Integrin-matrix Interactions Research Articles

TGF-β1 Drives Integrin-Dependent Pericyte Migration and Microvascular Destabilization in Fibrotic Disease

Interactions between endothelial cells (EC) and mural pericytes (PC) are critical to maintaining the stability and function of the microvascular wall. Abnormal interactions between these two cell types are a hallmark of progressive fibrotic diseases, such as systemic sclerosis (SSc, scleroderma). However, the role that PCs play in signaling microvascular dysfunction remains underexplored. It is hypothesized that integrin-matrix interactions contribute to PC migration from the vascular wall and conversion into interstitial myofibroblasts. Using pro-inflammatory TNF alpha (TNFα) or fibrotic growth factor TGF beta-1 (TGFβ1), human PC inflammatory and fibrotic phenotypes were evaluated by assessing their migration, matrix deposition, integrin expression, and subsequent effects on endothelial dysfunction. Both TNFα and TGFβ1 treatment altered integrin expression and matrix protein deposition, but only fibrotic TGFβ1 drove PC migration in an integrin-dependent manner. In addition, integrin-dependent PC migration was correlated to changes in EC angiopoietin-2 levels, a marker of vascular instability. Finally, there was evidence of changes in vascular stability corresponding to disease state in human SSc skin. The work presented demonstrates that TNFα and TGFβ1 induce changes in PC integrin expression and matrix deposition that facilitate migration and reduce vascular stability, providing evidence that microvascular destabilization can be an early indicator of tissue fibrosis.

RJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbβ3, a fundamental platelet glycoprotein, and αvβ3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of β-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbβ3 antagonist, capable of preventing arterial thrombosis.

Nanoarchitecture and molecular interactions of epithelial cell junction proteins revealed by super-resolution microscopy.

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin-matrix interactions. However, it is increasingly recognized that resident cell junction proteins can be found in varying locations and with previously unrecognized functions. Our study here presents the nanoarchitecture and nanocolocalization of cell junction proteins in culture and tissue by stochastic optical reconstruction microscopy (STORM). The Z-axial view of noncancerous MDCK-II and PZ-HPV-7 cell-cell junctions resolved β-catenin and p120ctn localizations to TJs and AJs, with p120ctn apical to β-catenin and colocalizing with TJ protein claudin-7. More basally, p120ctn and β-catenin become colocalized. This topography was lost in isogenic Ras-transformed MDCK cells and cancerous PC3 cells, where p120ctn becomes basally localized in relation to β-catenin. Claudin-7 gene conditional knockout (cKO) in mice also have altered polarity of p120ctn relative to β-catenin, like that seen in normal-to-cancer cell phenotypic transformation. Additionally, claudin-7 cKO resulted in redistribution and relocalization of other cell junction proteins, including claudin-1, zonula occludens-1, integrin α2, epithelial cell adhesion molecule, and focal adhesion kinase (FAK); specifically, integrin α2 and FAK were observed at the apical-lateral compartment. Our data show that STORM reveals regional cellular junction nanoarchitecture previously uncharacterized, providing new insight into potential trans-compartmental modulation of protein functions.

Prognostic Potential of Secreted Modular Calcium-Binding Protein 1 in Low-Grade Glioma.

Background: Secreted modular calcium-binding protein 1 (SMOC1) belongs to a family of matricellular proteins; it was involved in embryo development, endothelial cell proliferation, angiogenesis, integrin–matrix interactions, cell adhesion, and regulation of glucose metabolism. Previous studies showed that the expression of SMOC1 was increased in some tumors. However, the prognostic value and the biological function of SMOC1 in tumor remain unclear. Methods: In this study, we explored the expression profile and prognostic value of SMOC1 in pan-cancers, especially glioma, via multiple databases, including Oncomine, Gene Expression Profiling Interactive 2, PrognoScan, Kaplan–Meier plotter, and the Chinese Glioma Genome Atlas database. Furthermore, LinkedOmics was used to identify the genes coexpressed with SMOC1 and to perform Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology analysis in low-grade glioma (LGG). Also, the Cancer Single-Cell State Atlas database was used to evaluate the correlation between SMOC1 expression and functional state activities in glioma cells. In addition, the Tumor Immune Estimation Resource and TISIDB databases were used to evaluate the correlations between SMOC1 expression and tumor-infiltrating immune cells in the tumor microenvironment. Results: Compared with normal brain tissues, the expression of SMOC1 was increased in LGG tissues. The higher expression of SMOC1 was significantly correlated with better survival of LGG patients. Additionally, functional analyses showed that the SMOC1 coexpressed genes were inhibited in processes such as response to type I interferon and interferon-gamma, lymphocyte-mediated immunity, leukocyte migration, adaptive immune response, neutrophil-mediated immunity, T cell activation, and pathways including EMC–receptor interaction, Th17 cell differentiation, and leukocyte trans-endothelial migration in LGG. Moreover, the expression of SMOC1 was correlated with stemness, hypoxia, EMT, and metastasis of glioma cells. Additionally, the expression of SMOC1 expression was negatively correlated with levels of infiltrating B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells, and gene markers of most immune cells in LGG. Conclusion: Our results suggest that SMOC1 could be a potential biomarker to determine prognosis and might play a specific role in the tumor microenvironment of glioma, thereby influencing the development and progression of glioma. These findings provide some new insights for further investigation.

Nanoarchitecture and Molecular Interactions of Epithelial Cell Junction Proteins Revealed by Super‐Resolution Microscopy

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin-matrix interactions. However, it is increasingly recognized that their characteristic resident proteins can be found in each other's territories and demonstrate previously unrecognized functions. This study presents the nanoarchitecture and nano-colocalization of cell junction proteins in culture and in intestinal tissue by Stochastic Optical Reconstruction Microscopy (STORM). STORM allows for resolution of approximately 20 nm in the xy plane and 50 nm in the z plane. The Z-axial view of non-cancerous MDCK-II cell AJ proteins, β-catenin and p120ctn, showed greatest expression density towards the apical milieu of AJ. This is accompanied by the distinctive displacement of p120ctn being apical to β-catenin. Towards the basal direction, p120ctn and β-catenin become increasingly colocalized. Interestingly, this topography is lost in metastatic prostate cancer PC3 cells, in that p120ctn localization becomes basal to β-catenin. The deletion of TJ protein claudin-7 is sufficient in altering the polarity of p120ctn to basal β-catenin localization, like that seen in PC3 cells compared to MDCK-II. Loss of claudin-7 also significantly altered localization and distribution of many cell junction proteins. Therefore, STORM revealed the regional nanoarchitecture of cellular junctions that were previously unavailable, providing new insights in their potential trans-compartmental modulation of protein functions.

Optogenetic control of integrin-matrix interaction

Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix–OptoIntegrin system may serve as a blueprint for rendering matrix–receptor interactions amendable to precise control with light.

Analysis of Collagen-Binding Integrin Interactions with Supramolecular Aggregates of the Extracellular Matrix.

Integrin-mediated interactions of cells with the extracellular matrix (ECM) are important for their activities. The ECM itself is a complex network of macromolecules forming aggregates or suprastructures. Moreover, the molecular composition is important for the macromolecular organization and, thereby, the functional properties of the ECM. In addition, collagen molecules lose their integrin-binding capabilities after incorporation into fibrils. Therefore, we have established detailed protocols for the analysis of integrin-matrix interactions at the supramolecular level.

Normal and Fibrotic Rat Livers Demonstrate Shear Strain Softening and Compression Stiffening: A Model for Soft Tissue Mechanics.

Tissues including liver stiffen and acquire more extracellular matrix with fibrosis. The relationship between matrix content and stiffness, however, is non-linear, and stiffness is only one component of tissue mechanics. The mechanical response of tissues such as liver to physiological stresses is not well described, and models of tissue mechanics are limited. To better understand the mechanics of the normal and fibrotic rat liver, we carried out a series of studies using parallel plate rheometry, measuring the response to compressive, extensional, and shear strains. We found that the shear storage and loss moduli G’ and G” and the apparent Young's moduli measured by uniaxial strain orthogonal to the shear direction increased markedly with both progressive fibrosis and increasing compression, that livers shear strain softened, and that significant increases in shear modulus with compressional stress occurred within a range consistent with increased sinusoidal pressures in liver disease. Proteoglycan content and integrin-matrix interactions were significant determinants of liver mechanics, particularly in compression. We propose a new non-linear constitutive model of the liver. A key feature of this model is that, while it assumes overall liver incompressibility, it takes into account water flow and solid phase compressibility. In sum, we report a detailed study of non-linear liver mechanics under physiological strains in the normal state, early fibrosis, and late fibrosis. We propose a constitutive model that captures compression stiffening, tension softening, and shear softening, and can be understood in terms of the cellular and matrix components of the liver.

Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate.

Stem cells respond to nanoscale surface features, with changes in cell growth and differentiation mediated by alterations in cell adhesion. The interaction of nanotopographical features with integrin receptors in the cells' focal adhesions alters how the cells adhere to materials surfaces, and defines cell fate through changes in both cell biochemistry and cell morphology. In this Review, we discuss how cell adhesions interact with nanotopography, and we provide insight as to how materials scientists can exploit these interactions to direct stem cell fate and to understand how the behaviour of stem cells in their niche can be controlled. We expect knowledge gained from the study of cell-nanotopography interactions to accelerate the development of next-generation stem cell culture materials and implant interfaces, and to fuel discovery of stem cell therapeutics to support regenerative therapies.

Abstract 379: Potential Protective Effects of Semaphorin3A on Macrophage Function

Semaphorin3A (SEMA3A) is a secreted member of the Semaphorin family, first found to play a role in axon guidance. Recently many studies revealed immune functions for this protein. However, how SEMA3A may regulate monocyte and macrophage functions associated with atherosclerosis is unknown. An early step in plaque formation is the adhesion of monocytes on activated endothelial cells expressing adhesion molecules. However, we found that 25-100 ng/ml SEMA 3A did not affect the adhesion of monocyes on ICAM-1- or VCAM-1-coated plates. Similarly, stimulation of mouse carotid arteries with SEMA3A in an ex-vivo perfusion system, either administered alone or in conjunction with TNF-α, did not increase adhesiveness of monocytes injected in the intraluminal compartment. Furthermore SEMA3A did not stimulate monocyte migration in a boyden chamber assay. However SEMA3A had significant effects on macrophages, the most abundant cells in the plaque. Inefficient apoptotic cell phagocytosis and macrophage retention in the artery wall are hallmarks of atherosclerosis progression. We found that SEMA3A increased the migration of M2 “wound healing” macrophages more than 4-fold (p<0.05), but had no such effect on M1 “inflammatory” macrophages. Increased M2 migration was associated with phosphorylation of FAK Y397, and could be inhibited by RGD peptide that prevents integrin-matrix interaction and by a blocking antibody targeting the SEMA3A receptor Neuropilin-1. SEMA3A (100ng/ml) also significantly increased phagocytosis of apoptotic cells by M2 macrophages (1.8-fold, p<0.01) but not M1 cells. Interestingly, Sema3A did not affect lipoprotein uptake by macrophages, which characterizes foam cell formation. Finally in an in vivo model of acute inflammation, SEMA3A plasmid pretreatment reduced redness, swelling, and macrophage infiltration associated with LPS injection.In summary, our data suggest that SEMA3A is unlikely to participate in atherosclerotic plaque formation. On the contrary, effects of SEMA3A on macrophage function in vitro and in vivo indicate a potential protective effect in the inflammatory setting.

Integrin-substrate interactions underlying shear-induced inhibition of the inflammatory response of endothelial cells

Conditioning of endothelial cells by shear stress suppresses their response to inflammatory cytokines. We questioned whether signalling through different integrin-matrix interactions, previously associated with the pathogenic effects of disturbed flow, supported the anti-inflammatory action of steady shear. Primary human endothelial cells were cultured on different substrates and exposed to shear stress (2.0Pa) for varying periods before stimulation with tumour necrosis factor-α (TNF). Shear-conditioning inhibited cytokine-induced recruitment of flowing neutrophils. However, the effect was similar for culture on collagen, laminin or fibronectin, even when seeding was reduced to 2 hours, and shear to 3 hours before TNF treatment (to minimise deposition of endothelial matrix). Nevertheless, in short- or longer-term cultures, reduction in expression of β(1)-integrin (but not β(3)-integrin) using siRNA essentially ablated the effect of shear-conditioning on neutrophil recruitment. Studies of focal adhesion kinase (FAK) phosphorylation, siRNA against FAK and a FAK-inhibitor (PF573228) indicated that FAK activity was an essential component downstream of β(1)-integrin. In addition, MAP-kinase p38 was phosphorylated downstream of FAK and also required for functional modification. Mechanotransduction through β(1)-integrins, FAK and p38 is required for anti-inflammatory effects of steady shear stress. Separation of the pathways which underlie pathological versus protective responses of different patterns of flow is required to enable therapeutic modification or mimicry, respectively.

VEGF-induced endothelial cell migration requires urokinase receptor (uPAR)-dependent integrin redistribution.

Vascular endothelial growth factor (VEGF)-initiated angiogenesis requires coordinated proteolytic degradation of extracellular matrix provided by the urokinase plasminogen activator/urokinase receptor (uPA/uPAR) system and regulation of cell migration provided by integrin-matrix interaction. In this study, we investigated the mechanisms underlying the uPAR-dependent modulation of VEGF-induced endothelial migration. We used flow cytometry to quantify integrins at the cell surface. Stimulation of human and murine endothelial cells with VEGF resulted in internalization of α5β1-integrins. Micropatterning and immunocytochemistry revealed co-clustering of uPAR and α5β1-integrins and retrieval via clathrin-coated vesicles. It was also contingent on receptors of the low-density lipoprotein receptor (LDL-R) family. VEGF-induced integrin redistribution was inhibited by elimination of uPAR from the endothelial cell surface or by inhibitory peptides that block the uPAR-integrin interaction. Under these conditions, the migratory response of endothelial cells upon VEGF stimulation was impaired both in vitro and in vivo. The observations indicate that uPAR is an essential component of the network through which VEGF controls endothelial cell migration. uPAR is a bottleneck through which the VEGF-induced signal must be funnelled for both focused proteolytic activity at the leading edge and for redistribution of integrins.

Integrins and Their Extracellular Matrix Ligands in Lymphangiogenesis and Lymph Node Metastasis

In the 1970s, the late Judah Folkman postulated that tumors grow proportionately to their blood supply and that tumor angiogenesis removed this limitation promoting growth and metastasis. Work over the past 40 years, varying from molecular examination to clinical trials, verified this hypothesis and identified a host of therapeutic targets to limit tumor angiogenesis, including the integrin family of extracellular matrix receptors. However, the propensity for some tumors to spread through lymphatics suggests that lymphangiogenesis plays a similarly important role. Lymphangiogenesis inhibitors reduce lymph node metastasis, the leading indicator of poor prognosis, whereas inducing lymphangiogenesis promotes lymph node metastasis even in cancers not prone to lymphatic dissemination. Recent works highlight a role for integrins in lymphangiogenesis and suggest that integrin inhibitors may serve as therapeutic targets to limit lymphangiogenesis and lymph node metastasis. This review discusses the current literature on integrin-matrix interactions in lymphatic vessel development and lymphangiogenesis and highlights our current knowledge on how specific integrins regulate tumor lymphangiogenesis.

Abstract 545: Laminin induces tamoxifen resistance through alfa 6 integrin in breast cancer cells

Abstract Even though it is now well established that the tumor microenvironment plays a central role in modulating tumor progression and response to therapy, its function as a regulator of endocrine resistance in breast cancer has not yet been thoroughly explored. We previously showed that fibronectin through beta 1 integrin induces tamoxifen resistance in breast cancer cells. The aim of this study was to determine whether other extracellular matrix components induce tamoxifen resistance and what mechanisms are involved. We found that laminin, but not collagen IV, induced tamoxifen resistance in both estrogen receptor positive (ER+) human MCF-7 and mouse LM05-E breast cancer cells (P<0.001). To understand what signaling pathways were involved, cells were treated for 48 hours with estradiol (10−8M) and 4-OH-tamoxifen (10−6M) with the addition of either the MAPK/ERK1/2 inhibitor PD98059 (10µM) or the PI3K/AKT inhibitor LY294002 (10µM). Only LY294002 reversed the protective effect of laminin (P<0.001). Next to determine what integrin was mediating endocrine resistance, cells were pre-incubated with either an alfa 6 blocking antibody (GoH3, 0.15µM) or a beta 1 integrin blocking antibody (AIIB2, 0.15µM). The alfa 6 blocking antibody interfered with tamoxifen resistance induced by laminin (P<0.01), but not the beta 1 integrin blocking antibody. To establish whether laminin or fibronectin regulated the transcriptional activity of ER we carried out luciferase reporter assays by transiently transfecting the cells with 0.2µg of a pSV40-ERE-Luc plasmid together with 0.04µg pRL-TK-Renilla as a control. As expected, estradiol 10−8M induced a significant stimulation of ER activity (P<0.001) which was counteracted by the addition of 10−6M 4-OH-tamoxifen (P<0.001). Cells were then additionally treated with either laminin or fibronectin (30µg/ml) to test whether on their own, or in the presence of estradiol and/or tamoxifen they could regulate ER activity. No regulation of the transcriptional activity of ER was found, even though we have previously shown that fibronectin stimulates the phosphorylation of ER-α in serine-118. Our results show that laminin, like fibronectin, induces tamoxifen resistance in breast cancer cells through the specific binding to alfa 6 integrin, and that the PI3K/AKT pathway mediates the protective effect downstream. However, the mechanism does not seem to involve regulation of the transcriptional activity of ER. These results suggest that integrin-matrix interactions could be a valuable molecular target for breast cancer patients that develop tamoxifen resistance. This work is supported by grants from the Susan G. Komen Foundation for the Cure (BCTR0600341), and FONCYT (PICT 2008-0325) to M.S. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 545. doi:10.1158/1538-7445.AM2011-545

Impact of integrin–matrix interaction and signaling on insulin gene expression and the mesenchymal transition of human β‐cells

A critical shortage of donor pancreata currently prevents the development of a universal cell-based therapy for type I diabetes. The ex vivo expansion of insulin-producing beta-cells offers a potential solution but is problematic due to the inherent tendency of these cells to transition into mesenchymal-like cells that are devoid of function. Here, we demonstrate for the first time that exposure to elements of the extracellular matrix (ECM) directly potentiates the mesenchymal transition of cultured fetal beta-cells and causes associated declines in insulin gene expression. Individual ECM constituents varied in their ability to induce such responses, with collagen-IV (C-IV) and fibronectin inducing strong responses, whereas laminin-1 had no significant effect. Mesenchymal transition and concomitant losses in insulin gene expression observed on C-IV were found to be dependent on beta(1)-integrin ligation and were augmented in the presence of hepatocyte growth factor. Importantly, selective inhibition of c-Src, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) prior to exposure to C-IV prevented mesenchymal transition and effectively preserved insulin expression. Fetal beta-cells undergoing mesenchymal transition were found to acquire alpha(1)beta(1) expression, and ligation of this integrin then promotes declines in insulin gene expression and a marked increase in beta-cell motility. Inhibition of Src-, ERK-, or JNK-dependent signaling combined with the selective regulation of matrix exposure may ultimately facilitate the development of more effective beta-cell expansion protocols.

Formation of endothelial lumens requires a coordinated PKCϵ-, Src-, Pak- and Raf-kinase-dependent signaling cascade downstream of Cdc42 activation

In this study, we present data showing that Cdc42-dependent lumen formation by endothelial cells (ECs) in three-dimensional (3D) collagen matrices involves coordinated signaling by PKCepsilon in conjunction with the Src-family kinases (SFKs) Src and Yes. Activated SFKs interact with Cdc42 in multiprotein signaling complexes that require PKCepsilon during this process. Src and Yes are differentially expressed during EC lumen formation and siRNA suppression of either kinase, but not Fyn or Lyn, results in significant inhibition of EC lumen formation. Concurrent with Cdc42 activation, PKCepsilon- and SFK-dependent signaling converge to activate p21-activated kinase (Pak)2 and Pak4 in steps that are also required for EC lumen formation. Pak2 and Pak4 further activate two Raf kinases, B-Raf and C-Raf, leading to ERK1 and ERK2 (ERK1/2) activation, which all seem to be necessary for EC lumen formation. This work reveals a multicomponent kinase signaling pathway downstream of integrin-matrix interactions and Cdc42 activation involving PKCepsilon, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to control EC lumen formation in 3D collagen matrices.

Differential Wnt pathway activation in triple negative breast cancers comparative to HER2 and hormone positive breast cancers identified from formalin-fixed, paraffin-embedded tissues.

Abstract Abstract #43 Background: Patients afflicted with triple negative (TN) breast cancer (BC) are typically younger and have the worst prognosis of any BC subtype as a result of its aggressive and invasive phenotype. TN BC has been well studied with respect to the associative and prognostic values of its mRNA expression profiles. We have used the Illumina DASL (cDNA mediated, Annealing, Selection, Extension, and Ligation) platform to characterize 502 genes from 96 BC patients sourced from formalin-fixed, paraffin-embedded (FFPE) tissues. RNA extracted from FFPE tissues is highly fragmented and typically unsuited for expression platforms, but recent advances have allowed for accurate expression profiling of RNA from FFPE tissues. This 96 patient cohort included 21 TN BC patients identified by immunohistochemistry (IHC); differential analysis of TN expression was integrated with known pathway mechanisms and identified activation of the Wnt pathway in TN tumors.
 Material and Methods: The Illumina DASL assay was used to measure mRNA expression levels from BC patients obtained from St. Mary's Hospital, Montreal, Quebec. RNA was extracted from three 5µm tissue sections from FFPE blocks using the RNA High Pure Kit (Roche). Differential mRNA regulation was identified by Significance Analysis of Microarrays (SAM) software using a false discover rate (FDR, q-value < 0.01) or a Bonferroni corrected two-tailed student's t-test (p-value < 0.05). IHC concordance was measured by mRNA expression fold-change for positive versus negative IHC categories.
 Results: 165 samples from 96 patients were characterized for mRNA expression on 1,488 probes across 502 genes in the DASL assay. Quality control limited the analysis to 150 samples from 91 patients over 500 genes. Comparison of the TN subtype relative to HER2 positive and hormone positive breast carcinomas identified up-regulation [fold-change] of MMP7 [2.9], MMP9 [2.0], MMP1 [1.8], NOTCH1 [1.5], FZD7 [1.5], PPARD [1.3], CCND2 [1.2], MET [1.3], MYC [1.5], and MYCN [2.0] in the TN subtype. Most of these genes are either a component or downstream target of the Wnt signaling pathway. Down-regulated relevant genes included ESR1 [-8.4], PGR [-3.9], ERBB2 [-3.4] as expected, as well as ERRB3, ERBB4, GLI3, TFF1, LAF4/AFF3, and AR.
 Discussion: Down-regulation of TFF1 suggests inhibition of the ERK/MAPK pathways which have complex effects on cell-cell adhesion. In combination with over-expression of MMPs 1, 7, and 9, these changes may contribute to the invasive nature of TN BCs. GLI3 down-regulation may attenuate Hedgehog signaling potentially contributing to Wnt activation, further leading to cell migration, invasion, and decrease in cell adhesion to cognate integrin-matrix interaction Up-regulation of both the FZD7 receptor as well as the Wnt targets MMP7, MYC, MYCN, PPARD, and CCND2 strongly suggest Wnt pathway activation in the TN subtype. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 43.

Integrin Trafficking Regulated by Rab21 Is Necessary for Cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

Differential regulation of the lateral mobility of plasma membrane phospholipids by the extracellular matrix and cholesterol

In this study, we compared qualitative and quantitative changes in the lateral mobility of phospholipid molecules in the plasma membrane of intact cells under various conditions of specific interaction of integrins in the cell membrane with two extracellular matrix (ECM) components viz. fibronectin (FN) and laminin (LN). We found a strong and specific correlation between the lower lateral mobility of phosphatidylcholine (PC) and higher lateral mobility of phosphatidylethanolamine (PE) when cells were expressing high levels of alpha5beta1 integrin and thus were adherent and motile on FN. The interaction between PC and FN in alpha5 integrin expressing cells was aided by the strong affinity of alpha5 integrin to the FN matrix. Cholesterol was involved in regulating the lateral mobility of PC to a great extent and of PE to a lesser extent without affecting the overall microviscosity of the plasma membrane or the distribution of caveolin-marked domains. The distribution and mobility of PC and PE molecules in the lamellipodial regions differed from that in the rest of the membrane and also in the more motile and in the less motile cells. We propose that these differences in distribution of PC and PE in different regions of cell membrane and their respective lateral mobility are observed due to the specific interaction of PC molecules with FN molecules in the ECM. Our results outline a new role of integrin-matrix interactions in the regulation of membrane phospholipid behavior.

Understanding the Molecular Basis for Differential Binding of Integrins to Collagen and Gelatin

Integrin-mediated cell adhesion plays a central role in cell migration and signaling. Overexpression of integrins is also associated with cancer invasion and metastasis. Although a number of problems in integrin-matrix interactions have been studied in detail, the molecular specificity, which increases integrin adhesion to native collagen but results in poor integrin-gelatin interaction, is not understood. In this report, we study the role of individual amino acids in integrin-collagen and integrin-gelatin interactions using long-term (>100 ns) molecular simulations. The results, which are force-field independent, show that denatured collagen induces helical conformations in integrin amino acids and significantly reduces the poly-proline II content, which stabilizes the integrin-collagen interactions. Our simulations provide a possible explanation of the molecular specificity in integrin binding and suggest new targets for regulating integrin-mediated invasion and metastasis.