Journal of Young Investigators RESEARCH R E V I E W

Targeting the -8 Signaling Pathway in Colorectal Cancer: A Mini-review

Ishita Aggarwal1*

Colorectal cancer (CRC), commonly called colon cancer, is a cancer of the colon or rectum. Although colon cancer is highly treatable, the National Cancer Institute of Canada (NCIC) acknowledges it as the third most common cancer and the second most common cause of cancer-related deaths among Canadians. Scientific studies have shown that colon cancer is caused, in part, by the overproduction of a molecule called Interleukin-8 (IL-8), which is released from the surface of tumor cells. Once generated, IL-8 binds to specialized , called CXCR2 receptors, on the surface of nearby cancer cells. Binding of IL-8 to CXCR2 produces signals within tumor cells that activate molecules called transcription factors. The activation of various transcription factors, including NF-B and AP-1, via the Akt and MAPK signaling pathways, ultimately causes the growth and survival of colon cancer cells. Hence, reducing the expression of IL-8 by cancer cells may have therapeutic implications for patients suffering from colon cancer. Although various treatment methods have been developed to inhibit the production of IL-8, most techniques pose a safety risk to patients because they may interact with the human immune system in unpredictable ways. This review suggests that the safest treatment method to target IL-8 is the use of nano-particles, specifically quantum dots (QDs), to transport small interfering RNA (siRNA) into colon cancer cells. Once delivered, siRNA can silence IL-8 expression, reducing the risk of cancerous growth.

INTRODUCTION Colorectal cancer (CRC) is a cancer of the colon or rectum and is Wilson, 2008), the proliferation and of human the second most common cause of cancer-related deaths in colon cancer cells is likely dependent on IL-8 binding to the Canadian men and women (Jemal, Siegel, Xu, & Ward, 2010). latter (Raman, Baugher, Thu, & Richmond, 2007; Hoffmann, Studies have shown that (proteins secreted by cells) Dittrich-Breiholz, Holtmann, & Kracht, 2002). and their receptors are regulators of metastatic diseases, This review will discuss the importance of two well- including CRC (Balkwill, 2004; Waugh & Wilson, 2008). By characterized signaling pathways that are induced downstream of activating intracellular signaling pathways in target cells, IL-8 receptors in CRC. Furthermore, it will evaluate current chemokines can promote the proliferation and survival of cancer methods of targeting IL-8 and IL-8 receptors to suppress tumor cells within the tumor microenvironment (Balkwill, 2004). progression and development. Finally, it will offer support for Hence, chemokines and their receptors may be important targets the use of QDs in the treatment of human colon cancer cells. for novel drug and intervention therapies (Balkwill, 2004; Waugh & Wilson, 2008). DISCUSSION IL-8, also called CXCL8, belongs to a superfamily of chemically related chemokines that stimulate IL-8 signaling pathways and degranulation. Levels of IL-8 are low in healthy The increased synthesis and secretion of IL-8 from tumor cells tissues. However, IL-8 is rapidly generated in the presence of and the overexpression of the CXCR2 receptor in endothelial pro-inflammatory , including TNF-α and IL-1β cells and indicate that IL-8 and CXCR2 play a crucial (Hoffmann, Dittrich-Breiholz, Holtmann, & Kracht, 2002). The role in the development of CRC (Waugh & Wilson, 2008; 77-amino acid exerts its biological effects by binding to Raman, Baugher, Thu, & Richmond, 2007). Binding of ligand to two receptors, CXCR1 (IL-8RA) and CXCR2 (IL- CXCR2 stimulates multiple signaling cascades, two of which 8RB), which are members of the seven transmembrane G- promote the activation of Akt and mitogen-activated protein protein-coupled receptor (GPCR) family (Raman, Baugher, Thu, kinase (MAPK) (Figure 1), contributing to CRC metastasis & Richmond, 2007; Heidemann, Ogawa, & Dwinell, 2003). (Knall et al., 1996; Knall, Worthen, & Johnson, 1997). Signals are transmitted across the membrane via ligand-induced conformational changes, activating downstream pathways that Activation of Akt signaling \cascade promote the malignant development of cancer (Waugh & Binding of IL-8 to CXCR2 activates intracellular Wilson, 2008; Raman, Baugher, Thu, & Richmond, 2007; phosphatidylinositol-3 kinase (PI3-K) (Knall, Worthen, & Heidemann, Ogawa, & Dwinell, 2003). Although CXCR1 and Johnson, 1997). Knall et al. (1997) demonstrated that activation CXCR2 show considerable structural similarity (Waugh & of PI3-K results in the phosphorylation of Akt, a serine/threonine kinase also called Protein Kinase B (PKB). They concluded that

1 the phosphorylation of Akt increases its activity (Knall, Worthen, University of Toronto, 27 King’s College Circle, Toronto ON & Johnson, 1997). Upregulation of Akt has been detected in Canada M5S1A1. various forms of cancer, including CRC, and its role in mediating cell survival has been documented in multiple studies (Knall, *To whom correspondence should be addressed. Worthen, & Johnson, 1997; MacManus et al., 2007). Active Akt

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promotes cancer development via two pathways. In one cascade, exchange its GDP for GTP, activating the Ras-GTPase (Knall et Akt directly stimulates the activity of NF-B (Huang, al., 1996). According to Sparmann et al., Ras-GTP stimulates DeGuzman, Bucana, & Fidler, 2000; Karin, Cao, Greten, & Li, MAP3K (specifically Raf), a serine-threonine kinase which 2002; Richmond, 2002). Huang et al. (2000) showed that this further activates MAP2K (specifically MEK) (Knall et al., 1996; transcription factor regulates the expression of that Sparmann & Bar-Sagi, 2004). MAP2K in turn stimulates MAPK, promote cell proliferation, invasion, and angiogenesis. By which activates the transcription factor AP-1 (Knall et al., 1996; transfecting metastatic human melanoma variant cells with an Collins, Lee, & Ting, 2000). A study by Collins et al. (2000) inhibitor of NF-B, they documented a decrease in tumor growth demonstrated that AP-1upregulates the expression of pro- and lung metastasis in mice (Huang, DeGuzman, Bucana, & inflammatory cytokines, resulting in cell proliferation, cell Fidler, 2000). In a second cascade, phosphorylated Akt activates survival, and invasion. Hence, the MAPK signaling cascade, mTOR, a transcription factor that ultimately overexpresses prompted by IL-8 binding to its receptor, contributes to the onset various oncogenes, including rS6K and S6, resulting in an of CRC. Thus, inhibiting IL-8 signaling may affect the treatment increase in protein synthesis and cell proliferation (Waugh & of CRC. Wilson, 2008; Huang, DeGuzman, Bucana, & Fidler, 2000). As a result, the Akt signaling cascade, induced by IL-8 binding to TARGETING IL-8 AND CXCR2 FOR THERAPEUTIC CXCR2, contributes to the onset of CRC. Thus, targeting IL-8 PURPOSES IN COLORECTAL CANCER and CXCR2 may have important therapeutic implications for As demonstrated above, the expression of IL-8 and CXCR2 by colon cancer patients. cancer cells significantly contributes to the tumor microenvironment. Therapies that aim to suppress the IL-8 signaling pathway, which include the use of small molecule antagonists, humanized monoclonal antibodies, small interfering RNA (siRNA) vectors, and nano-particles, can reduce cell proliferation, decreasing the risk of tumorigenicity. As a result, inhibiting the effects of IL-8 signaling may have therapeutic implications for colon cancer patients.

Small molecule antagonists/humanized monoclonal antibodies Currently, small molecule antagonists and humanized monoclonal antibodies are used to attenuate the effects of IL-8 signaling. A study conducted by Ning et al. (2012) found that a

small molecule inhibitor of CXCR2, namely SCH-527123, can successfully treat metastatic CRC. SCH-527123 was orally administered to colon cancer mice models, which then exhibited a reduction in tumor volume when compared to control groups (Ning et al., 2012). It was concluded that SCH-527123 can inhibit cell proliferation and can cause programmed cell death in colon cancer cells (Ning et al., 2012). The result was validated in a study conducted by Singh et al. (2009). Additional small molecule inhibitors, including SB 225002, SB 656933, and SCH- Figure 1. IL-8 signalling pathways in colorectal cancer. Binding of IL- 479833, have been shown to suppress inflammatory diseases as 8 to CXCR1 and/or CXCR2 activates heterotrimeric G proteins, which well (Singh et al., 2009; White et al., 1998). In addition to small stimulate effector proteins, including phosphatidyl-inositol-3-kinase. molecule antagonists, humanized monoclonal antibodies, such as Activation of primary effectors promotes the activation of Akt and/or ABX-IL-8, have been developed to suppress the effects of IL-8 MAPK signalling pathways, causing protein translation, cell survival, signaling (Mian et al., 2003). A study by Mian et al. (2003) cell proliferation, and/or angiogenesis. Solid lines indicate transcription factors whose activity is upregulated by IL-8 signalling. Dashed lines demonstrated that ABX-IL-8 reduces tumor growth and show pathways through which IL-8 regulates transcription factors. angiogenesis in bladder cancer xenograft models. It is probable Image From: Waugh, D. J., & Wilson, C. (2008). The interleukin-8 that humanized monoclonal antibodies can be used as a treatment pathway in cancer. Clin Cancer Res., 14, 6735-6741. for patients with CRC as well. However, a disadvantage of using small molecule inhibitors and humanized monoclonal antibodies to suppress the effects of IL-8 is that they may interact with the immune system via unpredictable and harmful mechanisms Activation of MAPK signaling cascade (Ning et al., 2012; Mian et al., 2003). IL-8 signaling has been shown to induce the activation of the classic MAPK pathway in CRC. When bound to IL-8, CXCR2 siRNA vectors undergoes a conformational change that allows Ras-GTPase to Knockdown of IL-8, using siRNA vectors, has been shown to

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decrease migration and invasion of colon cancer cells. In a study nanotechnology were used in conjunction with siRNA silencing by Cao et al. (2005), downregulation of IL-8 expression was strategies. Regardless, mechanisms of IL-8 signaling in CRC and achieved by constructing viral vectors that were used to deliver potential treatment methods to control overexpression of the siRNA to human airway epithelial cells. Similar results have chemokine need to be furthermore investigated. been achieved in various cancers using a non-viral vector-based siRNA delivery approach as well (Chen, Du, Zhang, & Liang, 2005). siRNA-based treatment techniques can be used effectively REFERENCES in low concentrations and can inhibit expression with Ali, H. M., Urbanati, G., Raouane, M., & Massaad-Massade, L. increased specificity (Ali, Urbanati, Raouane, & Massaad- (2012). Significance and applications of nano-particles for Massade, 2012). 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