Integrin Signaling Pathways Research Articles

PCGF1 is a prognostic biomarker and correlates with tumor immunity in gliomas.

BackgroundPolycomb group factor 1 (PCGF1) plays a vital role in the self-renewal of cancer stem cells (CSCs). However, the prognostic value and potential function of PCGF1 in glioma progression, especially in tumor immunity, remain unclear.MethodsThis study investigated PCGF1 expression in pan-cancers and glioma using The Cancer Genome Atlas Project data, and conducted a logistic regression analysis to analyze the association between PCGF1 expression and the clinicopathological features of glioma patients. Kaplan-Meier and Cox regression analyses were used to assess the prognostic roles of PCGF1. The functional analysis using gene ontology and Kyoto Encyclopedia of Genes and Genomes databases and a gene set enrichment analysis (GSEA) were conducted to examine the PCGF1-related biological processes and signaling pathways. Finally, the roles of PCGF1 in immune infiltration were analyzed by a single sample GSEA (ssGSEA).ResultsPCGF1 expression was upregulated in glioma tissues. The high expression of PCGF1 was significantly associated with an age >60 years (P<0.001), an increased World Health Organization grade (P<0.001), and wildtype isocitrate dehydrogenase (IDH) status (P<0.001), and predicted poor overall survival (OS) [hazards ratio (HR) =2.90, 95% confidence interval (CI): 2.25–3.74; P<0.001], the progression-free interval (PFI) (HR =1.99, 95% CI: 1.60–2.46; P<0.001), and disease specific survival (DSS) (HR =2.84, 95% CI: 2.18–3.71; P<0.001). The multivariate regression analysis confirmed that PCGF1 expression was an independent prognostic factor of OS (HR =1.581, 95% CI: 1.161–2.153; P=0.004). Based on the functional enrichment analysis, we identified the PCGF1-related differentially expressed genes (DEGs), and found that PCGF1 was involved in regulating many oncogenic signaling pathways, such as the phosphatidylinositol 3-kinase and protein kinase B pathway, the Janus kinase/signal transducers and activators of transcription (JKT-STAT) signaling pathway, notch signaling and integrin signaling pathway. In addition, we found that PCGF1 expression was positively associated with the abundance of Th2, but negatively associated with T follicular helper, T central memory, and T gamma delta cells. Additionally, PCGF1 participated in the regulation of numerous immune-related processes in glioma.ConclusionsPCGF1 is a promising prognostic biomarker, and as it governs cancer-related pathways and tumor immunity, it plays an important role in glioma development.

Mechanism of Carthami Flos and Lepidii Semen drug pair in inhibition of myocardial fibrosis by improving cardiac microenvironment based on network pharmacology and animal experiment

Previously, Carthami Flos and Lepidii Semen(CF-LS) drug pair has been proved effective in inhibiting myocardial fibrosis(MF) by blunting the activity of cardiac fibroblasts. The present study explored the underlying mechanism of CF-LS in inhibiting MF by improving the cardiac microenvironment based on network pharmacology combined with experimental verification. Active compounds and potential targets of CF-LS were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the potential targets of MF were obtained from GeneCards, Online Mendelian Inheritance in Man(OMIM), and Pharmacogenetics and Pharmacogenomics Knowledge Base(PharmGKB). The &quot;active component-target-MF&quot; network was constructed and analyzed by Cytoscape 3.8.1. The protein-protein interaction(PPI) network was constructed by STRING. The Gene Ontology(GO) biological process enrichment analysis was performed by CluoGO plug-in. Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analysis was performed by R 4.0.2 and Funrich. Subsequently, the inhibitory effect of CF-LS on MF was investigated based on angiotensin Ⅱ(Ang Ⅱ)-induced MF rats. RT-PCR and ELISA were used to verify the effect of CF-LS on the targets of signaling pathways related to vascular endothelial cells predicted by the network pharmacology. Thirty-one active components and 204 potential targets of CF-LS, 4 671 MF-related targets, and 174 common targets were obtained. The network analysis showed that the key targets of CF-LS against MF included RAC-alpha serine/threonine-protein kinase(AKT1), transcription factor AP-1(JUN), mitogen-activated protein kinase 1(MAPK1), cellular tumor antigen p53(TP53), transcription factor p65(RELA), and mitogen-activated protein kinase 8(MAPK8). Biological processes mainly involved regulation of blood vessel diameter, regulation of blood vessel endothelial cell migration, cell death in response to oxidative stress, etc. Advanced glycation end products(AGE)-receptor for advanced glycation end products(RAGE) signaling pathway, phosphoinositide 3-kinase(PI3 K)-serine/threonine protein kinase(AKT) signaling pathway, hypoxia-inducible factor-1(HIF-1) signaling pathway, integrin signaling pathway, transforming growth factor-β(TGF-β) signaling pathway, etc. were involved in signaling pathway enrichment. Literature retrieval confirmed that some of these signaling pathways were closely related to vascular endothelial cells, including AGE-RAGE, PI3 K-AKT, HIF-1α, p53, the transcription factor activator protein-1(AP-1), integrin, p38 MAPK, and TGF-β. Animal experiments showed that CF-LS inhibited MF induced by Ang Ⅱ in rats by suppressing the expression of RAGE, HIF-1α, integrin β6, and TGF-β1. The inhibitory effect of CF-LS on MF has the characteristics of multiple components, multiple targets, and multiple pathways. CF-LS can inhibit MF by regulating the activity of vascular endothelial cells in the cardiac microenvironment.

Comparative Proteomics of the Acanthopagrus schlegelii Gonad in Different Sex Reversal.

A substantial proportion of Acanthopagrus schlegelii individuals change sex from male to female during their lifetime. However, the mechanisms underlying sex change are unknown. In this research, iTRAQ analyses of proteins obtained from A. schlegelii gonads in four different stages of development were compared. In total, 4692 proteins were identified, including common sex-specific proteins, such as sperm-associated antigen 6 and cilia- and flagella-associated proteins in males, and zona pellucida sperm-binding proteins in females. Furthermore, proteins involved in the integrin signaling pathway, inflammation mediated by the chemokine and cytokine signaling pathways, pyruvate metabolism, CCKR signaling map, de novo purine biosynthesis and the ubiquitin proteasome pathway were upregulated in female gonads, whereas proteins implicated in DNA replication, the heterotrimeric G-protein signaling pathway, Gi alpha- and Gs alpha-mediated pathways, wnt signaling pathway, and hedgehog signaling pathway were upregulated in male gonads. Interestingly, cathepsins were only identified in ovaries, indicating their potential involvement in rapid ovarian development. Apoptosis-related proteins expressed in ovaries (such as MAPK and Cdc42) may protect them from cancer. This is the first report on the gonad proteome from A. schlegelii in different stages of sex reversal, and the results provide important fundamental data for studying the molecular mechanisms of sex reversal.

Dietary zinc restriction affects the expression of genes related to immunity and stress response in the small intestine of pigs.

Zinc (Zn) is an essential mineral and its deficiency manifests in non-specific clinical signs that require long time to develop. The response of swine intestine to Zn restriction was evaluated to identify early changes that can be indicative of Zn deficiency. Twenty-seven pigs (body weight = 77⋅5 ± 2⋅5 kg) were assigned to one of three diets: diet without added Zn (Zn-restricted diet, ZnR), and ZnR-supplemented with either 50 (Zn50) or 100 mg of Zn/kg of diet (Zn100) of Zn supplied by ZnCl2. After 32 d consuming the diets, serum Zn concentration in ZnR pigs was below the range of 0⋅59-1⋅37 μg/ml considered sufficient, thereby confirming subclinical Zn deficiency. Pigs showed no obvious health or growth changes. RNA-seq analysis followed by qPCR showed decreased expression of metallothionein-1 (MT1) (P < 0⋅05) and increased expression of Zn transporter ZIP4 (P < 0⋅05) in jejunum and ileum of ZnR pigs compared with Zn-supplemented pigs. Ingenuity pathway analysis revealed that Zn50 and Zn100 induced changes in genes related to nucleotide excision repair and integrin signalling pathways. The top gene network in the ZnR group compared with Zn100 was related to lipid and drug metabolism; and compared with Zn50, was related to cellular proliferation, assembly and organisation. Dietary Zn concentrations resulted in differences in genes related to immune pathways. Our analysis showed that small intestine presents changes associated with Zn deficiency after 32 d of Zn restriction, suggesting that the intestine could be a sentinel organ for Zn deficiency.

Membrane-bound IL-6R is upregulated on Th17 cells and inhibits Treg cell migration by regulating post-translational modification of VASP in autoimmune arthritis

Autoimmune arthritis is characterized by impaired regulatory T (Treg) cell migration into inflamed joint tissue and by dysregulation of the balance between Treg cells and Th17 cells. Interleukin-6 (IL-6) is known to contribute to this dysregulation, but the molecular mechanisms behind impaired Treg cell migration remain largely unknown. In this study, we assessed dynamic changes in membrane-bound IL-6 receptor (IL6R) expression levels on Th17 cells by flow cytometry during the development of collagen-induced arthritis (CIA). In a next step, bioinformatics analysis based on proteomics was performed to evaluate potential pathways affected by altered IL-6R signaling in autoimmune arthritis. Our analysis shows that membrane-bound IL-6R is upregulated on Th17 cells and is inversely correlated with IL-6 serum levels in experimental autoimmune arthritis. Moreover, IL-6R expression is significantly increased on Th17 cells from untreated patients with rheumatoid arthritis (RA). Interestingly, CD4+ T cells from CIA mice and RA patients show reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Bioinformatics analysis based on proteomics of CD4+ T cells with low or high phosphorylation levels of VASP revealed that integrin signaling and related pathways are significantly enriched in cells with low phosphorylation of VASP. Specific inhibition of p-VASP reduces the migratory function of Treg cells but has no influence on effector CD4+ T cells. Importantly, IL-6R blockade restores the phosphorylation level of VASP, thereby improving the migratory function of Treg cells from RA patients. Thus, our results establish a link between IL6R signaling and phosphorylation of VASP, which controls Treg cell migration in autoimmune arthritis.

Coordinated regulation of WNT/\u03b2-catenin, c-Met, and integrin signalling pathways by miR-193b controls triple negative breast cancer metastatic traits

BackgroundTriple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Treatment options for TNBC patients are limited and further insights into disease aetiology are needed to develop better therapeutic approaches. microRNAs’ ability to regulate multiple targets could hold a promising discovery approach to pathways relevant for TNBC aggressiveness. Thus, we address the role of miRNAs in controlling three signalling pathways relevant to the biology of TNBC, and their downstream phenotypes.MethodsTo identify miRNAs regulating WNT/β-catenin, c-Met, and integrin signalling pathways, we performed a high-throughput targeted proteomic approach, investigating the effect of 800 miRNAs on the expression of 62 proteins in the MDA-MB-231 TNBC cell line. We then developed a novel network analysis, Pathway Coregulatory (PC) score, to detect miRNAs regulating these three pathways. Using in vitro assays for cell growth, migration, apoptosis, and stem-cell content, we validated the function of candidate miRNAs. Bioinformatic analyses using BC patients’ datasets were employed to assess expression of miRNAs as well as their pathological relevance in TNBC patients.ResultsWe identified six candidate miRNAs coordinately regulating the three signalling pathways. Quantifying cell growth of three TNBC cell lines upon miRNA gain-of-function experiments, we characterised miR-193b as a strong and consistent repressor of proliferation. Importantly, the effects of miR-193b were stronger than chemical inhibition of the individual pathways. We further demonstrated that miR-193b induced apoptosis, repressed migration, and regulated stem-cell markers in MDA-MB-231 cells. Furthermore, miR-193b expression was the lowest in patients classified as TNBC or Basal compared to other subtypes. Gene Set Enrichment Analysis showed that miR-193b expression was significantly associated with reduced activity of WNT/β-catenin and c-Met signalling pathways in TNBC patients.ConclusionsIntegrating miRNA-mediated effects and protein functions on networks, we show that miRNAs predominantly act in a coordinated fashion to activate or repress connected signalling pathways responsible for metastatic traits in TNBC. We further demonstrate that our top candidate, miR-193b, regulates these phenotypes to an extent stronger than individual pathway inhibition, thus emphasizing that its effect on TNBC aggressiveness is mediated by the coordinated repression of these functionally interconnected pathways.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 β1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

PINCH1 knockout aggravates myocardial infarction in mice via mediating the NF‑κB signaling pathway

Myocardial infarction (MI), the leading cause of death among patients with cardiovascular diseases, is characterized by acute cardiac muscle injury due to severe impairment of the coronary blood supply, which may lead to cardiogenic shock and cardiac arrest. Particularly interesting new cysteine histidine rich 1 (PINCH1) protein, a key component of the integrin signaling pathway, interacts with several proteins and serves a vital role in numerous cellular processes, including cytoskeleton remodeling, cell proliferation and cell migration. To investigate the role of PINCH1 in heart injury in the present study, PINCH1 was knocked out in the myocardial tissue of mice (age, 18 weeks) to induce MI. In addition, cell viability, migration and apoptosis, as well as the expression levels of NF-κB-associated proteins were determined in murine HL1 cardiomyocytes with a conditional PINCH1 shRNA using Cell Counting Kit-8, Transwell, flow cytometry and western blot assays, respectively. Furthermore, the cardiac expansion and myocardial fibrosis in PINCH1 knockout mice was investigated in vivo by performing morphological and histological examinations. Additionally, the murine ventricular myocardial ultrastructure was evaluated using an electron microscope, and the cardiomyocyte apoptotic rate and expression levels of NF-κB-related proteins were determined using TUNEL and western blot assays, respectively. The results showed that the apoptotic rate in the in vivo PINCH1 knockdown group was significantly increased. In addition, the protein expression levels of NF-κB signaling pathway-related proteins, including NF-κB, myeloid differentiation factor 88, TNF-α and caspase-3, were significantly increased in the in vivo PINCH1 knockdown group compared with the wild-type group, but the protein expression of MMP2 and MMP9 were the opposite. Overall, the in vitro and in vivo results revealed that PINCH1 knockout in mice significantly aggravated MI via the NF-κB signaling pathway.

Abstract 10717: Role of Integrin Signaling in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by progressive obstruction and decreased compliance of pulmonary arteries (PA), leading to right ventricular (RV) failure and premature death. It is now recognized that, like cancer cells, PA smooth muscle cells (PASMCs) exhibit exaggerated proliferation and resistance to apoptosis in response to increased PA stiffness caused by extracellular matrix (ECM) remodeling. Integrins (members of the cell adhesion receptors superfamily) are known to promote cell proliferation, survival, hypertrophic growth and fibrosis, which are key elements in the progression of PAH. Therefore, we explored integrins expression and its effects on PA remodeling and RV failure. By RNA sequencing, we demonstrated a significant upregulation of integrin signaling pathways. Among all integrins, the fibronectin binding integrin (FiBI) family was the most abundant in PASMCs and RV fibroblasts (RVFbs) isolated from PAH patients (n=5). By western blot (WB) we showed that integrin expression is significantly changed in distal PAs, PASMCs and RV from PAH patients (n=6-8) compared to controls (n=5-8). Similarly, increased expression was found in monocrotaline (MCT) and pulmonary artery banding (PAB) rats (n=20, p&lt;0.05.) In vitro , pharmacological inhibition of FiBI decreases PAH-PASMCs proliferation (WB: PCNA and Ki67 labeling; p&lt;0.05) and resistance to apoptosis (WB: Survivin and Annexin V labeling; p&lt;0.05). These effects were associated with a decreased activation of the AKT/mTOR pathway. In cardiomyocytes and human RVFbs, FiBI inhibition decreased phenylephrine-induced hypertrophy (Phalloidin labeling; p&lt;0.001) and TGFβ1-induced RVFbs activation (WB: αSMA, CTGF, FN and Col1; p&lt;0.05) and proliferation (Ki67; p&lt;0.05). In vivo , pharmacological FiBI inhibition improved PAs remodeling (EVG staining) and RV function (SV, CO, TAPSE, S-Wave, as assessed by echocardiography and right heart catheterization) in MCT rats with established PAH (n=20; p&lt;0.05). We demonstrated that upregulation of fibronectin binding integrins and broad spectrum integrin inhibition resulted in improvements in distal PAs remodeling and RV failure. These results suggest that integrins play an important role in the pathophysiology of PAH.

Recent Advances in the Role of Discoidin Domain Receptor Tyrosine Kinase 1 and Discoidin Domain Receptor Tyrosine Kinase 2 in Breast and Ovarian Cancer.

Discoidin domain receptor tyrosine kinases (DDRs) are a class of receptor tyrosine kinases (RTKs), and their dysregulation is associated with multiple diseases (including cancer, chronic inflammatory conditions, and fibrosis). The DDR family members (DDR1a-e and DDR2) are widely expressed, with predominant expression of DDR1 in epithelial cells and DDR2 in mesenchymal cells. Structurally, DDRs consist of three regions (an extracellular ligand binding domain, a transmembrane domain, and an intracellular region containing a kinase domain), with their kinase activity induced by receptor-specific ligand binding. Collagen binding to DDRs stimulates DDR phosphorylation activating kinase activity, signaling to MAPK, integrin, TGF-β, insulin receptor, and Notch signaling pathways. Abnormal DDR expression is detected in a range of solid tumors (including breast, ovarian, cervical liver, gastric, colorectal, lung, and brain). During tumorigenesis, abnormal activation of DDRs leads to invasion and metastasis, via dysregulation of cell adhesion, migration, proliferation, secretion of cytokines, and extracellular matrix remodeling. Differential expression or mutation of DDRs correlates with pathological classification, clinical characteristics, treatment response, and prognosis. Here, we discuss the discovery, structural characteristics, organizational distribution, and DDR-dependent signaling. Importantly, we highlight the key role of DDRs in the development and progression of breast and ovarian cancer.

Multi-omics analysis to reveal disorders of cell metabolism and integrin signaling pathways induced by PM2.5

Atmospheric fine particle pollution is known to cause many adverse health effects. However, the potential mechanisms of PM2.5-induced cytotoxicity still needs further understanding. Herein, we integrated cytotoxicity, component profiling, metabolomics and proteomics data to deeply explain the biological responses of human bronchial epithelial cells exposed to PM2.5. We observed that PM2.5 caused cell cycle arrest, calcium influx, cell damage and further induced cell apoptosis. The contents of heavy metals and 4–6 rings PAHs in PM2.5 were positively correlated with intracellular ROS, indicating that they might be the important components to induce the above cytotoxicity. Integrated metabolomics and proteomics analysis revealed the significant alterations of many metabolic processes, such as glycolysis, the citric acid cycle, amino acid metabolism and lipid metabolism. Notably, we found that PM2.5 inhibited the integrin signaling pathway, including down-regulating the protein expression of integrins and the phosphorylation of downstream signaling kinases, which might ultimately affect cell cycle progression, cell metabolism and apoptosis. This study provided a comprehensive data resource for the deep understanding of biological toxicity mechanisms caused by atmospheric fine particles in human lung-bronchial epithelium cells.

Physiological Functions of Thiol Peroxidases (Gpx1 and Prdx2) during Xenopus laevis Embryonic Development.

Glutathione peroxidase 1 (Gpx1) and peroxiredoxin 2 (Prdx2) belong to the thiol peroxidase family of antioxidants, and have been studied for their antioxidant functions and roles in cancers. However, the physiological significance of Gpx1 and Prdx2 during vertebrate embryogenesis are lacking. Currently, we investigated the functional roles of Gpx1 and Prdx2 during vertebrate embryogenesis using Xenopus laevis as a vertebrate model. Our investigations revealed the zygotic nature of gpx1 having its localization in the eye region of developing embryos, whereas prdx2 exhibited a maternal nature and were localized in embryonic ventral blood islands. Furthermore, the gpx1-morphants exhibited malformed eyes with incompletely detached lenses. However, the depletion of prdx2 has not established its involvement with embryogenesis. A molecular analysis of gpx1-depleted embryos revealed the perturbed expression of a cryba1-lens-specific marker and also exhibited reactive oxygen species (ROS) accumulation in the eye regions of gpx1-morphants. Additionally, transcriptomics analysis of gpx1-knockout embryos demonstrated the involvement of Wnt, cadherin, and integrin signaling pathways in the development of malformed eyes. Conclusively, our findings indicate the association of gpx1 with a complex network of embryonic developmental pathways and ROS responses, but detailed investigation is a prerequisite in order to pinpoint the mechanistic details of these interactions.

Loss of proximal tubular transcription factor Krüppel-like factor 15 exacerbates kidney injury through loss of fatty acid oxidation

Loss of fatty acid β-oxidation (FAO) in the proximal tubule is a critical mediator of acute kidney injury and eventual fibrosis. However, transcriptional mediators of FAO in proximal tubule injury remain understudied. Krüppel-like factor 15 (KLF15), a highly enriched zinc-finger transcription factor in the proximal tubule, was significantly reduced in proximal tubule cells after aristolochic acid I (AAI) treatment, a proximal tubule-specific injury model. Proximal tubule specific knockout of Klf15 exacerbated proximal tubule injury and kidney function decline compared to control mice during the active phase of AAI treatment, and after ischemia-reperfusion injury. Furthermore, along with worsening proximal tubule injury and kidney function decline, knockout mice exhibited increased kidney fibrosis as compared to control mice during the remodeling phase after AAI treatment. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in immune system and integrin signaling pathways and decreased transcripts encompassing metabolic pathways, specifically FAO, and PPARα signaling, in knockout versus control mice after AAI treatment. In silico and experimental chromatin immunoprecipitation studies collectively demonstrated that KLF15 occupied the promoter region of key FAO genes, CPT1A and ACAA2, in close proximity to transcription factor PPARα binding sites. While the loss of Klf15 reduced the expression of Cpt1a and Acaa2 and led to compromised FAO, induction of KLF15 partially rescued loss of FAO in AAI-treated cells. Klf15, Ppara, Cpt1a, and Acaa2 expression was also decreased in other mouse kidney injury models. Tubulointerstitial KLF15 independently correlated with eGFR, PPARA and CPT1A appearance in expression arrays from human kidney biopsies. Thus, proximal tubule-specific loss of Klf15 exacerbates acute kidney injury and fibrosis, likely due to loss of interaction with PPARα leading to loss of FAO gene transcription.

Effects of thyroxine on apoptosis and proliferation of mammary tumors

Hypothyroidism is a protective factor against breast cancer but long-term exposure or overdoses of thyroid replacement therapy with thyroxine (T4) may increase breast cancer risk. Objective: to study, in vivo and in vitro, the effects of T4 on the proliferation and apoptosis of mammary tumors of hypo- and euthyroid rats, and the possible mechanisms involved in these effects. Material and Methods: Female Sprague-Dawley rats were treated with a single dose of dimethylbenzathracene (15 mg/rat) at 55 days of age and were divided into three groups: hypothyroidism (HypoT; 0.01% 6-N-propyl-2-thiouracil -PTU- in drinking water, n = 20), hypothyroidism treated with T4 (HypoT + T4; 0.01% PTU in drinking water and 0.25 mg/kg/day T4 via sc; n = 20) and EUT (untreated control, n = 20). At sacrifice, tumor explants from HypoT and EUT rats were obtained and treated either with 10−10 M T4 in DMEM/F12 without phenol red with 1% Charcoalized Fetal Bovine Serum or DMEM/F12 only for 15 min to evaluate intracellular signaling pathways associated with T4, and 24 h to evaluate changes in the expression of hormone receptors and proteins related to apoptosis and proliferation by immunohistochemistry and Western Blot. Results: In vivo, hypothyroidism retards mammary carcinogenesis but its treatment with T4 reverted the protective effects. In vitro, the proliferative and anti-apoptosis mechanisms of T4 were different regarding the thyroid status. In EUT tumors, the main signaling pathway involved was the cross-talk with other receptors, such as ERα, PgR, and HER2. In HypoT tumors, the non-genomic signaling pathway of T4 was the chief mechanism involved since αvβ3 integrin, HER2, β-catenin and, downstream, PI3K/AKT and ERK signaling pathways were activated. Conclusion: T4 can regulate mammary carcinogenesis by mainly activating its non-genomic signaling pathway and by interacting with other hormone or growth factor pathways endorsing that overdoses of thyroid replacement therapy with T4 can increase the risk of breast cancer.

Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-Inflammatory Cytokines.

BackgroundMicroglia safeguard the CNS against injuries and pathogens, and in the presence of certain harmful stimuli are capable of inducing a disease-dependent inflammatory response. When exposed to anti-inflammatory cytokines, however, these cells possess the ability to switch from an inflammatory to an immunosuppressive phenotype. Cancer cells exploit this property to evade the immune system, and elicit an anti-inflammatory microenvironment that facilitates tumor attachment and growth.ObjectiveThe tumor-supportive biological processes that are activated in microglia cells in response to anti-inflammatory cytokines released from cancer cells were explored with mass spectrometry and proteomic technologies.MethodsSerum-depleted and non-depleted human microglia cells (HMC3) were treated with a cocktail of IL-4, IL-13, IL-10, TGFβ, and CCL2. The cellular protein extracts were analyzed by LC-MS/MS. Using functional annotation clustering tools, statistically significant proteins that displayed a change in abundance between cytokine-treated and non-treated cells were mapped to their biological networks and pathways.ResultsThe proteomic analysis of HMC3 cells enabled the identification of ~10,000 proteins. Stimulation with anti-inflammatory cytokines resulted in the activation of distinct, yet integrated clusters of proteins that trigger downstream a number of tumor-promoting biological processes. The observed changes could be classified into four major categories, i.e., mitochondrial gene expression, ECM remodeling, immune response, and impaired cell cycle progression. Intracellular immune activation was mediated mainly by the transducers of MAPK, STAT, TGFβ, NFKB, and integrin signaling pathways. Abundant collagen formation along with the expression of additional receptors, matrix components, growth factors, proteases and protease inhibitors, was indicative of ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that link ECM reorganization with cytoskeletal re-arrangements supportive of cell migration. Antigen processing/presentation was represented by HLA class I histocompatibility antigens, and correlated with upregulated proteasomal subunits, vesicular/viral transport, and secretory processes. Immunosuppressive and proangiogenic chemokines, as well as anti-angiogenic factors, were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors, signaling and ECM proteins indicated the presence of such capabilities.ConclusionsComprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a spectrum of microglia phenotypes supportive of cancer development in the brain via microenvironment-dependent biological mechanisms.

Prognostic role of DFNA5 in head and neck squamous cell carcinoma revealed by systematic expression analysis

BackgroundThe gasdermin E gene (GSDME, also known as DFNA5) is mutated in familial aging-related hearing loss. Recent studies have also revealed that the expression of DFNA5 is suppressed in many cancer types; however, little is known about the function of DFNA5 in head and neck squamous cell carcinoma (HNSCC). Accordingly, the aim of the present study was to evaluate the expression of DFNA5 and explore its prognostic value in HNSCC.ResultWe used a set of bioinformatics tools, including Oncomine, TIMER, TISIDB, cBioPortal, and GEPIA, to analyze the expression of DFNA5 in patients with HNSCC from public databases. Kaplan-Meier plotter was used to evaluate the potential prognostic significance of DFNA5. DFNA5 mRNA levels were significantly higher in HNSCC tissues than in normal tissues, and high DFNA5 expression was correlated with worse survival. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that DFNA5 expression has a strong positive correlation with cell adhesion and the integrin signaling pathway, whereas its expression was negatively correlated with the levels of infiltrating B cells (cor = − 0.223, P = 8.57e-07) and CD8 T cells (cor = − 0.223, P = 2.99e-07).ConclusionThis study demonstrates that DFNA5 expression has prognostic value for HNSCC patients. Moreover, these results suggest that regulation of lymphocyte infiltration is the mechanism underlying the function of DFNA5 in HNSCC.

Loss of Integrin-Linked Kinase Leads to Dysplasia of the Colon in Mice

Normal development of the colon is essential for well-being during the life span. Integrin linked kinase (ILK), which is a key part of the integrin signaling pathway, connects intracellular and extracellular signaling pathways and is evolutionally conserved. It has been shown that ILK knockouts can lead to abnormal formation of various organs. In our study, we found that deletion of ILK by platelet-derived growth factor receptor B (PDGFR[Formula: see text]-Cre leads to the abnormal migration of neural crest cells (NCCs) and the shortening of the gastrointestinal tract, along with the expansion of the colon cavity and dysplasia of the intestinal nerve fibers. In conclusion, ILK is required for normal colon development.

Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration.

Epidermal growth factor receptor (EGFR) plays a critical role in the promotion of epithelial cell proliferation and migration. Previous studies have suggested a cooperative role between EGFR and integrin signalling pathways that enable efficient adhesion and migration but the mechanisms controlling this remain poorly defined. Here, we show that EGFR forms a complex with focal adhesion kinase in epithelial cells. Surprisingly, this complex enhances local Src activity at focal adhesions to promote phosphorylation of the cytoskeletal adaptor protein ezrin at Y478, leading to actomyosin contractility, suppression of focal adhesion dynamics and slower migration. We further demonstrate this regulation of Src is due to the suppression of PTP1B activity. Our data provide new insight into EGF-independent cooperation between EGFR and integrins and suggest transient interactions between these kinases at the leading edge of cells act to spatially control signalling to permit efficient motility.

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/−, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/− embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Thrombospondin-1 mediates Rho-kinase inhibitor-induced increase in outflow-facility.

Rho‐kinase (ROCK) inhibitors, a novel class of anti‐glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin‐1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine–serine–lysine–leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose‐dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1.