The EGF/CSF-1 paracrine invasion loop can be triggered by heregulin beta1 and CXCL12 - PubMed (original) (raw)

The EGF/CSF-1 paracrine invasion loop can be triggered by heregulin beta1 and CXCL12

Lorena Hernandez et al. Cancer Res. 2009.

Abstract

An important step in the process of metastasis from the primary tumor is invasive spread into the surrounding stroma. Using an in vivo invasion assay, we have previously shown that imposed gradients of epidermal growth factor (EGF) or colony-stimulating factor-1 (CSF-1) can induce invasion through an EGF/CSF-1 paracrine loop between cancer cells and macrophages. We now report that invasion induced by other ligands also relies on this EGF/CSF-1 paracrine invasive loop. Using an in vivo invasion assay, we show that MTLn3 breast cancer cells overexpressing ErbB3 exhibit enhanced invasion compared with control MTLn3 cells in response to the ErbB3 ligand HRG-beta1. The invasive response of both MTLn3-ErbB3 and transgenic MMTV-Neu tumors to HRG-beta1 is inhibited by blocking EGF receptor, CSF-1 receptor, or macrophage function, indicating that invasiveness to HRG-beta1 is dependent on the EGF/CSF-1 paracrine loop. Furthermore, we show that CXCL12 also triggers in vivo invasion of transgenic MMTV-PyMT tumors in an EGF/CSF-1-dependent manner. Although the invasion induced by HRG-beta1 or CXCL12 is dependent on the EGF/CSF-1 paracrine loop, invasion induced by EGF is not dependent on HRG-beta1 or CXCL12 signaling, showing an asymmetrical relationship between different ligand/receptor systems in driving invasion. Our results identify a stromal/tumor interaction that acts as an engine underlying invasion induced by multiple ligands.

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Figures

Figure 1

Figure 1. HRGβ1 induces in vivo invasion of MTLn3-ErbB3 cells

A. Dose-response for HRGβ1-stimulated in vivo invasion in MTLn3-ErbB3 primary tumors (p<.002 by ANOVA). B. In vivo invasion of MTLn3-ErbB3 (light gray) and MTLn3-pLXSN (dark gray) primary tumors in response to 50nM HRGβ1 and 25nM EGF (p<.0004 for pLXSN and p<.02 for ErbB3 by ANOVA). Means and standard deviations are shown.

Figure 2

Figure 2. EGF/CSF-1 signaling is required for in vivo invasion of MTLn3-ErbB3 tumors in response to HRGβ1 stimulation

A. In vivo invasion in response to 25 nM EGF or 50 nM HRGβ1 in the presence of a control antibody, (IgG Ab), or a CSF-1 receptor-blocking antibody (CSF-1R Ab). B. The effect of 1μM Iressa or vehicle (DMSO) on in vivo invasion of MTLn3-ErbB3 primary tumors in response to 50 nM HRGβ1. C. In vivo invasion in response to 25 nM EGF or 50 nM HRGβ1 in the presence of a control antibody, (IgG Ab), or an EGF-binding antibody (EGF Ab). D. The effect of an EGF neutralizing antibody on chemotaxis of MTLn3-ErbB3 cells to 5 nM EGF or 12.5 nM HRGβ1 determined using a Boyden chamber. Results are shown as the percent of the chemotaxis response for each ligand remaining in the presence of the inhibitor compared to the chemotactic response for that ligand in the absence of the inhibitor. Means and standard deviations are shown. Pairwise comparisons by t-test are provided in the text.

Figure 3

Figure 3. MMTV-Neu tumors show in vivo invasion in response to HRGβ1 which requires EGF/CSF-1 signaling

A. MMTV-PyMT (dark gray) and MMTV-Neu (light gray) tumors were tested for their ability to invade in vivo in response to 50 nM HRGβ1. B. MMTV-Neu tumor in vivo invasive response to 50 nM HRGβ1 in the presence of either control antibody (IgG Ab), an EGF neutralizing antibody (EGF Ab) or a CSF-1 R blocking antibody (CSF-1R Ab) (p<5×10−13 by ANOVA). Means and standard deviations are shown.

Figure 4

Figure 4. MMTV-PyMT tumors invade in vivo in response to CXCL12 stimulation and this invasion is dependent on EGF/CSF-1 signaling and macrophages

In vivo invasion of MMTV-PyMT tumors in response to 62.5 nM CXCL12 in the presence of a control antibody (IgG Ab), a CSF-1 receptor antibody (CSF-1R Ab), or an EGF neutralizing antibody (EGF Ab) (p< 2 × 10−8 by ANOVA). Means and standard deviations are shown.

Figure 5

Figure 5. EGF induced in vivo invasion is not dependent on CXCL12 or HRGβ1 signaling

A. The effect of CXCR4 inhibition (AMD3100, 100 nM) on invasion of MMTV-PyMT tumors in response to 62.5 nM CXCL12. B. The effect of CXCR4 inhibition (AMD3100, 100 nM) on invasion of MMTV-PyMT tumors in response to EGF. C. In vivo invasion of MTLn3-ErbB3 tumors to 50 nM HRGβ1 in the presence of a control antibody (IgG Ab), or an ErbB3 blocking antibody (ErbB3 Ab). D. In vivo invasion of MTLn3-ErbB3 tumors to 25 nM EGF in the presence of a control antibody (IgG Ab), or an ErbB3 blocking antibody (ErbB3 Ab). Means and standard deviations are shown. Pairwise comparisons by t-test are provided in the text.

Figure 6

Figure 6. Model for in vivo invasion

The primary tumor expresses receptors for a number of chemoattractants, including the EGF receptor. Chemoattractants present in the tumor microenvironment can feed into the EGF/CSF-1 paracrine loop, which then drives invasion in vivo.

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