Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo

Abstract

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

Figure 1. Validation of HBEGF expression and EGFR activation

A, HBEGF concentrations in supernatants collected from empty vector control transductants (control) or HBEGF transductants (HBEGF) as measured by an HBEGF ELISA. N = 3, data are means and SEM; *** p < 0.001 by t-test. B, representative cropped bands from western blots of total EGFR, phospho-Tyr, and total and phospho-ERK expression. Beta-tubulin was used as a loading control. C, quantitation of relative EGFR and ERK phosphorylation normalized to total EGFR or ERK. D, representative cropped bands from western blots of total and phospho-ERK expression in HBEGF expressing transductants treated with 2ug/mL CRM197 for 15 minutes. Beta-tubulin was used as a loading control. E, quantitation of relative ERK phosphorylation normalized to total ERK. Data are means and SEM; *p < 0.05, ***p < 0.001 by z-test.

Figure 2. HBEGF expression enhances intravasation and metastasis with no effect on primary tumor growth

HBEGF transductants (HBEGF) or empty vector controls (Control) were injected into the mammary fat pads of SCID/NCr mice, and at 4 weeks (for MTLn3) and 14 weeks (for MDA-MB 231) primary tumor size, metastasis and intravasation were measured. A, primary tumor size B, spontaneous lung metastasis. C, intravasation efficiency of MTLn3 tumor cells in vivo. D, in vitro transendothelial migration (iTEM) of MDA-MB 231 transductant cell lines. Data are means and SEM; *p < 0.05, ***p < 0.001 by t-test.

Figure 3. HBEGF expression increases basal motility and invasion

A, in vivo invasion of MTLn3 ErbB1 or MDA-MB 231 tranductant primary tumors in response to buffer or 25nM EGF. B, intravital motility (IVI) using multiphoton imaging of GFP-labeled tumor cells. Total cell motility was quantified per 100um z-stack using 5um steps for 30 minute intervals. N = 5 – 10 mice per transductant cell line, data are means and SEM; *p < 0.05 by Mann-Whitney analysis. C,D, dose-response for EGF-stimulated chemotaxis for MTLn3 ErbB1 (C) and MDA-MB 231 (D) transductant cell lines. E, invasion in vitro in response to buffer or 25nM EGF. Samples were fixed and stained as described in the Materials and Methods and invasion was quantitated as the percent area of the filter covered by invading cells. Data are means and SEM; n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001 by t-test.

Figure 4. Invasion in vivo no longer requires the CSF-1/CSF-1R paracrine loop upon HBEGF expression

A,B, MTLn3 ErbB1 control (A) and HBEGF (B) primary tumors were tested for their ability to invade in vivo upon inhibition of the paracrine loop of invasion by using 1uM JnJ, a CSF-1R inhibitor. C, PBS-containing control or clodronate-containing liposomes were injected i.v. 48 h and 24 h prior to analysis into the tail veins of animals bearing MTLn3 ErbB1 HBEGF tumors. Four hours prior to analysis, 70kDa Texas Red dextran was injected intravenously, and the uptake of dextran by macrophages in the spleen and tumor was assessed using multiphoton imaging. Representative images of the spleen (left columns) and primary tumor (right columns) are shown. Magenta, collagen fibers; red, macrophages; green, tumor cells. Scale bar, 50 um. D, Needles containing buffer or 25nM EGF were inserted into the primary tumors of animals that were treated with PBS-containing control liposomes (vehicle) or clodronate-containing liposomes (clodronate) and the number of cells invading into the needle was determined. E, 3D in vitro invasion of MTLn3 ErbB1 and MDA-MB 231 transductant cell lines in the absence and presence of BAC macrophages measured as the fraction of carcinoma cells invading ≥ 20um into collagen gel. Data are means and SEM; **p < 0.01, ***p < 0.001 by t- test.

Figure 5. HBEGF expression increases basal invadopodium formation and matrix degradation

Representative images of invadopodia and matrix degradation for MTLn3 ErbB1 (A) and MDA-MB 231 (B) transductants. Invadopodia are defined as areas of cortactin and Tks5 co-localized puncta, examples of which are indicated by the red arrows. Scale bar, 25um. C, quantitation of the total number of invadopodia per cell for MTLn3 and MDA-MB 231 transductants. D, quantitation of total degradation area per field of view for MTLn3 (16 hours) and MDA-MB 231 (4 hours) transductants. All values were normalized to the corresponding control. E, cortactin activation measured as relative level of phosphorylation as described in Materials and Methods. F, quantitation of total number of invadopodia per cell after treatment with DMSO or 1uM Iressa for 15 minutes. G, quantitation of total number of invadopodia per cell after treatment with vehicle or 2ug/mL CRM197 for 15 minutes. H, quantitation of total number of invadopodia per cell after treatment with DMSO or 20uM SRCI1 for the duration of the invadopodium formation and matrix degradation assay (16 hours for MTLn3s and 4 hours for MDA-MB 231s). I, quantitation of total degradation area per field of view for HBEGF expressing transductants after treatment with DMSO or 20uM SrcI1. Three independent experiments were performed for each condition (N = 3), and for each independent experiment, eight to ten random fields of view were imaged for further data analysis. Data are means and SEM; *p < 0.05, **p < 0.01, ***p < 0.001 by t- test.

Figure 6. HBEGF expression enhances invadopodium function and carcinoma cell invasion via upregulation of MMP2 and MMP9 expression

A, relative fold change in MMP2, MMP9 and MMP14 mRNA expression in MTLn3 ErbB1 (left) and MDA-MB 231 (right) transductants as determined by qPCR. B, relative fold change in MMP2 and MMP9 mRNA expression in HBEGF expressing MTLn3 ErbB1 (left) and MDA-MB 231 (right) transductants after treatment with 10uM MEK inhibitor (MEKi) overnight as determined by qPCR. GAPDH was used as a housekeeping gene, and all values were normalized to the corresponding control. Data are means and SEM; **p < 0.01, ***p < 0.001 by z-test. C, invasion in vitro in response to DMSO or 0.5uM BiPS. D, quantitation of total degradation area per field of view for HBEGF expressing transductants after treatment with 0.5uM BiPS. All values were normalized to the corresponding control. Data are means and SEM; *p < 0.05, ***p < 0.001 by t-test.