Patent Application
20150079113
Throughout this application various publications, books, patents and patent application publications are referred to. The disclosures of all of these are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
The extracellular matrix (ECM) is a three dimensional network of proteins secreted, assembled and remodeled dynamically by cells that it contacts (Hynes and Naba, 2012; Wickstrom et al., 2011). Cell migration, differentiation and other processes are controlled by the ECM as it engages adhesion receptors and presents matrix-bound growth factors to their cell surface receptors. One of the best-characterized ECM proteins is fibronectin (FN), an abundant, ubiquitous component of the interstitial matrix (Singh et al., 2010). Outside of the bloodstream, FN typically functions in multimeric fibrils assembled by cells from soluble FN dimers and organized into complex meshworks (Schwarzbauer and DeSimone, 2011). These elaborate FN matrices surround and connect cells, providing a supporting scaffold capable of delivering complex sets of multivalent, spatially organized biochemical and mechanical signals that influence many aspects of cell behavior (Hynes, 2009; Huttenlocher and Horwitz, 2011; Geiger and Yamada, 2011).
The predominant ECM receptors proteins are integrins, a family of heterodimeric transmembrane proteins comprised of α and β subunits that link the ECM to the cytoskeleton and transmit signals and mechanical forces bi-directionally across the plasma membrane (Hynes, 2002). Integrins are regulated by clustering and conformational changes triggered either “outside in” by binding to their specific ECM ligands, or “inside out” by interaction between the intracellular tails of integrin subunits and cytoplasmic proteins (Margadant et al., 2011). The β subunit cytoplasmic tails share significant sequence similarity; several cytoplasmic proteins directly bind most β subunits to regulate integrin activation, trafficking and signaling (Moser et al., 2009; Calderwood, 2004). In contrast, the a integrin subunit tails share only a short, conserved membrane-proximal sequence that interacts directly with the β subunit and with proteins that regulate integrin trafficking (Ivaska and Heino, 2011) and with Sharpin, a negative regulator of integrin activation (Rantala et al., 2011). Less is known, however, about the potential unique functions conferred by the distal, divergent cytoplasmic tails of the 18 α subunits.
The two major FN receptors are αVβ3 and α5β1 (Hynes, 2002). α5β1 is the primary receptor for soluble FN and plays the predominant role in assembling FN into fibrils, though αVβ3 can assemble fibrils in cells lacking α5β1 (Yang et al., 1999). While αVβ3 and α5β1 can substitute for one another partially, typically they exert distinct effects on cell motility, invasion, signaling and matrix remodeling (Clark et al., 2005; Wickstrom et al., 2011; Caswell et al., 2009). For example, αVβ3 suppresses recycling of the epidermal growth factor receptor (EGFR), while inhibition or absence of αVβ3 drives α5β1 into a protein complex with EGFR mediated by Rab coupling protein (RCP) that drives coordinate recycling of the two receptors, dysregulates their signaling and promotes tumor cell invasion (Caswell et al., 2008; Muller et al., 2009).
Integrin-based ECM adhesions are dynamic, complex structures that turn over continually while changing their composition and morphology (Geiger and Yamada, 2011). Typically, new adhesions form as small integrin-rich punctae near the leading edge of spreading or migrating cells with associated cytoplasmic proteins bound to integrin tails that recruit additional signaling, adaptor or actin-binding proteins (Vicente-Manzanares and Horwitz, 2011). Nascent adhesions enlarge into focal complexes (FXs), more elongated, transient structures that mature into focal adhesions (FAs), larger structures that vary in composition and size that connect to the distal ends of Factin bundles. In some cell types, including fibroblasts, α5β1 exits from FAs and moves toward the cell interior along stress fibers (Pankov et al., 2000) into mature fibrillar adhesions (FBs), stable internal adhesions that mediate the critical process of FN fibrillogenesis. FBs are enriched for FN, α5β1 and tensin, the latter of which is not found in FXs and only weakly in FAs (Zaidel-Bar et al., 2007; Pankov et al., 2000; Zamir et al., 2000). FBs lack many abundant FA components, including phosphotyrosine-containing proteins, vinculin, FAK and zyxin. Fibrillogenesis begins as α5β1 translocates bound to FN out of FAs to FBs. This movement generates contractile forces on the α5β1-connection between the cytoskeleton and FN leading to conformational changes in α5β1 that strengthen and prolong FN binding (Margadant et al., 2011). The tensile forces also drive conformational changes in FN that expose self-association sites and align the nascent FN fibrils with intracellular actin bundles (Schwarzbauer and DeSimone, 2011).
The Ena/VASP family of actin-regulatory proteins plays diverse roles in cell movement and morphogenesis (Drees and Gertler, 2008; Bear and Gertler, 2009; Homem and Peifer, 2009). Ena/VASP influences membrane protrusion dynamics by promoting formation of longer, less-branched F-actin networks. Ena/VASP proteins increase F-actin elongation rates by promoting transfer of actin monomer from profilin to free barbed ends while protecting growing filaments from capping proteins that terminate polymerization (Hansen and Mullins, 2010; Bear and Gertler, 2009; Dominguez, 2009). Ena/VASP proteins are concentrated in sites of rapid actin assembly such as the tips of lamellipodia and filopodia. They also localize prominently to cell:cell and cell:matrix adhesions and interact with several FA components, including vinculin, zyxin, RIAM and palladin (Pula and Krause, 2008). While the function of Ena/VASP in FAs is not well understood, they are known to regulate integrin activation. For example, VASP negatively regulates αIIbβ3 activation in platelets (Aszodi et al., 1999; Hauser et al., 1999).
The three vertebrate Ena/VASP proteins Mena, VASP, and EVL share conserved domains (Gertler et al., 1996), including: 1) an N-terminal EVH1 domain that binds to proteins that typically contain one or more EVH1-binding sites with an optimal core motif of “FPPPP” (FP4) (Ball et al., 2002), though unconventional EVH1 ligands have been identified (Boeda et al., 2007); 2) a proline-rich center containing binding sites for SH3- and WW-domains, and the actin-monomer binding protein profilin (Ferron et al., 2007); 3) a C-terminal EVH2 domain that contains both G and F-actin binding sites and a coiled-coil that mediates their tetramerization (Barzik et al., 2005; Zimmermann et al., 2002) (
For example, a Mena isoform produced by alternate splicing, MenaINV (Gertler and Condeelis, 2011), promotes carcinoma metastasis by potentiating chemotactic responses to EGF (Roussos et al., 2011a; Philippar et al., 2008); but neither VASP nor EVL produce isoforms equivalent to MenaINV. Mena also has a unique low-complexity region of unknown function containing 13 repeats of α5-residue motif within a 91-residue span, termed the LERER-repeat (Gertler et al., 1996) (
The present invention provides novel treatments and assays based on the discovery of the interaction of Mena with integrins as disclosed hereinbelow.
A method is provided of treating invasion of a tumor in a subject or inhibiting metastasis of a tumor in a subject comprising administering to the subject an agent which inhibits the interaction of Mena with an alpha5 integrin in an amount effective to treat invasion or inhibit metastasis of a tumor.
A method is also provided of treating a fibronectin deposition disease in a subject or a fibroproliferative disease in a subject comprising administering to the subject an agent which inhibits the interaction of Mena with an alpha5 integrin in an amount effective to treat fibronectin deposition or fibroproliferative disease.
A method for identifying an agent as an inhibitor of an interaction of Mena with an alpha5 integrin, the method comprising contacting the alpha5 integrin with Mena (a) in the presence of and (b) in the absence of the agent under conditions permitting Mena to interact with the alpha5 integrin and quantifying the interaction of Mena with the alpha5 integrin in the presence and in the absence of the agent, and identifying the agent as an inhibitor or not of an interaction of Mena with an alpha5 integrin, wherein quantification of a decreased interaction of Mena with the alpha5 integrin in the presence of the agent compared to in the absence of the agent indicates that the agent is an inhibitor of the interaction of Mena with the alpha5 integrin, and wherein quantification of no change in interaction, or an increased interaction, of Mena with the alpha5 integrin in the presence of the agent compared to in the absence of the agent indicates that the agent is not an inhibitor of the interaction of Mena with the alpha5 integrin.
Additional objects of the invention will be apparent from the description which follows.
A fibronectin deposition disease is a disease which has symptoms or pathologies involving abnormal fibronectin deposition, for example a fibronectin glomerulopathy.
A fibroproliferative disease is a disease characterized by excessive accumulation of connective material in a critical location, such as fibroproliferative cardiovascular disease, pulmonary fibrosis, progressive kidney disease, systemic sclerosis, liver cirrhosis and fibroproliferative inflammatory bowel disease.
A method is provided of treating invasion of a tumor or inhibiting metastasis of a tumor in a subject comprising administering to the subject an agent which inhibits the interaction of Mena with an alpha5 integrin in an amount effective to treat invasion or inhibit metastasis of a tumor.
A method is also provided of treating a fibronectin deposition disease or a fibroproliferative disease in a subject comprising administering to the subject an agent which inhibits the interaction of Mena with an alpha5 integrin in an amount effective to treat fibronectin deposition or fibroproliferative disease.
In an embodiment, the agent inhibits the interaction of Mena with the C-terminal 5 residues of the alpha5 integrin C-terminal cytoplasmic tail. In an embodiment, the agent inhibits the interaction of a LERER repeat region of Mena with the alpha5 integrin. In an embodiment, the tumor is a breast cancer tumor. In an embodiment, the alpha5 integrin is part of an alpha5 beta1 integrin complex. In an embodiment, the alpha5 beta1 integrin is a fibronectin receptor. In an embodiment, the agent is a small organic molecule, an antibody, a fragment of an antibody, a peptide or an oligonucleotide aptamer. In an embodiment, the agent competes for binding to the alpha5 integrin with a LERER repeat region of Mena. In an embodiment, the Mena is human Mena. In an embodiment, the Mena is MenaINV. In an embodiment, the MenaINV is human MenaINV.
A method for identifying an agent as an inhibitor of an interaction of Mena with an alpha5 integrin, the method comprising contacting the alpha5 integrin with Mena (a) in the presence of and (b) in the absence of the agent under conditions permitting Mena to interact with the alpha5 integrin and quantifying the interaction of Mena with the alpha5 integrin in the presence and in the absence of the agent, and identifying the agent as an inhibitor or not of an interaction of Mena with an alpha5 integrin, wherein quantification of a decreased interaction of Mena with the alpha5 integrin in the presence of the agent compared to in the absence of the agent indicates that the agent is an inhibitor of the interaction of Mena with the alpha5 integrin, and wherein quantification of no change in interaction, or an increased interaction, of Mena with the alpha5 integrin in the presence of the agent compared to in the absence of the agent indicates that the agent is not an inhibitor of the interaction of Mena with the alpha5 integrin.
In an embodiment, quantifying the interaction of Mena with the alpha5 integrin in the presence of and in the absence of the agent comprises quantifying the amount of Mena bound to alpha5 integrin. In an embodiment, quantifying the interaction of Mena with the alpha5 integrin in the presence and in the absence of the agent comprises quantifying the activity of alpha5 integrin. In an embodiment, the alpha5 integrin is part of an alpha5 beta1 integrin complex. In an embodiment, the agent is a small organic molecule, an antibody, a fragment of an antibody, a peptide or an oligonucleotide aptamer.
Assay techniques for use in the methods of the invention can comprise, in non-limiting examples, immunoprecipitation, protein purification, blots, and/or proximity ligation assays.
In an embodiment, the agent inhibits the interaction of Mena with the C-terminal 5 residues of the alpha5 integrin C-terminal cytoplasmic tail. In an embodiment, the agent is based on a LERER repeat region of Mena. In an embodiment, the agent is based on a LERER repeat region of Mena. In an embodiment, the agent competes for binding to the alpha5 integrin with a LERER repeat region of Mena. In an embodiment, the agent comprises a peptide having the sequence of the C-terminal 5 residues of the alpha5 integrin C-terminal cytoplasmic tail. In an embodiment, the Mena is human Mena. In an embodiment, the Mena is MenaINV. In an embodiment, the MenaINV is human MenaINV.
A method is also provided for identifying an agent that binds to the LERER repeat region of Mena, the method comprising contacting the alpha5 integrin with Mena in the presence of an agent under conditions permitting Mena to interact with the LERER repeat region of alpha5 integrin and quantifying the interaction of Mena with the alpha5 integrin, wherein an agent that binds is identified as an agent that binds the LERER repeat region of alpha5 integrin, and wherein an agent that does not bind is identified as an agent that does not bind the LERER repeat region of alpha5 integrin.
In an embodiment of the methods disclosed herein, the Mena is a human Mena. In an embodiment, the Mena has the sequence set forth in Uniprot Q8N8S7. In another embodiment, the Mena is MenaINV. In a further embodiment, the MenaINV is human MenaINV. In an embodiment, the MenaINV is encoded by a Mena gene encoded mRNA but which contains the +++ exon and lacks the 11a exon.
In an embodiment of the methods disclosed herein, the alpha5 integrin is a human alpha5 integrin. In a further embodiment, the alpha5 beta1 integrin is human alpha5 beta1 integrin.
As used herein, “treating” a invasion of a tumor means that one or more symptoms of the invasion are inhibited, reduced, ameliorated, prevented, placed in a state of remission, or maintained in a state of remission. As used herein, “inhibiting” metastasis of a tumor in a subject” means that one or more symptoms or one or more other parameters by which the disease is characterized, are reduced, ameliorated, or prevented. Non-limiting examples of such parameters include uncontrolled degradation of the basement membrane and proximal extracellular matrix, and travel of tumor cells through the bloodstream or lymphatics, invasion, dysregulated adhesion, and proliferation at secondary site.
In an embodiment of the methods disclosed herein, the agent is a small organic molecule of 2000 daltons or less, an antibody, an antibody fragment, a peptide, a fusion protein or peptide, an RNAi agent or an oligonucleotide aptamer. In an embodiment of the methods disclosed herein, the agent is an RNAi agent and is an siRNA or a shRNA.
In an embodiment of the methods disclosed herein, the tumor is a mammary tumor. In an embodiment, the tumor is a tumor of a nasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus, testes, ovary, uterus, endometrium, liver, small intestine, appendix, colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast, cervix, vagina, vulva, prostate, thyroid or skin, or is a glioma.
As used herein a “small organic molecule” is an organic compound which contains carbon-carbon bonds, and has a molecular weight of less than 2000. The small organic molecule may also comprise inorganic atoms. The small molecule may be a substituted hydrocarbon or an substituted hydrocarbon. In an embodiment, the small molecule has a molecular weight of less than 1500. In an embodiment, the small molecule has a molecular weight of less than 1000.
As used herein “under conditions permitting Mena to interact with the alpha5 integrin” means conditions, for example as described herein, that permit Mena to interact with the alpha5 integrin excepting the presence of the tested agent.
All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Here it is disclosed that the Mena LERER repeat interacts directly with the cytoplasmic tail of α5 integrin, mediating a robust adhesion-modulated interaction between Mena and α5β1. The Mena: α5 interaction contributes to key α5β1 functions that include FN fibrillogenesis, cell spreading and motility. Given their established roles in EGFR signaling responses, tumor cell motility, invasion and metastasis, a direct link between Mena and α5β1 is understood to play an important role in tumor-cell invasion and metastasis.
Relocalization of Mena to mitochondrial also recruits α5. While investigating Ena/VASP- and integrin-mediated neuritogenesis a serendipitous observation was made that the subcellular distribution of α5β1 could be influenced by Ena/VASP. A strategy was used to block Ena/VASP function by depleting them from their normal locations and sequestering them on the mitochondrial surface by expressing a construct containing EVH1-binding sites (FPPPP; “FP4”) fused to a mitochondrial-targeting motif (FP4-Mito)(Bear et al., 2000). FP4-Mito expression phenocopies defects arising from loss of Ena/VASP function in fibroblasts, endothelial cells, neurons and in Drosophila, where transgenic expression of FP4-Mito phenocopies axon-guidance and epithelial defects observed in Ena mutants (Bear et al., 2002; Furman et al., 2007; Dent et al., 2007; Gates et al., 2007). While FP4-Mito redistributes Ena/VASP proteins to the mitochondrial surface, it has no detectable effects on localization Ena/VASP-binding partners such as the FA proteins zyxin and vinculin, and causes no evident defects when expressed in Ena/VASP-deficient fibroblasts (Bear et al., 2000).
Primary meningeal fibroblasts (present in cortical neuronal preparations) transfected GFP-tagged FP4-Mito and stained with anti-Mena and anti-α5 antibodies were examined and the expected redistribution of Mena observed (not shown) along with an unanticipated recruitment of α5 integrin to the mitochondrial surface (
The observed recruitment to mitochondria likely involves capture of α5β1-containing vesicles with their cytoplasmic tails accessible to bind the mitochondrial tethered Ena/VASP proteins directly or indirectly. Integrin trafficking is exquisitely regulated and has been studied extensively during biosynthesis, adhesion disassembly, integrin redistribution and regulation of growth factor receptor trafficking among other processes (Caswell et al., 2009; Margadant et al., 2011). Whether the putative α5β1-containing vesicles were captured by Ena/VASP during a particular stage of trafficking was investigated. FP4-Mito expressing cells were stained with antibodies that recognize vesicle populations involved in some of the known α5β1 trafficking pathways including: EEA1, an early endosomal marker, Rab7, a marker for vesicles containing activated β1 integrins (Arjonen et al., 2012) and Rab11, which decorates α5β1-containing vesicles as they pass through the perinuclear recycling compartment and is retained during integrin recycling to the plasma membrane (Margadant et al., 2011). No notable enrichment of these markers was observed on the α5β1-coated mitochondria of FP4-Mito expressing cells.
FP4-Mito recruits α5 to mitochondria through Mena. Next, it was investigated whether Mena, VASP and EVL could each recruit α5 to mitochondria in FP4-Mito expressing cells. As before, FP4-Mito expression in MVD7 cells failed to recruit α5 integrin to mitochondria (
The LERER repeat mediates Mena:α5 interaction. Having determined that Mena and α5 associate within cells, next the regions in Mena required to interact with α5 integrin were mapped by transfecting FP4-Mito into cells expressing a series of previously characterized GFP-tagged Mena deletion mutants (Loureiro et al., 2002). As expected, GFP-tagged EVH1 domain of Mena was recruited to FP4-Mito labeled mitochondria, however α5 integrin localization was unaffected (
α5 integrin binds directly to the LERER repeat region. Since the LERER repeat is necessary for the Mena:α5 complex, it was investigated whether it was sufficient to mediate the interaction. When the isolated LERER repeat from Mena was expressed as a GFP fusion (GFP-LERER) in MVD7 cells expressing mCherry-Mena, GFP signal appeared enriched in peripheral FAs containing both α5 integrin and mCherry-Mena, but was weak or undetectable in adhesions containing either α5 or mCherry-Mena, but not both (
It was next asked whether the LERER repeat could bind directly to the α5 cytoplasmic tail. Purified His-tagged LERER repeat protein (His-LERER) was mixed with purified GST-α5 cytoplasmic tail (GST α5 tail) immobilized on glutathione beads (
Next, the sequences within the α5 tail that bind Mena were delineated. First, it was asked whether the free C-terminal end of the α5 tail was required for the interaction using an α5 construct fused to GFP tag at its C-terminus (α5-GFP). NIH3T3 cells cotransfected with α5-GFP and FP4-Mito exhibited no detectable enrichment of α5-GFP on the mitochondrial surface, while endogenous α5 (detected by immunofluorescence) was clearly recruited to FP4-Mito (
The Mena LERER repeat region modulates the subcellular distribution of α5. Like all components of cell:matrix adhesions, Mena and α5β1 levels vary dynamically within these structures as they mature during cell spreading and migration (Zaidel-Bar et al., 2003). Whether the Mena:α5 interaction influences the distribution of either molecule to the different types of adhesive structures was studied. In fibroblasts cultured on FN, α5β1 is found typically in nascent FXs, FAs and FBs. MVD7 cells expressing GFP-Mena exhibited extensive co-localization of Mena, α5 and paxillin in peripheral FAs, while the cell center displayed robust α5 signal typical of FBs that contained little, if any detectable GFP-Mena (
To confirm these results in a second cell type, primary fibroblasts were isolated from perinatal mice homozygous for a conditional Mena allele (MenaFloxed) to examine formation of α5-containing FBs after Mena deletion in culture. To excise the MenaFloxed allele, cells were infected with adenovirus expressing either GFP-Cre recombinase or GFP alone (FIGS. 6A,C). In GFP-infected control fibroblasts, Mena and α5 co-localized at the leading edge and in peripheral FAs, while α5, but not Mena, was also present in central FBs (
The effects of α5 deletion on Mena were tested. Primary fibroblasts isolated from perinatal mice homozygous for an α5floxed allele (van der Flier et al., 2010) were infected with Cre-expressing or control adenovirus (
Adhesion to FN increases the amount Mena in complex with α5. The activation state of integrins often modulates interactions with their cytosolic binding partners. To determine whether the Mena:α5 interaction is sensitive to α5β1 activation, α5 complexes were immunoprecipitated from adherent, suspended and spreading cells. Interestingly, compared to adherent cells in steady-state conditions, significantly more Mena was detected in complex with α5 30 min after plating cells on FN (
MVD7 cells expressing GFP-Mena were significantly more spread (p<0.01 ANOVA LSD) compared to both MVD7 cells and MVD7+GFP-MenaΔLERER cells, which spread equivalently. Therefore, the increased amount of α5:Mena in complex during cell spreading correlates with increased cell spreading. Fibroblasts spread on FN in distinct steps initiated as integrins bind to FN and trigger rapid actin-polymerization-driven, adhesion-independent membrane extension followed by a distinct phase during which adhesions form dynamically, providing traction required for further spreading (Zhang et al., 2008). As fibroblasts attach to, and spread on FN, Mena localizes to the leading edge and to nascent β1-positive peripheral adhesions as they appear (Zhang et al., 2008).
Whether the adhesion dependent increase in Mena interaction with α5 affects its stability in FAs during spreading was investigated also. FRAP (Fluorescence Recovery After Photobleaching) analysis was used to measure the recovery dynamics after photobleaching of GFP-Mena or GFP-MenaΔLERER in nascent, peripheral adhesions within cells plated for 30 minutes on FN (
The Mena:α5 interaction is required for normal FN fibrillogenesis. α5β1 remains attached to FN as it moves centripetally along stress fibers towards the cell center, forming FBs and generating the tension required to initiate fibrillogenesis (Danen et al., 2002; Pankov et al., 2000). The absence of central α5β1-positive FBs in MVD7 and MenaΔLERER cells (
The Mena:α5 interaction influences cell motility. Both Mena and α5β1 exert context-dependent effects on cell motility, prompting investigation of whether disrupting their interaction would affect cell migration. MVD7 cells exhibit a hypermotile phenotype, migrating roughly twice as fast as MVD7 cells expressing GFPMena at levels typical for fibroblasts (Bear et al., 2000). Time-lapse movies of MVD7 cells and derivative lines expressing GFP-Mena and GFP-MenaΔLERER were analyzed to determine cell speed and directional persistence (
Cell motility and morphogenesis are dynamic, highly regulated processes that require continual remodeling of the cytoskeleton as well as cell:cell and cell:matrix adhesions. Requirements for Ena/VASP in all of these processes have been demonstrated in a wide range of systems. While Ena/VASP influences the formation, morphology and dynamics of cellular protrusions by regulating actin polymerization through a mechanism that is now coming into focus (Bear and Gertler, 2009; Hansen and Mullins, 2010), exactly how Ena/VASP affects adhesion is not well understood. This study identifies a direct connection between Mena and α5 integrin required during cell spreading and migration on FN and for FB formation and proper FN fibrillogenesis. Along with promoting α5β1 function inside-out, the Mena:α5 interaction is enhanced outside-in by FN binding to α5β1.
When validating the findings in primary fibroblasts isolated from α5FLOXED or MenaFLOXED animals, it was found that acute depletion of Mena protein caused a loss of central α5-containing FB-like adhesions. Interestingly, acute α5 depletion resulted in loss of Mena protein without affecting Mena mRNA levels. Therefore, in primary fibroblasts that normally express both α5 and Mena, loss of α5 causes a reduction in Mena levels either by blocking Mena translation or inducing its degradation. Consistent with this idea, integrins and FA proteins form complexes with the mRNA translation machinery (de Hoog et al., 2004; Humphries et al., 2009), and adhesion to FN triggers α5β1-dependent translation (Gorrini et al., 2005; Chung and Kim, 2008). FA proteins are also regulated by proteolytic enzymes (Franco and Huttenlocher, 2005) and by ubiquitinmediated proteosome degradation (Huang et al., 2009). Interestingly, however, Mena and α5 are each normally expressed in cells that lack the other, for example cultured cortical neurons contain Mena but lack detectable α5 (Gupton and Gertler, unpublished). Therefore, cells that normally express both proteins must have specific regulatory mechanisms that coordinate Mena levels with α5.
This direct, specific Mena:α5 interaction requires the C-terminal 5 of the 28 residue α5 cytoplasmic tail and is blocked by tagging the tail at its C-terminus Interestingly the tight junction protein ZO1 was recently identified as an α5 interacting protein (Tuomi et al., 2009), and binds residues next to those required for Mena binding. ZO1 interactions help target α5β1 to the lamellae of lung cancer cells. Whether Mena and ZO1 bind to α5 simultaneously is unknown, but it is interesting that complexes containing both VASP and ZO1 have been reported (Comerford et al., 2002), suggesting that Ena/VASP:ZO1 interactions may have specific functions. Mena binding to α5 requires the LERER repeat, a region spanning 91 or 121 amino acids with 13 or 15 repeats of the 5-residue LERER motif in mouse and human, respectively. Whether each repeat can bind an α5 tail is unknown, however, it is possible that multiple α5 tails could bind LERER repeats within each subunit of a Mena tetramer, raising the interesting possibility that Mena clusters α5β1, thereby strengthening FN binding by increased avidity. Mena promotes actin polymerization in cell protrusions (Bear and Gertler, 2009), within FAs and in sarcomeric units along Factin bundles attached to FAs of endothelial cells (Furman et al., 2007). The contractile forces exerted by endothelial cells and myosin light chain phosphorylation levels are proportional to the total level of Ena/VASP function (Furman et al., 2007) and VASP regulates smooth muscle cell contractility (Defawe et al., 2010). Therefore, Mena might contribute to contractile forces that generate conformational changes that permit highaffinity catch bonds between α5β1 and FN (Friedland et al., 2009; Kong et al., 2009). In this model, cells incapable of forming Mena:α5 complexes would remodel FN less efficiently because of reduced ability to generate forces needed to expose sites buried within FN, including the synergy site which enhances α5β1 binding and the self-association sites that dimerize FN (Schwarzbauer and DeSimone, 2011).
Despite its role in fibrillogenesis, Mena is barely detectable in FBs compared to FAs, as are two other molecules important for fibrillogenesis: FAK (Hie et al., 2004) and ILK (Vouret-Craviari et al., 2004; Stanchi et al., 2009; Zamir et al., 2000). Mena may cluster α5β1 and strengthen FN binding within FAs before α5β1:FN complexes begin moving towards central FBs. Alternatively, Mena:α5 interactions could target FAs for maturation by altering α5 dynamics and stability within FAs. Consistent with this possibility, deletion of the LERER repeat increased turnover of Mena in nascent adhesions formed during cell spreading.
The inability of α5-GFP to bind Mena may perturb α5 function in some contexts. The original description of α5-GFP included a comprehensive, convincing set of controls demonstrating that α5-GFP functioned equivalently to untagged α5 in migration and spreading when expressed in α5-deficient CHO B2 cells (Laukaitis et al., 2001). Some CHO cell lines (Benz et al., 2009) including CHO B2 lack detectable Mena protein (Riquelme and Gertler, unpublished), therefore perturbation of Mena-dependent α5 function by GFP tagging would not be expected. The consequences of disrupting the Mena:α5 interaction by GFP-tagging will likely be cell-type- and context-dependent. Along with the potential limitations of α5-GFP, we found that use of the FP4-Mito system to block Ena/VASP function can also block α5 function, an effect that must be considered when using this tool in α5-expressing cells. Our lab and others have used FP4-Mito to study Ena/VASP function in a variety of systems and thus far, most of the conclusions from these studies have been validated by experiments conducted in MVD7 cells (Loureiro et al., 2002; Bear et al., 2002), primary neurons isolated from Mena/VASP/EVL triple-null embryos (Dent et al., 2007) or Ena mutant Drosophila (which lack both α5 and the LERER-repeat) (Gates et al., 2007). The Peifer lab, however, has demonstrated that FP4-Mito expression in flies causes a partial co-depletion of Dia through association with Ena, raising the possibility that it could induce phenotypic effects that may be more severe than the Ena null state (Homem and Peifer, 2009). The LERER repeat is not found in VASP, EVL or the invertebrate and Dictyostelium Ena/VASP orthologs. Interestingly, fibronectin, α5β1 and the Mena LERER repeat are all vertebrate-specific adaptations (Whittaker et al., 2006), raising the possibility that they co-evolved. The Mena:α5 interaction is highly regulated: loss of adhesion reduces the interaction while acute FN binding during cell spreading increases both levels of the complex and the residence time of Mena within FAs. Interestingly, though VASP is not known to bind any integrin subunit directly, it promotes inside-out activation of β1- and β2-containing integrins indirectly through adaptor or signaling intermediates (Deevi et al., 2010). VASP functions in cross-regulation between αVβ3 and α5β1 (Worth et al., 2010): loss of P3 function reduces phosphorylation of a PKAdependent site within VASP near its EVH1 domain, allowing it to bind FPPPP-repeats within RIAM, an adaptor that mediates Rap-GTPase-driven integrin activation (Lafuente et al., 2004). The VASP:RIAM complex associates with the β subunit-binding protein talin (Anthis and Campbell, 2011) causing α5β1 activation at peripheral adhesions (Worth et al., 2010). Others, however, find that RIAM can promote integrin activation by talin independently of Ena/VASP (Lafuente et al., 2004; Lee et al., 2009). The Mena EVH1 domain binds many of the same ligands as VASP (Ball et al., 2002) connecting it to integrins through RIAM or other FA proteins containing EVH1-binding sites, such as vinculin and zyxin, that associate with β subunits indirectly. Juxtaposition of its EVH1 domain and LERER repeat may enable Mena to connect directly to α5 and indirectly to β1 simultaneously. In addition, Ena/VASP proteins can form mixed tetramers (Ahern-Djamali et al., 1998) that could combine Mena:α5 binding with VASP- or EVL-specific properties while diluting potential LERER-repeat clustering of α5β1. Here we found that rescue of the MVD7 hypermotile phenotype by GFP-Mena required the LERER repeat; however, previously we found that GFP-Mena and GFP-MenaΔLERER rescued the MVD7 hypermotility phenotype equivalently as did GFPVASP or GFP-EVL (Loureiro et al., 2002). That GFP-MenaΔLERER was expressed stably and exhibited subcellular distribution similar to GFP-Mena, as previously observed (Loureiro et al., 2002), was verified. The divergent results may have arisen from differences in methods and reagents used in the 10-year old study that cannot be tested, including FN or other reagents, or use of cells adapted to CO2-independent media as opposed to the current enclosed environmental chamber used for live-cell imaging. In addition, the current sample size is much larger: 372 MVD7 cells expressing GFP-MenaΔLERER from 4 separate 12-hour time-lapse movies were analyzed compared to 22 cells from 2 separate 4-hour experiments in the older study.
Why is the LERER repeat required for Mena to rescue MVD7 cell spreading and motility? Ena/VASP deficiency reduces cellular capacity to generate actin-driven protrusive forces that drive lamellipodial and filopodial extension and propulsion of the intracellular pathogen Listeria monocytogenes, even though the actin networks formed during these process are organized differently. In general, expression of Mena, VASP or EVL each rescue the actin polymerization-dependent phenotypes evident in the absence of Ena/VASP in MVD7 cells or in primary neurons from triple Mena/VASP/EVL null embryos (Loureiro et al., 2002; Geese et al., 2002; Applewhite et al., 2007; Dent et al., 2007). GFP-Mena expression in MVD7 cells produces rapidly extending, but shortlived lamellipodia that cannot contribute efficiently to locomotion (Bear et al., 2002).
Conversely, lamellipodia in parental MVD7 cells protrude slowly but are more stable and can contribute to productive translocation likely by adhering to the substratum before the protrusion cycle ends (Bear et al., 2002). These differences probably arise from changes in the actin network: high Ena/VASP activity produces longer, sparsely branched filament networks that, absent stabilizing interconnections, become increasingly prone to buckle against the membrane as they elongate due to their inherent flexibility (Mogilner and Oster, 2003). Importantly, the net effect of Ena/VASP on actin polymerization leads to context-dependent morphological outputs contingent on variables including location, density, and strength of adhesion sites along with the relative amounts of actin bundling and crosslinking proteins (Mogilner and Keren, 2009). By coupling its stimulatory effect on barbed end elongation with the ability to bind and cluster α5β1, Mena could present activated but unbound integrins right at the tips of lamellipodia and filopodia consistent with the proposed “sticky fingers” mechanism for haptotaxis (Galbraith et al., 2007). In addition, through its role in FN remodeling, Mena may help form the interstitial fibrillar network that serves both as a migration substrate and template that organizes growth factors and other ECM components into spatially organized cues that elicit complex, coordinated responses (Hynes and Naba, 2012) when touched by the sticky fingers of cells in transit. Over the past several years, new evidence has implicated both α5β1 (Muller et al., 2009; Caswell et al., 2008; Valastyan et al., 2009) and Mena (Robinson et al., 2009; Philippar et al., 2008; Roussos et al., 2010) in breast cancer invasion and metastasis through effects on EGFR (Gertler and Condeelis, 2011). Many carcinomas types exhibit elevated levels of Mena that are, at least in breast cancer, critical for metastatic progression (Gertler and Condeelis, 2011; Roussos et al., 2010) and could involve interaction with α5. In breast cancer patients, risk of distant metastasis correlates with density of a tripartite microanatomical structure called TMEM composed of a carcinoma cell expressing Mena, a macrophage and a blood vessel all contacting each other (Robinson et al., 2009). During breast cancer progression, changes in alternative splicing produce additional Mena protein isoforms co-expressed with the canonical isoform. Primary tumor cells express Mena11a, normally an epithelial-specific isoform lost when cells undergo epithelial to mesenchymal transition (Shapiro et al., 2011; Warzecha et al., 2009). Invasive tumors stop expressing Mena11a, while a subpopulation of highly invasive, motile and chemotactic tumor cells express an invasion-specific Mena isoform, MenaINV (Goswami et al., 2009). MenaINV has been detected in breast cancer patients with invasive ductal carcinomas at levels proportionate to their TMEM density (Roussos et al., 2011b). Interestingly, the exon encoding the 19 amino acid INV sequence is inserted between the EVH1 domain and the LERER repeat region.
Both MenaINV and α5β1 modulate EGFR function. MenaINV sensitizes tumor cells to EGF, allowing invasive or chemotactic responses to 25-50 fold lower EGF concentrations than in cells lacking this isoform, and leads to substantially increased metastatic burden (Philippar et al., 2008; Roussos et al., 2011a). Upon inhibition of αVβ3 or in cells expressing the mutant form of the p53 tumor suppressor, α5β1 forms complexes with EGFR through their mutual cytosolic binding partner, RCP (Caswell et al., 2008; Muller et al., 2009). Association of α5β1-RCP with EGFR leads to coordinated recycling that targets α5β1 and EGFR to the front of cells and promotes 3D invasion. Complex formation between α5β1 and EGFR also dysregulates signaling downstream of both receptors. Interestingly, a recent report demonstrated that increased expression of Mena and mutant p53 were highly correlated in patients with infiltrating ductal carcinomas (Toyoda et al., 2011).
Western Blotting/Immunoprecipitation. Standard procedures were used for protein electrophoresis, western blotting and immunoprecipitations. Western blots were developed using HRP secondary antibodies and ECL reagent (Amersham). For α5 integrin immunoprecipitation, cells were lysed at 4° C. in CSK buffer (Humphries et al., 2009) with intermittent agitation for 20 minutes, passed through a 23.5 gauge needle, and the supernatant was kept after spinning 15 minutes at 21,000×g. Lysates were precleared with protein A beads for two hours, incubated with an α5 integrin antibody (Millipore, 1928) for two hours at 4° C., and then captured with BSA blocked protein A beads for two hours. Beads were washed three times in lysis buffer, and proteins were eluted in sample buffer. Western blots were probed for α5 integrin (Santa Cruz sc-166681) Mena (Lebrand et al., 2004), Paxillin (Signal Transduction laboratories), p34 (Millipore, 07-227), β1 integrin (Millipore, 1949), GFP (Clontech, JL-8), GAPDH(Signal trandsduction laboratories, 2118), porin (Molecular Probes, A-21317), tubulin (DM1A), His tag (Sigma, H1029), and VASP polyclonal (Lanier et al., 1999). The function blocking α5 integrin antibody BIIG2 was purchased from Iowa University Developmental Studies Hybridoma bank, and used at 20 μg/ml.
Mitochondrial Purification: Mitochondria were isolated from NIH3T3 cells expressing either FP4-Mito or DP4-Mito using paramagnetic beads conjugated to an antibody specific for mitochondrial protein Tom34 (Miltenyi Biotec, according to manufacturer instructions).
Binding Assays: GST-α5 integrin constructs and His-tagged variants of the LERER repeat region were expressed and purified from E. Coli. 10 nM α5 integrin cytoplasmic tail was immobilized on Glutathione beads and incubated for 1 hour, 4° C. with 200 nM His-LERER variants at constant agitation in PBS with 0.1% TritonX-100 and 2 mM βME. Beads were washed three times, and proteins were eluted in sample buffer, and assayed by western blot.
Microscopy—Cells were fixed in 4% paraformaldehyde in PHEM buffer warmed to 37° C. for 20 minutes. Cells were permeabilized in 0.2% TX-100 and blocked in 10% Donkey Serum. Primary antibodies used for immunofluorescence include α5 integrin (Millipore 1928), integrin α4 [PS/2] (Abcam ab25247), integrin αv [RMV-7] (Abcam ab63490), integrin α6 [GoH3] (ab105669), vinculin (Sigma), Mena, GFP (Clontech, JL-8), paxillin (BD Transduction, 610052), Rab7 (Cell Signaling, 9367S), Rab11 (Cell Signaling 5589), and EEA1 (Cell Signaling, 3288S). F-actin was stained with AlexaFluor Phalloidin (Invitrogen). Z series of images were taken on an Olympus microscope with a 60× plan apo objective. Images were deconvolved using Deltavision Softworx software. FRAP was performed on a Olympus microscope using DeltaVision software and solid state 405 laser in TIRF mode with a depth of 100 nm. Images were acquired pre and post bleach with 488 and 561 solid state laser with 63×1.4 NA Plan Apochromatic objective lens (Olympus). A pre-bleach series of ten images was collected at 10 s interval, the area of interest was bleached with 50% laser power. The acquisition settings were returned to pre-bleach settings, and images were taken at adaptive time frame. Total elapsed time between the end of the pre-bleach series and the beginning of the post-bleach series was 40-90 s (median 50 s).
Sequence analysis—The murine Mena (ENAH) from UniProt (mouse: Q03173) to identify the repeat region as residues 175-252. By visual inspection, these sequence regions were divided into chunks fitting one of several motifs: a five amino acid motif roughly consistent with the form “L/M/Q-E-R/Q-E-R/Q” (SEQ ID NO:1), a seven amino acid motif roughly consistent with the 5-mer motif with the last two amino acids of the motif repeated (SEQ ID NO:2), and an eight amino acid motif roughly consistent with the 5-mer motif preceded by a repetition of the first three amino acids of the motif (SEQ ID NO:3). All sequence in the region of interest fell into one of these three motifs, with no sequence unused. A motif logo was generated for each species using each instance of the 5-mer motif, the first five amino acids of the 7-mer motif, and the last five amino acids of the 8-mer motif using the program WebLogo (http://weblogo.berkeley.edu/)
Image analysis—Cell masks of cell area were made by threshholding phalloidin images. Subsequently, threshholding was done to evenly include adhesive structures between cells within these masks, and intensity and area of these regions were measured. For analysis of photobleaching data, images were first corrected for overall photobleaching, and the integrated fluorescence intensity (Fr) inside a region that was smaller than the original bleached region by 4 pixels in x and y in each image was measured in the pre-bleach and recovery image series. Calculation of the t1/2 of recovery and percent fluorescence recovery was performed as described (Bulinski et al., 2001).
Cell Culture and Plasmids. Coverslips were coated with 10 μg/ml bovine FN (Sigma) for 2 hours at 37° C. Primary meningeal fibroblasts were cultured with cortical neurons, isolated from embryonic day 14.5 mice as described (Dent et al., 2007). Perinatal fibroblasts were isolated from postnatal day 1 mice that harbored either floxed α5 integrin (van der Flier et al., 2010) or floxed Mena (will be described in a separate publication). NIH3T3 cells, Rat2 cells, and perinatal fibroblasts were cultured in DME supplemented with 10% fetal bovine serum. Parental MVD7 cells and MVD7 cells expressing GFP-tagged Mena and Mena mutants were cultured as described (Bear et al., 2000). Mcherry FP4-Mito, GFP-LERER, and GFP-α5 integrin were introduced into MVD7 cells using Lonza nucleofection per the manufacturer protocol. pMVSCV-GFPLERER, pMVSCV-GFP-MenaΔLERER, pGEX-GST-α5 cytoplasmic tail, pGEX-GST α5 cytoplasmic tail ACOOH, pQE80L-His-LERER, pQE80L-His-LERER-CoCo, pQE80LHis-LERER-EVH2, and pQE80L-His-EVH2 were cloned using standard cloning procedures. mCherry-FP4-Mito was previously described (Bear et al., 2000). GFPtensin was a kind gift from Ken Yamada and was introduced into Rat2 cells with Lipofectamine 2000 (Invitrogen) following manufacturer's directions. GFP-α5 integrin (Laukaitis et al., 2001) was purchased from Addgene.
FN Fibrillogenesis—FN-depleted medium was prepared as described (Pankov and Momchilova, 2009). FN was fluorescently labeled with 549—NHS ester from Thermo-Scientific (46407), as directed by the manufacturer. MVD7 cells were seeded on coverslips coated with vitronectin (10 μg/ml) from Sigma (V9881) and allowed to adhere overnight. Medium was replaced with FN-depleted growth medium containing 10 μg/ml fluorescently labeled FN and incubated at 32° C. for four hours. Cells were then fixed and stained as indicated above.
Motility analysis—MVD7 cells were stained with 1 μM CMFDA (Invitrogen) and seeded overnight in growth medium at 2000 cells/cm2 on FN (10 μg/mL) coated coverglass. Media was replenished directly before imaging to facilitate addition of 10 ρg/mL of α5 blocking antibody [BD Pharmingen, 5H10-27 (MFRS)] where applicable. Two-dimensional migration was quantified by recording cell centroid displacement after live-cell imaging for 12 hrs (1 image/10 min) using a Zeiss Axiovert inverted microscope equipped with automatic stage positioning, a 5% CO2-37° C. environmental chamber, fluorescent light source, and 10× plan-fluor objective. Resulting images were semi-automatically tracked using Imaris software (Bitplane, Inc). A custom Matlab (Mathworks) script was used to calculate migration parameters and create wind-rose plots. Cell speed is reported for the final 6 hours of the experiment to ensure steady-state. α5 integrin surface levels For assessment of α5 integrin surface levels, MVD7 fibroblasts were incubated on ice in 1% BSA, 2 mM EDTA in PBS with biotinylated α5 integrin antibody (BD Pharmingen, 557446) or biotinylated rat IgG (Jackson ImmunoResearch, 012-060-003) for 30 mins. Cells were washed, and incubated for 30 mins on ice with APC streptavidin (BD Pharm 554067) and propidium iodide. Cells were washed, resuspended and directly analyzed on a FACSCalibur (BD Biosciences). Biotinylation and analysis of surface levels of α5 integrin was performed as described (Caswell et al., 2008).
Yang, J. T., B. L. Bader, J. A. Kreidberg, M. Ullman-Culleré, J. E. Trevithick, and R. O. Hynes. 1999. Overlapping and independent functions of fibronectin receptor integrins in early mesodermal development. Developmental Biology. 215:264-277.
This application claims benefit of U.S. Provisional Application No. 61/645,782, filed May 11, 2012, and of U.S. Provisional Application No. 61/788,411, filed Mar. 15, 2013, the contents of each of which are hereby incorporated by reference.
This invention was made with Government support under Grant Nos. R01 GM58801 and U54-CA112967 awarded by the National Institutes of Health. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US13/40274 | 5/9/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61788411 | Mar 2013 | US | |
| 61645782 | May 2012 | US |
