Role of chemokine receptor CXCR2 expression in mammary tumor growth, angiogenesis and metastasis

Abstract

Background: Chemokines and their receptors have long been known to regulate metastasis in various cancers. Previous studies have shown that CXCR2 expression is upregulated in malignant breast cancer tissues but not in benign ductal epithelial samples. The functional role of CXCR2 in the metastatic phenotype of breast cancer still remains unclear. We hypothesize that the chemokine receptor, CXCR2, mediates tumor cell invasion and migration and promotes metastasis in breast cancer. The objective of this study is to investigate the potential role of CXCR2 in the metastatic phenotype of mouse mammary tumor cells.

Materials and methods: We evaluated the functional role of CXCR2 in breast cancer by stably downregulating the expression of CXCR2 in metastatic mammary tumor cell lines Cl66 and 4T1, using short hairpin RNA (shRNA). The effects of CXCR2 downregulation on tumor growth, invasion and metastatic potential were analyzed in vitro and in vivo.

Results: We demonstrated knock down of CXCR2 in Cl66 and 4T1 cells (Cl66-shCXCR2 and 4T1-shCXCR2) cells by reverse transcriptase polymerase chain reaction (RT-PCR) at the transcriptional level and by immunohistochemistry at the protein level. We did not observe a significant difference in in vitro cell proliferation between vector control and CXCR2 knock-down Cl66 or 4T1 cells. Next, we examined the invasive potential of Cl66-shCXCR2 cells by in vitro Matrigel invasion assay. We observed a significantly lower number (52 ± 5) of Cl66-shCXCR2 cells invading through Matrigel compared to control cells (Cl66-control) (182 ± 3) (P < 0.05). We analyzed the in vivo metastatic potential of Cl66-shCXCR2 using a spontaneous metastasis model by orthotopically implanting cells into the mammary fat pad of female BALB/c mice. Animals were sacrificed 12 weeks post tumor implantation and tissue samples were analyzed for metastatic nodules. CXCR2 downregulation significantly inhibited tumor cell metastasis. All the mice (n = 10) implanted with control Cl66 cells spontaneously developed lung metastasis, whereas a significantly lower number of mice (40%) implanted with Cl66-shCXCR2 cells exhibited lung metastases.

Conclusions: Together, these results suggest that CXCR2 may play a critical role in breast cancer invasion and metastasis.

Keywords: CXC chemokines; CXCR2; metastasis; tumor growth.

Figure 1

CXCR2 expression in parent, vector control and CXCR2 knock-down cells, (a) mRNA expression in mammary tumor cell lines showing constitutive expression of CXCR2 and its ligand CXCL-1 as analyzed by semi-quantitative RT-PCR, (b) RT-PCR analysis of CXCR2 transcript levels in Cl66-shCXCR2 and Cl66-control vector transfected cells and quantitative analysis of CXCR2 expression reveals more than 50% reduction in shCXCR2 Cl66 cells (n =3, P<0.05). CXCR2 expression was normalized to GAPDH, (c) CXCR2 protein expression by immunofluorescence using a CXCR2 specific antibody. Confocal images show that CXCR2 protein expression was significantly reduced in Cl66-shCXCR2 cells compared to control cells (×200), (d) mRNA and protein expression of CXCR2 in 4T1-control and 4T1-shCXCR2 cells determined by RT-PCR and immunofluorescence, (×200)

Figure 2

Analysis of cell proliferation and invasion in CXCR2 knock-down cells. (a) CXCR2 down regulation in Cl66 tumor cells did not affect cell proliferation as determined by MTT assay. The values are mean absorbance + SEM (standard error of mean) (n =3, P<0.05), (b) CXCR2 down regulation significantly reduced the invasive capacity of Cl66 cells in an in vitro Matrigel invasion assay. A significantly lower number (52 ± 5) of Cl66-shCXCR2 cells invaded through Matrigel as compared to control cells (Cl66-control) (182± 3)(n =3, P<0.05) (×200)

Figure 3

Tumor growth kinetics of CXCR2 knock-down mammary tumor cells. Cl66-control and Cl66-shCXCR2 cells were implanted into the MFP. Tumor growth was monitored twice weekly. The values are mean tumor volume ± SEM. No significant difference in growth tumor kinetics in mammary fat pad tumors was observed

Figure 4

Inhibition of spontaneous lung metastasis following CXCR2 knock-down. The spontaneous metastatic potential of Cl66-control and Cl66-shCXCR2 cells was monitored as described in materials and methods. (a) A photomicrograph showing gross lung metastatic nodules in animals bearing Cl66-control tumors or Cl66-shCXCR2 tumors, (b) Incidence of metastasis in Cl66-control or Cl66-shCXCR2 tumor-bearing mice, (c) Number of lung metastatic nodules in animals bearing Cl66-control or Cl66-shCXCR2 cells. The values are mean ± SEM. CXCR2 down regulation in Cl66 cells significantly inhibited development of metastatic nodules in mice. (P = 0.002)

Figure 5

In situ cell proliferation, apoptosis and microvessel density in Cl66-control and Cl66-shCXCR2 tumors (a) Cell proliferation was examined using immunohistochemistry using PCNA antibody. The values are mean PCNA positive cells ± SEM. The number of PCNA positive cells in Cl66-control and Cl66-shCXCR2 tumors was not statistically different (×200), (b) In situ apoptosis was monitored by counting cleaved caspase-3 positive apoptotic cells. The values are mean caspase-3 positive apoptotic cells ± SEM. A significant increase in the number of apoptotic cells was observed in Cl66-shCXCR2 tumors as compared to Cl66-control tumors (×100), (c) CXCR2 down regulation in Cl66 cells inhibited tumor-induced angiogenesis. Isolectin B4 immunostaining demonstrates the reduction in microvessel density in Cl66-shCXCR2 tumors compared to Cl66-control tumors. The values are mean microvessel density ± SEM (×200)