Chemokines: manipulators of metastasis

Allison Bierly, Technical and Marketing Writer
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As drivers of immunity, embryonic development, wound healing, and other important biological processes, chemokines are essential to maintaining health. However, they have also been implicated in the progression of various cancers. Recently, CXCL1 and CXCL2 were revealed to be at the center of a chemotherapy-triggered process leading to chemoresistance and metastasis, while CCL25/CCR9 signaling was shown to restrict colon cancer metastasis. These disparate findings highlight the unique roles that chemokines can play in different types of cancer, and their potential as therapeutic targets.
Highlights
  • Chemokines play key roles in driving and preventing cancer progression
  • CXCL1/2 promote chemoresistance and metastasis
  • CCL25/CCR9 blocks colorectal cancer metastasis, and CCR9 loss links to cancer spread
Introduction
CXCL1/2, MDSCs, and chemoresistance
CCL25 signaling prevents colorectal cancer metastasis
Conclusions
References
Back to top Introduction
Chemokines are 8- to 12-kD polypeptides that signal through G protein-coupled receptors, triggering signaling cascades that lead to cell movement (1–2). Chemokine-guided cell migration is vital for a variety of functions in the organism; for example, chemokines direct the movement of trophoblasts into endometrial epithelia, and are also involved in development of the heart, central nervous system, and skeletal muscle. Additionally, chemokines are intimately involved in wound healing, and can have positive and negative effects on angiogenesis through manipulation of endothelial cells (2).

Chemokines play a critical role in the immune response, guiding immune cells to regions of the secondary lymphoid organs as well as sites of inflammation (2). Recent work has suggested that keratinocyte-produced chemokines regulate the recruitment of Langerhans cells to hair follicles (3), and a study in protozoan parasite infection showed that CXCL10 not only recruits CD8+ T cells to areas of infection, but also influences the speed of the T cells as they search for target cells (4). Thus, new roles for chemokines in immunity continue to be revealed.

Many chemokines and receptors are associated with cancer progression. CXCR4 is frequently overexpressed in cancer cells, and CXCL12-CXCR4 or CXCL12-CXCR7 interactions are thought to underlie invasiveness in a variety of cancers. CCR7 and CXCR3 have been linked to metastasis, as has CCR9 (2). The abundance of chemokines and receptors involved in cancer invasiveness and metastasis suggests that cancer therapies may be improved if combined with modulation of chemokine signaling. Two recent studies provide greater insights into the role of chemokine signaling in breast cancer and colorectal cancer, respectively, emphasizing pivotal functions for these chemokines in metastasis.
Back to top CXCL1/2, MDSCs, and chemoresistance
Acharyya and colleagues recently elucidated how chemotherapy triggers chemoresistance and metastasis in breast cancer cells (5). Treatment with common chemotherapeutic agents led to a chain reaction beginning with stromal cells, with CXCL1/2 at its center, that resulted in cancer cells receiving pro-survival factors (see figure Chemokines and cancer).
To pinpoint the characteristics of cancer cells that led to metastatic capability and chemoresistance, the team first established that knocking down CXCL1 and CXCL2 in mouse breast cancer models led to lower tumor growth and metastasis of tumors to the lungs. CXCL1/2 knockdown also led to a diminished population of tumor-associated CD11b+Gr-1+ myeloid-derived suppressor cells, which express the CXCL1/2 receptor CXCR2. This suggested a potential role for this population in promoting tumor growth and metastasis.

The team used gene expression analysis to identify factors that the CD11b+Gr-1+ cells may be producing to promote tumor survival. They determined that S100A8 and S100A9 may be the responsible factors, and mice receiving transplants of S100A9-negative bone marrow did show reduced tumor growth and metastasis from cancer cell line implants compared to mice receiving wild-type bone marrow. Moreover, clinical samples of human lung metastases revealed correlation between higher levels of S100A8/9 and shorter survival time.

S100A8/9 not only promoted tumor growth and metastasis, but also chemoresistance. Deleting S100A9 led to greater apoptosis of tumor cells in vivo both with and without chemotherapy, and ablated the ability of CD11b+ Gr-1+ cells to protect cancer cells from doxorubicin in vitro. S100A8/9 are known to signal through TLR4 and RAGE, and inhibitor experiments suggested that their pro-survival, chemoprotective functions were effected through activation of ERK1/2 and p70S6K pathways in cancer cells.

The team had established that CXCL1/2 production attracts tumor-associated CD11b+Gr-1+ cells, which promote chemoresistance and metastasis through production of S100A8/9. They next investigated whether the selective pressure of chemotherapy itself could be triggering this sequence of events. Indeed, tumors treated with doxorubicin and cyclophosphamide upregulated CXCL1 and CXCL2, and showed increased recruitment of CD11b+Gr-1+ cells and S100A8/9-expressing cells. In vitro experiments revealed that chemotherapy induced TNF-a production from endothelial cells, which caused CXCL1/2 upregulation in cancer cells via NFkB activation.

In light of these results, the team tested the ability of CXCR2 inhibitors to reduce metastasis in xenograft mice, and found that the CXCR2 inhibitor had no effect on its own. However, when added to doxorubicin-cyclophosphamide treatment, it reduced lung metastases when compared to chemotherapy alone, suggesting that targeting chemokine signaling may be an effective strategy for fighting metastasis in breast cancer.
Back to top CCL25 signaling prevents colorectal cancer metastasis
In contrast to the CXCL1/2 findings in breast cancer, chemokine signaling in colorectal cancer (CRC) may actually hold tumors back from metastasizing. The epithelia of the small intestine and colon produce CCL25, which is the ligand for CCR9. Chen and colleagues found that expression of CCR9 was increased in early-stage CRC cells and tissue, and reduced in those from invasive, later-stage CRC (6). They investigated this loss to clarify the potential role of CCR9/CCL25 signaling in metastasis.

The team first showed that CCR9-expressing cancer cells preferentially form tumors in the colon and small intestine. They injected mice systemically with either CRC cell lines lacking CCR9 expression, or colon cancer-initiating cells (CCIC) derived from early-stage tumors, which expressed CCR9. Early-stage CCICs generated tumors largely in the small intestine and colon, but tumors from the CCR9-negative cell lines and late-stage CCICs were entirely outside of these areas.

To investigate whether CCR9/CCL25 signaling was involved in this difference, the team examined its effect at two timepoints: before and after tumor formation. shRNA knockdown of CCR9 in CCICs led to reduced tumor incidence in the small intestine and colon, and increased tumors in other regions. Antibody treatment against CCL25 before or alongside CCIC injection also reduced tumor multiplicity in the colon and intestine. Importantly, blocking CCR9/CCL25 signaling after tumor formation caused greater tumor incidence and multiplicity outside the GI tract, mimicking metastasis.

Gene expression analysis and in vitro cell activation revealed activation of AKT signaling in these cells in response to CCL25. Using CCICs that expressed GFP under the control of a NOTCH-responsive promoter, the team showed that NOTCH signaling downregulated CCR9 expression and AKT activation, and that the CCR9 regulation occurred at the protein level through proteasomal degradation. Consequently, CCICs expressing high levels of NOTCH formed more tumors outside of the colon and intestine. Therefore, NOTCH signaling appears to trigger colon cancer metastasis by downregulating CCR9, disrupting the CCR9-CCL25 axis that restricts cancer cells to the colon and intestine in early stages of the disease.
Back to top Conclusions
The intimate involvement of chemokines in the movement of cells makes them key players in cancer, whether promoting metastasis as the CXCL1/2 network does in breast cancer, or preventing it as suggested by the CCL25/CCR9 findings in colon cancer. In light of these studies as well as others, treatments augmenting or inhibiting chemokine signaling are likely to play an important role in cancer therapy in the future. Discovering which chemokines are responsible for both normal and disease-related phenomena, therefore, is vital.
Studying gene expression, signaling pathway activation, and the presence of chemokines in serum or cell culture supernatant are all useful methods for determining which chemokines are active in a particular system. QIAGEN provides solutions for each of these aspects of chemokine activity, with emphasis on a whole-pathway approach. RT2 Profiler PCR Arrays provide real-time PCR assays for the 84 most relevant genes in certain biological or disease-related pathways, while Cignal Finder Reporter Arrays provide cell-based reporter assays for 10 related signaling pathways in one array. Multi-Analyte ELISArrays allow simultaneous analysis of 12 related chemokines or cytokines at once, in a convenient traditional ELISA format.
Back to top References
  1. Patel, S.J., et al. (2012) Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure. Nat. Biotech. 30, 179.
  2. Abbas, A.K. and Lichtman, A.H. (2003) Cellular and Molecular Immunology. 5th ed. Philadelphia, PA: Saunders.
  3. Ramen, D., Sobolik-Delmaire, T., and Richmond, A. (2011) Chemokines in health and disease. Exp. Cell Res.l 317, 575.
  4. Nagao, K. et al. (2012) Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin. Nat. Immunol. 13, 744.
  5. Harris, T.H. et al. (2012) Generalized Levy walks and the role of chemokines in migration of effector CD8+ T cells. Nature 486, 545.
  6. Acharyya, S. et al. (2012) A CXCL1 paracrine network links cancer chemoresistance and metastasis. Cell 150, 165.
  7. Chen, H.J. et al. (2012) Chemokine 25-induced signaling suppresses colon cancer invasion and metastasis. J. Clin. Invest. Epub ahead of print.




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