COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer

Abstract

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors.

Figure 1

Expression of VEGF-A, -B, -C and -D mRNAs and VEGF-A, -C, and –D protein secretion by human breast cancer cell lines MDA-MB-231 and MCF-7. (A) RT–PCR-based amplification of VEGF mRNAs, showing that message for each VEGF class is detectable in both cell lines. (B) Accumulation of secreted VEGF-A, -C and -D proteins in serum-free culture media of both cell lines at 24 h showing high VEGF-C production by MDA-MB-231 cells. (C) Differences in expression of VEGF-C mRNA by MDA-MB-231 and MCF-7 cells as revealed by real time qPCR. (D) Kinetics of accumulation of VEGF-A, -C and -D protein in culture media of MDA-MB-231 cells. Data in (B, C, and D) represent mean±s.d. (n=4).

Figure 2

Relationship between COX-2 expression and VEGF-C synthesis/expression in human breast cancer cell lines and tissues. (A) Expression of COX-2 and GAPDH genes in MCF-7, T-47D, Hs578T and MDA-MB-231 human breast cancer cells as detected by RT–PCR. COX-2 expression is not detectable in MCF-7, weak in T-47D, moderate in Hs578T, and high in MDA-MB-231 cells. (B) Accumulation of VEGF-C in culture media of the above breast cancer cell lines at a 24 h correlates with their levels of COX-2 expression. Data represent mean±s.d. (n=4). (C) Correlation between relative levels of COX-2 mRNA and VEGF-C mRNA (standardized against GAPDH mRNA) expression in human breast cancer tissues (n=10) measured with real-time qPCR reveals a strong positive association between the two parameters (r=0.94, P=0.0002). Numbers on the graph present patient identifiers, and dotted lines present 95% confidence intervals of the regression line.

Figure 3

Relationship between LYVE-1 and COX-2 (A) or VEGF-C (B) mRNA expression in the same breast cancer tissues (n=10) used for data in Figure 2C. A positive association (r=0.75, P=0.017 and r=0.78, P=0.013, respectively) is revealed in both cases; dotted lines present 95% confidence interval of the regression line.

Figure 4

Effects of COX inhibitors on VEGF-C and VEGF-A secretion and VEGF-C expression in breast cancer cells. (A) Effects of COX-1 inhibitor SC-560, COX-1/-2 inhibitor indomethacin, and COX-2 inhibitor NS-398 on VEGF-C or VEGF-A production by COX-2 expressing MDA-MB-231 cells. (B) Effects of same inhibitors on VEGF-C production by COX-2 expressing Hs578T cells. COX-1 inhibitor caused a minor (but significant; P<0.05) reduction, whereas COX-2 or COX-1/-2 inhibitors caused a substantial (P<0.01) and similar reduction in VEGF-C production by both cell lines. However, these inhibitors have no inhibitory effect on VEGF-A production. Con A, known to stimulate PGE₂ production by MDA-MB-231 cells, also stimulated VEGF-C as well as VEGF-A production, which were substantially (P<0.01) blocked with COX-1/2 inhibitor. (C) Effect of 24 h treatment with COX inhibitors (20 μM indomethacin or 50 μM NS-398 in serum-free culture) on VEGF-C mRNA expression in MDA-MB-231 cells. Data represent mean±s.d. (n=4). ^*P<0.05 and ^**P<0.001 in comparison to control untreated cells.

Figure 5

Effects of COX-2 siRNA treatment on COX-2 expression and VEGF-C production by MDA-MB-231 cells. The first bar in each series is control, that is, the treatment with scrambled siRNA, and the second bar is experimental, that is, the treatment with COX-2 siRNA. siRNA treatment was conducted in serum containing medium for 48 h. Cells were washed and cultured in serum free media for another 24 h to measure VEGF-C protein accumulation in the cell free media, and COX-2 mRNA levels in cells at both 48 and 72 h following the siRNA treatment. A significant suppression of COX-2 mRNA expression as well as VEGF-C secretion was noted in COX-2 siRNA treated cells. ^*P<0.01, ^**P<0.001.

Figure 6

Roles of EP receptors in the regulation of VEGF-C production and expression by COX-2 expressing human breast cancer cells. Cells were treated for 24 h with antagonists of EP1 receptors (SC-51322) and EP4 receptors (AH-23848B and L-161982) in serum-free DMEM. (A and B) Effects of EP receptor antagonists on VEGF-C accumulation in culture media of MDA-MB-231 cells (A) and Hs538T cells (B). A consistent and significant inhibition of VEGF-C production was observed with highly specific EP1 receptor antagonist SC-51322 and EP4 receptor antagonist L-161982. (C) Quantification of VEGF-C mRNA levels in MDA-MD-231 cells after the 24 h exposure to EP receptor antagonists. Data represent mean±s.d. (n=4). ^*P<0.05, ^**P<0.01.

Figure 7

Reduction of VEGF-C expression in MDA-MB-231 cells induced by kinase inhibitors for Her2/Neu (PD153035), Src (PP1) and p38 MAPK (SB203580). MDA-MB-231 cells were cultured for 24 h in serum-free DMEM supplemented with 0.2 % BSA. (A) imunohistochemistry; all the kinase inhibitors at the concentrations employed inhibited cytoplasmic immunostaining for VEGF-C. Scale bar represents 60 μm. (B) cell proliferation/survival (MTT test) and VEGF-C secretion; VEGF-C secretion, but not proliferation/survival, was inhibited by all the inhibitors. Data represent mean±s.d. (n=4), ^*P<0.001.