Mechanisms of breast cancer metastasis

Invasive breast cancer tends to metastasize to lymph nodes and systemic sites. The management of metastasis has evolved by focusing on controlling the growth of the disease in the breast/chest wall, and at metastatic sites, initially by surgery alone, then by a combination of surgery with radiation, and later by adding systemic treatments in the form of chemotherapy, hormone manipulation, targeted therapy, immunotherapy and other treatments aimed at inhibiting the proliferation of cancer cells. It would be valuable for us to know how breast cancer metastasizes; such knowledge would likely encourage the development of therapies that focus on mechanisms of metastasis and might even allow us to avoid toxic therapies that are currently used for this disease. For example, if we had a drug that targeted a gene that is critical for metastasis, we might even be able to cure a vast majority of patients with breast cancer. By bringing together scientists with expertise in molecular aspects of breast cancer metastasis, and those with expertise in the mechanical aspects of metastasis, this paper probes interesting aspects of the metastasis cascade, further enlightening us in our efforts to improve the outcome from breast cancer treatments

Fibroblast growth factor receptor 1 activation in mammary tumor cells promotes macrophage recruitment in a CX3CL1-dependent manner.

Tumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for growth and progression of the primary tumor. Previous studies have shown that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. These studies also demonstrated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium where macrophages contributed to iFGFR1-mediated epithelial cell proliferation and angiogenesis. The studies presented here further utilize this model to identify the mechanisms that regulate FGFR1-induced macrophage recruitment. Results from this study elucidate a novel role for the inflammatory chemokine CX3CL1 in FGFR1-induced macrophage migration. Specifically, we illustrate that activation of both the inducible FGFR1 construct in mouse mammary epithelial cells and endogenous FGFR in the triple negative breast cancer cell line, HS578T, leads to expression of the chemokine CX3CL1. Furthermore, we demonstrate that FGFR-induced CX3CL1 is sufficient to recruit CX3CR1-expressing macrophages in vitro. Finally, blocking CX3CR1 in vivo leads to decreased iFGFR1-induced macrophage recruitment, which correlates with decreased angiogenesis. While CX3CL1 is a known target of FGF signaling in the wound healing environment, these studies demonstrate that FGFR activation also leads to induction of CX3CL1 in a tumor setting. Furthermore, these results define a novel role for CX3CL1 in promoting macrophage recruitment during mammary tumor formation, suggesting that the CX3CL1/CX3CR1 axis may represent a potential therapeutic approach for targeting breast cancers associated with high levels of tumor-associated macrophages

Mechanisms of breast cancer metastasis

Invasive breast cancer tends to metastasize to lymph nodes and systemic sites. The management of metastasis has evolved by focusing on controlling the growth of the disease in the breast/chest wall, and at metastatic sites, initially by surgery alone, then by a combination of surgery with radiation, and later by adding systemic treatments in the form of chemotherapy, hormone manipulation, targeted therapy, immunotherapy and other treatments aimed at inhibiting the proliferation of cancer cells. It would be valuable for us to know how breast cancer metastasizes; such knowledge would likely encourage the development of therapies that focus on mechanisms of metastasis and might even allow us to avoid toxic therapies that are currently used for this disease. For example, if we had a drug that targeted a gene that is critical for metastasis, we might even be able to cure a vast majority of patients with breast cancer. By bringing together scientists with expertise in molecular aspects of breast cancer metastasis, and those with expertise in the mechanical aspects of metastasis, this paper probes interesting aspects of the metastasis cascade, further enlightening us in our efforts to improve the outcome from breast cancer treatments.ISSN:1573-7276ISSN:0262-089

FGFR activation in mammary epithelial cells and breast cancer cells promotes macrophage recruitment.

<p>A) Conditioned media from HC-11/R1 cells containing soluble factors induced following treatment of cells with either B/B to activate iFGFR1 or ethanol as a solvent control for 24 were used to assess migration of RAW 264.7 cells. A significant induction in macrophage recruitment was observed in response to media containing iFGFR1-induced soluble factors. ***p<0.001. B) Conditioned media from HS578T cells treated with either PD173074, which inhibits FGFR activity, or DMSO as a solvent control were used to assess recruitment of THP-1 cells. A significant decrease in macrophage recruitment was observed in response to media from cells treated with an FGFR1 inhibitor. *p<0.05. Error bars represent SEM. C) Expression of CX3CR1 in macrophages isolated from mammary glands of mice treated with either B/B or solvent control for either 48 hours or 4 weeks. Expression levels were assessed using microarray analysis and expression levels are normalized to levels of GAPDH.</p

Proteomic analysis of soluble proteins after iFGFR1 activation.

a<p>The statistics listed in the table for each identified sequence represent those from the best matching spectrum.</p>*<p>Carbamidomethyl modified cysteine.</p><p>HC-11/R1 cells were treated with B/B for 24 hours. Following treatment, conditioned media were collected and analyzed by mass spectrometry for soluble protein expression. Proteomic analysis indicated that several peptides representing CX3CL1 were present in the media following iFGFR1 activation.</p

Human breast cancer cells secrete CX3CL1 in an FGF-dependent manner to promote macrophage cell migration.

<p>FGF-dependent production and secretion of CX3CL1 by HS578T cells mediates recruitment of human macrophages. A) HS578T cells treated with 50ng/mL bFGF demonstrated significant induction of CX3CL1 gene expression after 4 hours relative to PBS solvent control-treated cells. B) Soluble CX3CL1 protein concentrations were significantly upregulated in HS578T cells treated with bFGF for 8 hours in comparison to control-treated cells. C) HS578T cells, which produce high levels of endogenous bFGF, were treated with the PD173074 for 8 hours to inhibit autocrine FGFR activation. A significant reduction in CX3CL1 gene expression was observed relative to DMSO-treated cells as measured by qRT-PCR. D) THP1 cells that had been differentiated into macrophages using PMA demonstrated increased migratory potential when exposed to conditioned medium from CX3CL1-expressing HS578T cells in comparison to serum free medium. Furthermore, this enhanced cell migration was significantly reduced when THP1 cells were treated with a CX3CL1 blocking antibody (aCX3CL1) relative to IgG-treated THP1 cells. *p<0.05, **p<0.001. Results in each figure panel are representative of a minimum of three different experiments. Error bars represent SEM.</p

iFGFR1-induced CX3CL1 secretion by epithelial cells mediates macrophage migration.

<p>siRNA techniques were used to directly target CX3CL1 in order to determine the role of CX3CL1 in iFGFR1-mediated macrophage recruitment. A) CX3CL1 gene expression levels were significantly elevated in HC-11/R1 cells transfected with non-targeting (NT) siRNA. In comparison, CX3CL1 gene expression levels were significantly reduced in CX3CL1siRNA HC-11/R1 cells treated with B/B after 24 hours. B) ELISA was performed to verify reduction in CX3CL1 protein secretion in HC-11/R1 cells transfected with CX3CL1siRNA. Results indicated that soluble CX3CL1 protein concentrations were significantly induced in HC-11/R1 cells transfected with non-targeting (NT) siRNA. In comparison, soluble protein concentrations were reduced in CX3CL1siRNA HC-11/R1 cells treated with B/B after 24 hours. C) Conditioned medium from non-targeting HC-11/R1 cells treated with B/B significantly increased migration of RAW 264.7 cells relative to conditioned medium from cells treated with solvent alone. Exposure to conditioned medium from CX3CL1 siRNA HC-11/R1 cells significantly decreased RAW 264.7 cell migration. Furthermore, addition of 50ng/mL rmCX3CL1 to CX3CL1siRNA HC-11/R1 cells at the time of B/B treatment rescued the loss of RAW 264.7 cell migration in cells exposed to conditioned medium from CX3CL1siRNA HC-11/R1 cells. These results indicate that CX3CL1 is a key mediator of macrophage recruitment. *p<0.05, **p<0.001, ***p<0.0001. Results in each figure panel are representative of a minimum of three different experiments. Error bars represent SEM.</p