SM's extensive history, an effective antidote does not exist. The effects of SM are predominantly based on its ability to alkylate important biomolecules. Also, with the potential for a fraction of SM to remain unreacted up to days after initial contact, a window of opportunity exists for direct neutralization of unreacted SM over the days following exposure. In this study, we evaluated the structure-activity relationship of multiple nucleophilic molecules to neutralize the toxic effects of 2-chloroethyl ethyl sulfide (CEES), a monofunctional analogue of SM, on human keratinocyte (HaCaT) cells. Cell viability, relative loss of extracellular matrix adhesions, and apoptosis caused by CEES were measured via MTT, cell-matrix adhesion (CMA), and apoptosis protein marker assays, respectively. A set of five two-carbon compounds with various functional groups served as a preliminary group of first-generation neutralizing agents to survey the correlation between mitigation of CEES's toxic effects and functional group nucleophilicity. Apart from thioacids, which produced additive toxicity, we generally observed the trend of increasing protection from cytotoxicity with increasing nucleophilicity. We extended this treatment strategy to second-generation agents which contained advantageous structural features identified from the first-generation molecules. Our results show that methimazole (MIZ), a currently FDA-approved drug used to treat hyperthyroidism, effectively reduced cytotoxicity, increased CMA, and decreased apoptosis resulting from CEES toxicity. MIZ selectively reacts with CEES to produce 2-(2-(ethylthio)ethylthio)-1-methyl-1H-imidazole (EEMI) in media and cell lysate treatments resulting in the reduction of toxicity. Based on these results, future development of MIZ as an SM therapeutic may provide a viable approach to reduce both the immediate and long-term toxicity of SM and may also help mitigate slower developing SM toxicity due to residual intact SM.

Janus kinase inhibitors in autoimmune bullous diseases.

Autoimmune bullous disease (AIBD) is a severe skin disorder caused by autoantibodies that target intercellular or cell-matrix adhesion proteins. Currently, the preferred treatment for AIBD involves the use of glucocorticoids or traditional immunosuppressants. Additionally, the utilization of biological agents such as rituximab, omalizumab, and dupilumab is on the rise. However, effectively managing AIBD remains a challenge. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway has been implicated in various inflammatory diseases. In recent years, a range of drugs known as JAK inhibitors, which target this pathway, have been developed. Several studies have explored the efficacy and safety of JAK inhibitors for treating AIBD. Consequently, this review begins by examining the role of the JAK/STAT pathway in AIBD, summarizing the application of different JAK inhibitors in AIBD treatment, and emphasizing the importance of disease management in treating AIBD with JAK inhibitors. Furthermore, it highlights the need for a better understanding of the JAK/STAT pathway's role in AIBD, as well as the effectiveness and safety of JAK inhibitors for treating this disease.

RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell–cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development

BackgroundSecreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations.Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes.ResultsCultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell–cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient’s keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control.ConclusionsAltogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

Cell-Matrix Interactions Contribute to Barrier Function in Human Colon Organoids.

The importance of cell-matrix adhesion to barrier control in the colon is unclear. The goals of the present study were to: (i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control measurement and (ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes can mitigate the effects of cell-matrix disruption. Human colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions and in the presence of Aquamin®, a multi-mineral product. A function-blocking antibody directed at the C-terminal region of the laminin α chain was used in parallel. The effects of Aquamin® on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. Aquamin® increased the expression of multiple basement membrane, hemidesmosomal and focal adhesion proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin® than they were under control conditions. The blocking antibody reduced TEER values under both conditions but was most effective in the absence of Aquamin®, where expression of cell-matrix adhesion proteins was lower to begin with. These findings provide evidence that cell-matrix interactions contribute to barrier control in the colon.

Substrate rigidity modulates traction forces and stoichiometry of cell-matrix adhesions.

In cell-matrix adhesions, integrin receptors and associated proteins provide a dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. This allows bidirectional transmission of forces between the ECM and the cytoskeleton, which tunes intracellular signaling cascades that control survival, proliferation, differentiation, and motility. The quantitative relationships between recruitment of distinct cell-matrix adhesion proteins and local cellular traction forces are not known. Here, we applied quantitative super-resolution microscopy to cell-matrix adhesions formed on fibronectin-stamped elastomeric pillars and developed an approach to relate the number of talin, vinculin, paxillin, and focal adhesion kinase (FAK) molecules to the local cellular traction force. We find that FAK recruitment does not show an association with traction-force application, whereas a ∼60 pN force increase is associated with the recruitment of one talin, two vinculin, and two paxillin molecules on a substrate with an effective stiffness of 47kPa. On a substrate with a fourfold lower effective stiffness, the stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same ∼60 pN traction force. The relative change in force-related vinculin recruitment indicates a stiffness-dependent switch in vinculin function in cell-matrix adhesions. Our results reveal a substrate-stiffness-dependent modulation of the relationship between cellular traction-force and the molecular stoichiometry of cell-matrix adhesions.

E-cadherin is a robust prognostic biomarker in colorectal cancer and low expression is associated with sensitivity to inhibitors of topoisomerase, aurora, and HSP90 in preclinical models.

Cell–cell and cell–matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial‐to‐mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E‐cadherin (ECAD), integrin β4 (ITGB4), zonula occludens 1 (ZO‐1), and cytokeratins in a single‐hospital series of Norwegian patients with colorectal cancer (CRC) stages I–IV (n = 922) using multiplex fluorescence‐based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO‐1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E‐cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.

Collagen XVII inhibits breast cancer cell proliferation and growth through deactivation of the AKT/mTOR signaling pathway.

Collagen XVII (COL17), a cell-matrix adhesion protein, has been found to be suppressed in breast cancer. Our previous data demonstrated a preventive role of COL17 in breast cancer invasiveness. The present study used the stable COL17-overexpressing MCF7 and MDA-MB-231 cells to reveal an anti-proliferative effect of COL17 on breast cancer cell through mTOR deactivation. Cell proliferation was negatively correlated with the expression level of COL17 in a concentration-dependent manner in both conventional and three-dimensional (3D) culture systems. The correlation was confirmed by decreased expression of the proliferative marker Ki67 in COL17-expressing cells. In addition, overexpression of COL17 reduced the clonogenicity and growth of the cells. We demonstrated that COL17 affects the AKT/mTOR signaling pathway by deactivation of AKT, mTOR and downstream effectors, particularly 4EBP1. Moreover, mice xenografted with high COL17-expressing cells exhibited delayed tumor progression and prolonged survival time. The high expression of COL17A1 gene encoding COL17 is associated with low-proliferation tumors, extended tumor-free period, and overall survival of breast cancer patients. In conclusion, our results revealed the novel function of COL17 using in vitro and in vivo models and elucidated the related pathway in breast cancer cell growth and proliferation.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Ras suppressor-1 (RSU-1) promotes cell invasion in aggressive glioma cells and inhibits it in non-aggressive cells through STAT6 phospho-regulation

Most gliomas are invasive tumors formed from glial cells and associated with high mortality rates. In this study, we characterized four glioma cell lines of varying degree of aggressiveness (H4, SW1088, A172 and U87-MG) in terms of morphology, cytoskeleton organization and stiffness, and evaluated their invasive potential by performing invasion, colony forming and spheroid invasion assays. Cells were divided into two distinct groups: aggressive cell lines (A172 and U87-MG) with more elongated, softer and highly invasive cells and less aggressive cells (H4 and SW088). Interestingly, we found that Ras Suppressor-1 (RSU-1), a cell-matrix adhesion protein involved in cancer cell invasion, was significantly upregulated in more aggressive glioma cells compared to less aggressive. Importantly, RSU-1 silencing had opposing effects on glioma cell invasion depending on their aggressiveness, inhibiting migration and invasion of aggressive cells and promoting those of less aggressive cells. Finally, we found that RSU-1 silencing in aggressive cells led to decreased Signal Transducer and Activator of Transcription6 (STAT6) phosphorylation and Matrix Metalloproteinase13 (MMP13) expression in contrast to less invasive cells. Our study demonstrates that RSU-1 promotes invasion of aggressive glioma cells and inhibits it in the non-aggressive cells, indicating that it could serve as a predictor of gliomas progression.

The effect of physiological stretch and the valvular endothelium on mitral valve proteomes.

This work is important to the field of heart valve pathophysiology as it provides new insights into molecular markers of mechanically induced valvular degeneration as well as the protective role of the valvular endothelium. These discoveries reported here advance our current knowledge of the valvular endothelium and how its removal essentially takes valve leaflets into an environmental shock. In addition, it shows that static conditions represent a mild pathological state for valve leaflets, while 10% cyclic stretch provides valvular cell quiescence. These findings impact the field by informing disease stages and by providing potential new drug targets to reverse or slow down valvular change before it affects cardiac function.

Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs.

Extracellular vesicles (EVs) are important mediators of cell-cell communication due to their cargo content of proteins, lipids, and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics analyses revealed that SEVs are enriched in proteins associated with cell-cell junctions, cell-matrix adhesion, exosome biogenesis machinery, and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis, processing, and metabolism. Western blot analysis of EVs purified from two different cancer cell types confirmed the enrichment of cell-matrix and cell-cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.

Cerebrovascular Injury After Serial Exposure to Chronic Stress and Abstinence from Methamphetamine Self-Administration

Cerebrovascular damage caused by either exposure to stress or the widely abused drug, methamphetamine (Meth) is known but stress and drug abuse frequently occur in tandem that may impact their individual cerebrovascular effects. This study examined their co-morbid cerebrovascular effects during abstinence from self-administered Meth after the exposure to chronic unpredictable stress (CUS). Exposure to CUS prior to unrestricted Meth self-administration had no effect on Meth intake in rats; however, the pro-inflammatory mediator cyclooxygenase-2 (COX-2) and the breakdown of cell-matrix adhesion protein β-dystroglycan in isolated cerebral cortical capillaries were increased after 3 days of abstinence and persisted for 7 days. These changes preceded decreases in occludin, a key structural protein component of the blood-brain barrier. The decrease in occludin was blocked by the COX-2 specific inhibitor nimesulide treatment during abstinence from Meth. The changes in COX-2, β-dystroglycan, and occludin were only evident following the serial exposure to stress and Meth but not after either one alone. These results suggest that stress and voluntary Meth intake can synergize and disrupt cerebrovasculature in a time-dependent manner during abstinence from chronic stress and Meth. Furthermore, COX-2 inhibition may be a viable pharmacological intervention to block vascular changes after Meth exposure.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

SummaryWe show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Effects of oxymatrine on the adhesion of human hepatoma cell line QGY-7703 and the expression of integrin β1

Background: To explore the effect of Oxymatrine (OM) on cell-matrix adhesion of human hepatoma cell line QGY-7703 and the expression of integrin β1. Methods: The effect of OM on cell-matrix adhesion of human hepatoma cell line QGY-7703 was determined with cell adhesion assay and the effect on the mRNA and protein expression of integrin β1 in QGY-7703 cells was detected by quantitative PCR and Western blot. Results: Compared with the control group, QGY-7703 cell-matrix adhesion in the group treated with 1 mg.mL-1 OM was not inhibited, while the groups treated with 2.5 mg.mL-1 and 5 mg.mL-1 OM treated cells (P<0.05) was significantly inhibited (p<0.05), accompanied by the decreased mRNA and protein expression of integrin β1 (P <0.05). Conclusion: In vitro OM can inhibit the cell-matrix adhesion and mRNA and protein expression of integrin β1 of human hepatoma cell line QGY-7703.

Age-Related Changes in the Interaction of Acute Myeloid Leukemia with the Bone Marrow Microenvironment Correlate with Response to Treatment and Survival: A Role for L-Selectin

Introduction: Acute myeloid leukemia (AML) arises in the bone marrow microenvironment (BME), where cell-cell and cell-matrix adhesion promote AML survival. There is increased incidence of AML in older adults and age-related changes in the BME such as the senescence associated secretory phenotype (SASP) may promote myeloid disorders and cancer. Our objective is to further elucidate the influence of the BME on AML pathobiology.Methods: Using flow cytometry, we prospectively characterized 17 cell adhesion receptors expressed on AML blasts derived from 106 patient blood and bone marrow samples on an IRB approved protocol. For 55 patients, we correlated expression of these individual adhesion receptors with age, cytogenetics, complete remission (CR), relapse free survival (RFS) and overall survival (OS). We correlated expression of adhesion receptors with basal levels of DNA damage in AML blasts, as measured by phosphorylation of histone H2AX (γ-H2AX) by flow cytometry. mRNA microarray gene expression profiling (GEP) on 30 patients, we examined for age-associated differences in the gene expression of cell-cell and cell-matrix adhesion proteins. For 6 patients, we evaluated for changes in GEP that occur in AML blasts with adhesion to plates coated with alternative spliced fibronectin peptide CH296, RetronectinTM (RN) vs. bovine serum albumin (BSA) control.Results: We found a significant positive correlation between L-selectin cell surface expressed protein levels with advancing age (Spearman r = 0.35, p = 0.009) (Figure 1). The response to treatment (CR/CRi) rate, but not RFS, increased with greater surface expression of the following adhesion proteins: CD11a (αL), CD29 (β1), CD44, CD49a (α1) and CD49e (α5). OS improved with greater cell surface expressed protein levels of CD11a (αL) and CD29 (β1).There was a significant negative correlation in L-selectin mRNA levels with basal DNA damage in AML blasts, as measured by γ-H2AX (Spearman r = -0.6, p = 0.005). To further investigate the relationship between L-selection expression and DNA damage in AML, we cultured AML blasts on an L-selectin ligand, P-selectin glycoprotein-1 (PSGL-1) vs. BSA control. We observed increased blast survival and reduced apoptosis when adherent on PSGL-1 compared to BSA after treatment with cytarabine arabinoside (Ara-C) in 4/15 (27%) of samples tested (Figure 2).Ingenuity Pathway Analysis (IPA) results on the gene expression data showed age-associated changes in the expression of L-selectin and cell-matrix proteins involved in the SASP, such as tissue inhibitor of metalloproteinases (TIMP). Adhesion of AML blasts to RN vs. BSA control resulted in up-regulation of genes involved in protein ubiquitination and the PI3K/AKT, nuclear factor-kappa beta (NF-kβ) and IL-8 signaling pathways.Conclusions: Age-related changes in the expression of L-selectin, cell-cell and cell-matrix proteins in AML blasts appear to influence DNA damage signaling/response (DDR), promote Ara-C resistance, and influence patient response to treatment and survival. Adhesion of AML blasts to the BME results in up-regulation of cell survival pathways that confer resistance to apoptosis and maintenance of the SASP. Our results support disruption of L-selectin and other BME-mediated interactions as potential targets to enhance cytotoxicity to chemotherapy and improve age-specific outcomes in AML. [Display omitted] [Display omitted] DisclosuresBecker:Invivoscribe: Honoraria; CVS Caremark: Other: Accordant Health Services Medical Advisory Board; Millennium: Research Funding; Amgen: Research Funding; Abbvie: Research Funding; Glycomimetics: Research Funding; Pfizer: Other: Scientific Steering Committee for a post marketing study; JW Pharmaceutical: Research Funding.

Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis

Migfilin is a novel cell–matrix adhesion protein known to interact with Vasodilator Stimulated Phosphoprotein (VASP) and be localized both at cell–matrix and cell–cell adhesions. To date there is nothing known about its role in hepatocellular carcinoma (HCC). As matrix is important in metastasis, we aimed to investigate the Migfilin׳s role in HCC metastasis using two human HCC cell lines that differ in their metastatic potential; non-invasive Alexander cells and the highly invasive HepG2 cells. We silenced Migfilin by siRNA and studied its effect on signaling and metastasis-related cellular properties. We show that Migfilin׳s expression is elevated in HepG2 cells and its silencing leads to upregulation of actin reorganization-related proteins, namely phosphor-VASP (Ser157 and Ser239), Fascin-1 and Rho-kinase-1, promoting actin polymerization and inhibiting cell invasion. Phosphor-Akt (Ser473) is decreased contributing to the upregulation of free and phosphor-β-catenin (Ser33/37Thr41) and inducing proliferation. Migfilin elimination upregulates Extracellular Signal–regulated kinase, which increases cell adhesion in HepG2 and reduces invasiveness. This is the first study to reveal that Migfilin inhibition can halt HCC metastasis in vitro, providing the molecular mechanism involved and presenting Migfilin as potential therapeutic target against HCC metastasis.

RhoA activation by CNFy restores cell–cell adhesion in kindlin‐2‐deficient keratinocytes

Kindlins are a family of integrin adapter and cell-matrix adhesion proteins causally linked to human genetic disorders. Kindlin-2 is a ubiquitously expressed protein with manifold functions and interactions. The contribution of kindlin-2 to integrin-based cell-matrix adhesions has been extensively explored, while other integrin-independent roles emerge. Because of the early involvement of kindlin-2 in development, no viable animal models with its constitutional knockout are available to study its physiological functions in adult skin. Here, we uncovered a critical physiological role of kindlin-2 in the epidermis by using a skin-equivalent model with shRNA-mediated knock-down of kindlin-2 in keratinocytes. Kindlin-2-deficient keratinocytes built stratified epidermal layers, but displayed impaired dermal-epidermal and intra-epidermal adhesion and barrier function. Co-immunoprecipitation studies demonstrated that kindlin-2 interacts with both integrin- and cadherin-based adhesions. In kindlin-2-deficient keratinocytes, reduced cell-cell adhesion was associated with abnormal cytoplasmic distribution of adherens junctions and desmosomal proteins, which was dependent on RhoA activation. Direct activation of RhoA with recombinant bacterial cytotoxic necrotizing factor y (CNFy) reverted the abnormal phenotype and barrier function of kindlin-2-deficient keratinocytes and skin equivalents. These findings have physiological and pathological significance, since kindlin-2 expression modulates the phenotype in Kindler syndrome, a skin fragility disorder caused by kindlin-1 deficiency. Our results suggest that pharmacological regulation of RhoGTPase activity may represent a therapeutic option for skin fragility.