Migration-promoting role of VEGF-C and VEGF-C binding receptors in human breast cancer cells

Abstract

Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic factor over-expressed in highly metastatic, cyclooxygenase (COX)-2 expressing breast cancer cells. We tested the hypothesis that tumour-derived VEGF-C may play an autocrine role in metastasis by promoting cellular motility through one or more VEGF-C-binding receptors VEGFR-2, VEGFR-3, neuropilin (NRP)-1, NRP-2, and integrin alpha9beta1. We investigated the expression of these receptors in several breast cancer cell lines (MDA-MB-231, Hs578T, SK-BR-3, T-47D, and MCF7) and their possible requirement in migration of two VEGF-C-secreting, highly metastatic lines MDA-MB-231 and Hs578T. While cell lines varied significantly in their expression of above VEGF-C receptors, migratory activity of MDA-MB-231 and Hs578T cells was linked to one or more of these receptors. Depletion of endogenous VEGF-C by treatments with a neutralising antibody, VEGF-C siRNA or inhibitors of Src, EGFR/Her2/neu and p38 MAP kinases which inhibited VEGF-C production, inhibited cellular migration, indicating the requirement of VEGF-C for migratory function. Migration was differentially attenuated by blocking or downregulation of different VEGF-C receptors, for example treatment with a VEGFR-2 tyrosine kinase inhibitor, NRP-1 and NRP-2 siRNA or alpha9beta1 integrin antibody, indicating the participation of one or more of the receptors in cell motility. This novel role of tumour-derived VEGF-C indicates that breast cancer metastasis can be promoted by coordinated stimulation of lymphangiogenesis and enhanced migratory activity of breast cancer cells.

Figure 1

(A and B) Effects of different concentrations of a polyclonal, function blocking antibody against human Vascular endothelial growth factor (VEGF)-C on migration of VEGF-C-secreting MDA-MB-231 and Hs578T cells cultured for 24 h in serum-free DMEM supplemented with 0.1% FBS. A strong inhibition of migration (P<0.02), noted at all antibody concentrations ranging between 5 and 20 μg ml⁻¹, and no inhibition with similar concentrations of control goat immunoglobulin G (IgG), indicated an autocrine migration-promoting role of VEGF-C in these cells. (C) Effects of siVEGF-C (100 nM) treatments on the expression of VEGF-C gene in MDA-MB-231 cells (48 h). (D) VEGF-C secretion from MDA-MB-231 cells transfected with siVEGF-C (100 nM, 48 h) which were incubated in serum-free DMEM for other 24 h. (E) Migration of MDA-MB-231 cells is inhibited by transfection with siVEGF-C (100 nM) as measured at 24 h time point in DMEM supplemented with 0.1% FBS. (F) Effects of kinase inhibitors for EGFR/Her2/Neu (PD153035), Src (PP1), and p38 MAPK (SB203580) on migration of MDA-MB-231 cells cultured for 24 h in DMEM supplemented with 0.1% FBS. Data represent mean±s.d. (n=4). ^*P<0.01. All the above kinase inhibitors at the tested non-toxic concentrations had no significant effect on cell proliferation/survival and inhibited VEGF-C production, as we reported earlier (Timoshenko et al, 2006).

Figure 2

(A) Expression of VEGFR-2 and VEGFR-3 mRNA in human breast cancer cells (MCF-7, T-47D, Hs578T, and MDA-MB-231) and a human trophoblast cell line (HTR-8/SVneo, used as a positive control for VEGFR-3) as revealed by RT–PCR. MDA-MB-231 cells express VEGFR-2 (binding to VEGF-A and -C) but not VEGFR-3 (binding to VEGF-C and -D). MCF-7 and Hs578T cells expressed none of these receptors, whereas T-47D expressed VEGFR-3. (B) Immunostaining of MDA-MB-231 cells for VEGFR-2 (image on the left) and its control (image on the right), cells counterstained with hematoxylin and eosin (H&E). (C) Expression of neuropilin (NRP)-1 and NRP-2 mRNAs in human breast cancer cell lines MCF-7, MDA-MB-231, Hs578T, SK-BR-3, and T-47D as revealed by RT–PCR. (D) Flow cytometry analysis of various human breast cancer cells (468LN, MDA-MB-231, Hs578T, SK-BR-3, MCF7, and T-47D) labelled with α9β1 integrin antibody. In comparison with strongly α9β1 integrin positive 468LN cells (15), other cell lines demonstrated relatively low levels of α9β1 integrin expression (filled profiles) overlapping partially with non-specific isotype control staining (opened profiles). Approximately 20 000 cells were analysed in each case.

Figure 3

Effect of SU5416, a selective inhibitor of VEGFR-2 (Flk-1/KDR) tyrosine kinase, on migration of MDA-MB-231 and Hs578T cells cultured for 24 h in DMEM supplemented with 0.1% FBS. Data represent mean±s.d. (n=3). ^*P<0.02. The migration of VEGFR-2 expressing MDA-MB-231 cells was strongly inhibited at inhibitor concentrations ⩾2 μM, while only a very minor effect was noted with VEGFR-2-negative Hs578T cells.

Figure 4

(A) Silencing of neuropilin (NRP)-1 and neuropilin (NRP)-2 mRNA expression in MDA-MB-231 and Hs578T cells transfected with 100 nM of respective siRNAs for 48 h (see Material and Methods). The RT–PCR data demonstrated a highly specific knock-down of both genes. (B) Migration of untreated (mock-transfected control) and NRP-1 and NRP-2 siRNA-transfected MDA-MB-231 and Hs578T cells. Whereas both siRNAs inhibited (^*P<0.05) migration of VEGFR-2-expressing MDA-MB-231 cells, the migratory activity of transfected Hs578T cells was not impaired.

Figure 5

Effects of α9β1 integrin function-blocking antibody on migration of MDA-MB-231 and Hs578T breast cancer cells at a 24 h. The antibody (1 and 5 μg ml⁻¹) caused a concentration dependent inhibition of migration of Hs578T but not MDA-MB-231 cells. ^*P<0.05.