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Essentials Cysteine oxidation to sulfenic acid plays a key role in redox regulation and signal transduction. Platelet sulfenylome was studied by quantitative proteomics in pathogen inactivated platelets. One hundred and seventy-four sulfenylated proteins were identified in resting platelets. Pathogen inactivation oxidized integrin βIII, which could activate the mitogen-activated protein kinases pathway. ABSTRACT: Background Cysteine-containing protein modifications are involved in numerous biological processes such redox regulation or signal transduction. During the preparation and storage of platelet concentrates, cell functions and protein regulations are impacted. In spite of several proteomic investigations, the platelet sulfenylome, ie, the proteins containing cysteine residues (R-SH) oxidized to sulfenic acid (R-SOH), has not been characterized. Methods A dimedone-based sulfenic acid tagging and enrichment coupled to a mass spectrometry identification workflow was developed to identify and quantify the sulfenic acid-containing proteins in platelet concentrates treated or not with an amotosalen/ultraviolet A (UVA) pathogen inactivation technique. Results One hundred and seventy-four sulfenylated proteins were identified belonging mainly to the integrin signal pathway and cytoskeletal regulation by Rho GTPase. The impact on pathogen inactivated platelet concentrates was weak compared to untreated ones where three sulfenylated proteins (myosin heavy chain 9, integrin βIII, and transgelin 2) were significantly affected by amotosalen/UVA treatment. Of particular interest, the reported oxidation of cysteine residues in integrin βIII is known to activate the receptor αIIbβIII. Following the pathogen inactivation, it might trigger the phosphorylation of p38MAPK and explain the lesions reported in the literature. Moreover, procaspase activating compound-1 (PAC-1) binding assays on platelet activation showed an increased response to adenosine diphosphate exacerbated by the tagging of proteins with dimedone. This result corroborates the hypothesis of an oxidation-triggered activation of αIIbβIII by the pathogen inactivation treatment. Conclusions The present work completes missing information on the platelet proteome and provides new insights on the effect of pathogen inactivation linked to integrin signaling and cytoskeleton regulation.

Bufalin is an anticancer drug extract from traditional Chinese medicine. Several articles about bufalin have been published. However, the literature on bufalin has not yet been systematically studied. This study aimed to identify the study status and knowledge structures of bufalin and to summarize the antitumor mechanism. Data were retrieved and downloaded from the PubMed database. The softwares of BICOMB, gCLUTO, Ucinet 6.0, and NetDraw2.084 were used to analyze these publications. The bufalin related genes were recognized and tagged by ABNER software. Then these BF-related genes were performed by Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, and protein-protein interaction (PPI) network analysis. A total of 474 papers met the search criteria from 2000 to 2019. By biclustering clustering analysis, the 50 high-frequency main MeSH terms/subheadings were classified into 5 clusters. The clusters of drug therapy and the mechanism of bufalin were hotspot topics. A total of 50 genes were identified as BF-related genes. PPI network analysis showed that inducing apoptosis was the main effect of bufalin, and apoptosis-related gene Caspase 3 was the most reported by people. Bufalin could inhibit the proliferation, invasion, and metastasis of cancer cells through multiple signaling pathways, such as PI3K/AKT, Hedgehog, MAPK/JNK, Wnt/[Formula: see text]-catenin, TGF-[Formula: see text]/Smad, Integrin signaling pathway, and NF-KB signaling pathway via KEGG analysis. Through the quantitative analysis of bufalin literature, we revealed the research status and hot spots in this field and provided some guidance for further research.

Breast cancer brain metastasis has been recognized as one of the central issues in breast cancer research. The elucidation of the processes and pathways that mediate this step will provide important clues for a better understanding of breast cancer metastasis. Increasing evidence suggests that aberrant glycosylation patterns greatly contribute to cell invasion and cancer metastasis. Herein, we combined next-generation RNA sequencing with liquid chromatography-tandem mass spectrometry-based proteomic and N-glycomic analysis from five breast cancer cell lines and one brain cancer cell line to investigate the possible mechanisms of breast cancer brain metastasis. The genes/proteins associated with cell movement were highlighted in breast cancer brain metastasis. The integrin signaling pathway and the up-regulation of α-integrin (ITGA2, ITGA3) were associated with the brain metastatic process. 12 glycogenes showed unique expression in 231BR, which could result in an increase of sialylation during brain metastasis. In agreement with the changes of glycogenes, 60 out of 63 N-glycans that were identified exhibited differential expression among cell lines. The correlation between glycogenes and glycans revealed the importance of sialylation and sialylated glycans in breast cancer brain metastasis. Highly sialylated N-glycans, which were up-regulated in brain-seeking cell line 231BR, likely play a role in brain metastasis.

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.

Background/purpose: Mechanical stretch plays a key role in promoting proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) in distraction osteogenesis (DO). A better understanding of how the extracellular biomechanical stimulation is transferred to intracellular signal expression will benefit DO. Focal adhesion kinase (FAK) is a key factor in integrin signaling pathway. However, little is known about the effect of integrin-FAK signaling during the process of stretch induced osteogenic differentiation of BMSCs. Materials and methods: A specific short hairpin RNAs (shRNAs) lentiviral expression vector was used to silence Fak gene and a well-established in vitro uniaxial dynamic stretching device was applied to stimulate DO. Fak silencing was confirmed by fluorescence microscopy and the detection of Fak mRNA and FAK, p-FAK protein expression. Alkaline phosphatase (ALP) activity, expression of osteogenic differentiation markers - runt-related transcription factor 2 (RUNX2/Runx2) and alkaline phosphatase (Alp) together with integrin upstream signal transduction molecules integrin beta-1 (ITGB1/Itgb1) and downstream signal transduction molecules integrin-linked kinase (ILK) were detected after the stretch. Results: The results showed that mechanical stretch in control groups significantly induced the osteogenic differentiation of BMSCs with increased ALP activity, expression of RUNX2/Runx2 and Alp, together with upregulated ITGB1/Itgb1 and ILK, which all vanished in Fak silencing group. Conclusion: Silencing of the Fak gene inhibited the osteogenic differentiation of rat BMSCs induced by in vitro mechanical stretch through integrin signaling pathway. Keywords: Distraction osteogenesis, Focal adhesion kinase, Integrin signaling pathway, Mesenchymal stem cells, Mechanical stretch

AimAnalysis of the pathophysiology of mesenteric fibrosis (MF) in small intestinal neuroendocrine tumors (SI-NETs) in an in vitro paracrine model and in human SI-NET tissue samples.MethodsAn indirect co-culture model of SI-NET cells KRJ-I and P-STS with stromal cells HEK293 was designed to evaluate the paracrine effects on cell metabolic activity, gene expression by RT2 PCR Profilers to analyse cancer and fibrosis related genes, and RNA sequencing. The integrin signaling pathway, a specific Ingenuity enriched pathway, was further explored in a cohort of human SI-NET tissues by performing protein analysis and immunohistochemistry.ResultsRT Profiler array analysis demonstrated several genes to be significantly up- or down-regulated in a cell specific manner as a result of the paracrine effect. This was further confirmed by employing RNA sequencing revealing multiple signaling pathways involved in carcinogenesis and fibrogenesis that were significantly affected in these cell lines. A significant upregulation in the expression of various integrin pathway – related genes was identified in the mesenteric mass of fibrotic SI-NET as confirmed by RT-qPCR and immunohistochemistry. Protein analysis demonstrated downstream activation of the MAPK and mTOR pathways in some patients with fibrotic SI-NETs.ConclusionThis study has provided the first comprehensive analysis of the crosstalk of SI-NET cells with stromal cells. A novel pathway – the integrin pathway – was identified and further validated and confirmed in a cohort of human SI-NET tissue featured by a dual role in fibrogenesis/carcinogenesis within the neoplastic fibrotic microenvironment.

Background Heart failure (HF) is an end stage heart condition with poor prognosis which brings about tremendous social medical cost. Along decades, mechanism and treatments of HF have been under restless research. Methods In the present study, we first established pressure overload induced HF model using transaortic arch constriction (TAC) method in mice. The global expression profiles of long noncoding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) were obtained by microarray probes, which were further confirmed by quantitative PCR (qPCR). Bioinformatics analysis was performed using multiple methods including volcano plotting, heatmapping and hierarchical clustering, Gene Ontology (GO) and pathway enrichment analysis, and competing endogenous RNA (ceRNA) regulatory network construction. Results Totally, 1,139 differentially expressed mRNAs (DEmRNAs), 3,830 lncRNAs (DElncRNAs) and 13 miRNAs (DEmiRNAs) were identified in HF group compared to control group, which could distinctly differentiate HF from normal control and were potential candidate biomarkers for HF. GO and pathway enrichment analysis revealed that multiple significant biological processes and pathways were involved in HF pathogenesis, such as extracellular matrix structural constituent, proteinaceous extracellular matrix, positive regulation of apoptotic process and integrin signaling pathway. Nine DElncRNAs, 3 DEmiRNAs and 25 DEmRNAs were filtrated out to construct a ceRNA network, which visually displayed their regulatory roles with therapeutic target potential. Conclusions The present study identified differentially expressed RNAs that might be involved in the pathogenesis and progression of HF. The outcomes shed lights into the underlying mechanisms for HF and provided candidate biomarkers and intervention targets for further research.

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.

Despite the brilliant bioactive performance of tantalum as an orthopedic biomaterial verified through laboratory researches and clinical practice in the past decades, scarce evidences about the essential mechanisms of how tantalum contributes to osteogenesis were systematically discussed. Up to now, a few studies have uncovered preliminarily the biological mechanism of tantalum in osteogenic differentiation and osteogenesis; it is of great necessity to map out the panorama through which tantalum contributes to new bone formation. This minireview summarized current advances to demonstrate the probable signaling pathways and underlying molecular cascades through which tantalum orchestrates osteogenesis, which mainly contain Wnt/β-catenin signaling pathway, BMP signaling pathway, TGF-β signaling pathway, and integrin signaling pathway. Limits of subsistent studies and further work are also discussed, providing a novel vision for the study and application of tantalum.

The proto-oncogene YAP /Yki, a transcription co-factor of the Hippo pathway, has been linked to many cancers. YAP interacts with DNA-binding TEAD/Sd proteins to regulate expression of its transcriptional targets. Disruption of YAP-TEAD therefore offers a potential therapeutic strategy. The mammalian Vestigial Like (VGLL) protein, specifically its TONDU domain, has been shown to competitively inhibit YAP-TEAD interaction and a TONDU peptide can suppress YAP-induced cancer. As TONDU could potentially be developed into a therapeutic peptide for multiple cancers, we evaluated its efficacy in Yki-driven adult Intestinal Stem Cell (ISC) tumors in Drosophila. We show that oral uptake of the TONDU peptide is highly effective at inhibiting Yki-driven gut tumors by suppressing YAP-TEAD interaction. Comparative proteomics of early and late stage Yki-driven ISC tumors revealed enrichment of a number of proteins, including members of the integrin signaling pathway, such as Talin, Vinculin and Paxillin. These, in turn displayed a decrease in their levels in TONDU-peptide treated tumors. Further, we show that Sd binds to the regulatory region of integrin-coding gene, mew, which codes for αPS1, a key integrin of the ISCs. In support to a possible role of integrins in Yki-driven ISC tumors, we show that genetic downregulation ofmewarrests Yki-driven ISC proliferation, reminiscent of the effects of TONDU peptide. Altogether, our findings present a novel platform for screening therapeutic peptides and provide insights into tumor suppression mechanisms.SIGNIFICANCE STATEMENTDiscovering novel strategies to inhibit oncogene activity is a priority in cancer biology. As signaling pathways are widely conserved between mammals and Drosophila, these questions can be effectively addressed in this model organism. Here, we show that progression ofDrosophilaIntestinal Stem Cell (ISC) tumors induced by gain of an oncogenic form of the transcription co-factor Yki can be suppressed by feeding a peptide corresponding to the conserved TONDU domain of Vestigial (Vg), which blocks binding of Yki to the Sd transcription factor. Further, we show that down regulation of the integrin signaling pathway is causally linked to TONDU-peptide-mediated ISC tumor suppression. Our findings reveal thatDrosophilacan be successfully used to screen peptides for their therapeutic applications.

Background: Tetraspanins constitute a family of transmembrane spanning proteins that function mainly by organizing the plasma membrane into micro-domains. CD82, a member of tetraspanins, is a potent inhibitor of cancer metastasis in numerous malignancies. CD82 is a highly glycosylated protein, however, it is still unknown whether and how this post-translational modification affects CD82 function and cancer metastasis. Methods: The glycosylation of CD82 profiles are checked in the paired human ovarian primary and metastatic cancer tissues. The functional studies on the various glycosylation sites of CD82 are performed in vitro and in vivo. Results: We demonstrate that CD82 glycosylation at Asn157 is necessary for CD82-mediated inhibition of ovarian cancer cells migration and metastasis in vitro and in vivo. Mechanistically, we discover that CD82 glycosylation is pivotal to disrupt integrin α5β1-mediated cellular adhesion to the abundant extracellular matrix protein fibronectin. Thereby the glycosylated CD82 inhibits the integrin signaling pathway responsible for the induction of the cytoskeleton rearrangements required for cellular migration. Furthermore, we reveal that the glycosyltransferase MGAT3 is responsible for CD82 glycosylation in ovarian cancer cells. Metastatic ovarian cancers express reduced levels of MGAT3 which in turn may result in impaired CD82 glycosylation. Conclusions: Our work implicates a pathway for ovarian cancers metastasis regulation via MGAT3 mediated glycosylation of tetraspanin CD82 at asparagine 157.

Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Enterotoxigenic Escherichia coli (ETEC) that expresses F4ac fimbriae is the major pathogenic microorganism responsible for bacterial diarrhea in neonatal piglets. The susceptibility of piglets to ETEC F4ac is determined by a specific receptor on the small intestinal epithelium surface. We performed an iTRAQ-labeled quantitative proteome analysis using a case-control design in which susceptible and resistant full-sib piglets were compared for the protein expression levels. Two thousand two hundred forty-nine proteins were identified, of which 245 were differentially expressed (fold change > 1.5, FDR-adjusted P < 0.05). The differentially expressed proteins fell into four functional classes: (I) cellular adhesion and binding, (II) metabolic process, (III) apoptosis and proliferation, and (IV) immune response. The integrin signaling pathway merited particular interest based on a pathway analysis using statistical overexpression and enrichment tests. Genomic locations of the integrin family genes were determined based on the most recent porcine genome sequence assembly (Sscrofa11.1). Only one gene, ITGB5, which encodes the integrin β5 subunit that assorts with the αv subunit to generate integrin αvβ5, was located within the SSC13q41 region between 13:133161078 and 13:139609422, where strong associations of markers with the ETEC F4ac susceptibility were found in our previous GWAS results. To identify whether integrin αvβ5 is the ETEC F4acR, we established an experimental model for bacterial adhesion using IPEC-J2 cells. Then, the ITGB5 gene was knocked out in IPEC-J2 cell lines using CRISPR/Cas9, resulting in a biallelic deletion cell line (ITGB5−/−). Disruption of ITGB5 significantly reduced ETEC F4ac adhesion to porcine intestinal epithelial cells. In contrast, overexpression of ITGB5 significantly enhanced the adhesion. A GST pull-down assay with purified FaeG and ITGB5 also showed that FaeG binds directly to ITGB5. Together, the results suggested that ITGB5 is a key factor affecting the susceptibility of piglets to ETEC F4ac.

Melanoma is the leading cause of death from skin disease due to its propensity for metastasis. Studies have shown that integrin-mediated focal adhesion kinase (FAK) signal pathway is implicated in cell proliferation, survival and metastasis of tumor cells. Our previous results indicated that diallyl trisulfide (DATS) provided its antimelanoma activity via inducing cell cycle arrest and apoptosis. The aim of this study was to explore DATS mediated antimetastatic effect and the corresponding mechanism in human melanoma A375 cells. We found that DATS exhibited an inhibitory effect on the abilities of migration and invasion in A375 cells under noncytotoxic concentrations analyzed by wound healing assays and Matrigel invasion chamber system. DATS attenuated invasion of A375 cells with characteristic of decreased activities and protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, DATS exerted an inhibitory effect on cell adhesion of A375 cells, which is in correlation with the change in integrin signaling pathway. Results of Western blotting showed that DATS decreased the levels of several integrin subunits, including α4, α5, αv, β1, β3 and β4. Subsequently, DATS induced a strong decrease in total FAK, phosphorylated FAK Tyr-397,-576, -577, and disorganized F-actin stress fibers, resulting in a nonmigratory phenotype. These results suggest that the antimetastatic potential of DATS for human melanoma cells might be due to the disruption of integrin/FAK signaling pathway.

Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a pivotal role in the regulation of estrogens and androgens that promote the growth of hormone-dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not clear. To elucidate the role of STS in cancer cell proliferation, we investigated whether STS is able to regulate the integrin signaling pathway. We found that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), one of the main metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. Further, STS expression and DHEA treatment enhanced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Moreover, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS activates the MAPK/ ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up-regulation of integrin β1 and activation of FAK.

Experimental studies have demonstrated several effects of statins in acute coronary syndrome (ACS) that may extend their clinical benefit beyond the lipid profile modification itself. However, the precise underlying mechanism remains to be elucidated. microRNAs (miRNAs) serve significant roles in the pathophysiology of atherosclerotic plaque progression. The present study investigated the protective role of statins in patients with unstable angina (UA) by regulating the circulating miRNA network. miRNA array results demonstrated that there were 21 differentially expressed miRNAs in non-statin-treated patients with UA (n=8) compared with non-coronary artery disease controls (n=8), and 33 differentially expressed miRNAs in statin-treated patients with UA (n=8) compared with non-statin patients. TargetScan and miRanda programs were used to predict miRNAs target genes. miRNAs target genes in vascular endothelial cells and monocytes were clustered based on the CGAP SAGE library via the Database for Annotation, Visualization and Integrated Discovery (DAVID) platform, and miRNA target genes in platelets were clustered based on a UP tissue-specific library via the DAVID platform. The PANTHER database via DAVID platform was used to perform signaling pathway analysis. The miRNA-gene/pathway network was visualized by Cytoscape software. Bioinformatic analysis suggested that statin-induced miRNAs functions were primarily enriched in angiogenesis, integrin and platelet derived growth factor signaling pathways in UA patients. In endothelial cells and platelets, statin-induced miRNAs primarily targeted the integrin signaling pathway, and in monocytes primarily targeted cytoskeletal regulation by the Rho GTPase signaling pathway. These results revealed that statins may serve systematic protective roles in UA patients by influencing the circulating miRNA regulatory network. Further studies are required to verify the functions of statin-induced miRNAs in endothelial cells, platelets and monocytes.

Abdominal aortic aneurysm (AAA) is a serious condition of unclear pathogenesis and progression. Two samples were collected from 48 patients during AAA surgery. One sample was collected from the aneurysm, the other from the aneurysm proximal neck where the tissue did not exhibit any aneurysmal changes. Subsequently, gene expression profiles using microarrays (Illumina) were compared in RNA extracted from the samples. Overall, 2,185 genes were found to be upregulated and 2,100 downregulated; from which 158 genes had a different expression with FDR

Sex differences in the incidence and outcome of human disease are broadly recognized but, in most cases, not sufficiently understood to enable sex-specific approaches to treatment. Glioblastoma (GBM), the most common malignant brain tumor, provides a case in point. Despite well-established differences in incidence and emerging indications of differences in outcome, there are few insights that distinguish male and female GBM at the molecular level or allow specific targeting of these biological differences. Here, using a quantitative imaging-based measure of response, we found that standard therapy is more effective in female compared with male patients with GBM. We then applied a computational algorithm to linked GBM transcriptome and outcome data and identified sex-specific molecular subtypes of GBM in which cell cycle and integrin signaling are the critical determinants of survival for male and female patients, respectively. The clinical relevance of cell cycle and integrin signaling pathway signatures was further established through correlations between gene expression and in vitro chemotherapy sensitivity in a panel of male and female patient-derived GBM cell lines. Together, these results suggest that greater precision in GBM molecular subtyping can be achieved through sex-specific analyses and that improved outcomes for all patients might be accomplished by tailoring treatment to sex differences in molecular mechanisms.

Ocular melanoma research, the most common primary intraocular malignancy in adults, is hindered by limited in vivo models. In a series of experiments using melanoma cells injected intraocularly into mouse eyes, we developed a model for ocular melanoma. Inoculation of 5 × 105 B16F10 cells led to rapid tumor growth, extensive lung metastasis, and limited animal survival, while injection of 102 cells was sufficient for intraocular tumors to grow with extended survival. In order to improve tumor visualization, 102 melanoma cells (B16F10 or B16LS9) were inoculated into Balb/C albino mouse eyes. These mice developed intraocular tumors that did not metastasize and exhibited extended survival. Next, we studied the therapeutic potential of inhibitor of the thyroid hormones-αvβ3 integrin signaling pathway in ocular melanoma. By utilizing tetraiodothyroacetic acid (tetrac), a thyroid hormone derivative, a delay in tumor onset in the B16F10 (integrin+) arm was observed, compared to the untreated group, while in the B16LS9 cells (integrin-) a similar rate of tumor onset was noticed in both experimental and control groups. In summary, following an optimization process, the mouse ocular melanoma model was developed. The models exhibited an extended therapeutic window and can be utilized as a platform for investigating various drugs and other treatment modalities.

Background Ovarian cancer is a significant cancer-related cause of death in women worldwide. The most used chemotherapeutic regimen is based on carboplatin (CBDCA). However, CBDCA resistance is the main obstacle to a better prognosis. An in vitro drug-resistant cell model would help in the understanding of molecular mechanisms underlying this drug-resistance phenomenon. The aim of this study was to characterize cellular and molecular changes of induced CBDCA-resistant ovarian cancer cell line A2780. Methods The cell selection strategy used in this study was a dose-per-pulse method using a concentration of 100 μM for 2 h. Once 20 cycles of exposure to the drug were completed, the cell cultures showed a resistant phenotype. Then, the ovarian cancer cell line A2780 was grown with 100 μM of CBDCA (CBDCA-resistant cells) or without CBDCA (parental cells). After, a drug sensitivity assay, morphological analyses, cell death assays and a RNA-seq analysis were performed in CBDCA-resistant A2780 cells. Results Microscopy on both parental and CBDCA-resistant A2780 cells showed similar characteristics in morphology and F-actin distribution within cells. In cell-death assays, parental A2780 cells showed a significant increase in phosphatidylserine translocation and caspase-3/7 cleavage compared to CBDCA-resistant A2780 cells (P

Background and Objective: Non-small cell lung cancer (NSCLC) accounts for 80-85% of all patients with lung cancer and 5-year relative overall survival (OS) rate is less than 20%, so that identifying novel diagnostic and prognostic biomarkers is urgently demanded. The present study attempted to identify potential key genes associated with the pathogenesis and prognosis of NSCLC. Methods: Four GEO datasets (GSE18842, GSE19804, GSE43458, and GSE62113) were obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between NSCLC samples and normal ones were analyzed using limma package, and RobustRankAggreg (RRA) package was used to conduct gene integration. Moreover, Search Tool for the Retrieval of Interacting Genes database (STRING), Cytoscape, and Molecular Complex Detection (MCODE) were utilized to establish protein-protein interaction (PPI) network of these DEGs. Furthermore, functional enrichment and pathway enrichment analyses for DEGs were performed by Funrich and OmicShare. While the expressions and prognostic values of top genes were carried out through Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan Meier-plotter (KM) online dataset. Results: A total of 249 DEGs (113 upregulated and 136 downregulated) were identified after gene integration. Moreover, the PPI network was established with 166 nodes and 1784 protein pairs. Topoisomerase II alpha (TOP2A), a top gene and hub node with higher node degrees in module 1, was significantly enriched in mitotic cell cycle pathway. In addition, Interleukin-6 (IL-6) was enriched in amb2 integrin signaling pathway. The mitotic cell cycle was the most significant pathway in module 1 with the highest P-value. Besides, five hub genes with high degree of connectivity were selected, including TOP2A, CCNB1, CCNA2, UBE2C, and KIF20A, and they were all correlated with worse OS in NSCLC. Conclusion: The results showed that TOP2A, CCNB1, CCNA2, UBE2C, KIF20A, and IL-6 may be potential key genes, while the mitotic cell cycle pathway may be a potential pathway contribute to progression in NSCLC. Further, it could be used as a new biomarker for diagnosis and to direct the synthesis medicine of NSCLC.

Simple Summary The extracellular matrix (ECM) has emerged as a critical part of the tumor microenvironment. This glycoprotein- and proteoglycan-rich part of the tumor serves as a niche for the enrichment of cancer stem cells that can drive resistance to therapy and metastasis. Additionally, the ECM can act as a barrier to drug delivery, thereby physically contributing to resistance to therapy. This review summarizes the role of the ECM in enriching for cancer stem cells and how it contributes to therapy resistance in cancer. Finally, it discusses the attempts to develop molecules that can target the ECM as potential therapy options. Abstract The extracellular matrix (ECM) has remained an enigmatic component of the tumor microenvironment. It drives metastasis via its interaction with the integrin signaling pathway, contributes to tumor progression and confers therapy resistance by providing a physical barrier around the tumor. The complexity of the ECM lies in its heterogeneous composition and complex glycosylation that can provide a support matrix as well as trigger oncogenic signaling pathways by interacting with the tumor cells. In this review, we attempt to dissect the role of the ECM in enriching for the treatment refractory cancer stem cell population and how it may be involved in regulating their metabolic needs. Additionally, we discuss how the ECM is instrumental in remodeling the tumor immune microenvironment and the potential ways to target this component in order to develop a viable therapy.

Gα13 directly binds to the cytoplasmic-domain ExE motif of the integrin β1 subunit. Gα13–β1 interaction mediates β1 integrin–dependent Src activation and transient RhoA inhibition after adhesion. This binding is critical for cell migration on β1 integrin ligands., Heterotrimeric G protein Gα13 is known to transmit G protein–coupled receptor (GPCR) signals leading to activation of RhoA and plays a role in cell migration. The mechanism underlying the role of Gα13 in cell migration, however, remains unclear. Recently we found that Gα13 interacts with the cytoplasmic domain of integrin β3 subunits in platelets via a conserved ExE motif. Here we show that a similar direct interaction between Gα13 and the cytoplasmic domain of the integrin β1 subunit plays a critical role in β1-dependent cell migration. Point mutation of either glutamic acid in the Gα13-binding 767EKE motif in β1 or treatment with a peptide derived from the Gα13-binding sequence of β1 abolished Gα13–β1 interaction and inhibited β1 integrin–dependent cell spreading and migration. We further show that the Gα13-β1 interaction mediates β1 integrin–dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Gα13 in mediating GPCR-dependent RhoA activation. These data indicate that Gα13 plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on β1 integrin ligands.

Endocrine disrupting chemicals (EDCs) can induce abnormalities in organisms via alteration of molecular pathways and subsequent disruption of endocrine functions. Bisphenol A (BPA) and 17α-ethinylestradiol (EE2) are ubiquitous EDCs in the environment. Many aquatic organisms, including fish, are often exposed to varying concentrations of BPA and EE2 throughout their lifespan. Both BPA and EE2 can activate estrogenic signaling pathways and cause adverse effects on reproduction via alteration of pathways associated with steroidogenesis. However, transcriptional pathways that are affected by chronic exposure to these two ubiquitous environmental estrogens during embryonic, larval, and juvenile stages are not clearly understood. In the present study, we examined transcriptional alterations in the testis of medaka fish (Oryzias latipes) chronically exposed to a low concentration of BPA or EE2. Medaka were exposed to BPA (10 μg/L) or EE2 (0.01 μg/L) from 8 h post-fertilization (as embryos) to adulthood 50 days post fertilization (dpf), and transcriptional alterations in the testis were examined by RNA sequencing (RNA-seq). Transcriptomic profiling revealed 651 differentially expressed genes (DEGs) between BPA-exposed and control testes, while 1475 DEGs were found between EE2-exposed and control testes. Gene ontology (GO) analysis showed a significant enrichment of "intracellular receptor signaling pathway", "response to steroid hormone" and "hormone-mediated signaling pathway" in the BPA-induced DEGs, and of "cilium organization", "microtubule-based process" and "organelle assembly" in the EE2-induced DEGs. Pathway analysis showed significant enrichment of "integrin signaling pathway" in both treatment groups, and of "cadherin signaling pathway", "Alzheimer disease-presenilin pathway" in EE2-induced DEGs. Single nucleotide polymorphism (SNP) and insertion-deletion (Indel) analysis found no significant differences in mutation rates with either BPA or EE2 treatments. Taken together, global gene expression differences in testes of medaka during early stages of gametogenesis were responsive to chronic BPA and EE2 exposure.

37 p.-6 fig., Purpose: New targets are required for the control of advanced metastatic disease. We investigated the use of cadherin RGD motifs, which activate the α2β1integrin pathway, as targets for the development of therapeutic monoclonal antibodies (mAb)., Experimental Design: Cadherin 17 (CDH17) fragments and peptides were prepared and used for immunization and antibody development. Antibodies were tested for inhibition of β1 integrin and cell adhesion, proliferation, and invasion assays using cell lines from different cancer types (colorectal, pancreatic, melanoma, and breast cancer). Effects of the mAbs on cell signaling were determined by Western blot analysis. Nude mice were used for survival analysis after treatment with RGD-specific mAbs and metastasis development., Results: Antibodies against full-length CDH17 failed to block the binding to α2β1 integrin. However, CDH17 RGD peptides generated highly selective RGD mAbs that blocked CDH17 and vascular-endothelial (VE)-cadherin-mediated β1 integrin activation in melanoma and breast, pancreatic, and colorectal cancer cells. Antibodies provoked a significant reduction in cell adhesion and proliferation of metastatic cancer cells. Treatment with mAbs impaired the integrin signaling pathway activation of FAK in colorectal cancer, of JNK and ERK kinases in colorectal and pancreatic cancers, and of JNK, ERK, Src, and AKT in melanoma and breast cancer. In vivo, RGD-specific mAbs increased mouse survival after inoculation of melanoma and colorectal cancer cell lines to cause lung and liver metastasis, respectively., Conclusions: Blocking the interaction between RGD cadherins and α2β1 integrin with highly selective mAbs constitutes a promising therapy against advanced metastatic disease in colon cancer, melanoma, and, potentially, other cancers., This research was supported by grants BIO2015-66489-R from the MINECO, Foundation Ramón Areces and PRB2 (IPT13/0001-ISCIII-SGEFI/FEDER).

Graphene oxide (GO), with good hydrophilicity and biocompatibility, is widely explored as a carrier for various factors in the field of stem cell differentiation. However, its function of sustaining the stemness of mouse embryonic stem cells (mESCs) and the underlying mechanisms of this process remains undiscovered. Herein, we explored the biofunction of GO on mESCs and revealed the involved signaling pathways and key gene. The alkaline phosphatase activity detection, pluripotency genes quantification and the teratomas formation in vivo confirmed that GO nanosheets could sustain the self-renewal ability of mESCs instead of influencing its pluripotency. The underlying signaling pathways were uncovered by RNA-seq that integrin signaling pathway was involved in the biofunction of GO on mESCs and Vinculin turned to be a key gene for the effect of GO. Further experiments confirmed that the downregulation of Vinculin influenced the fate of mESCs through decreasing the expression of MEK1. Altogether, the study demonstrated for the first time that GOs hold the potential in sustaining the self-renewal of mESCs and clarified the mechanism of this function, which make it play a new role in stem cell research and regenerative medicine.

In the past few decades, there has been a global decrease in dairy cow reproductive performance. An activated inflammatory system, due to uterine infection, has been associated with decreased cow fertility and as such, there is a need to detect uterine disease earlier. Early detection could be achieved by identifying biomarkers for uterine disease. Exosomes are small nanovesicles known to package and deliver protein, mRNA, and miRNAs to near and distant sites. Therefore, the content of circulating exosomes may have the potential to carry biomarkers for earlier diagnosis of disease. We hypothesized that circulating exosomes from cows with and without uterine infection may contain information representative of endometrial health or disease. We compared the proteomic content of circulating exosomes derived from plasma of dairy cows with (n = 10) or without (n = 10) induced uterine infection, using high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS). Our results demonstrate that there were a total of 103 bovine and 9 Trueperella pyogenes proteins found in plasma exosomes derived from infected cows (infected exosomes), and 90 bovine and 5 T. pyogenes proteins found in exosomes derived from plasma of non-infected cows (non-infected exosomes). 71 bovine proteins were found to be unique to the infected exosomes while only 4 bovine proteins were found to be unique to the non-infected exosomes. 8 unique T. pyogenes proteins were identified in infected exosomes and 4 were found to be unique to the non-infected exosomes. Pathway analysis showed that infected exosomes had more proteins involved in structural molecule activity and immune system processes than non-infected exosomal protein. Additionally, proteins from infected exosomes were involved in unique pathways: angiogenesis and integrin signaling pathway. Our data provide preliminary evidence of a potential role for exosomes in the early diagnosis of uterine infection in dairy cows.

Sex differences in the incidence and outcome of human disease are broadly recognized but in most cases not adequately understood to enable sex-specific approaches to treatment. Glioblastoma (GBM), the most common malignant brain tumor, provides a case in point. Despite well-established differences in incidence, and emerging indications of differences in outcome, there are few insights that distinguish male and female GBM at the molecular level, or allow specific targeting of these biological differences. Here, using a quantitative imaging-based measure of response, we found that temozolomide chemotherapy is more effective in female compared to male GBM patients. We then applied a novel computational algorithm to linked GBM transcriptome and outcome data, and identified novel sex-specific molecular subtypes of GBM in which cell cycle and integrin signaling were identified as the critical determinants of survival for male and female patients, respectively. The clinical utility of cell cycle and integrin signaling pathway signatures was further established through correlations between gene expression and in vitro chemotherapy sensitivity in a panel of male and female patient-derived GBM cell lines. Together these results suggest that greater precision in GBM molecular subtyping can be achieved through sex-specific analyses, and that improved outcome for all patients might be accomplished via tailoring treatment to sex differences in molecular mechanisms.One Sentence SummaryMale and female glioblastoma are biologically distinct and maximal chances for cure may require sex-specific approaches to treatment.

The objective of the present study was to investigate the long non-coding RNA (lncRNA) and mRNA expression profiles that are associated with the invasion and metastasis of papillary thyroid carcinoma (PTC). Transwell invasion assays were used to screen three highly invasive sub-strains of the human PTC IHH4 cell line: IHH4-M1, IHH4-M2 and IHH4-M3. In addition, tumor-bearing nude mice were used to identify the invasive and metastatic capacity of the three sub-strains. Agilent lncRNA microarray chips were used to screen 795 differentially expressed lncRNAs and 788 differentially expressed mRNAs. A total of 10 lncRNAs and 10 mRNAs were randomly selected for RT-qPCR validation to confirm that the results were consistent with the microarray chips, suggesting that the results of the microarray chip analysis were relatively accurate. Gene ontology enrichment-based cluster analysis revealed that the differentially expressed genes were mainly associated with steroid biosynthesis, bioadhesion, intercellular adhesion and other metastasis-associated biological processes. The results of the pathway cluster analysis identified that the differentially expressed genes were associated with tumor metastasis-associated signaling pathways, including the cholesterol metabolic signaling pathway, the sterol regulatory element-binding protein signaling pathway and the integrin signaling pathway, suggesting that lncRNA may regulate PTC metastasis through various signaling pathways. The present study screened and constructed PTC metastasis-associated lncRNA and mRNA expression profiles, and it provides a molecular basis for the future study of high-risk molecular markers of PTC.

Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-β1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-β1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn(2+). In mice, conditional inactivation of integrin-β1 in collecting ducts resulted in a dramatic inhibition of Pkd1-dependent cystogenesis with a concomitant suppression of fibrosis and preservation of normal renal function. Our data provide genetic evidence that a functional integrin-β1 is required for the early events leading to renal cystogenesis in ADPKD and suggest that the integrin signaling pathway may be an effective therapeutic target for slowing disease progression.

High-density fibrillar collagen matrix induces invadopodia formation in both fibroblasts and carcinoma cell lines through a kindlin2-dependent mechanism that drives local ECM remodeling., Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.

Background: Nogo-A, a major myelin-associated inhibitor, can inhibit injured optic nerve regeneration. However, whether Amino-Nogo is the most important functional domain of Nogo-A remains unknown. This study aimed to identify the role of Amino-Nogo following optic nerve injury, and the mechanism of the Amino-Nogo-integrin αv signaling pathway in vivo. Methods: Sprague-Dawley rats with optic nerve crush injury were injected with Nogo-A siRNA (Nogo-A-siRNA), the Nogo-66 functional domain antagonist peptide of Nogo-A (Nep1-40) or a recombinant rat Amino-Nogo-A protein (∆20) into the vitreous cavity to knock down Nogo-A, inhibit Nogo-66 or activate the Amino-Nogo, resparately. Retinal ganglion cell (RGC) density, axon regeneration and the pattern of NPN of visual electrophysiology (flash visual evoked potentials [F-VEP]) at different times post-injury were investigated. Results: Our study revealed a lower RGC survival rate; shorter axonal outgrowth; longer N1, P1 and N2 waves latencies; and lower N1-P1 and P1-N2 amplitudes in the Δ20 group, and Δ20 treatment significantly attenuated integrin αv expression and phosphorylated focal adhesion kinase (p-FAK) levels. In the Nep1-40 and Nogo-A siRNA groups, there were higher RGC survival rates, longer axonal outgrowth, shorter N1 and P1 wave latencies, and higher N1-P1 and P1-N2amplitudes. Nogo-A siRNA treatment significantly increased integrin αv expression and p-FAK levels. Nepl-40 treatment did not alter integrin αv expression. In addition, there was no significant change in integrin α5 in any group. Conclusion: These results suggest that the integrin signaling pathway is regulated by Amino-Nogo, which inhibits optic nerve regeneration and functional recovery, and that the integrin subunit involved might be integrin αv but not integrin α5.

The identification of biomarkers for toxicity prediction is crucial for drug development and safety evaluation. The selective and specific biomarkers for antihistamines-induced cardiotoxicity is not well identified yet. In order to evaluate the mechanism of the life-threatening effects caused by antihistamines, we used DNA microarrays to analyze genomic profiles in H9C2 rat cardiomyocytes that were treated with antihistamines. The gene expression profiles from drug-treated cells revealed changes in the integrin signaling pathway, suggesting that cardiac arrhythmias induced by antihistamine treatment may be mediated by changes in integrin-mediated signaling. It has been reported that integrin plays a role in QT prolongation that may induce cardiac arrhythmia. These results indicate that the integrin-mediated signaling pathway induced by antihistamines is involved in various biological mechanisms that lead to cardiac QT prolongation. Therefore, we suggest that genomic profiling of antihistamine-treated cardiomyocytes has the potential to reveal the mechanism of adverse drug reactions, and this signal pathway is applicable to prediction of in vitro cardiotoxicity induced by antihistamines as a biomarker candidate.

Following axon injury, a cascade of signaling events is triggered to initiate axon regeneration. However, the mechanisms regulating axon regeneration are not well understood at present. In Caenorhabditis elegans, axon regeneration utilizes many of the components involved in phagocytosis, including integrin and Rac GTPase. Here, we identify the transthyretin (TTR)-like protein TTR-11 as a component functioning in axon regeneration upstream of integrin. We show that TTR-11 binds to both the extracellular domain of integrin-α and phosphatidylserine (PS). Axon injury induces the accumulation of PS around the injured axons in a manner dependent on TTR-11, the ABC transporter CED-7, and the caspase CED-3. Furthermore, we demonstrate that CED-3 activates CED-7 during axon regeneration. Thus, TTR-11 functions to link the PS injury signal to activation of the integrin pathway, which then initiates axon regeneration., Apoptotic cells display surface signals such as phosphatidlyserines that are recognized by phagocytes via engulfment signal receptors. Here, the authors show how one such receptor, transthyretin-like protein 11, plays a role in initiating axon regeneration in the peripheral nervous system.

We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked the binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway.

Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human β1 integrin or α2β1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of β1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.

Background: To understand the function of β6 integrin and elucidate its signaling pathways in TGF-β-induced EMT in breast cancer. Methods: The interactions between TGF-β1 and β6 integrin were measured by coimmunoprecipitation. The EMT responses, phospherlation of PI3K/Akt and COX-2 expression were determined by real-time PCR, transwell assay, and western blot after the blockage of β6 integrin. Results: TGF-β1 and β6 integrin could bind with each other. Blockage of β6 integrin rescued TGF-β1-induced EMT phenotype and reduced expression of COX-2 via dephosphorylation of PI3K/Akt. Conclusions: β6 integrin plays a critical role in TGF-β1-induced EMT and overexpression of COX-2 in breast cancer.

The aim of our study was to investigate the osteoinductive potential of a titanium (Ti) surface with nanotopography, using mesenchymal stem cells (MSCs) and the mechanism involved in this phenomenon. Polished Ti discs were chemically treated with H2 SO4 /H2 O2 to yield nanotopography and rat MSCs were cultured under osteogenic and non-osteogenic conditions on both nanotopography and untreated polished (control) Ti surfaces. The nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of key bone markers of cells grown under both osteogenic and non-osteogenic conditions. Additionally, the gene expression of α1 and β1 integrins was higher in cells grown on Ti with nanotopography under non-osteogeneic condition compared with control Ti surface. The higher gene expression of bone markers and Alp activity induced by Ti with nanotopography was reduced by obtustatin, an α1β1 integrin inhibitor. These results indicate that α1β1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. This finding highlights a novel mechanism involved in nanosurface-mediated MSCs fate and may contribute to the development of new surface modifications aiming to accelerate and/or enhance the process of osseointegration.

The study aims to investigate the effects of Periostin gene silencing on tumor microenvironment and bone metastasis via the integrin-signaling pathway in lung cancer (LC). LC patients were divided into bone metastasis and non-bone metastasis groups; Healthy volunteers were selected as normal group. ELISA was performed to detect serum Periostin levels and plasma calcium ion concentration. SBC-5 cells were assigned into blank group (without transfection), negative control (NC) group (transfected with empty plasmid), si-Periostin group (transfected with si-Periostin plasmid), si-Integrin-αvβ3 group (transfected with Integrin-αvβ3 siRNA plasmid) and si-Periostin+si-Integrin-αvβ3 group (transfected with si-Periostin and si-Integrin-αvβ3 plasmid). qRT-PCR and Western blotting were performed to determine mRNA and protein expression of Periostin, metastasis-associated factors of tumor microenvironment and integrin signaling pathway-related proteins. CCK-8, scratch test and transwell assay were applied to detect cell proliferation, migration and invasion respectively. Nude mouse models of LC bone metastasis were established. TRAP Staining was employed to measure the number of osteoclasts. Bone metastasis group exhibited higher levels of Periostin compared to normal and non-bone metastasis groups. Si-Periostin, si-Integrin-αvβ3 and si-Periostin+si-Integrin-αvβ3 groups showed decreased Periostin expression, proliferation rate, migration distance, invasive cells, and expressions of metastasis-associated factors of tumor microenvironment and integrin signaling pathway-related proteins compared to blank and NC groups. Similarly, number of osteoclasts and expression of integrin signaling pathway-related proteins were decreased, and bone injury and calcium ion concentration were reduced. The study demonstrated that down-regulation of Periostin expression modulated tumor microenvironment and inhibited bone metastasis by blocking integrin-signaling pathway in LC.

Citrus unshiu peel has been used empirically as a traditional medicine to improve bronchial asthma and blood circulation in northeast Asian nations, including Korea, Japan, and China. In this study, we report the proangiogenic effects of the aqueous extract of Citrus unshiu peel (AECUP). In human umbilical vein endothelial cells, AECUP significantly induced cellular migration and capillary tube formation. We also demonstrated that AECUP markedly increased the phosphorylation of FAK and ERK1/2 through the integrin signaling pathway. Additionally, we identified that narirutin and hesperidin were major constituents of AECUP and both showed proangiogenic effects, but at different levels. Collectively, these results suggest that the AECUP may have potential as a therapeutic agent for improving angiogenic functions with reduced harmful side effects.

In this paper we provide a mean-field Boolean network model for a signal transduction network of a generic fibroblast cell. The network consists of several main signaling pathways, including the receptor tyrosine kinase, the G-protein coupled receptor, and the Integrin signaling pathway. The network consists of 130 nodes, each representing a signaling molecule (mainly proteins). Nodes are governed by Boolean dynamics including canalizing functions as well as totalistic Boolean functions that depend only on the overall fraction of active nodes. We categorize the Boolean functions into several different classes. Using a mean-field approach we generate a mathematical formula for the probability of a node becoming active at any time step. The model is shown to be a good match for the actual network. This is done by iterating both the actual network and the model and comparing the results numerically. Using the Boolean model it is shown that the system is stable under a variety of parameter combinations. It is also shown that this model is suitable for assessing the dynamics of the network under protein mutations. Analytical results support the numerical observations that in the long-run at most half of the nodes of the network are active.

Cell adhesion plays an important role in the steps of cancer metastasis. Regulation of cell-cell (intercellular) and cell-matrix adhesion is a promising strategy for cancer progression. Gambogic acid is a xanthone derived from the resin of the Chinese plant Garciania hanburyi, with potent anti-metastasis activity on highly metastatic cells. The aim of this study was to investigate the function and mechanism of gambogic acid on tumor adhesion. We found that gambogic acid strongly inhibited the adhesion of human cancer cells to fibronectin. This inhibition was associated with the deformation of focal adhesion complex, which was mediated by suppressing the expression of integrin β1 and integrin signaling pathway. In vitro, cell lipid rafts clustering was inhibited following treatment of gambogic acid, which induced the suppression of integrin β1 and focal adhesion complex proteins colocalization within rafts. Moreover, gambogic acid significantly decreased cellular cholesterol content, whereas cholesterol replenishment lessened the inhibitory effect of gambogic acid on cell adhesion. Real-time PCR analysis showed that gambogic acid reduced mRNA levels of hydroxymethylglutaryl-CoA reductase and sterol regulatory element binding protein-2, while increased acetyl-CoA acetyltransferase-1/2. Taken together, these results demonstrate that gambogic acid inhibits cell adhesion via suppressing integrin β1 abundance and cholesterol content as well as the membrane lipid raft-associated integrin function, which provide new evidence for the anti-cancer activity of gambogic acid.