**2. NF-κB – A key player**

Recent studies have implicated an inflammation-induced protein called nuclear factor kappa B (NF-κB) as a central figure in the link between cancer and inflammation. In 1986, Baltimore et al., originally discovered NF- κB as a factor in the nucleus of B cells that binds to the enhancer of the kappa light chain of immunoglobulin (Sen, 1986). NF- κB activity is considered a hallmark of inflammation. It is shown that NF-κB manipulation can convert inflammation-driven tumour growth into inflammation-induced tumour regression (Luo, 2004). Furthermore, many oncogenes and carcinogens can cause activation of NF-κB, whereas chemicals with chemo-protective properties can interfere with NF-κB activation (Bharti, 2002).

In unstimulated cells, the majority of NF-κB complexes are kept predominantly cytoplasmic and in an inactive form by binding a family of inhibitors known as Inhibitor-κB (IκB). The NF-κB pathway can be activated by numerous stimuli including bacteria, viruses and proinflammatory cytokines especially tumour necrosis factor (TNF). Phosphorylation of NF-κB bound I-kappa-B kinases (IκK) on two conserved serine residues within the N-terminal

The Role of Inflammation in Cancer 393

NF-κB activating ability. In addition to preventing the expression of anti-apoptotic proteins, inhibition of NF-κB also led to increased expression of the Trail receptor DR5. Type I and II interferons (IFN) are efficient inducers of Trail. The authors postulate that IFN based therapy with anti-TNF alpha medications may reduce inflammation associated toxicities, block inflammation induced tumour growth and potentiate Trail dependent tumour killing. The bcl-2 family of proto-oncogenes are a critical regulator of apoptosis and are frequently deregulated in a wide variety of cancers. They have recently been identified as having an NF-κB binding site in the bcl-2 p2 promoter (Catz, 2001). Thus chemotherapeutic drugs that activate NF-κB can also activate bcl-2 family proteins in various cancer cell lines. Activated NF-κB binds to specific DNA sequences in target genes, designated as kB elements and regulates transcription of over 400 genes involved in immunoregulation, growth,

Several chemotherapeutic agents including 5-fluorouracil (5-FU) have been reported to induce NF-κB activation in various cell lines (Cusack, 1999). Cytotoxic drugs induce NF-κB with delayed kinetics. This delay is due to the time required to induce nuclear DNA damage and relay the damage signal to the cytoplasmic IκK complex. Treatment with 5-FU can also induce activation of NF-κB in colorectal cancer cells (Wang, 2003). Using RKO human colorectal cell line and two NF-κB signalling deficient RKO mutants, Fukuyama et al demonstrated that 5-FU stimulates NF-κB and RKO cell survival through induction of IκK activity (Fukuyama, 2007). Several studies have shown that inhibition of NF-κB activation results in reversal of chemoresistance (Jones, 2000; Arlt, 2001; Cusack, 2001). It was shown that the inhibition of inducible NF-κB by a NF-κB decoy could induce apoptosis and reduce chemoresistance against 5-FU (Uetsuka, 2003). Inhibition of NF-κB activity reduces chemoresistance to 5-fluorouracil (5-FU) in human stomach cancer. Ionising radiation (IR) has been reported to activate NF-κB in both in vitro and in vivo studies (Rithidech, 2005; Ahmed, 2006). Laszlo et al recently reported that IkB-alpha depletion in the late phase of IR is a result of a combined regulation at both transcription and translation levels (Laszlo, 2008).

Inflammation is initiated through many cellular transmembrane receptors of which the best characterised are the TLR family. Toll like receptors (TLRs) have been the subject of extensive investigation since their discovery in 1996. The first Toll receptor was discovered in Drosophila and it was revealed that the innate immune system may be activated once the receptor was bound by an extracellular ligand (Belvin, 1996). Subsequent research has revealed a total of 13 mammalian TLRs, 11 of which are expressed in humans. They are involved in the recognition by immune and non-immune cells of stimuli such as lipopolysaccharides (LPS) and dsRNA. They signal through the use of adapter proteins such as TRIF-related adaptor molecule (TRAF) and myeloid differentiation factor 88 (Myd88) (Killeen, 2009). Recognition of pathogen–associated molecular patterns (PAMPS) through TLRs, either alone or in heterodimerization with other TLRs or non-TLRs, triggers signals responsible for activation of the innate and adaptive immune responses. Most TLRs are found in innate immune cells such as polymorphonuclear neutrophils, monocytes/macrophages and dendritic cells where they trigger an immediate response. More recently TLRs have been shown to be expressed in a number of different cancer cells (Cheadle, 2002; Huang, 2008). Recent experimental evidence shows that TLRs display both

inflammation, carcinogenesis and apoptosis.

**3. Toll-like receptors** 

**3.1 Overview** 

domain of the IκB proteins, which allows for proteasomal degradation and resultant NF-κB liberation. Activation of the NF-κB signalling cascade results in complete degradation of IκB allowing translocation of NF-κB to the nucleus where it induces transcription. The NF-κB family of transcription factors is composed of homodimers and heterodimers derived from 5 subunits - including REL-A(p65), c-REL, REL-B, p50(NF-κB1) and p52 (NF-κB2). When these subunits enter the nucleus, they can mediate transcription of target genes (Hayden, 2004). All family members share a highly conserved Rel homology domain responsible for DNA binding, dimerization domain and interaction with IκBs, the intracellular inhibitor of NF-κB. Cellular stresses including ionising radiation and chemotherapeutic agents can also activate NF-kB. The subsequent release of pro-inflammatory mediators (CSF-1, COX-2, IL-6, IL-1, VEGF, TNF) (Luo, 2004; Marx, 2004) and known anti-apoptotic regulators (BCL-2 and GADD45β) are thought to potentiate tumour growth. In 2004, Karin et al discovered the mechanism that NF-κB contributes to tumourigenesis (Luo, 2004). Prior to this study evidence implicating NF-κB and cancer was mostly circumstantial. Using a murine model of colitis-associated cancer, researchers were able to use genetically altered mice which had the NF-κB activator enzyme IκK knocked out of intestinal epithelial cells and macrophages. In the absence of the IκK gene, neither cell could activate NF-κB. Interestingly, loss of NF-κB activity in both macrophages and epithelial cells individually reduced tumour incidence. They also found that this occurred through two different mechanisms. Tumour incidence decreased by approximately 50% with the loss of NF-κB activity in macrophages through a reduction in growth factors produced by inflammatory cells normally induced by NF-κB activation. Secondly, the loss of NF-κB activity in intestinal epithelial cells resulted in an 80% drop in tumour incidence. The mechanism seen here was different to that seen with the macrophages. Inflammation was not reduced as with the macrophages, instead, apoptosis was no longer inhibited in intestinal cells (Karin, 2004). Pikarsky et al had similar findings in a murine model of inflammation-associated liver cancer where they inhibited NF-κB by adding a gene encoding for IκB, a natural NF-κB inhibitor (Pikarsky, 2004).

The NF-κB family of transcription factors plays a key role in regulation of immune and inflammatory responses including apoptosis and oncogenesis (Baldwin, 2001). NF-κB regulated gene products including those encoding ICAM-1, the extracellular matrix protein tenascin C, vascular endothelial growth factor (VEGF), the chemokine IL-8, the proinflammatory enzyme COX2 and matrix metalloprotease 9 (MMP9) are associated with tumour progression and metastasis (Karin, 2002). However NF-κB may also control the expression of apoptosis promoting cytokines such as TNF alpha and FAS ligand (Kasibhatla, 1998).

NF-κB activation is required for endotoxin induced tumour growth. Lou et al showed both tumour nodule numbers and lung weights in the Lipopolysaccharide (LPS) challenged CT26 and CT26 vector groups were significantly higher than the controls (<0.05) (Luo, 2004). This demonstrates that inhibition of NF-κB activity in cancer cells converts the LPS-induced proliferative response to an apoptotic response. LPS was shown to induce NF-κB dependent genes in tumour cells. TNF alpha was shown to mediate LPS induced tumour growth and NF-κB activation. TRAIL mediates LPS induced regression of NF-κB deficient tumours. Overall, NF-κB in CT26 cells is responsible for induction of several anti-apoptotic proteins including Bcl-2 and Bcl-X. Most importantly the inhibition of NF-κB converted the growth promoting effect of LPS mediated by TNF alpha into a cytocidal effect. Trail is a weak inducer of inflammation which is an important characteristic, most likely related to its poor

domain of the IκB proteins, which allows for proteasomal degradation and resultant NF-κB liberation. Activation of the NF-κB signalling cascade results in complete degradation of IκB allowing translocation of NF-κB to the nucleus where it induces transcription. The NF-κB family of transcription factors is composed of homodimers and heterodimers derived from 5 subunits - including REL-A(p65), c-REL, REL-B, p50(NF-κB1) and p52 (NF-κB2). When these subunits enter the nucleus, they can mediate transcription of target genes (Hayden, 2004). All family members share a highly conserved Rel homology domain responsible for DNA binding, dimerization domain and interaction with IκBs, the intracellular inhibitor of NF-κB. Cellular stresses including ionising radiation and chemotherapeutic agents can also activate NF-kB. The subsequent release of pro-inflammatory mediators (CSF-1, COX-2, IL-6, IL-1, VEGF, TNF) (Luo, 2004; Marx, 2004) and known anti-apoptotic regulators (BCL-2 and GADD45β) are thought to potentiate tumour growth. In 2004, Karin et al discovered the mechanism that NF-κB contributes to tumourigenesis (Luo, 2004). Prior to this study evidence implicating NF-κB and cancer was mostly circumstantial. Using a murine model of colitis-associated cancer, researchers were able to use genetically altered mice which had the NF-κB activator enzyme IκK knocked out of intestinal epithelial cells and macrophages. In the absence of the IκK gene, neither cell could activate NF-κB. Interestingly, loss of NF-κB activity in both macrophages and epithelial cells individually reduced tumour incidence. They also found that this occurred through two different mechanisms. Tumour incidence decreased by approximately 50% with the loss of NF-κB activity in macrophages through a reduction in growth factors produced by inflammatory cells normally induced by NF-κB activation. Secondly, the loss of NF-κB activity in intestinal epithelial cells resulted in an 80% drop in tumour incidence. The mechanism seen here was different to that seen with the macrophages. Inflammation was not reduced as with the macrophages, instead, apoptosis was no longer inhibited in intestinal cells (Karin, 2004). Pikarsky et al had similar findings in a murine model of inflammation-associated liver cancer where they inhibited NF-κB by

adding a gene encoding for IκB, a natural NF-κB inhibitor (Pikarsky, 2004).

1998).

The NF-κB family of transcription factors plays a key role in regulation of immune and inflammatory responses including apoptosis and oncogenesis (Baldwin, 2001). NF-κB regulated gene products including those encoding ICAM-1, the extracellular matrix protein tenascin C, vascular endothelial growth factor (VEGF), the chemokine IL-8, the proinflammatory enzyme COX2 and matrix metalloprotease 9 (MMP9) are associated with tumour progression and metastasis (Karin, 2002). However NF-κB may also control the expression of apoptosis promoting cytokines such as TNF alpha and FAS ligand (Kasibhatla,

NF-κB activation is required for endotoxin induced tumour growth. Lou et al showed both tumour nodule numbers and lung weights in the Lipopolysaccharide (LPS) challenged CT26 and CT26 vector groups were significantly higher than the controls (<0.05) (Luo, 2004). This demonstrates that inhibition of NF-κB activity in cancer cells converts the LPS-induced proliferative response to an apoptotic response. LPS was shown to induce NF-κB dependent genes in tumour cells. TNF alpha was shown to mediate LPS induced tumour growth and NF-κB activation. TRAIL mediates LPS induced regression of NF-κB deficient tumours. Overall, NF-κB in CT26 cells is responsible for induction of several anti-apoptotic proteins including Bcl-2 and Bcl-X. Most importantly the inhibition of NF-κB converted the growth promoting effect of LPS mediated by TNF alpha into a cytocidal effect. Trail is a weak inducer of inflammation which is an important characteristic, most likely related to its poor NF-κB activating ability. In addition to preventing the expression of anti-apoptotic proteins, inhibition of NF-κB also led to increased expression of the Trail receptor DR5. Type I and II interferons (IFN) are efficient inducers of Trail. The authors postulate that IFN based therapy with anti-TNF alpha medications may reduce inflammation associated toxicities, block inflammation induced tumour growth and potentiate Trail dependent tumour killing.

The bcl-2 family of proto-oncogenes are a critical regulator of apoptosis and are frequently deregulated in a wide variety of cancers. They have recently been identified as having an NF-κB binding site in the bcl-2 p2 promoter (Catz, 2001). Thus chemotherapeutic drugs that activate NF-κB can also activate bcl-2 family proteins in various cancer cell lines. Activated NF-κB binds to specific DNA sequences in target genes, designated as kB elements and regulates transcription of over 400 genes involved in immunoregulation, growth, inflammation, carcinogenesis and apoptosis.

Several chemotherapeutic agents including 5-fluorouracil (5-FU) have been reported to induce NF-κB activation in various cell lines (Cusack, 1999). Cytotoxic drugs induce NF-κB with delayed kinetics. This delay is due to the time required to induce nuclear DNA damage and relay the damage signal to the cytoplasmic IκK complex. Treatment with 5-FU can also induce activation of NF-κB in colorectal cancer cells (Wang, 2003). Using RKO human colorectal cell line and two NF-κB signalling deficient RKO mutants, Fukuyama et al demonstrated that 5-FU stimulates NF-κB and RKO cell survival through induction of IκK activity (Fukuyama, 2007). Several studies have shown that inhibition of NF-κB activation results in reversal of chemoresistance (Jones, 2000; Arlt, 2001; Cusack, 2001). It was shown that the inhibition of inducible NF-κB by a NF-κB decoy could induce apoptosis and reduce chemoresistance against 5-FU (Uetsuka, 2003). Inhibition of NF-κB activity reduces chemoresistance to 5-fluorouracil (5-FU) in human stomach cancer. Ionising radiation (IR) has been reported to activate NF-κB in both in vitro and in vivo studies (Rithidech, 2005; Ahmed, 2006). Laszlo et al recently reported that IkB-alpha depletion in the late phase of IR is a result of a combined regulation at both transcription and translation levels (Laszlo, 2008).
