A number of signaling pathways regulate expression of and response to pro-angiogenic factors. Because the protein tyrosine kinase, Src has been implicated in both of these processes, we hypothesized that Src is a critical mediator of angiogenesis induced by colorectal carcinoma cells. To examine Src-mediated angiogenic properties, temperature-sensitive SV40 Tag immortalized endothelial cells from both the colon (CEC) and liver (HEC) were stimulated with conditioned media from HT29 human colorectal carcinoma cells previously treated with a selective Src kinase inhibitor, PP2, or DMSO control. Endothelial proliferation was determined by MTT; migration was determined by modified Boyden chamber assay. To asses the ability of Src to regulate growth factor-induced endothelial activation, the above assays were repeated in the presence of 1) 2% FBS, 2) EGF, 3) bFGF, 4) VEGF-A or 5) HGF either in the presence or absence of a selective Src inhibitor. For protein analysis, HEC pretreated with PP2 or DMSO were stimulated with EGF and lysed at various time points. Total and phosphorylated forms of Akt, Erk, and Src were assessed by western blotting and densitometry. Conditioned media from HT29 colorectal carcinoma cells induced a ∼2-fold increase in proliferation and migration in the CEC and >3-fold increase in proliferation and migration HEC respectively. The conditioned media from the Src-inhibited colorectal carcinoma cells failed to induce significant proliferation or migration in either cell line. Likewise, EGF, bFGF, VEGF, and HGF induced a 5.0-, 3.7-, 4.4-, and 3.9-fold increase in migration in HEC respectively and a 3.3-, 2.4-, 2.7- and 4.0-fold increase in migration in CEC respectively. In contrast, while EGF and bFGF caused ∼2-fold increase in proliferation in both HEC and CEC, VEGF failed to increase proliferation above control values. HGF induced a 1.5-fold increase in CEC proliferation, but failed to induce proliferation in HEC. Pretreatment with the selective Src inhibitor abrogated endothelial proliferation and migration induced by all factors. EGF treatment of HEC induced a 2-fold increase in Src kinase activity, a >5-fold increase in phosphorylated Erk and ∼3 fold increase in phosphorylated Akt, all of which were effectively blocked by Src inhibition. We therefore conclude that angiogenesis induced by colorectal carcinoma cells is regulated by Src kinase and that heterogeneous endothelial cells from distinct anatomic sites may be differentially activated by colorectal carcinoma cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]