Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts.

A possible link between oncogenes and tumor angiogenesis has been implicated by the finding that expression of various oncogenes, particularly mutant ras, can lead to a marked induction of a potent paracrine stimulator of angiogenesis, vascular endothelial growth factor (VEGF). We sought to determine how oncogenic ras induction of VEGF is mediated at the molecular level and whether the mechanisms involved differ fundamentally between transformed epithelial cells and fibroblasts. Our results suggest that in a subline (called RAS-3) of immortalized nontumorigenic rat intestinal epithelial cells (IEC-18) that acquired a tumorigenic phenotype upon transfection of mutant ras, up-regulation of VEGF occurs in the absence of an autocrine growth factor circuit. The expression of VEGF mRNA and protein by RAS-3 cells was strongly suppressed in the presence of LY294002, an inhibitor of phosphatidylinositol 3'-kinase, but remained largely unaffected in the same cells treated with an inhibitor (PD98059) of mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MKK/MEK-1). This is consistent with the observation that overexpression of a constitutively activated mutant of MEK-1 (AN3/ S222D) in the parental IEC-18 cells did not result in up-regulation of VEGF production. The impact of mutant ras on VEGF expression was also significantly amplified at high cell density, conditions under which RAS-3 cells became less sensitive to LY294002-induced VEGF down-regulation. In marked contrast to cells of epithelial origin, ras-transformed murine fibroblasts (3T3RAS) up-regulated VEGF in a manner that was strongly inhibitable by MEK-1 blockade (ie. treatment with PD98059), whereas these cells were relatively unaffected by treatment with the phosphatidylinositol 3'-kinase inhibitor LY294002. In addition, VEGF was up-regulated by 2-3-fold in NIH3T3 cells overexpressing mutant MEK-1. Collectively, the data suggest that the stimulatory effect of mutant ras on VEGF expression is executed in a nonautocrine and cell type-dependent manner and that it can be significantly exacerbated by physiological/ environmental influences such as high cell density.

[1]  L. Chin,et al.  Essential role for oncogenic Ras in tumour maintenance , 1999, Nature.

[2]  R. Kerbel,et al.  Collateral expression of proangiogenic and tumorigenic properties in intestinal epithelial cell variants selected for resistance to anoikis. , 1999, Neoplasia.

[3]  G. Goodall,et al.  Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements. , 1999, Molecular biology of the cell.

[4]  D. Hanahan,et al.  Tumor-derived expression of vascular endothelial growth factor is a critical factor in tumor expansion and vascular function. , 1999, Cancer research.

[5]  R. Kerbel,et al.  Normoxic and hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells is mediated by Ras , 1999, International journal of cancer.

[6]  C. Dinney,et al.  Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[7]  N. Ferrara,et al.  OncogenicrasFails to Restore anin VivoTumorigenic Phenotype in Embryonic Stem Cells Lacking Vascular Endothelial Growth Factor (VEGF) , 1999 .

[8]  M. J. Newman,et al.  Downregulation of the pro-apoptotic protein Bak is required for the ras-induced transformation of intestinal epithelial cells , 1998, Current Biology.

[9]  J. Pouysségur,et al.  p42/p44 MAP Kinase Module Plays a Key Role in the Transcriptional Regulation of the Vascular Endothelial Growth Factor Gene in Fibroblasts* , 1998, The Journal of Biological Chemistry.

[10]  Shu-Yun Le,et al.  Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription , 1998, Oncogene.

[11]  P. Einat,et al.  Translation of Vascular Endothelial Growth Factor mRNA by Internal Ribosome Entry: Implications for Translation under Hypoxia , 1998, Molecular and Cellular Biology.

[12]  A. Harris,et al.  Differential expression of vascular endothelial growth factor mRNA vs protein isoform expression in human breast cancer and relationship to eIF-4E. , 1998, British Journal of Cancer.

[13]  Robert S. Kerbel,et al.  Establishing a Link between Oncogenes and Tumor Angiogenesis , 1998, Molecular medicine.

[14]  Kelly Gs.  Clinical applications of N-acetylcysteine. , 1998 .

[15]  R. Kerbel,et al.  Impact of oncogenes in tumor angiogenesis: mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  L. Ellis,et al.  Down-regulation of Vascular Endothelial Growth Factor in a Human Colon Carcinoma Cell Line Transfected with an Antisense Expression Vector Specific for c-src * , 1998, The Journal of Biological Chemistry.

[17]  G. Semenza,et al.  V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression. , 1997, Cancer research.

[18]  N. Goldstein,et al.  Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. , 1997, The American journal of pathology.

[19]  A. Giaccia,et al.  Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element. , 1997, Blood.

[20]  S. Oldham,et al.  A Raf-independent Epidermal Growth Factor Receptor Autocrine Loop Is Necessary for Ras Transformation of Rat Intestinal Epithelial Cells* , 1997, The Journal of Biological Chemistry.

[21]  Douglas Hanahan,et al.  Signaling Vascular Morphogenesis and Maintenance , 1997, Science.

[22]  K. Alitalo,et al.  Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia , 1997, Oncogene.

[23]  P. Warne,et al.  Role of Phosphoinositide 3-OH Kinase in Cell Transformation and Control of the Actin Cytoskeleton by Ras , 1997, Cell.

[24]  K. Dameron,et al.  Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenicity , 1997, Oncogene.

[25]  J. Zweier,et al.  Mitogenic Signaling Mediated by Oxidants in Ras-Transformed Fibroblasts , 1997, Science.

[26]  E. Pennisi.  Superoxides Relay Ras Protein's Oncogenic Message , 1997, Science.

[27]  R. Swerlick,et al.  Transforming growth factor‐α‐induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP‐2‐dependent DNA binding and transactivation , 1997, The EMBO journal.

[28]  J. Folkman,et al.  Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  N. Ferrara,et al.  The biology of vascular endothelial growth factor. , 1997, Endocrine reviews.

[30]  W. Kaelin,et al.  Regulation of vascular endothelial growth factor by hypoxia and its modulation by the von Hippel-Lindau tumor suppressor gene. , 1997, Kidney international.

[31]  G. Fontanini,et al.  Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A. , 1996, Journal of the National Cancer Institute.

[32]  F. Larcher,et al.  Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels. , 1996, Cancer research.

[33]  E. Voest,et al.  Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. , 1996, The Journal of clinical investigation.

[34]  L. Ellis,et al.  Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by cell density. , 1996, Cancer research.

[35]  G. Semenza,et al.  Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1 , 1996, Molecular and cellular biology.

[36]  D. Hanahan,et al.  Patterns and Emerging Mechanisms of the Angiogenic Switch during Tumorigenesis , 1996, Cell.

[37]  A. Giaccia,et al.  Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression. , 1996, Cancer research.

[38]  S. Oldham,et al.  Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Kenneth J. Hillan,et al.  Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene , 1996, Nature.

[40]  A. De Benedetti,et al.  Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: Implications for tumor angiogenesis , 1996, International journal of cancer.

[41]  D. Shima,et al.  The Mouse Gene for Vascular Endothelial Growth Factor , 1996, The Journal of Biological Chemistry.

[42]  M. Goldberg,et al.  Post-transcriptional Regulation of Vascular Endothelial Growth Factor by Hypoxia (*) , 1996, The Journal of Biological Chemistry.

[43]  Philip R. Cohen,et al.  PD 098059 Is a Specific Inhibitor of the Activation of Mitogen-activated Protein Kinase Kinase in Vitro and in Vivo(*) , 1995, The Journal of Biological Chemistry.

[44]  D. Marmé,et al.  Both v-Ha-Ras and v-Raf Stimulate Expression of the Vascular Endothelial Growth Factor in NIH 3T3 Cells (*) , 1995, The Journal of Biological Chemistry.

[45]  R. Kerbel,et al.  Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. , 1995, Cancer research.

[46]  M. Neeman,et al.  Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes , 1995, Molecular and cellular biology.

[47]  A. Bridges,et al.  A synthetic inhibitor of the mitogen-activated protein kinase cascade. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[48]  D. Mukhopadhyay,et al.  Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation , 1995, Nature.

[49]  H. Dvorak,et al.  Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. , 1995, The American journal of pathology.

[50]  P. Bornstein,et al.  Transformation by v-src causes transient induction followed by repression of mouse thrombospondin-1. , 1994, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[51]  P. Steeg,et al.  Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines , 1994, International journal of cancer.

[52]  K. Dameron,et al.  Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. , 1994, Science.

[53]  Michael J. Fry,et al.  Phosphatidylinositol-3-OH kinase direct target of Ras , 1994, Nature.

[54]  N. Ahn,et al.  Transformation of mammalian cells by constitutively active MAP kinase kinase. , 1994, Science.

[55]  C. Marshall,et al.  Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells , 1994, Cell.

[56]  J. Hancock,et al.  Activation of Raf as a result of recruitment to the plasma membrane. , 1994, Science.

[57]  Sally J. Leevers,et al.  Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane , 1994, Nature.

[58]  A. Brunet,et al.  Constitutively active mutants of MAP kinase kinase (MEK1) induce growth factor-relaxation and oncogenicity when expressed in fibroblasts. , 1994, Oncogene.

[59]  K Y Hui,et al.  A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). , 1994, The Journal of biological chemistry.

[60]  J. Filmus,et al.  Activated ras and src induce CD44 overexpression in rat intestinal epithelial cells. , 1994, Oncogene.

[61]  Y. Ogiso,et al.  Biological activity of a K-ras mutant that contains the 12R/59T/116Y mutations. , 1993, Cancer letters.

[62]  S. Shirasawa,et al.  Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. , 1993, Science.

[63]  J. Filmus,et al.  Role of transforming growth factor alpha (TGF-alpha) in the transformation of ras-transfected rat intestinal epithelial cells. , 1993, Oncogene.

[64]  J. Fiddes,et al.  The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. , 1991, The Journal of biological chemistry.

[65]  A. Quaroni,et al.  Activated H-ras transforms rat intestinal epithelial cells with expression of α-TGF☆ , 1987 .

[66]  C. Marshall,et al.  Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor , 1986, Molecular and cellular biology.

[67]  D. Schmitt,et al.  The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell lines is reduced by a farnesyl transferase inhibitor. , 1999, Anticancer research.

[68]  C. Der,et al.  Increasing complexity of Ras signal transduction: involvement of Rho family proteins. , 1998, Advances in cancer research.

[69]  N. Bouck,et al.  How tumors become angiogenic. , 1996, Advances in cancer research.

[70]  Goldman,et al.  Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology. , 1993, Molecular biology of the cell.