Integrin alpha2 mediates selective metastasis to the liver.
ABSTRACT: Cancers display distinct patterns of organ-specific metastasis. Comparative analysis of a broad array of cell membrane molecules on a liver-metastasizing subline of B16 melanoma versus the parental B16-F0 revealed unique up-regulation of integrin alpha2. The direct role of integrin alpha2 in hepatic metastasis was shown by comparison of high versus low-expressing populations, antibody blockade, and ectopic expression. Integrin alpha2-mediated binding to collagen type IV (highly exposed in the liver sinusoids) and collagen type IV-dependent activation of focal adhesion kinase are both known to be important in the metastatic process. Analysis of primary colorectal cancers as well as coexisting liver and lung metastases from individual patients suggests that integrin alpha2 expression contributes to liver metastasis in human colorectal cancer. These findings define integrin alpha2 as a molecule conferring selective potential for formation of hepatic metastasis, as well as a possible target to prevent their formation.
Project description:Integrin alpha2beta1 is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the alpha2-I domain (alpha2-I), which binds fibrillar collagen via Mg(2+)-bridged interactions. The crystal structure of a truncated form of the alpha2-I domain, bound to a triple helical collagen peptide, revealed conformational changes suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex. Collagen binding by alpha2-I and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptides. Here we describe the inhibitory effect of a short cyclic peptide derived from the snake toxin metalloprotease jararhagin, with specific amino acid sequence RKKH, on the ability of alpha2-I to bind triple helical collagen. Isothermal titration calorimetry measurements showed that the interactions of alpha2-I with collagen or RKKH peptide have similar affinities, and NMR chemical shift mapping experiments with (15)N-labeled alpha2-I, and unlabeled RKKH peptide, indicate that the peptide competes for the collagen-binding site of alpha2-I but does not induce a large scale conformational rearrangement of the I domain.
Project description:The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a beta1 integrin, rather than alphav beta3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to alpha2 and beta1 integrin subunits and to IAP. A combination of antialpha2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antialphav beta3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between alpha2 beta1 and IAP was detected by the coimmunoprecipitation of both alpha2 and beta1 integrin subunits with IAP. These data suggest that IAP can associate with alpha2 beta1 integrin and modulate its function.
Project description:Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.
Project description:Rhodocetin is a snake venom protein that binds to alpha2beta1 integrin, inhibiting its interaction with its endogenous ligand collagen. We have determined the mechanism by which rhodocetin inhibits the function of alpha2beta1. The interaction of alpha2beta1 with collagen and rhodocetin differed: Ca(2+) ions and slightly acidic pH values increased the binding of alpha2beta1 integrin to rhodocetin in contrast with their attenuating effect on collagen binding, suggesting that rhodocetin preferentially binds to a less active conformation of alpha2beta1 integrin. The alpha2A-domain [von Willebrand factor domain A homology domain (A-domain) of the integrin alpha2 subunit] is the major site for collagen binding to alpha2beta1. Recombinant alpha2A-domain bound rhodocetin, demonstrating that the A-domain is also the rhodocetin-binding domain. Although the interaction of alpha2beta1 with rhodocetin is affected by altering divalent cations, the interaction of the A-domain was divalent-cation-independent. The rhodocetin-binding site on the alpha2A-domain was mapped first by identifying an anti-alpha2 antibody that blocked rhodocetin binding and then mapping the epitope of the antibody using human-mouse alpha2A-domain chimaeras; and secondly, by binding studies with alpha2A-domain, which bear point mutations in the vicinity of the mapped epitope. In this way, the rhodocetin-binding site was identified as the alpha3-alpha4 loop plus adjacent alpha-helices. This region is known to form part of the collagen-binding site, thus attaining a mainly competitive mode of inhibition by rhodocetin.
Project description:Background:The liver is the most common target for metastatic colorectal cancer. Changes of the local hepatic niche due to hepatic diseases such as cirrhosis decrease the incidence of colorectal cancer liver metastasis. Hepatic niche heterogeneity could influence the risk of hepatic metastasis. Materials and Methods:We simulated changes of the hepatic niche via prophylactical liver irradiation with a safe dose of 6 Gy. GEO dataset and GO analysis revealed a difference in the expression of matrix metalloproteinase 1 (MMP1) in primary colorectal cancer versus liver metastasis, as well as synchronous versus metachronous liver metastasis. Western blotting, Immunofluorescence and qRT-PCR were conducted to measure protein expressions, location and RNA expressions. Colony formation, wound-healing, transwell assays experiments were performed to determine the malignant biological properties of colorectal cancer cells. shRNA transfection was used to conduct stable transfected cell lines. Results:Tissue inhibitor of metalloproteinases 1 (TIMP1) expression was significantly higher in metastases lesions than primary tumors. In vivo, TIMP1 expression in the hepatic niche increased after a safe dose of 6 Gy irradiation, along with MMP1 decreased, leading to collagen fiber deposition and impairment of hepatic microcirculation. In vitro, irradiated hepatic stellate cells-conditioned media reduced the migration and clone formation ability of colon cancer cells SW480 and HCT116. Low TIMP1 expression in hepatic stellate cells reduced tumor cell invasion and migration. Conclusion:Prophylactical 6 Gy whole-liver irradiation could inhibit colorectal cancer liver metastasis by regulating TIMP1/MMP1 balance in the hepatic niche before liver metastatic lesion formed.
Project description:Capillaries expressing the laminin alpha2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin alpha2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin alpha2 chain rather than through the laminin beta1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the alpha6beta1 integrin, which is one of the laminin alpha2 chain receptors. EGF up-regulated alpha6beta1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-alpha6 integrin chain monoclonal antibody. These data indicate that laminin alpha2 chain and alpha6beta1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin alpha2 chain-positive vessels may favor metastasis of EGF-dependent tumors.
Project description:NADPH oxidase 1 (Nox1) is expressed mainly in colon epithelial cells and produces superoxide ions as a primary function. We showed that Nox1 knockdown inhibits directional persistence of migration on collagen I. This paper dissects the mechanism by which Nox1 affects the direction of colonic epithelial cell migration in a two-dimensional model. Transient activation of Nox1 during cell spreading on collagen 1 temporarily inactivated RhoA and led to efficient exportation of alpha2beta1 integrin to the cell surface, which supported persistent directed migration. Nox1 knockdown led to a loss of directional migration which takes place through a RhoA-dependent alpha2/alpha3 integrin switch. Transient RhoA overactivation upon Nox1 inhibition led to transient cytoskeletal reorganization and increased cell-matrix contact associated with a stable increase in alpha3 integrin cell surface expression. Blocking of alpha3 integrin completely reversed the loss of directional persistence of migration. In this model, Nox1 would represent a switch between random and directional migration through RhoA-dependent integrin cell surface expression modulation.
Project description:Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.
Project description:Homing of colorectal cancer (CRC) cells to the liver is a non-random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis-specific peptide ligand (CGIYRLRSC) that binds a complex of E-cadherin and ?(6) integrin on the surface of CRC cells. We identify angiopoietin-like 6 protein as a peptide-mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin-like 6 and tumoural ?(6) integrin/E-cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed ?(6) integrin and E-cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC.
Project description:Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.