Chapter 5 Expression of Chemokines and Chemokine Receptors in Human Colon Cancer

Abstract

Human colorectal cancer (CRC), the second largest cause of tumor‐related death in Western countries, represents a paradigm for the now well‐established connections between inflammation and cancer. In this study, we investigated which inflammatory mediators are mostly expressed in the microenvironment of human CRC. The RNA profile of a large panel of inflammatory genes, in particular chemokines and chemokine receptors, was analyzed in eight surgical tumor samples and in paired normal tissues from CRC patients. We employed an “inflammatory gene card” (TaqMan Low Density Array by Applied Biosystem), designed by our group, containing probes for 24 chemokines and 17 chemokine receptors. Several chemokines were strongly upregulated in the tumor microenvironment, most frequently CCL4 and CCL5, chemotactic for monocytes/macrophages and T cells, and the corresponding receptors CCR1 and CCR5; the angiogenic chemokines CXCL1 and CXCL8, and the receptor CXCR2. The antiangiogenic chemokines CXCL9 and CXCL10 were also expressed, but in the absence of the receptor CXCR3. Selected results have been confirmed in a larger number of samples. The levels of mRNA CXCL8 were significantly associated with the levels of osteopontin, a matrix‐associated protein that shares with chemokines important functions such as induction of cell migration and survival, and modulation of the neoangiogenesis. Overall these results could be helpful to identify the most relevant inflammatory pathways present in CRC tumors and to build a solid rationale for future therapeutic interventions based on anti‐inflammatory strategies.

Introduction

Links between cancer and inflammation were first suggested in the 19th century on the basis of observations that tumors often arise at sites of chronic inflammation and that inflammatory cells are present in the biopsied samples from tumors (Mantovani et al., 2001). Epidemiological studies have shown that chronic inflammation predisposes individuals to various types of cancer, including microbial infections, autoimmune diseases, and inflammatory conditions of unknown origin. The hallmarks of cancer‐related inflammation include the presence in tumor tissues of inflammatory cells and soluble mediators such as chemokines, cytokines and prostaglandins, tissue remodeling, and angiogenesis (Coussens and Werb, 2002, Karin, 2006, Mantovani, 2005, Mantovani et al., 2008).

Chemokines are chemotactic cytokines that cause the direct migration of leukocytes and are induced by inflammatory cytokines, growth factors, and pathogenic stimuli. Many human cancers have a complex chemokine network that regulates the extent and phenotype of the infiltrating leukocytes, as well as have an effect on tumor growth, survival, migration, and angiogenesis (Balkwill, 2004). The pattern of chemokine‐receptor and ligand expression in a tissue is generally correlated with the numbers and types of infiltrating cells that are present in the tumor microenvironment (Balkwill, 2004, Karin and Greten, 2005, Rossi and Zlotnik, 2000).

The influence of the immune response in the behavior of neoplasia has been extensively investigated. There is little doubt that adaptive immune cells, especially cytotoxic CD8⁺ T‐cell effectors, have the potential to limit tumor progression (Dunn et al., 2004). The protective function of CD8⁺ T cells has been demonstrated in patients with melanoma, ovarian, and colorectal cancer (CRC) (Coukos et al., 2005, Taylor et al., 2007). Recently, Galon and colleagues (Galon et al., 2006, Pages et al., 2005) demonstrated that the presence of a strong immune‐cell infiltrate is associated with the absence of early metastatic processes, which include vascular emboli, lymphatic invasion, and perineural invasion, demonstrating a beneficial effect of the host's immune response.

On the other hand, the persistence of active innate immune responses (i.e., chronic inflammation) at tumor sites has been more frequently associated with poor clinical outcome (Balkwill, 2004, Coussens and Werb, 2002, Dunn et al., 2004, Karin, 2006, Mantovani, 2005, Mantovani et al., 2001, Mantovani et al., 2008).

The links between inflammation and cancer promotion are especially strong in human colorectal carcinoma (CRC), the second largest cause of cancer‐related death in Western countries. Patients with inflammatory bowel disease, both ulcerative colitis and Crohn's disease, are at increased risk of developing colorectal cancer. Even precancerous tissues show signs of inflammation. Accordingly, treatment with nonsteroidal anti‐inflammatory agents decreases the incidence of colon cancer, and the mortality that results from it (Bertagnolli, 2003).

The contribution of macrophages to CRC development is quite controversial. Bailey et al. (2007) demonstrated that the increase of macrophages number in all areas within the tumor correlated with advanced tumor stage. In contrast, Forssell et al. (2007) showed that in CRC, macrophages are localized principally at the tumor front and positively influenced prognosis. These contrasting results may be explained by the “macrophages balance hypothesis,” proposed by our group, to convey the idea that macrophages may inhibit or stimulate tumor growth according to their functional polarization and state of activation (Mantovani et al., 2004, Mantovani et al., 2002, Pollard, 2004). While M1‐polarized macrophages, activated by IFNγ and bacterial products such as LPS, usually have tumoricidal activity, M2 macrophages, differentiated in the presence of Th2 cytokines (IL‐4, IL‐13) or IL‐10, most frequently are not cytotoxic, favor tumor cell proliferation and the angiogenic switch, and lead to tumor progression and invasion (Mantovani et al., 2008). In this context, it is clear that inflammatory mediators, such as chemokines, cytokines, and growth factors play a pivotal role in the recruitment of the inflammatory infiltrate and in the buildup of the tumor microenvironment.

In this study, we investigated the expression of chemokines and chemokine‐receptors in surgical samples of human CRC. We analyzed the mRNA profile using a customized TaqMan Low Density Array (Lu et al., 2008). The results show a strong upregulation of chemokines and chemokine‐receptors, indicating the pivotal role of these inflammatory mediators in the growth and progression of CRC.