CXC Chemokine Receptors 1621 CXCR4

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Muller et al. performed a comprehensive examination of chemokine receptor expression on a series of breast cancer and melanoma cell lines (2). Using quantitative RT-PCR and specific probes for CXCR1-5, CCR1-10, CX3CR1, and XCR1, seven breast cancer cell lines expressed mRNA primarily for CXCR4, CXCR2, and CCR7. In comparison with normal mammary epithelial cultures, CXCR4 and CCR7 were consistently elevated in malignant cell lines. Like breast cancer cell lines, melanoma cell lines also expressed high levels of CXCR4 and CCR7, but in addition, these cells expressed high levels of CCR10 compared with normal primary melanocytes. Examination of pleural effusions from breast cancer patients, primary invasive lobular carcinomas, or ductal carcinomas also revealed heightened expression of both CXCR4 and CCR7 mRNA in malignant tissues compared with normal mammary gland. CXCR4 expression in primary tumors, axillary lymph nodes, and pulmonary and hepatic metastases was confirmed by immunohistochemistry. Using in vitro migration and invasion assays as measures of metastatic potential, breast cancer cells were responsive to CXCL12 and CCL21, respectively the ligands for CXCR4 and CCR7. Chemotaxis and invasion induced by CXCL12 were both blocked by neutralizing CXCR4 and CXCL12 antibodies.

These studies led the authors to hypothesize that, like hematopoietic cells, malignant cells employ chemokine receptors to migrate toward chemokine gradients. Using a panel of human tissues, CXCL12 mRNA was detected primarily in lymph nodes, lungs, liver, and bone marrow, which are preferred sites of breast cancer metastasis. Finally, treatment with antibody to CXCR4 inhibited metastasis in severe combined immunodeficient (SCID) mice bearing xenografts of human breast cancer cell line MDA-MB-231.

A global gene expression study also detected increased CXCR4 mRNA in invasive breast cancers (4). Likewise, invasive ductal carcinomas metastasizing to lymph nodes, but not other sites, expressed more CXCR4 (5). Several laboratories have shown that inhibition of CXCR4 signaling using either small interfering RNA (siRNA) (6), small-molecular-weight peptides (7), or neutralizing antibody (2) blocked migration of breast cancer cell lines to CXCL12. Conversely, forced overexpression of CXCR4 in B16 melanoma cells increased metastatic spread to the lungs (8). Interestingly, in melanoma, CXCR4 expression mediated metastasis to lungs, but not to the lymph nodes. Taken together, these findings are consistent with a mechanism whereby breast tumor cells expressing CXCR4 migrate in response to chemokine gradients toward lymph nodes expressing higher levels of the CXCR4-specific ligand, CXCL12, whereas melanoma cells migrate toward lungs. CXCR4-CXCL12 interactions may direct chemotaxis to only particular sites, and this organ specificity may differ depending on the cancer type.

CXCR4 receptor function has also been characterized in ovarian cancer. Unlike breast cancer, in which several receptors are detected, CXCR4 appears to be the only chemokine receptor expressed in ovarian cancers (3). Expression of CXCR4 and migration to CXCL12 has also been documented in many other cancer types, including neuroblastoma, glioblastoma, non-Hodgkin lymphoma, small cell lung carcinoma, renal cell carcinoma, and pancreatic carcinoma (9-14), among others, suggesting that this may be an important mechanism common to many cancers.

In addition to mediating tumor cell migration and invasion, CXCR4 may play a role in other critical functions that affect metastatic success, and the mechanisms are beginning to be elucidated. Proliferation of ovarian carcinoma cells was stimulated in vitro by recombinant CXCL12 (15), an effect associated with Akt activation. The effects of CXCL12 on proliferation were blocked with AMD3100, a specific inhibitor of CXCR4. Likewise, neutralizing antibody to CXCR4 inhibited proliferation and induced apoptosis of non-Hodgkin lymphoma cells (10). Studies in vivo have confirmed that CXCR4 can play a role in local tumor growth. Non-Hodgkin lymphoma tumor-bearing mice treated with antibody to CXCR4 experienced a reduction in tumor weight and increased survival time. Knockdown of CXCR4 with siRNA also limited growth of orthotopically transplanted 4T1 mammary tumor cells (16). Mice transplanted with control tumor cells died from pulmonary metastases, whereas those treated with CXCR4-silenced tumor cells survived without developing macroscopic metastases, confirming a role for CXCR4 in promoting metastasis.

Although it seems clear that CXCR4 is often highly expressed in malignancies, the mechanism responsible for upregulation of CXCR4 have not been completely elucidated. CXCR4 on breast cancer cell lines MDA-MB-361 and SKBR3 has been implicated in CXCL12-mediated transactivation of HER2-neu, an effect associated with Src kinase activation (17). Inhibitors of CXCR4, epidermal growth factor receptor (EGFR)/HER2-neu tyrosine kinase, or Src kinase all blocked CXCL12-induced HER2-neu phosphorylation and tumor cell migration. Hypoxia has also been implicated in increased CXCR4 expression by tumor cells (18,19). Tumors rapidly outgrow the local vasculature and become hypoxic. In order to continue expanding, tumors must induce new blood vessel formation. Vascular endothelial growth factor (VEGF) is suspected to regulate CXCR4 expression on MDA-MB-231 breast cancer cells, because VEGF antisense oligodeoxynucleotides blocked migration of the cells to exogenous CXCL12 (20). This finding was confirmed in ovarian cancer, where VEGF and CXCL12 synergistically induced in vivo angiogenesis (18). Specifically, VEGF upregulated CXCR4 expression on vascular endothelial cells, facilitating tumor cell migration, and CXCR4 upregulated VEGF on vascular endothelial cells, facilitating tumor cell growth and escape from oxygen starvation-induced apoptosis. Hypoxia was identified as the trigger for this angiogenic synergism. Constitutively active nuclear factor kappa B (NFkB), the extracellular signal-activated transcription factor, is a common feature of malignancies, and it was also examined as a potential regulator of CXCR4 expression (21). NFkB p65 and p50 were reported to bind and activate the CXCR4 promoter in breast cancer cells. Other studies have shown that the tumor suppressor von Hippel-Lindau gene, which is linked to the development of several tumor types, negatively regulated expression of CXCR4 (22,23). This may represent an additional regulatory mechanism for those cancers in which the von Hippel-Lindau gene is inactivated.

Thus, expression of CXCR4 on tumor cells, which is common to many breast cancers as well as other cancer types, may contribute to malignant behavior in several ways. CXCR4 expression by tumor cells may interact with CXCL12 to facilitate tumor cell growth and escape from oxygen starvation-induced apop-tosis and as a mechanism to home (metastasize) to secondary sites.

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