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ABSTRACT: Interactions with the extracellular matrix (ECM) provide cells with physical and chemical cues that act in concert with growth factors to support survival and proliferation. Transmembrane receptors of the integrin family mediate ECM attachment and play important roles in sensing and responding to ECM properties. Integrin signaling involves large integrin-associated intracellular protein complexes that act as anchors for the cytoskeleton and as signaling hotspots where enzymes and substrates are concentrated. Moreover, many different growth factor signaling cascades are amplified when cells are attached to the ECM. Integrins are involved in many pathologies; here we focus on their roles in cancer. Although "anchorage-independence" is a hallmark of cancer cells, genetic studies clearly show that integrins and associated proteins provide essential support for early tumor development and growth. Integrins also provide support during later stages of tumor progression but in some scenarios they appear to have suppressive activity, which is currently not understood.
The international journal of biochemistry & cell biology 02/2013; · 4.89 Impact Factor
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Jordi Carreras Puigvert,
Louise von Stechow,
Ramakrishnaiah Siddappa,
Alex Pines,
Mahnoush Bahjat,
Lizette C J M Haazen,
Jesper V Olsen,
Harry Vrieling,
John H N Meerman,
Leon H F Mullenders,
Bob van de Water, Erik H J Danen
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ABSTRACT: In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.
Science Signaling 01/2013; 6(259):ra5. · 7.50 Impact Factor
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ABSTRACT: A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.
PLoS ONE 01/2012; 7(2):e31281. · 4.09 Impact Factor
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ABSTRACT: Cell spheroids (CS) embedded in 3D extracellular matrix (ECM) serve as in vitro mimics for multicellular structures in vivo. Such cultures, started either from spontaneous cell aggregates or single cells dispersed in a gel are time consuming, applicable to restricted cell types only, prone to high variation, and do not allow CS formation with defined spatial distribution required for high-throughput imaging. Here, we describe a method where cell-polymer suspensions are microinjected as droplets into collagen gels and CS formation occurs within hours for a broad range of cell types. We have automated this method to produce CS arrays in fixed patterns with defined x-y-z spatial coordinates in 96 well plates and applied automated imaging and image analysis algorithms. Low intra- and inter-well variation of initial CS size and CS expansion indicates excellent reproducibility. Distinct cell migration patterns, including cohesive strand-like - and individual cell migration can be visualized and manipulated. A proof-of-principle chemical screen is performed identifying compounds that affect cancer cell invasion/migration. Finally, we demonstrate applicability to freshly isolated mouse breast and human sarcoma biopsy material - indicating potential for development of personalized cancer treatment strategies.
Biomaterials 01/2012; 33(1):181-8. · 7.40 Impact Factor
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Alex Pines,
Christian D Kelstrup,
Mischa G Vrouwe,
Jordi C Puigvert,
Dimitris Typas,
Branislav Misovic,
Anton de Groot,
Louise von Stechow,
Bob van de Water, Erik H J Danen,
Harry Vrieling,
Leon H F Mullenders,
Jesper V Olsen
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ABSTRACT: Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response.
Molecular and cellular biology 12/2011; 31(24):4964-77. · 6.06 Impact Factor
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Hildegonda P H Naber,
Eliza Wiercinska,
Evangelia Pardali,
Theo van Laar,
Ella Nirmala,
Anders Sundqvist,
Hans van Dam,
Geertje van der Horst,
Gabri van der Pluijm,
Bertrand Heckmann, Erik H J Danen,
Peter Ten Dijke
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ABSTRACT: The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.
In this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay.
BMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin α(v)β(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β(3) negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β(3) counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion.
Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β(3).
Cellular oncology (Dordrecht). 09/2011; 35(1):19-28.
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ABSTRACT: Apoptosis is important for embryonic development, tissue homeostasis, and removal of cells with (potentially transforming) DNA lesions or other types of injuries. Functional genomics screens performed to unravel apoptotic signaling cascades in the context of toxicant-induced cell injury commonly use apoptosis as an end-point. Here, a method to detect the accumulation of apoptotic cells in real time that is well suited for high-throughput screens is described. The method uses automated microscopy in a 96-well format setting to visualize binding of fluorescent annexin V to the outer membrane leaflet of apoptotic cells. The automated image acquisition is followed by quantitative analysis using bioinformatics software. A protocol for each of the steps in this kinetic method is described, which includes the caspase-dependent apoptotic response to toxic compounds in multiple cell types and demonstrates that RNAi-based gene silencing of candidate apoptotic regulators affects the apoptosis kinetics as expected. This protocol will be useful for functional genomics as well as chemical (drug) screens.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 06/2010; Chapter 18:Unit 18.10.1-13.
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ABSTRACT: Activation of Src family kinases is an important event downstream of integrin adhesion signaling in many cell types. A particularly intriguing connection between an integrin and a Src family kinase was first discovered in platelets, where the selective direct interaction of alphaIIbbeta3 integrins with c-Src promotes full kinase activation of c-Src through its local clustering by the cytoplasmic tail of the beta3 integrin subunit. The same integrin beta3-c-Src interaction not only drives platelet aggregation, but it also promotes the oncogenic potential of c-Src and drives tumor growth by alphavbeta3-expressing tumor cells, which may explain why increased activity of c-Src and elevated levels of integrin alphavbeta3 are often found in the same tumor types. Moreover, recent evidence from patient material and in vivo studies strongly indicate that this oncogenic signaling complex, consisting of c-Src and alphavbeta3, underlies tumor progression of human tumors. Here, we give an overview of the beta3-c-Src interaction and its implications for signaling in platelets and tumor cells, and we mention the possibilities for therapeutic intervention that is aimed at disrupting the beta3-c-Src interaction for antithrombotic and anticancer purposes.
TheScientificWorldJOURNAL 01/2010; 10:1100-5. · 1.66 Impact Factor
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ABSTRACT: PURPOSE: Tumours are composed of a heterogeneous cell population. Cancer stem cells, which make up a minor fraction of a tumour, may be the cells that initiate and sustain tumour growth. Cancer stem cells are believed to share many properties with normal stem cells that render them relatively insensitive to classical radio- and chemotherapy. CONCLUSIONS: We discuss what those (cancer) stem cell properties are and how the interactions with the microenvironment--'the niche'--control those aspects of (cancer) stem cell biology. We also describe possible strategies to target cancer stem cells in order to prevent cancers from escaping therapy.
International Journal of Radiation Biology 11/2009; 85(11):955-62. · 2.28 Impact Factor
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ABSTRACT: The adult, virgin mammary gland is a highly organized branched ductal network comprising two major cell types: myoepithelial and luminal epithelial cells. To study the role and mechanism of focal adhesion kinase (FAK)-mediated signaling in mammary gland development and differentiation, we used a conditional Fak-knockout mammary epithelial cell (MEC) transplantation model. Conditional Cre recombinase (Cre)-mediated Fak deletion in primary cultured MECs isolated from FAK(lox/lox)/Rosa26Cre-ERT2 donor mice caused loss of FAK in all mammary cells. Transplantation of Fak-knockout MECs in a cleared mammary fat pad of immune-deficient recipient mice resulted in development of new but dilated virgin ducts with a disrupted myoepithelial and luminal epithelial cell multilayer and aberrant ductal morphogenesis during pregnancy. In the absence of FAK, MECs spread poorly, showed enhanced Rho kinase (ROCK)-mediated cytoskeletal contractility, and failed to respond to receptor-mediated cytoskeletal remodeling. Likewise, FAK deficiency fully inhibited branching morphogenesis of mammary gland organoids in a ROCK-dependent manner. Altogether these data suggest a model in which FAK coordinates contractile forces in MECs to maintain the bilayered cellular organization of myoepithelial and luminal epithelial cells in ducts, thus allowing proper mammary gland development and function.
The FASEB Journal 08/2009; 23(10):3482-93. · 5.71 Impact Factor
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ABSTRACT: Interactions between cells and the extracellular matrix coordinate signaling pathways that control various aspects of cellular behavior. Integrins sense the physical properties of the extracellular matrix and organize the cytoskeleton accordingly. In turn, this modulates signaling pathways that are triggered by various other transmembrane receptors and augments the cellular response to growth factors. Over the past years, it has become clear that there is extensive crosstalk between integrins, Src-family kinases and Rho-family GTPases at the heart of such adhesion signaling. In this Commentary, we discuss recent advances in our understanding of the dynamic regulation of the molecular connections between these three protein families. We also discuss how this signaling network can regulate a range of cellular processes that are important for normal tissue function and disease, including cell adhesion, spreading, migration and mechanotransduction.
Journal of Cell Science 05/2009; 122(Pt 8):1059-69. · 6.11 Impact Factor
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ABSTRACT: Chemotherapy often relies on cancer cell death resulting from DNA damage. The p53 tumor suppressor pathway that is an important player in DNA damage response is frequently inactivated in cancer. Genotoxicants also activate DNA damage-independent stress pathways and activity of oncogenic signaling and adhesive interactions with the cancer microenvironment can have a strong impact on chemosensitivity. Here, we have investigated how two different oncogenes modulate the response to genotoxicants in the context of two classes of integrin adhesion receptors. Epithelial cells expressing either beta1 or beta3 integrins, in which p53 activity is suppressed, undergo G(2) arrest but show little apoptosis after treatment with cisplatin or other genotoxicants. The apoptotic response is strongly enhanced by the c-Src[Y530F] oncogene in cells expressing beta1 integrins, whereas such sensitization is reduced when these cells are engineered to express beta3 integrins instead. The H-Ras[G12V] oncogene fails to sensitize, regardless of the integrin expression profile. The enhanced sensitivity induced by c-Src[Y530F] in the context of beta1 integrins does not rely on p53-mediated DNA damage signaling but instead involves increased endoplasmic reticulum stress and caspase-3 activation. Our data implicate that the expression profiles of oncogenes and integrins strongly affect the response to chemotherapeutics and may thus determine the efficacy of chemotherapy.
Molecular pharmacology 02/2009; 75(4):947-55. · 4.53 Impact Factor
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Erik H J Danen
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ABSTRACT: Integrin transmembrane receptors orchestrate signaling cascades by recruiting cytoskeletal linker proteins and enzymes to sites of cell adhesion. A proteomics-based view of such integrin-associated signaling networks is now available. Besides the usual suspects, the interactomes contain several proteins that were not previously connected to integrins. One of these, regulator of chromosome condensation-2 (RCC2), represents an unexpected molecular connection between integrins and the cell-migration machinery.
Science Signaling 01/2009; 2(89):pe58. · 7.50 Impact Factor
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ABSTRACT: Focal adhesions are randomly distributed across the ventral surface or along the edge of epithelial cells. In fibroblasts they orient centripetally and concentrate at a few peripheral sites connecting long F-actin stress fibers, causing a typical elongated, contractile morphology. Extensive remodeling of adhesions in fibroblasts also takes part in fibronectin fibrillogenesis, a process that depends on Rho-mediated contractility and results in the formation of a fibronectin matrix. Our current study shows that all these fibroblast characteristics are controlled by the ability of integrin alpha5 beta1 to bind soluble fibronectin molecules in their compact inactive conformation. The hypervariable region of the ligand-binding I-like domain of integrin alpha5 beta1 supports binding of soluble fibronectin. This supports the distribution of centripetally orientated focal adhesions in distinct peripheral sites, Rho activation and fibronectin fibrillogenesis through a mechanism that does not depend on Syndecan-4. Integrin alpha v beta3, even when locked in high affinity conformations for the RGD recognition motif shows no appreciable binding of soluble fibronectin and, consequently, fails to support the typical fibroblast focal adhesion distribution, Rho activity and fibronectin fibrillogenesis in the absence of integrin alpha5 beta1. The ability of alpha5 beta1 integrin to interact with soluble fibronectin may thus drive the cell-matrix adhesion and cytoskeletal organization required for a contractile, fibroblast-like morphology, perhaps explaining why alpha5 beta1 integrin, similarly to fibronectin, is essential for development.
Journal of Cell Science 08/2008; 121(Pt 15):2452-62. · 6.11 Impact Factor
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ABSTRACT: Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin alphavbeta3 strongly supports Src-mediated oncogenic transformation through an interaction at the beta3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, beta1 and beta3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of beta3, but not beta1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the beta3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of beta3 phosphorylation. Instead, phosphorylation of beta1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.
Journal of Biological Chemistry 06/2008; 283(19):13243-51. · 4.77 Impact Factor
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ABSTRACT: Integrin-mediated adhesion regulates multiple signaling pathways. Our group previously showed that ectopic expression of different integrin beta-subunits in the neuroepithelial cell line GE11, has distinct effects on cell morphology, actin cytoskeletal organization, and on focal contact distribution. In this report we have investigated changes in gene transcription levels resulting from overexpression of the integrin beta3 subunit. We found that beta3 overexpression leads to the transcriptional downregulation of MARCKS related protein (MRP) resulting in a decreased expression of the MRP protein. Furthermore, we show that the Ras/MAPK pathway controls the basal level of MRP expression but beta3 overexpression bypasses this pathway downstream of ERK to downregulate MRP. Further studies indicate that a region of the cytoplasmic tail of beta3 containing part of the NITY motif is responsible for increased cell spreading and MRP downregulation. However, MRP overexpression failed to inhibit the beta3-induced increase in cell spreading while the knock down of MRP expression in GE11 cells did not increase cell spreading. We suggest that the downregulation of MRP by beta3 is not required for increased cell spreading but instead that MRP downregulation is a secondary effect of increased cell spreading.
Experimental Cell Research 05/2007; 313(6):1260-9. · 3.58 Impact Factor
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ABSTRACT: Increased activity of the proto-oncogene c-Src and elevated levels of integrin alpha(v)beta(3) are found in melanomas and multiple carcinomas. Regulation of c-Src involves "priming" through disruption of intramolecular interactions followed by "activation" through phosphorylation in the kinase domain. Interactions with overexpressed receptor tyrosine kinases or mutations in the SRC gene can induce priming of c-Src in cancer. Here, we show that alpha(v)beta(3) promotes activation of primed c-Src, causing enhanced phosphorylation of established Src substrates, survival, proliferation, and tumor growth. The beta(3) cytoplasmic tail is required and sufficient for integrin-mediated stimulation of all these events through a mechanism that is independent of beta(3) tyrosine phosphorylation. Instead, experiments using Src variants containing the v-Src Src homology 3 (SH3) domain and using mutant beta(3) subunits indicate that a functional interaction of the beta(3) cytoplasmic tail with the c-Src SH3 domain is required. These findings delineate a novel integrin-controlled oncogenic signaling cascade and suggest that the interaction of alpha(v)beta(3) with c-Src may represent a novel target for therapeutic intervention.
Cancer Research 04/2007; 67(6):2693-700. · 7.86 Impact Factor
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ABSTRACT: During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins. Here, we show that beta1 integrins promote random migration, whereas beta3 integrins promote persistent migration in the same epithelial cell background. Adhesion to fibronectin by alpha(v)beta3 supports extensive actin cytoskeletal reorganization through the actin-severing protein cofilin, resulting in a single broad lamellipod with static cell-matrix adhesions at the leading edge. Adhesion by alpha5beta1 instead leads to the phosphorylation/inactivation of cofilin, and these cells fail to polarize their cytoskeleton but extend thin protrusions containing highly dynamic cell-matrix adhesions in multiple directions. The activity of the small GTPase RhoA is particularly high in cells adhering by alpha5beta1, and inhibition of Rho signaling causes a switch from a beta1- to a beta3-associated mode of migration, whereas increased Rho activity has the opposite effect. Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases.
The Journal of Cell Biology 06/2005; 169(3):515-26. · 10.26 Impact Factor
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Erik H J Danen
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ABSTRACT: Integrins are adhesion receptors that connect cells to components of the extracellular matrix or to counter receptors on other cells. Besides mediating stable adhesion, these receptors are implicated in the deposition of extracellular matrices and they are crucial for cell migration. Integrin-mediated adhesion also modulates signal transduction cascades downstream of other receptors and thereby regulates cell survival, proliferation, and the expression of differentiation-related genes. In this review, an overview of the evidence for roles of integrins in tissue development and function is given and the contribution of changes in integrin expression to cancer is discussed.
Current Pharmaceutical Design 02/2005; 11(7):881-91. · 3.87 Impact Factor
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ABSTRACT: Cell adhesion is indispensable for embryonic development and for proper tissue function. In metazoans, integrins are the major adhesion receptors that connect cells to components of the extracellular matrix. Integrins are implicated in assembly of extracellular matrices, cell adhesion and migration on extracellular matrices, and in vertebrates (in which the integrin family has expanded) they can also mediate cell-cell adhesion. Furthermore, integrin-mediated adhesion can modulate many different signal transduction cascades and support cell survival, proliferation, and influence the expression of differentiation-related genes. In this review we briefly explain how integrins can affect so many different aspects of cell behavior and discuss evidence for roles of integrins in tissue development, function, and disease.
The Journal of Pathology 01/2004; 201(4):632-41. · 6.32 Impact Factor
