CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature

Abstract

Chemokines and chemokine receptors have been posited to have important roles in several common malignancies, including breast and lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2), recently deorphanized as a chemokine receptor that binds chemokines CXCL11 and CXCL12, can regulate these two common malignancies. Using a combination of overexpression and RNA interference, we establish that CXCR7 promotes growth of tumors formed from breast and lung cancer cells and enhances experimental lung metastases in immunodeficient as well as immunocompetent mouse models of cancer. These effects did not depend on expression of the related receptor CXCR4. Furthermore, immunohistochemistry of primary human tumor tissue demonstrates extensive CXCR7 expression in human breast and lung cancers, where it is highly expressed on a majority of tumor-associated blood vessels and malignant cells but not expressed on normal vasculature. In addition, a critical role for CXCR7 in vascular formation and angiogenesis during development is demonstrated by using morpholino-mediated knockdown of CXCR7 in zebrafish. Taken together, these data suggest that CXCR7 has key functions in promoting tumor development and progression.

Fig. 1.

Surface expression of CXCR7 in breast and lung cancer cell lines. (A) Surface expression of CXCR7 on 4T1, LLC, and MDA MB 435s cell lines was quantified by flow cytometry, using CXCR7 antibody 11G8. (B) Binding of radiolabeled CXCL12 to 4T1, LLC, and MDA MB 435s cells was quantified in the presence or absence of nonradiolabeled chemokines and corresponds to levels of CXCR7 measured by flow cytometry. (C) Surface expression of CXCR4 on 4T1, LLC, and MDA MB 435s cell lines was quantified by flow cytometry using CXCR4 Ab 12G5. wt, wild type.

Fig. 2.

CXCR7 promotes growth of breast and lung cancer cell-derived tumors. (A–C) Tumor growth was monitored after WT and transfected, and control MDA MB 435s cells were implanted s.c. into the flank (A and B) of scid mice or mammary fat pad of nude mice (C). (D and E) 4T1 (D) and LLC (E) cells were implanted s.c. into the flank of BALB/c or C57Bl6 mice, respectively. Tumor progression was quantified by tumor volume over time and tumor weight at the end of the experiment. Studies were repeated twice with n ≥ 6 mice per group.

Fig. 3.

RNA interference of CXCR7 reduces growth of experimental lung metastases. WT and CXCR7 RNAi 4T1 lines were injected intravenously into BALB/c mice via tail vein. Tumor growth in lung was quantified by bioluminescence imaging. The study was repeated twice with n = 4 mice per group.

Fig. 4.

CXCR7 expression in human breast, lung, and other cancers. (A) (Left) Undetectable expression of CXCR7 in normal breast tissue from a reduction mammoplasty. (Right) Primary invasive ductal carcinoma of the breast with increased amounts of CXCR7. Isotype control (Left) versus CXCR7 (Right) staining of sections from lung adenocarcinoma (B), Rhabdomyosarcoma (C), or Cervix squamous cell carcinoma (D). Nuclei were counterstained with hematoxylin (blue). (Magnification: ×400.)

Fig. 5.

CXCR7 is expressed in tumor vasculature. (A) Tumors formed from MDA MB 435s cells were stained with antibodies to the vascular endothelial marker CD31 (green) or CXCR7 (red). Merged image shows colocalization of CXCR7 and CD31 in tumor blood vessels (yellow). (B) Intense CXCR7 staining is observed on the tumor vascular of sections from breast carcinoma (a), lung adenocarcinoma (b), ovary mucinous adenocarcinoma (c), breast adenocarcinoma (d), lung squamous cell carcinoma (e), liver hepatocellular carcinoma (f), bladder transitional cell carcinoma (g), kidney renal cell carcinoma (h), and liver cholangiocarcinoma (i). No staining was observed with isotype control antibody.