Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin.

Osteopontin (OPN) is a secreted adhesive glycoprotein with a gly-arg-gly-asp-ser (GRGDS) cell binding domain. Several independent studies have suggested that OPN functions in tumor growth and metastasis, and one likely possibility is that OPN facilitates tumor invasion by promoting tumor cell migration. Consistent with this hypothesis, immobilized OPN promoted concentration-dependent tumor cell migration (i.e., haptotaxis) in modified Boyden chambers. In particular, cleavage of OPN by thrombin, which likely occurs in the tumor microenvironment, resulted in enhancement of OPNs haptotactic activity; and assays performed with purified preparations of the two individual OPN thrombin-cleavage fragments demonstrated that all detectable activity was associated with the GRGDS-containing fragment. In contrast to the activity of both OPN and its GRGDS-containing fragment in promoting haptotaxis, neither of these proteins in solution promoted chemotaxis, indicating that each must be immobilized to promote cell migration. In haptotaxis assays, antibody LM609 to integrin alpha v beta 3 blocked > 80% cell migration towards the GRGDS-containing OPN fragment, implicating alpha v beta 3 as its principal functional receptor. In comparison with equimolar quantities of other adhesive proteins, the GRGDS-containing OPN thrombin-cleavage fragment was not only > 2-fold more effective than intact OPN at promoting haptotaxis, but also > 8-fold and > 6-fold more effective than fibrinogen and vitronectin, respectively, indicating that this OPN fragment is highly active relative to other alpha v beta 3 ligands.

Osteopontin expression and distribution in human carcinomas.

Osteopontin (OPN), a secreted adhesive glycoprotein, is significantly overexpressed in a variety of experimental models of malignancy. Moreover, increased levels of OPN have been detected in the blood of patients with metastatic carcinoma. To investigate OPN expression and distribution in human carcinomas directly, we studied a wide variety of common tumors by Northern analysis, in situ hybridization, and immunohistochemistry. All 14 tumors studied by Northern analysis showed very substantial increases in OPN messenger (m)RNA when compared to corresponding normal tissues. Moreover, intense labeling for OPN mRNA was detected in 71 of 76 carcinomas studied by in situ hybridization. In most of the carcinomas studied (colon, stomach, duodenum, pancreas, breast, lung, bladder, prostate, ovary, thyroid, and melanoma), tumor cells did not label detectably for OPN mRNA; however, macrophages intimately associated with tumor cells labeled strongly for the OPN transcript. In carcinomas of the kidney and endometrium, both tumor cells and host macrophages labeled strongly for OPN mRNA. The presence of OPN mRNA in macrophages was particularly pronounced at the edge of tumors (ie, the tumor/stroma interface) and in areas of tumor necrosis. Although in most cases tumor cells did not label detectably for OPN mRNA, both tumor cells and macrophages stained for OPN protein, suggesting that OPN secreted by macrophages may bind to tumor cells, possibly through the glycine-arginine-glycine-aspartate-serine cell binding domain in OPN. Collectively, these data suggest that OPN functions in adhesive interactions at the tumor/host interface and thereby may influence processes such as invasion and metastasis.

Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.

Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces.

Osteopontin, a glycoprotein with a glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain, has been described in bone and is also known to be expressed in other organs, particularly kidney. The goal of the present work was to define the distribution of osteopontin synthesis and deposition in a wide variety of normal adult human tissues using a multifaceted approach that included immunohistochemistry, in situ hybridization, and Northern analysis. Immunohistochemical studies have revealed the unexpected finding that osteopontin is deposited as a prominent layer at the luminal surfaces of specific populations of epithelial cells of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung, breast, salivary glands, and sweat glands. Northern analyses identified gallbladder as a major site of osteopontin gene transcription comparable in magnitude with that of kidney, and immunoblotting identified osteopontin in bile. In situ hybridization localized osteopontin gene transcripts predominantly to the epithelium of a variety of organs as well as to ganglion cells of bowel wall. Osteopontin of epithelial cell origin, like bone-derived osteopontin, promoted GRGDS-dependent cell spreading in attachment assays. We postulate that osteopontin secreted by epithelium binds integrins on luminal surfaces. Collectively, these findings suggest an important role for osteopontin on many luminal epithelial surfaces communicating with the external environment.

Elevated expression of secreted phosphoprotein I (osteopontin, 2ar) as a consequence of neoplastic transformation.

Several lines of evidence have recently been presented indicating that proteins previously referred to as Mr 62,000-67,000 transformation-associated secreted phosphoprotein, 2ar, osteopontin, bone sialoprotein I, and 44K Dal bone phosphoprotein are all very likely encoded by the same gene. We have found that markedly elevated expression of this protein, which henceforth is referred to as secreted phosphoprotein I (Sppl) closely correlates with neoplastic transformation of a variety of cell types and that patients with advanced stage metastatic cancers have elevated levels of Sppl in their blood. Moreover, Sppl expression is induced in mouse epidermal cells in vitro and mouse epidermis in vivo with the tumor promoter TPA (Craig et al, J. Biol. Chem. 264: 9682-9689, 1989). Sppl amino acid sequence deduced from cDNA nucleotide sequence (Oldberg et al, Proc. Natl. Acad Sci. U.S.A. 83: 8819-8823, 1986) contains the GRGDS cell-binding sequence which is known to be important for cell attachment to several adhesive proteins found in extracellular matrices. Because of the presence of the GRGDS cell-binding sequence in Sppl, it is probable that abnormally high expression of this soluble protein by tumor cells has important consequences for interactions between tumor cells and the host tissue matrix.

Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin.

A wide variety of rodent and human tumor cells secrete antigenically related phosphoproteins with molecular weights (Mr) of approximately 58,000 (hamster), 62,000 (rat, mouse), 67,000 (human) (Senger, D.R. and Perruzzi, C.A. (1985) Cancer Res. 45, 5818-5823). Expression of these phosphoproteins is transformation-related; tumor cells produce at least 10-fold or more of this protein as compared to their normal or untransformed counterparts. N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein indicate that it is identical to rat osteopontin, a bone protein with an Arg-Gly-Asp cell-binding sequence (Oldberg, A., Franzen, A. and Heinegard, D. (1986) Proc. Natl. Acad. Sci. USA 83, 8819-8823). Antibody raised to the Mr 62,000 rat tumor-secreted phosphoprotein was found to bind Mr 75,000 and Mr 35,000 components of human milk, indicating that milk contains antigenically related proteins. The Mr 75,000 protein, which is present in human milk at concentrations ranging from 3 to 10 micrograms/ml, has been purified to homogeneity. The Mr 35,000 component is apparently derived from the Mr 75,000 protein by proteolytic cleavage, and this cleavage also occurs in vitro in the presence of thrombin. N-terminal and internal amino acid sequences were derived from the Mr 75,000 milk protein and found to be similar (12/21 residues) to N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein and osteopontin. Moreover, sequence derived from the N-terminus of the human milk protein is identical to that of human bone sialoprotein I (the likely human homolog of rat osteopontin) (Fisher, L.W., Hawkins, G.R., Tuross, N. and Termine, J.D. (1987) J. Biol. Chem. 262, 9702-9708).

Secreted phosphoproteins associated with neoplastic transformation: close homology with plasma proteins cleaved during blood coagulation.

We have previously demonstrated that a wide variety of rodent and human tumor cells secrete markedly elevated levels of a transformation-related phosphoprotein with a molecular weight (depending on animal species) of 58,000 to 69,000. With antibody raised to the tumor-secreted phosphoprotein (rat), we have now identified an antigenically related protein in normal rat and human plasma. The rat plasma protein and the rat tumor-secreted phosphoproteins comigrated on polyacrylamide gels and were identically cleaved during blood coagulation as well as by purified thrombin. Mouse macrophages expressed a similar or identical phosphoprotein suggesting that monocytic cells may be a source of the normal plasma protein. Consistent with elevated secretion of this protein by tumor cells, plasma and sera from cancer patients contained elevated levels of this protein, raising the possibility that this circulating marker may prove useful for monitoring tumor burden. Amino acid sequence derived from the amino terminus of the rat tumor phosphoprotein indicates that it is distinct from previously sequenced proteins, but that it may be related to protein-tyrosine kinases encoded by viral and proto-onc genes.

T24 human bladder carcinoma cells with activated Ha-ras protooncogene: nontumorigenic cells susceptible to malignant transformation with carcinogen.

A comparative analysis of T24 human bladder carcinoma cells and N-methyl-N'-nitro-N-nitrosoguanidine (MeNNG)-transformed derivatives (MeNNG-T24 cells) revealed the following: (i) The presence of an activated c-Ha-ras gene (in the absence of the normal allele) is insufficient to confer upon T24 cells a tumor-associated phenotype. (ii) MeNNG-transformed T24 cells not only acquire tumor-associated (in vitro) traits (growth in soft agar and rhodamine retention) but, are highly tumorigenic in nude mice. (iii) It is possible to render T24 cells tumorigenic by chemical transformation; therefore, the reason that T24 cells lack tumorigenicity is not because of possible incompatibilities between these cells and nude mice but, in fact, because T24 cells are not malignant. (iv) The loss of expression of a transformation-related Mr 67,000 phosphoprotein by MeNNG-T24 cells after explantation of these cells from nude mouse tumors to in vitro culture indicates that culture conditions can be responsible for rapid phenotypic conversion of human tumor cell lines.

A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines.

We have previously reported that rodent tumor cell lines secrete a potent vascular permeability factor with a molecular weight of 34,000-42,000 (Senger et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science (Wash. DC), 219: 983-985, 1983). This tumor-secreted vascular permeability factor (VPF) causes a rapid and completely reversible increase in microvascular permeability in the species (guinea pig or rat) from which the tumors were derived without causing mast cell degranulation or endothelial cell damage or exciting an inflammatory cell infiltrate. This VPF may be responsible, at least in part, for the increased permeability which is commonly displayed by solid and ascites tumor vessels. We have now examined 7 human tumor cell lines and have determined that 5 of them also secrete this same VPF. Antibody raised to guinea pig line 10 VPF neutralized more than 90% of the vascular permeability-increasing activity secreted by these 5 human tumor lines. Furthermore, VPFs from both guinea pig and human tumor sources bound to and were eluted similarly from immobilized heparin and comigrated identically on sodium dodecyl sulfate-polyacrylamide gels. Finally, 2 tumorigenic (in nude mice) human cell lines were found to secrete at least 14-fold more VPF than their directly matched, nontumorigenic counterparts, suggesting that elevated expression of this permeability factor may correlate with neoplastic transformation. These data suggest that a broad spectrum of tumor cells from several species, including humans, secretes a highly conserved molecule that enhances local vascular permeability and that this function may be important for tumor growth.

Secreted phosphoprotein markers for neoplastic transformation of human epithelial and fibroblastic cells.

A wide variety of rodent tumor cells of both fibroblastic and epithelial origins secrete a major transformation-related phosphoprotein with a molecular weight of approximately 62,000. Tumorigenic cells, regardless of the transforming agent, secrete 10-fold or more of this 32P-labeled protein as compared with their nontumorigenic counterparts. In this study we have extended these previous findings to tumorigenic human cells of diverse origins (both sarcomas and carcinomas). Metabolic labeling of cells with [32P]orthophosphate and immunoprecipitation with antibody specifically directed against the rat transformation-dependent secreted phosphoprotein have been used to identify antigenically related human phosphoproteins (Mr 66,000-69,000). Of the 14 human cell lines examined, all 8 of the lethal tumorigenic cell populations secreted these phosphoproteins either in continuous culture or as fresh explants from nude mice while the six nonmalignant cell lines did not (tumorigenicity in all cases was assayed in nude mice). Included in our study were three tumorigenic human cell lines (two sarcomas, one carcinoma), each with a matched, nontumorigenic control. The very close correlation between secretion of these phosphoproteins and the tumor cell phenotype of both rodents and humans raises the possibility that they may be important for tumor growth in vivo.

A secreted phosphoprotein marker for neoplastic transformation of both epithelial and fibroblastic cells.

A wide variety of virally and spontaneously transformed fibroblasts secrete a major transformation-related phosphoprotein with a molecular weight (MW), depending on the species of origin, of about 62,000 (62K). Markedly elevated extracellular levels of this major 32P-labelled protein are not simply linked to exponential growth but instead are associated directly with transformation. The phosphoprotein is not antigenically related to p60src, p60c-src or simian virus 40 (SV40) non-viral T antigen, and it is further distinguishable from SV40 non-viral T antigen (pp 53) on the basis of its electrophoretic mobility. In this study we have compared a variety of normal and transformed epithelial cells for secretion of this 32P-labelled protein and have found that this marker distinguishes neoplastic from preneoplastic and normal mouse mammary epithelium and also identifies highly tumorigenic cells derived from guinea pig bile duct epithelium and rat liver epithelium. Because the classical phenotypic properties commonly associated with transformation of fibroblasts cannot be generally used to discriminate tumorigenic from non-tumorigenic epithelial cells, this phosphoprotein, which identifies tumorigenic cells of both fibroblastic and epithelial origin, is likely to be of particular importance.

Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.

Tumor ascites fluids from guinea pigs, hamsters, and mice contain activity that rapidly increases microvascular permeability. Similar activity is also secreted by these tumor cells and a variety of other tumor cell lines in vitro. The permeability-increasing activity purified from either the culture medium or ascites fluid of one tumor, the guinea pig line 10 hepatocarcinoma, is a 34,000- to 42,000-dalton protein distinct from other known permeability factors.