Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation

Abstract

Hepatocyte growth factor/scatter factor (HGF/SF), acting through the Met receptor, plays an important role in most human solid tumors, and inappropriate expression of this ligand-receptor pair is often associated with poor prognosis. The molecular basis for the malignant potential of the HGF/SF-Met signal in cancer cells has mostly been attributed to its mitogenic and invasive properties. However, HGF/SF also induces angiogenesis, but the signaling mechanism has not been fully explained, nor has this activity been directly associated with HGF/SF-Met-mediated tumorigenesis. It is known that HGF/SF induces in vitro expression of vascular endothelial growth factor (VEGF), a key agonist of tumor angiogenesis; by contrast, thrombospondin 1 (TSP-1) is a negative regulator of angiogenesis. Here, we show that, in the very same tumor cells, in addition to inducing VEGF expression, HGF/SF dramatically down-regulates TSP-1 expression. We show that TSP-1 shut-off plays an important, extrinsic role in HGF/SF-mediated tumor development, because ectopic expression of TSP-1 markedly inhibits tumor formation through the suppression of angiogenesis. Interestingly, although VEGF-induced expression is sensitive to inhibitors of several pathways, including mitogen-activated protein kinase, phosphoinositide 3-kinase, and signal transducer and activator of transcription 3, TSP-1 shut-off by HGF/SF is prevented solely by inhibiting mitogen-activated protein kinase activation. These studies identify HGF/SF as a key switch for turning on angiogenesis. They suggest that TSP-1 is a useful antagonist to tumor angiogenesis and that it may have therapeutic value when used in conjunction with inhibitors of VEGF.

Fig. 1.

HGF/SF up-regulates VEGF and down-regulates TSP-1 expression in SK-LMS-1 cells (A) and MDA-MB-231 cells (B). Total RNA was prepared from SK-LMS-1 cells or MDA-MB-231 cells treated with recombinant human HGF/SF (200 units/ml) at the indicated time points. Total RNA was also prepared from the SK/HGF cell line, a long-term culture derivative of SK-LMS-1 cells that is autocrine for human HGF/SF (26). Northern blots were probed with ³²P-radiolabeled TSP-1, VEGF, or GAPDH cDNA fragments, respectively.

Fig. 2.

HGF/SF–Met signaling pathways regulate TSP-1 and VEGF expression. (A) HGF/SF induces activation of the MAPK and PI3-kinase pathways in SK-LMS-1 cells and MDA-MB-231 cells. Serum-starved cells were untreated ([–]HGF); treated with only HGF ([+]HGF, blank column); or treated with DMSO (control), PD98059 (80 μM), U0126 (40 μM), or LY294002 (40 μM) for 1 h, followed by HGF/SF stimulation for 15 min (a good time point for observing the status of all tyrosine phosphorylations). Whole-cell extracts were prepared, and the state of Met phosphorylation was detected by immunoprecipitation with anti-human Met antibody, followed by Western blot with anti-phosphotyrosine (and/or anti-human Met antibody). For detection of extracellular signal-related kinase (Erk) and Akt, Western blots were probed with anti-phospho p44/42 MAPK, anti-p44/42 MAPK, anti-phospho Akt (Ser-473), or anti-Akt antibodies, respectively. (B) Negative regulation of TSP-1 expression occurs primarily through the MAPK pathway, whereas positive regulation of VEGF expression occurs through MAPK and PI3-kinase pathways. Total RNA was prepared from cells with or without HGF/SF treatment and/or inhibitor treatment. Northern blot analyses were performed as described for Fig. 1. Down-regulation of TSP-1 by HGF/SF was inhibited by the MAPK inhibitors PD98059 or U0126 but was not affected by LY294002. Up-regulation of VEGF was inhibited by PD98059 and U0126 as well as LY294002. (C) VEGF (but not TSP-1) expression was regulated by Stat3 signaling. Total RNA was prepared from SK/HGF cells with or without overexpression of a dominant-negative form of Stat3 and Stat3β (27). Overexpression of Stat3β decreased VEGF expression but did not affect TSP-1 expression in SK/HGF cells.

Fig. 3.

TSP-1 inhibits HGF/SF-induced tumor growth in vivo. (A) TSP-1 was ectopically expressed in SK/HGF cells, establishing the SK/HGF-TSP1 cell line. The expression of TSP-1 in SK/HGF-TSP1 cells was confirmed by Northern blot analysis. (B) Tumor growth of SK-LMS-1 cells and the influence of TSP-1 overexpression. SK-LMS-1 control cells, SK/HGF control cells, and SK/HGF-TSP1 cells (clone 26) were s.c. implanted in athymic nude mice. The animals were monitored for tumor growth, and tumor volume was measured twice a week. The tumor volume values represent an average of four mice for each group (P < 0.025). (C) Visualization of the tumors after the mice were killed. (D) TSP-1 protein in tumor xenografts is derived from SK/HGF-TSP1 cells. Cell extracts were prepared from fresh tumors, and TSP-1 protein was detected by anti-TSP-1 antibody under denaturing conditions.

Fig. 4.

TSP-1 inhibits HGF/SF-induced tumor angiogenesis. (A) Decreased neovascularization in SK/HGF-TSP1 tumors: tissue sections prepared from tumors derived from SK/HGF and SK/HGF-TSP1 cells were immunohistochemically stained with anti-mouse CD31 antibody. Three fields (×10 magnification) from each stained tumor section were photographed, and the number of CD31-positive vessels (brown staining) was scored for each field. The values in the graph represent the average number of blood vessels in the three selected sections from each of four tumors for each cell type (P < 0.01). (B) Three representative fields from each group of tumors are displayed.

Fig. 5.

Schematic representation of tumor angiogenesis induced by HGF/SF–Met signaling. Intrinsically, HGF/SF activates the Met receptor on the surface of host endothelial cells, inducing proliferation and migration. Extrinsically, HGF/SF–Met signaling turns on the angiogenic switch by simultaneously up-regulating proangiogenic VEGF expression and down-regulating antiangiogenic TSP-1 expression from the tumor cells. We do not exclude the possibility that, in endothelial cells, HGF/SF–Met signaling may have extrinsic activity in addition to intrinsic activity.