The International Journal of Biochemistry & Cell Biology 42 (2010) 10611065

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The International Journal of Biochemistry & Cell Biology

journal homepage: www.elsevier.com/locate/biocel

Medicine in focus

Colon carcinogenesis: Learning from NF-B and AP-1

Aristides G. Vaiopoulos, Katerina K. Papachroni, Athanasios G. Papavassiliou
Department of Biological Chemistry, University of Athens Medical School, 75 Mikras Asias Street, Goudi, 11527 Athens, Greece

a r t i c l e

i n f o

a b s t r a c t
Colorectal cancer (CRC) is among the most common types of cancer attributed to genetic alterations. Its manifestation implicates NF-B and AP-1 signaling pathways by virtue of their regulative role on the genetic control of cell cycle and apoptosis as well as by their capacity to be constitutively activated or exogenously induced by growth factors, cytokines, stress signals and oncoproteins. In CRC, the positive impact of NF-B and AP-1 on the transcription of angiogenic and invasive factors strongly implicates these transcription factors in the transition of benign carcinomas towards a metastatic phenotype. Furthermore, the deregulated function of NF-B and AP-1 in CRC cells affects inammatory cascades, manifested by the ample production of inammatory mediators. In this perspective, inhibition of NF-B and AP-1 signaling mechanisms has become a rational target in the development of novel therapeutic approaches against CRC. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 28 January 2010 Received in revised form 15 March 2010 Accepted 22 March 2010 Available online 27 March 2010 Keywords: Colorectal cancer Inammation Signal transduction NF-B AP-1

1. Introduction Colorectal cancer (CRC) is a leading cause of morbidity and mortality. It is presented as a multistep genetic disorder, the development and progression of which are caused by characterised mutations and abnormalities in signal transduction pathways. Most cases of sporadic and familial CRC are associated with mutations in the molecules along the Wnt signaling pathway, which lead to aberrant -catenin activation and deregulated gene transcription. The development and progression of CRC, however, requires the concurrent failure of other protective mechanisms, including the p53 tumour suppressor gene, the pro-apoptotic protein Bcl-2-associated X protein (BAX), the anti-proliferative protein transforming growth factor beta (TGF-) or its downstream transcription factors, the SMAD proteins, as well as induction of oncogene pathways (Markowitz and Bertagnolli, 2009). Recently, the transcription factors nuclear factor-B (NF-B) and activator protein-1 (AP-1), which have an established role in the regulation of cell cycle and the progression of immune-mediated and inammatory diseases, have been implicated in the tumourigenesis of colon epithelium.

2. Pathogenesis 2.1. The role of NF-B in CRC NF-B, collectively referring to homo- and heterodimeric complexes of members of ve protein families [(RelA, RelB, c-Rel, p50/p105 (NF-B1), p52/p100 (NF-B2)] (Basseres and Baldwin, 2006). All NF-B members share a 300-aa Rel-homology domain responsible for DNA binding, dimerization and interaction with their inhibitors IBs (Shen and Tergaonkar, 2009). Upon receipt of a signal [cytokines, growth factors (GFs), stress signals, oncoproteins], IBs are phosphorylated and proteolyticaly removed and NF-B enters the nucleus to regulate genes involved in cell cycle, cell survival and innate and adaptive immune responses (Basseres and Baldwin, 2006). Evidence for involvement of NF-B or IB members in oncogenesis is based on several observations, namely that (i) NF-B proteins are members of a proto-oncogene family; (ii) the NF-B2 (p52/p100) gene and the Bcl-3 gene are translocated in certain lymphomas; (iii) NF-B gene is activated by viral transforming proteins; (iv) loss of NF-B1 (p50/p105) accelerates B-cell growth and turnover in vivo and (v) exposure of cells to IB antisense results in oncogenic transformation (Basseres and Baldwin, 2006). In CRC, activation of the noncanonical NF-B1 pathway in response to -catenin signaling is reported (Lauscher et al., 2010) as well as activation of distinct NF-B complexes (p50/p50, RelA/p52) upon release of endogenous ceramide, a bioactive lipid implicated in colon cancer (Colell et al., 2002). Activation of NF-B promotes tumour invasiveness and links the inammatory processes to carcinogenesis (Horst et al., 2009).

Corresponding author. Tel.: +30 210 7462 508/9; fax: +30 210 7791 207. E-mail addresses: papavas@med.uoa.gr, gpapavas@ath.forthnet.gr (A.G. Papavassiliou). 1357-2725/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.03.018

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In colitis-associated intestinal cancer (CAC) correlations have been made with Ras and p53-activated pathways (Karin, 2006; Basseres and Baldwin, 2006). NF-B induces cell proliferation by potentiating the phosphoinositide 3-kinase (PI3-K)-AKTmammalian target of rapamycin (mTOR) signaling pathway and the key cell-cycle regulatory genes including cyclin D1, c-myc, cyclin-dependent kinases (CDKs) (Shen and Tergaonkar, 2009). In CRC, NF-B suppresses apoptosis by inducing genes encoding antioxidant enzymes and by inhibiting the c-Jun N-terminal kinase (JNK) cascade (Bubici et al., 2006). In human colon cancer cell lines, curcumin, the principal substance of the spice turmeric, interacts with the NF-B-reactive oxygen species (ROS)JNK pathway and induces apoptosis in vitro (Collett and Campbell, 2004). NF-B also mediates the enhanced angiogenesis and invasiveness of CRC tumour cells, by up-regulating vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2), interleukine (IL)-6, cell adhesion molecules (ICAM-1, VCAM-1) and matrix metalloproteinases (MMPs), that collectively facilitate progression to a metastatic phenotype (Basseres and Baldwin, 2006; Chen and Castranova, 2007). Furthermore, NF-B confers anti-apoptotic advantage to CRC tumour cells via induction of the anti-apoptotic genes Bcl-2, Bcl-x and cIAPs (Chen and Castranova, 2007). In accord to these data, analysis of human CRC tissue revealed constitutive up-regulation of RelA, correlated with the expression of Bcl-2 and Bcl-x (Yu et al., 2004). However, while NF-B has emerged as a critical promoter of inammation-linked cancers, strong evidence also suggests that it suppresses chemically induced skin and liver cancers, by inhibition of cell-cycle progression or down-regulation of JNK activity (Karin, 2006; Chen and Castranova, 2007). Nevertheless, even when NF-B suppresses tumourigenesis, its inammatory activity promotes tumour development by the induction of pro-inammatory cytokines, tumour necrosis factor alpha (TNF-) and IL-6, which serve as GFs for premalignant cells as well as for already formed tumours (Karin, 2006). 2.2. The role of AP-1 in CRC The AP-1 group of transcription factors consists of dimers mainly of the Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) subfamilies that harbor a basic leucine zipper (bZIP) domain and can form duplexes between themselves and with other bZIP proteins. This advantage for complex formation allows AP-1 to target a broad range of DNA-binding sites and regulate genes engaged in cell cycle (cyclin D1, p53, p21, p19, p16) and inammation (cox-2). AP-1 contributes to basal gene expression but also participates in the immediate-early cellular response to a wide gamut of physiological and pathological stimuli, including, GFs, pro-inammatory cytokines, stress signals, infections and most importantly, oncogenic signals. AP-1 activation is induced by cis-elements in the promoters of AP-1-encoding genes, followed by rapid phosphorylation of the AP-1 proteins mainly through the mitogen-activated protein kinase (MAPK) cascade. GFs activate extracellular signalregulated kinase (ERK), pro-inammatory cytokines and genotoxic stress induce p38 MAPKs and JNKs, while oncoproteins (e.g. Src, Ras) induce the ERK or JNK pathway (see Fig. 2) (Karamouzis et al., 2007; Shaulian and Karin, 2001, 2002). Recently, enhanced activity of AP-1 in human colon adenocarcinoma grade II cell line has been demonstrated (Yao et al., 1994), where AP-1 contributes to transcriptional induction of redox-regulating enzymes in the areas of solid tumours. Immunohistochemical analysis on human colon adenocarcinoma revealed that AP-1 is expressed in the stromal myobroblasts surrounding the tumour in the majority of cases examined, and this expression correlates positively with the expression of VEGF, a downstream target of AP-1, epidermal growth factor receptor (EGFR) and COX-

2 (Konstantinopoulos et al., 2007a). In addition, data from studies on experimental models and examination of human tissue of CRC suggests that the presence of high concentrations of bile acids induces AP-1 expression, perhaps through protein kinase C (PKC) and ERK signaling, and results in COX-2 stimulation that mediates anti-apoptosis, motility and invasion (Debruyne et al., 2001). Data concerning a series of signaling cascades in CRC epithelial cells mediated by or resulting in AP-1 activity have been reported. These include activation of the Wnt/-catenin pathway which regulates c-jun and fra-1 genes (Mann et al., 1999), and also the Ras-GTPases cascades. Gain-of-function mutations in the K-ras gene stimulate the ERK and JNK pathway, potentiate AP-1 and have been documented to be crucial for the development of CRC (Ashida et al., 2005). Interestingly, AP-1 is likely to act as a homeostasis switch regulating the cell cycle. Depending on the duration and type of stimuli, AP-1 up-regulates cyclin D1 but can also act as an anti-apoptotic factor via negative modulation of p53 (Shaulian and Karin, 2001) and induction of the anti-apoptotic Bcl genes (Bcl-3, Bim). Moreover, depending on the stimulus (e.g. sustained JNK activation) AP-1 may encourage cell death by up-regulating Fas ligand (FasL) and thus promote apoptosis. Notably, AP-1 in human colon cancer cell lines mediates an anti-apoptotic response to the hypoxic conditions often encountered in the environment of solid tumours and thus contributes to chemo- and radiotherapy resistance (Shaulian and Karin, 2002). Similar to NF-B, AP-1 also controls the expression of angiogenic (VEGF) (Grau et al., 2006) and invasive factors (MMPs) of the cancer cells (Debruyne et al., 2001). 2.3. NF-B and AP-1: an intriguing link between cancer and inammation Chronic inammation has been correlated with carcinogenesis in a number of human malignancies. The production of ROS, the epigenetic changes and the release of cytokines at the site of inammation are events with documented carcinogenic capacity that create a vicious circuitry between the two processes (Fig. 1). This relationship has been particularly studied in malignancies of the gastrointestinal tract, where the risk of carcinogenesis increases in the presence of chronic inammation-inducing conditions (Maeda and Omata, 2008). Emphasis is given on the role of NF-B and AP-1 as mediators of the cross-talk between inammation and cancer, given their induction by inammatory cytokines (TNF-, IL-1, IL6, IL-8), the presence of regulatory elements for NF-B and AP-1 in the promoters of inammatory genes (cox-2, MMPs) and their known impact on cell proliferation. COX-2 expression is strongly correlated to intestinal tumourigenesis, as shown by the studies on animal models and humans. CAC is more frequent in patients with inammatory bowel disease (IBD) and epidemiologic studies show a reduction in CRC incidence among chronic users of non-steroidal anti-inammatory drugs (NSAIDs) (Sinicrope and Gill, 2004). In a murine model of familial adenomatous polyposis (FAP), deletion of the murine cox-2 gene in polyps-prone mice (Apc 716 ), was found to dramatically reduce the number of intestinal polyps in double knockout mice relative to cox2 wild-type animals (Oshima et al., 1996). Furthermore, in genetic or carcinogen-induced colon cancer model systems, treatment with traditional NSAIDs or selective COX-2 inhibitors reduces tumour size and multiplicity. The relevance of these ndings to human neoplasia was shown in patients with germline APC mutations and the FAP syndrome, where treatment with NSAID effectively regressed colorectal adenomas relative to placebo (Sinicrope and Gill, 2004). The promoter of cox-2 harbors binding sites for both AP-1 and NF-B (Konstantinopoulos et al., 2007b). Experimental data from animal models show that reduced activation of NF-B diminishes the risk of malignant transformation. In CAC animal model,

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Fig. 1. The vicious circuitry of inammation and cancer. Growth factors (GFs) and cytokines mediate the cross-talk between inammatory cells and myobroblasts by inducing NF-B and AP-1-mediated expression of angiogenic, invasive and inammatory mediators. These in turn act on the epithelial tumour cells and stimulate activation of MAPK cascades resulting in transcriptional activation of genes involved in proliferation and metastasis. Moreover, tumour cells produce IL-8 and necrotic by-products which favor a hypoxic environment. These events further potentiate NF-B in inammatory cells and trigger production of cytokines, COX-2, VEGF and MMPs. Tumour cells also mediate broblast transformation to myobroblasts by secretion of TGF-, and myobroblasts consecutively induce NF-B- and AP-1-mediated expression of VEGF and COX-2. Based on data from Vandoros et al. (2006), Konstantinopoulos et al. (2007a), Maeda and Omata (2008) and Sanchez-Munoz et al. (2008).

conditional inactivation of IKK gene, a signaling intermediate of NF-B pathway, revealed that the IKK-mediated NF-B activation contributes to CAC through two distinct cell-type specic mechanisms. In enterocytes it activates anti-apoptotic genes and thereby suppresses the apoptotic elimination of preneoplastic cells, whereas in myeloid cells it promotes production of cytokines that serve as GFs for premalignant enterocytes (Karin, 2006). Studies in the same system revealed that any single AP-1 protein is dispensable for CAC and perhaps several AP-1 proteins have to be targeted to impair carcinogenesis (Hasselblatt et al., 2008). In CRC, NF-B is activated both in the tumour cells and in the cells of the surrounding inammatory stroma (Grau et al., 2006; Karin, 2006; Konstantinopoulos et al., 2007a). Stromal cells generate oncogenic signals which favour remodeling towards a malignant phenotype of broblasts, namely the myobroblasts (Konstantinopoulos et al., 2007a). Inammatory cells and myobroblasts that congregate at tumour sites secret a variety of GFs and cytokines that enhance tumour cell proliferation and invasion. NF-B released by the tumour cells potentiates the secretion of VEGF, IL-8 and MMPs from the inammatory cells and acts in concert with AP-1 to regulate expression of VEGF by the stromal myobroblasts (Fig. 1). Expression of COX-2 in the CRC cells further stimulates pro-angiogenic prostaglandin release and expression of MMP-2 (Debruyne et al., 2001). Increased expression of COX-2 in both epithelial and stromal cells correlates with EGFR expression, c-Jun/AP-1 and NF-B activation. Moreover, peroxisome proliferator-activated receptor gamma (PPAR), a nuclear

receptor which inhibits the action of both AP-1 and NF-B, acts as an on/off switch for the production of COX-2. It is down-regulated in CRC tissue permitting the unopposed AP-1/NF-B transcriptional activity and up-regulation of their downstream target COX-2 (Konstantinopoulos et al., 2007a,b; Vandoros et al., 2006). 3. Therapy The plethora of experimental data supports the notion that NF-B and AP-1 exert a fundamental role in growth and progression of CRC and encourages the endeavor to employ them as rational targets for the development of novel therapeutic strategies. Indeed, more than 700 compounds, in the form of small molecules from natural/dietary sources (nutraceuticals), synthetic compounds and cell permeable peptides, reported to inhibit various steps of NF-B activation have entered preclinical and clinical trials (Shen and Tergaonkar, 2009). Antisense RNA and gene therapy (achieving over-expression of IB) capable of blocking IB kinase (IKK) and IKK upstream signaling, IB degradation and nuclear function of NF-B, are also tested (Sethi and Tergaonkar, 2009; Shen and Tergaonkar, 2009) (Fig. 2). Efforts have also been made towards use of already known natural agents with potent anti-AP-1 effect as well as towards the development of AP-1 novel signaling pathway inhibitors (Ashida et al., 2005) (Fig. 2). Moreover, NF-B and AP-1 being at the convergence point of several epithelial-to-mesenchymal transition (EMT) pathways, along with the AKT/mTOR axis, MAPK, -catenin and PKC, present valuable

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Fig. 2. NF-B and AP-1 pathways as putative targets for therapeutic intervention. NF-B inhibition on various levels of the signaling cascades involves the following: (i) IKK and IKK upstream signaling inhibitors, comprising small natural and synthesized molecules (e.g. curcumin), receptor blockers (e.g. anti-TNF); (ii) IB degradation inhibitors, including proteasome inhibitors; and (iii) NF-B nuclear translocation/DNA-binding inhibitors, including cell permeable peptides, antisense RNA. AP-1 inhibitors may target: (i) receptor components, e.g. anti-EGFR; (ii) constituents of the MAPK signaling cascade; (iii) AP-1 dimerisation; (iv) AP-1 DNA binding; and (v) AP-1 interaction with transcriptional co-factors. PPAR ligands [e.g. non-steroidal anti-inammatory drugs (NSAIDs), thiazolidinediones] impair COX-2 and VEGF production by blocking NF-B and AP-1 activity.

targets to control EMT and the progression of human epithelial cancers, including CRC. Several inhibitors targeting these signaling cascades as well as upstream EMT signaling pathways induced by receptor and nonreceptor tyrosine kinases [e.g. EGFR, IGF-R, VEGF-R, integrins/focal adhesion kinase (FAK), Src] and G-proteincoupled receptors (GPCR) are tested in preclinical and clinical trials or are currently used in the clinic for cancer treatment (Sabbah et al., 2008). The formulation of targeted therapy without severe sideeffects such as systemic toxicity or broad immunosuppression will provide additional weapons in the therapeutic arsenal against CRC. References
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