K-Ras promotes angiogenesis mediated by immortalized human pancreatic epithelial cells through mitogen-activated protein kinase signaling pathways

Mol Cancer Res. 2009 Jun;7(6):799-808. doi: 10.1158/1541-7786.MCR-08-0577. Epub 2009 Jun 9.

Abstract

Activating point mutations in the K-Ras oncogene are among the most common genetic alterations in pancreatic cancer, occurring early in the progression of the disease. However, the function of mutant K-Ras activity in tumor angiogenesis remains poorly understood. Using human pancreatic duct epithelial (HPDE) and K-Ras4B(G12V)-transformed HPDE (HPDE-KRas) cells, we show that activated K-Ras significantly enhanced the production of angiogenic factors including CXC chemokines and vascular endothelial growth factor (VEGF). Western blot analysis revealed that K-Ras activation promoted the phosphorylation of Raf/mitogen-activated protein kinase kinase-1/2 (MEK1/2) and expression of c-Jun. MEK1/2 inhibitors, U0126 and PD98059, significantly inhibited the secretion of both CXC chemokines and VEGF, whereas the c-Jun NH(2)-terminal kinase inhibitor SP600125 abrogated only CXC chemokine production. To further elucidate the biological functions of oncogenic K-Ras in promoting angiogenesis, we did in vitro invasion and tube formation assays using human umbilical vein endothelial cells (HUVEC). HUVEC cocultured with HPDE-KRas showed significantly enhanced invasiveness and tube formation as compared with either control (without coculture) or coculture with HPDE. Moreover, SB225002 (a CXCR2 inhibitor) and 2C3 (an anti-VEGF monoclonal antibody) either alone or in a cooperative manner significantly reduced the degree of both Ras-dependent HUVEC invasiveness and tube formation. Similar results were obtained using another pair of immortalized human pancreatic duct-derived cells, E6/E7/st and its oncogenic K-Ras variant, E6/E7/Ras/st. Taken together, our results suggest that angiogenesis is initiated by paracrine epithelial secretion of CXC chemokines and VEGF downstream of activated oncogenic K-Ras, and that this vascular maturation is in part dependent on MEK1/2 and c-Jun signaling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Pancreatic Ductal / blood supply
  • Carcinoma, Pancreatic Ductal / enzymology
  • Carcinoma, Pancreatic Ductal / genetics
  • Cell Line
  • Cell Transformation, Neoplastic / genetics
  • Cell Transformation, Neoplastic / metabolism*
  • Cells, Cultured
  • Chemokines, CXC / biosynthesis
  • Chemokines, CXC / metabolism
  • Epithelial Cells / metabolism
  • Epithelial Cells / pathology
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • Genes, ras
  • Humans
  • MAP Kinase Signaling System*
  • Neovascularization, Pathologic / enzymology
  • Neovascularization, Pathologic / genetics
  • Neovascularization, Pathologic / metabolism
  • Pancreatic Neoplasms / blood supply*
  • Pancreatic Neoplasms / enzymology
  • Pancreatic Neoplasms / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Vascular Endothelial Growth Factor A / biosynthesis
  • Vascular Endothelial Growth Factor A / metabolism
  • ras Proteins / genetics
  • ras Proteins / metabolism*

Substances

  • Chemokines, CXC
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Extracellular Signal-Regulated MAP Kinases
  • ras Proteins