C-X-C motif chemokine 12/C-X-C chemokine receptor type 7 signaling regulates breast cancer growth and metastasis by modulating the tumor microenvironment

Abstract

Introduction: Although C-X-C motif chemokine 12 (CXCL12) has been shown to bind to C-X-C chemokine receptor type 7 (CXCR7), the exact molecular mechanism regulations by CXCL12/CXCR7 axis in breast tumor growth and metastasis are not well understood. CXCR7 expression has been shown to be upregulated during pathological processes such as inflammation and cancer.

Methods: Breast cancer cell lines were genetically silenced or pharmacologically inhibited for CXCR7 and/or its downstream target signal transducer and activator of transcription 3 (STAT3). 4T1 or 4T1 downregulated for CXCR7 and 4T1.2 breast cancer cell lines were injected in mammary gland of BALB/c mice to form tumors, and the molecular pathways regulating tumor growth and metastasis were assessed.

Results: In this study, we observed that CXCL12 enhances CXCR7-mediated breast cancer migration. Furthermore, genetic silencing or pharmacologic inhibition of CXCR7 reduced breast tumor growth and metastasis. Further elucidation of mechanisms revealed that CXCR7 mediates tumor growth and metastasis by activating proinflammatory STAT3 signaling and angiogenic markers. Furthermore, enhanced breast tumorigenicity and invasiveness were associated with macrophage infiltration. CXCR7 recruits tumor-promoting macrophages (M2) to the tumor site through regulation of the macrophage colony-stimulating factor (M-CSF)/macrophage colony-stimulating factor receptor (MCSF-R) signaling pathway. In addition, CXCR7 regulated breast cancer metastasis by enhancing expression of metalloproteinases (MMP-9, MMP-2) and vascular cell-adhesion molecule-1 (VCAM-1). We also observed that CXCR7 is highly expressed in invasive ductal carcinoma (IDC) and metastatic breast tissue in human patient samples. In addition, high CXCR7 expression in tumors correlates with worse prognosis for both overall survival and lung metastasis-free survival in IDC patients.

Conclusion: These observations reveal that CXCR7 enhances breast cancer growth and metastasis via a novel pathway by modulating the tumor microenvironment. These findings identify CXCR7-mediated STAT3 activation and modulation of the tumor microenvironment as novel regulation of breast cancer growth and metastasis. These studies indicate that new strategies using CXCR7 inhibitors could be developed for antimetastatic therapy.

Figure 1

CXCL12 enhances CXCR7-mediated cell migration and signaling. (A) 4T1 Vec and CXCR7 shRNA-transfected cells were subjected to chemotaxis toward CXCL12 (50 and 100 ng/ml) by using the modified Boyden chamber assay, as described in Materials and methods. (B) 4T1 Vec and CXCR7 shRNA-transfected cells were grown to confluence in complete medium in six-well plates, and then a wound was made with a 200-μl pipette tip, and the closure of the wounds was monitored in the presence or absence of CXCL12 (50 and 100 ng/ml) by microscopy after 24 and 36 hours. (C) Quantitative analysis of percentage of wound closure. (D) 4T1 and (E) 4T1.2 cells were pretreated for 1 hour with vehicle or CCX771 (1 μM) and were subjected to chemotactic assay in the absence or presence of CXCL12 (100 ng/ml). (F) 4T1 Vec and 4T1 sh-CXCR7 cells were serum starved for 4 hours and stimulated with CXCL12 (100 ng/ml) for different times, as indicated, and incubated at 37°C. After treatment, cells were washed, lysed, and analyzed with Western blotting for Phospho STAT3, STAT3, Phospho-ERK (p-ERK), and GAPDH by Immunoblotting. (G, H) Densitometry analysis of Western blots shows quantitation of pSTAT3 and pERK levels. (I) 4T1.2 cells were serum starved for 12 hours and stimulated with CXCL12 (100 ng/ml) for different time points, as indicated, and incubated at 37°C. After treatment, cells were washed, lysed, and analyzed for Phospho STAT3, STAT3, Phospho-ERK (p-ERK), and ERK with immunoblotting. (J) Densitometry analysis of Western blots shows quantitation of pSTAT3 and ERK levels, *P < 0.05, **P < 0.01, **P < 0.001 versus none, and ^##P < 0.05, ^##P < 0.01 ^###P < 0.001 versus control.

Figure 2

CXCR7 regulates breast cancer tumor growth in vivo by regulating angiogenic, proliferative, and signaling pathways. Female BALB/c mice (6 to 8 weeks old, n = 5) were anesthetized and injected with 2.5 x 10⁵ breast cancer cells 4T1 Vec and 4T1 sh-CXCR7 (downregulated for CXCR7). (A) Tumors were measured by digital calipers weekly for 36 days (mice model inset). (B) Representative photographs of tumors, 36 days after the injection of cells. (C) Tumor weight after 36 days. Female BALB/c mice (6 to 8 weeks old) were anesthetized and injected with 1 × 10⁵ viable 4T1.2 cells into the fourth mammary fat pad. Mice were divided into three groups of five mice each and were injected intraperitoneally with either DMSO or CXCR7 inhibitor (CCX771) or STAT3 inhibitor (S31-201) at 5 mg/kg body weight 3 times a week for 21 days (inhibitors were injected in mice after tumors were palpable), (D, G) tumors were measured with digital calipers (mice model inset) (E, H) Representative photographs of tumors, 31 days after the injection of cells. (F, I) Tumor weight after 31 days. (J) 4T1 Vec, 4T1 sh-CXCR7, and 4T1.2 cell line-derived tumors from CCX771 or S31-201 treated or untreated group were subjected to immunohistochemical (40×) staining with anti-CD31 or Ki67. (K, L) The stained cells were counted in four different fields by using bright-field microscope in each experimental group and the average was calculated. Bars represent the mean ± SD of number of CD31-positive blood vessels and Ki67-positive cells to that of total cells. (M) 4T1 Vec and 4T1 sh-CXCR7 cell line-derived tumors were lysed and analyzed for Phospho STAT3, STAT3, Phospho-ERK (p-ERK), ERK, cyclin D1, and GAPDH with immunoblotting. Data represent the mean ± SD per experimental group. Scale bars, 0.03 mm. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.

Figure 3

CXCR7 promotes breast cancer metastasis. Lungs were removed from mice used in the experiment presented in Figure 2 and inflated with Bouin fixative and number of metastatic nodules on the lungs was counted with the aid of a dissecting microscope. (A) H&E (2.5×) staining of metastatic nodules in the lungs of mice bearing 4T1 Vec or 4T1 sh-CXCR7 (insets: 20× magnification of the areas selected by rectangles) (B) bar showing the number of metastatic lung nodules and (C) H&E (2.5X) staining of metastatic nodules in the lungs of mice bearing 4T1.2 tumors treated with either DMSO or CXCR7 inhibitor (CCX771) or STAT3 inhibitor (S31-201) insets: 5× magnification and (D) bar showing the number of metastatic lung nodules. Scale bars, 0.01 mm. **p < 0.01, ***p < 0.001 versus control.

Figure 4

CXCR7 enhances recruitment of M2 macrophages into tumor stroma. Tumors from mice used in experiment presented in Figure 2 were subjected to IHC (20×) staining for macrophage marker, F4/80. (A) Representative image of 4T1 Vec and 4T1 sh-CXCR7 (B) 4T1.2 tumors treated with either DMSO or CXCR7 inhibitor (CCX771) or STAT3 inhibitor (S31-201). The F4/80⁺-stained cells were counted in four different fields by using bright-field microscope in each experimental group, and the average was calculated. Bars represent the mean ± SD of number of F4/80 macrophages (C) 4T1 Vec, 4T1 sh-CXCR7 and (D) 4T1.2 tumors treated with either DMSO or CXCR7 inhibitor (CCX771) or STAT3 inhibitor (S31-201). CD11b⁺F4/80⁺CD206⁺ cells in tumors were quantified by flow cytometry (E, F) 4T1 and 4T1 sh-CXCR7 (G, H) 4T1.2 tumors treated with either DMSO or CXCR7 inhibitor (CCX771) or STAT3 inhibitor (S31-201). Lungs were removed and stained for Arginase-1 (5X) (I) 4T1 Vec or 4T1 sh-CXCR7 (insets: 40× magnification) and (J) 4T1.2 tumors treated with either DMSO or CXCR7 inhibitor (CCX771) or STAT3 inhibitor (S31-201) (2.5×; insets, 10× magnification). Data represent the mean ± SD per experimental group. Scale bars, 0.03 mm. *P < 0.05, ^**P < 0.01 versus vehicle or vector control.

Figure 5

CXCR7 regulates M-CSF, MMPs, and VCAM-1 expression. (A) Murine macrophages (RAW 264.7) were serum starved for 4 hours and plated on the top chamber of 8-μm-pore polycarbonate membrane filters and SFM containing 4T1 or 4T1 sh-CXCR7 CM (50 to 100 μg/ml) was placed in the lower chamber. After 12 hours of incubation, cells that migrated across the filter toward SFM with or without CM (50 to 100 μg/ml) were fixed, stained, and counted by using bright-field microscopy in five random fields. (B) Conditioned media (CM) obtained from Vector or CXCR7-downregulated cells were subjected to cytokine profiling. (C) The array data were quantitated by ImageJ to generate a protein profile (histogram) (D) Murine macrophages (RAW 264.7) were serum starved for 4 hours and treated with 50 and 100 μM ki20227 (MCSF-R inhibitor) and then plated on the top chamber of 8-μm-pore polycarbonate membrane filters. SFM, in the absence or presence of 4T1 CM (100 μg/ml), was placed in the lower chamber. After 12 hours of incubation, cells that migrated were fixed, stained, and counted by using bright-field microscopy in five random fields. (E) CM obtained from vector or CXCR7-downregulated cells was subjected to gelatin zymography for MMPs activity. (F) CM obtained from 4T1.2 cells treated with CXCR7-specific (CCX771) at 0.5 and 1 μM concentration or STAT3-specific inhibitors (S31-201) at 5 and 10 μM concentration in the presence and absence of CXCL12 (100 ng/ml) were subjected to gelatin zymography. (G) 4T1 and 4T1 shCXCR7 cell line-derived tumors were lysed and analyzed for VCAM-1, MMP-9, and GAPDH expression by Immunoblotting. The 4T1 Vec, 4T1 sh-CXCR7 (H), and 4T1.2 (I) cell line-derived tumors from CCX771 or S31-201 treated or untreated groups were subjected to immunohistochemical staining for MMP-9 expression (40×). Data represent the mean ± SD per experimental group. Scale bars, 0.03 mm. ^**P < 0.01, ^***P < 0.001 versus none and ^###P < 0.01 versus control.

Figure 6

CXCR7 expression in human breast tumors correlates with worse patient outcome and CXCR7 and STAT3 expression in breast cancer patients. (A) Overall survival (OS) of TCGA IDC patients with or without ackr3 overexpression (greater than 1.0-fold SD above mean). (B) Lung metastasis-free survival (LMFS) of IDC patients from study GSE2603. LMFS was compared between 20 high-CXCR7- and 20 low-CXCR7-expressing patients. (C) Breast tumors and adjacent normal tissue (n = 4) were lysed with RIPA buffer, and the lysates were analyzed with Western blotting by using specific antibodies against p-STAT3 (86 kDa), CXCR7 (52 kDa), and GAPDH (37 kDa) as loading control. (D, E) Representative tissue microarray cores of normal and cancerous breast tissue (20×, scale bar, 0.03 mm). Tissue microarray (TMA) samples containing 10 normal, 10 metastatic, and 38 invasive ductal carcinomas (IDCs) were analyzed with immunohistochemistry by using CXCR7 antibodies (kindly provided by ChemoCentryx).

Figure 7

Schematic representation of CXCR7-mediated signaling that regulates breast cancer growth and metastasis. CXCL12 binding to CXCR7 leads to activation of ERK and STAT3 and enhanced expression of VCAM-1. CXCR7 also, either directly or indirectly through STAT3, may enhance MMP-9 and TAMs recruitment to the tumor site. TAMs in turn enhance growth factor, chemokines, and MMPs secretion in a tumor microenvironment. These CXCR7-mediated mechanisms may regulate primary breast tumor growth and metastasis, especially to lungs.