Late SV40 factor (LSF) enhances angiogenesis by transcriptionally up-regulating matrix metalloproteinase-9 (MMP-9)

Abstract

The transcription factor late SV40 factor (LSF) is overexpressed in human hepatocellular carcinoma (HCC) fostering a highly aggressive and metastatic phenotype. Angiogenesis is an essential component of cancer aggression and metastasis and HCC is a highly aggressive and angiogenic cancer. In the present studies, we analyzed the molecular mechanism of LSF-induced angiogenesis in HCC. Employing human umbilical vein endothelial cells (HUVEC) differentiation assay and chicken chorioallantoic membrane (CAM) assay we document that stable LSF overexpression augments and stable dominant negative inhibition of LSF (LSFdn) abrogates angiogenesis by human HCC cells. A quest for LSF-regulated factors contributing to angiogenesis, by chromatin immunoprecipitation-on-chip (ChIP-on-chip) assay, identified matrix metalloproteinase-9 (MMP-9) as a direct target of LSF. MMP-9 expression and enzymatic activity were higher in LSF-overexpressing cells and lower in LSFdn-expressing cells. Deletion mutation analysis identified the LSF-responsive regions in the MMP-9 promoter and ChIP assay confirmed LSF binding to the MMP-9 promoter. Inhibition of MMP-9 significantly abrogated LSF-induced angiogenesis as well as in vivo tumorigenesis, thus reinforcing the role of MMP-9 in facilitating LSF function. The present findings identify a novel target of LSF contributing to its oncogenic properties.

FIGURE 1.

LSF induces differentiation of human umbilical vein endothelial cells (HUVEC). A, HUVECs were cultured in either basal media or differentiation media or treated with conditioned media (CM) from the indicated cells and tube formation was photographed. B, graphical representation of percentage of tube formation by HUVEC treated as in A when tube formation in differentiation media was considered as 100%. The data represent mean ± S.E. *, p < 0.05.

FIGURE 2.

LSF induces neovascularization in CAM assay. A, CAM was treated either with VEGF (positive control) or with CM from the indicated cells and neovascularization was photographed. B, graphical representation of percentage of new blood vessel formation in CAM treated as in A when VEGF-treated CAM was considered as 100%. Data represent mean ± S.E. *, p < 0.05.

FIGURE 3.

LSF induces MMP-9. A, human angiogenesis array was screened using conditioned media (CM) from the indicated cells. Representative expression levels of Angiogenin, PlGF, and IL-8 are shown. MMP-9 mRNA expression was detected in parental HepG3 cells and its LSF-overexpressing clones (B) and in parental QGY-7703 cells and its LSF dominant negative-expressing clones (C) by real-time PCR. Data represent mean ± S.E. *, p < 0.05. D, analysis of Pro- and active MMP-9 by Western blot analysis using conditioned media from the indicated cells. E, functionally active MMP-9 level was determined in the indicated cells by gelatin zymography.

FIGURE 4.

LSF transcriptionally up-regulated MMP-9. Control-8, LSF-1, and LSF-17 clones of HepG3 cells (A) and Control-1, LSFdn-8, and LSFdn-15 clones of QGY-7703 cells (B) were transfected with pGL3-basic vector or MMP-9-Prom-luc along with Renilla luciferase expression vector. Luciferase assay was performed 2 days later, and firefly luciferase activity was normalized by Renilla luciferase activity. Data represent mean ± S.E. *, p < 0.05. C, schematic representation of the N1 and N2 deletion mutants of MMP-9-Prom-luc (FL) construct. The numbers represent nucleotide position when the translation initiation site was regarded as +1. D, MMP-9-Prom-luc (FL) and its N1 and N2 deletion mutants were transfected in Control-8 and LSF-17 clones of HepG3 cells, and luciferase assay was performed as in A. Data represent mean ± S.E. *, p < 0.05. E, schematic diagram of MMP-9 promoter showing the location of potential LSF-binding sites in the promoter and primers designed for ChIP assay. Fragment A, B, and C represent 195, 192, and 204 bp fragments. F, ChIP assay to detect LSF binding to the MMP-9 promoter. G, MMP-9 induces HUVEC differentiation. HUVEC were treated with the indicated concentrations of MMP-9 or MMP-9 (1 μg/ml) that was inactivated by boiling and were subjected to tube formation assay. Photomicrographs of tube formation are shown. H, graphical representation of percentage of tube formation by HUVEC treated as in G when tube formation in differentiation media was considered as 100%. The data represent mean ± S.E. *, p < 0.05.

FIGURE 5.

Inhibition of MMP-9 abrogates augmentation of proliferation, invasion and angiogenesis by LSF. A, MMP-9sh-13 and MMP-9sh-15 clones with stable knockdown of MMP-9 were generated in LSF-17 clone of HepG3 cells. Con-11 is a control puromycin resistant-clone expressing scrambled shRNA. MMP-9 mRNA expression was determined by real-time PCR in the indicated cells. MMP-9 protein expression was detected by Western blot analysis (B) and gelatin zymography (C) using conditioned media from the indicated cells. Cell viability by standard MTT assay (D), colony formation assay (E), and Matrigel invasion assay (F) was performed using the indicated cells. Conditioned media from the indicated cells were subjected to HUVEC differentiation assay (G) and neovascularization assay in CAM (H). Data represent mean ± S.E. *, p < 0.05.

FIGURE 6.

Inhibition of MMP-9 abrogates LSF-induced tumorigenesis and metastasis. Control-11 and MMP-9sh-13 clones of LSF-17 cells were subcutaneously implanted into athymic nude mice. Animals were sacrificed after 3 weeks. A, representative photograph of tumor-bearing mice at the end of the study. Measurement of tumor volume (B) and tumor weight (C) at the end of the study. Data represent mean ± S.E. *, p < 0.05. D, tumor sections derived from Control-11 and MMP-9sh-13 clones were stained with H&E and for CD31. Arrows indicate blood vessels. E, indicated cells were intravenously injected into athymic nude mice and tumors were allowed to develop for 6 weeks. Sections of the lungs showing microscopic tumor nodules (arrow).