**3. Toll-like receptors**

#### **3.1 Overview**

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

The Role of Inflammation in Cancer 395

(poly I:C) is a stable synthetic dsRNA which acts as an agonist on the TLR3 receptor. It has been the subject of anti-cancer immunotherapy trials for decades. However its activity on the TLR3 receptor as well as RIG-1 and MDA-5 has only been recently identified. It activates the human innate system which subsequently regulates adaptive immunity. Poly I:C is recognised by both endosomal receptor TLR3 and cytosolic receptors including RNA helicases such as RIG-1 melanoma differentiation associated gene 5 (MDA5). TLR3 preferentially recognizes synthetic poly(I:C) rather than a virus derived dsRNA (Okahira, 2005). The important role played by TLR3 in poly I:C recognition was shown in one study which used TLR3 deficient mice. These mice demonstrated reduced responses to poly I:C and produced lower levels of inflammatory cytokines (Alexopoulou, 2001). Poly I:C has several side effects such as nephrotoxicity, coagulopathies and hypersensitivity reactions. In order to reduce this toxicity a modified form of poly I:C known as Poly I:C12U has been produced. This substitutes uridine for cytosine at a ratio of 1:12 and results of a clinical trial of patients with HIV have proven Poly I:C12u to be very safe. Studies using differing lengths of poly I:C showed that longer duplexes are better inducers of TLR3 signalling (Boone, 2004; Okahira, 2005). There are various alternatives to Poly I:C available. Poly I:C12U is a mismatched dsRNA helix in which uridine has replaced cytosine. It has a rapid half-life compared to Poly I:C which has opened the door for its use as a clinically useful drug. Poly I:C12U is more specific in its binding to TLR3 and this may account for its reduced toxicity and safe use in clinical trials (Mitchell, 2006). No evidence exists of dose limiting organ toxicity including haematological, liver or renal toxicity following intravenous administration to HIV positive patients (Thompson, 1996). Poly I:C mediates a potent adjuvant effect in cells expressing TLR3 and this strongly enhances antigen specific CD8+ Tcell responses, promotes antigen cross presentation by dendritic cells (Cui, 2006; Schulz, 2005) and directly acts on effector CD8+ T and natural killer cells to alter the release of IFN-γ (Tabiasco, 2006). It is the most potent type 1 interferon stimulant that is recognised by TLR3

TLR3 stimulation by dsRNA can cause direct apoptosis and inhibit cell proliferation in various types of cancer cells including colon, breast and melanoma (Taura, 2010; Salaun, 2006, 2007). It is now known that expression and function of TLR3 is dependent on p53 activation (Taura, 2008). Salaun et al recently used poly I:C to treat a breast cancer cell line by triggering apoptosis (Salaun, 2006). They showed that poly I:C can directly cause apoptosis without the involvement of the immune system. They also showed that poly I:C induced apoptosis occurred through the Fas-associated protein death domain-caspase 8 signalling pathway. The extrinsic apoptotic pathway was also shown to be involved by the same group in selected cancer cells (Salaun, 2007). Polyadenylic:polyuridylic acid (poly A:U) is another synthetic double stranded RNA. Poly A:U is unique as it only signals through TLR3. Poly A:U has been used with moderate success to treat breast and gastric cancer as a monotherapy (Laplanche, 2000; Jeung, 2010). Interferons (IFN) are a group of cytokines that can cause antiviral, antiproliferative and apoptotic effects through the Jak/STAT pathway signal transducers (Stark, 2007). In particular, IFN-α, a type I IFN has been shown to induce TLR3 expression and thus significantly enhance tumour cell apoptosis when combined with Poly I:C compared to each treatment alone (Taura 2010). The same study combined IFN-α, Poly I:C and 5-FU which resulted in a significant increase in cancer cell death in a colon cancer cell line. They also showed that by inhibiting the JAK/STAT pathway, induction of TLR3 by INF- α caused a reduced response of TLR3 to INF- α stimulation. Several clinical trials have examined the effect of combining a TLR3 agonist and INF-α with or without a

(Yoneyama, 2004; Matsumoto, 2008).

tumouricidal and tumourigenesis properties (Salaun, 2006; Killeen, 2008). Each TLR recognises a different ligand. All TLRs (except for TLR3) signal through the adapter protein MyD88. MyD88 has been found to play a key role in inflammation induced tumour growth, principally through the innate immune system (Rakoff-Nahoum S, 2007). MyD88 mediates NF-κB activation through the canonical pathway (O'Neill, 2008; Akira, 2006). MyD88 is capable of NF-κB signalling selectively via TLR2 (Fitzgerald, 2003; Horng, 2001). Hence, both TLR2 and TLR4 both of which are expressed on the plasma membrane of cells appear to be involved in NF-κB inflammation-associated tumourigenesis. TLR2 is stimulated by numerous pro-inflammatory stimuli and helps activate the innate immune system.

Several TLRs are responsible for NF-κB activation. TLR4 induces tumour growth through NF-κB activation mediated by TNF- (Luo, 2004). Interestingly, endotoxin or LPS, which binds specifically to TLR4 in cells is shown to be capable of inducing tumour growth (Pigeon, 1999). Endotoxin is a molecule found in the outer membrane of Gram-negative bacteria and elicits a strong inflammatory response in animals. Hence, it appears that the pro-tumorigenic effects of endotoxin occur through TLR4 mediated NF-κB activation. The focus of recent cancer immunology and medical oncology research has been aimed at activating the immune system in order to inhibit cancer cell growth and induce cancer cell apoptosis. Thus, TLRs offer a unique target for cancer therapy.

#### **3.2 Toll-like receptor-3**

TLR3 is a type 1 trans-membrane receptor protein located intracellularly on the endosome of eukaryotic cells. TLR3 has been shown to be an important "danger" signalling receptor that has a dual role in controlling the delicate balance between tolerance and inflammation on one hand and inflammation and disease on the other hand (Vercammen, 2008). TLR3 is composed of an ectodomain (ECD) containing multiple leucine rich repeats (LRRs), a transmembrane region and a cytoplasmic tail containing the Toll interleukin-1 receptor (TIR) domain (Vercammen, 2008). The LRRs form a horse shoe shaped solenoid structure which is capped at one end by a LRR N-terminal (LRR-NT) and at the other end by a LRR Cterminal (LRR-CT) (Bell, 2005). The TLR3 ectodomain (ECD) binds dsRNA. This can only occur in an acidic environment (pH<6.5) reflecting the endosomal location of the receptor. DsRNA binding initiates a signalling pathway that recruits the TIR domain containing adaptor protein (TRIF) to its cytoplasmic domain. TLR3 is the sole TLR to interact directly with TRIF. The TIr domain of TLR3 then binds to TRIF. This indirectly activates several transcription factors including NFκB and IRF3. TRIF knockout mice have shown impaired Interferon-B production in response to a TLR3 ligand which proves that TRIF is essential to the signalling pathway (Takeuchi, 2003). TRIF is an adaptor molecule which is essential for TLR-3 signalling pathways (Yamamoto, 2003). The activity of TRIF allows indirect activation of several transcription factors such as NF-κB and IRF-3.

TLR3 is the critical sensor of the dsRNA. In response to dsRNA stimulation, specific signalling pathways are activated leading to activation of transcription factors such as nuclear factor- κB (NF-κB) and interferon regulatory factor 3 (IRF-3). This response acts as a defence mechanism against a viral insult to the body. The dsRNA itself is produced during viral replication (Jacobs, 1996). Strong or sustained TLR-3 signalling is potentially harmful and in some cases fatal to the host cell. It appears that the mammalian cells have adapted to this using a negative feedback mechanism. PIK3 and RIP-1 binding to TRIF has been shown to inhibit the actions of TRIF signalling (Meylan, 2004). Polyribosinic:polyriboctidic acid

tumouricidal and tumourigenesis properties (Salaun, 2006; Killeen, 2008). Each TLR recognises a different ligand. All TLRs (except for TLR3) signal through the adapter protein MyD88. MyD88 has been found to play a key role in inflammation induced tumour growth, principally through the innate immune system (Rakoff-Nahoum S, 2007). MyD88 mediates NF-κB activation through the canonical pathway (O'Neill, 2008; Akira, 2006). MyD88 is capable of NF-κB signalling selectively via TLR2 (Fitzgerald, 2003; Horng, 2001). Hence, both TLR2 and TLR4 both of which are expressed on the plasma membrane of cells appear to be involved in NF-κB inflammation-associated tumourigenesis. TLR2 is stimulated by

Several TLRs are responsible for NF-κB activation. TLR4 induces tumour growth through NF-κB activation mediated by TNF- (Luo, 2004). Interestingly, endotoxin or LPS, which binds specifically to TLR4 in cells is shown to be capable of inducing tumour growth (Pigeon, 1999). Endotoxin is a molecule found in the outer membrane of Gram-negative bacteria and elicits a strong inflammatory response in animals. Hence, it appears that the pro-tumorigenic effects of endotoxin occur through TLR4 mediated NF-κB activation. The focus of recent cancer immunology and medical oncology research has been aimed at activating the immune system in order to inhibit cancer cell growth and induce cancer cell

TLR3 is a type 1 trans-membrane receptor protein located intracellularly on the endosome of eukaryotic cells. TLR3 has been shown to be an important "danger" signalling receptor that has a dual role in controlling the delicate balance between tolerance and inflammation on one hand and inflammation and disease on the other hand (Vercammen, 2008). TLR3 is composed of an ectodomain (ECD) containing multiple leucine rich repeats (LRRs), a transmembrane region and a cytoplasmic tail containing the Toll interleukin-1 receptor (TIR) domain (Vercammen, 2008). The LRRs form a horse shoe shaped solenoid structure which is capped at one end by a LRR N-terminal (LRR-NT) and at the other end by a LRR Cterminal (LRR-CT) (Bell, 2005). The TLR3 ectodomain (ECD) binds dsRNA. This can only occur in an acidic environment (pH<6.5) reflecting the endosomal location of the receptor. DsRNA binding initiates a signalling pathway that recruits the TIR domain containing adaptor protein (TRIF) to its cytoplasmic domain. TLR3 is the sole TLR to interact directly with TRIF. The TIr domain of TLR3 then binds to TRIF. This indirectly activates several transcription factors including NFκB and IRF3. TRIF knockout mice have shown impaired Interferon-B production in response to a TLR3 ligand which proves that TRIF is essential to the signalling pathway (Takeuchi, 2003). TRIF is an adaptor molecule which is essential for TLR-3 signalling pathways (Yamamoto, 2003). The activity of TRIF allows indirect activation

TLR3 is the critical sensor of the dsRNA. In response to dsRNA stimulation, specific signalling pathways are activated leading to activation of transcription factors such as nuclear factor- κB (NF-κB) and interferon regulatory factor 3 (IRF-3). This response acts as a defence mechanism against a viral insult to the body. The dsRNA itself is produced during viral replication (Jacobs, 1996). Strong or sustained TLR-3 signalling is potentially harmful and in some cases fatal to the host cell. It appears that the mammalian cells have adapted to this using a negative feedback mechanism. PIK3 and RIP-1 binding to TRIF has been shown to inhibit the actions of TRIF signalling (Meylan, 2004). Polyribosinic:polyriboctidic acid

numerous pro-inflammatory stimuli and helps activate the innate immune system.

apoptosis. Thus, TLRs offer a unique target for cancer therapy.

of several transcription factors such as NF-κB and IRF-3.

**3.2 Toll-like receptor-3** 

(poly I:C) is a stable synthetic dsRNA which acts as an agonist on the TLR3 receptor. It has been the subject of anti-cancer immunotherapy trials for decades. However its activity on the TLR3 receptor as well as RIG-1 and MDA-5 has only been recently identified. It activates the human innate system which subsequently regulates adaptive immunity. Poly I:C is recognised by both endosomal receptor TLR3 and cytosolic receptors including RNA helicases such as RIG-1 melanoma differentiation associated gene 5 (MDA5). TLR3 preferentially recognizes synthetic poly(I:C) rather than a virus derived dsRNA (Okahira, 2005). The important role played by TLR3 in poly I:C recognition was shown in one study which used TLR3 deficient mice. These mice demonstrated reduced responses to poly I:C and produced lower levels of inflammatory cytokines (Alexopoulou, 2001). Poly I:C has several side effects such as nephrotoxicity, coagulopathies and hypersensitivity reactions. In order to reduce this toxicity a modified form of poly I:C known as Poly I:C12U has been produced. This substitutes uridine for cytosine at a ratio of 1:12 and results of a clinical trial of patients with HIV have proven Poly I:C12u to be very safe. Studies using differing lengths of poly I:C showed that longer duplexes are better inducers of TLR3 signalling (Boone, 2004; Okahira, 2005). There are various alternatives to Poly I:C available. Poly I:C12U is a mismatched dsRNA helix in which uridine has replaced cytosine. It has a rapid half-life compared to Poly I:C which has opened the door for its use as a clinically useful drug. Poly I:C12U is more specific in its binding to TLR3 and this may account for its reduced toxicity and safe use in clinical trials (Mitchell, 2006). No evidence exists of dose limiting organ toxicity including haematological, liver or renal toxicity following intravenous administration to HIV positive patients (Thompson, 1996). Poly I:C mediates a potent adjuvant effect in cells expressing TLR3 and this strongly enhances antigen specific CD8+ Tcell responses, promotes antigen cross presentation by dendritic cells (Cui, 2006; Schulz, 2005) and directly acts on effector CD8+ T and natural killer cells to alter the release of IFN-γ (Tabiasco, 2006). It is the most potent type 1 interferon stimulant that is recognised by TLR3 (Yoneyama, 2004; Matsumoto, 2008).

TLR3 stimulation by dsRNA can cause direct apoptosis and inhibit cell proliferation in various types of cancer cells including colon, breast and melanoma (Taura, 2010; Salaun, 2006, 2007). It is now known that expression and function of TLR3 is dependent on p53 activation (Taura, 2008). Salaun et al recently used poly I:C to treat a breast cancer cell line by triggering apoptosis (Salaun, 2006). They showed that poly I:C can directly cause apoptosis without the involvement of the immune system. They also showed that poly I:C induced apoptosis occurred through the Fas-associated protein death domain-caspase 8 signalling pathway. The extrinsic apoptotic pathway was also shown to be involved by the same group in selected cancer cells (Salaun, 2007). Polyadenylic:polyuridylic acid (poly A:U) is another synthetic double stranded RNA. Poly A:U is unique as it only signals through TLR3. Poly A:U has been used with moderate success to treat breast and gastric cancer as a monotherapy (Laplanche, 2000; Jeung, 2010). Interferons (IFN) are a group of cytokines that can cause antiviral, antiproliferative and apoptotic effects through the Jak/STAT pathway signal transducers (Stark, 2007). In particular, IFN-α, a type I IFN has been shown to induce TLR3 expression and thus significantly enhance tumour cell apoptosis when combined with Poly I:C compared to each treatment alone (Taura 2010). The same study combined IFN-α, Poly I:C and 5-FU which resulted in a significant increase in cancer cell death in a colon cancer cell line. They also showed that by inhibiting the JAK/STAT pathway, induction of TLR3 by INF- α caused a reduced response of TLR3 to INF- α stimulation. Several clinical trials have examined the effect of combining a TLR3 agonist and INF-α with or without a

The Role of Inflammation in Cancer 397

Cox-2 expression and increased EGFR signalling (Fukata, 2007). Recent experimental studies have shown that TLR4 induced inflammatory factors may cause tumour cell escape from

One proposed mechanism by which tumour cells resist treatment involves an impairment of the immune response by the inflammatory microenvironment at the tumour site (Coussens, 2002). TLR4 upregulation has been shown to increase the secretion of immunosuppressive cytokines TGF-β, VEGF and pro-angiogenic chemokine IL-8 in human lung cancer cells and induces resistance of human lung cancer cells to TNF-α and TRAIL induced apoptosis (He, 2007). The same study demonstrated secretion of VEGF and IL-8 is p38MAPK dependent and the ERK1/2 and JNK1/2 proteins are not activated. NF-κB activation contributes to apoptosis resistance of human lung cancer cells after LPS stimulation and LPS-induced apoptosis is dependent on NF-κB activation in human lung cancer cells. NF-κB is an antiapoptotic transcriptional factor induced by cellular components such as LPS, radiation and

Contrasting evidence for the role of TLR4 in chemotherapy and radiotherapy resistance was reported by Apetoh et al who demonstrated reduced tumour growth and prolonged survival in immunocompetent wild type mice when treated with chemotherapeutic agents such as oxaliplatin and doxorubicin but this effect was significantly less in TLR4-/- mice and nu/nu mice (Apetoh, 2007). These findings were also demonstrated in TRIF-/- mice which behaved like the wild type mice but Myd88-/- mice behaved like TLR-/- mice. This suggests a certain dependence of tumour treatment resistance on the MyD88 dependent pathway in TLR4 signalling. These results show that when TLR4 itself is knocked out this will lead to a reduction in tumour growth when the tumour is exposed to an appropriate chemotherapeutic agent or radiotherapy. However, when TLR4 is up regulated in the presence of LPS, this results in resistance to chemotherapy and arguably radiotherapy

Various agents have been suggested as having a role in reversing TLR4 induced tumour apoptotic resistance. Rapamycin is one such agent. Rapamycin is a macrolide antifungal agent and is a potent immunosuppressive medication that is used as an anti-inflammatory and immunosuppressive drug for the treatment of autoimmune diseases such as Systemic lupus erythematosus (SLE) and transplantation rejection (Fernandez, 2006; Hackstein, 2003). In terms of cancer treatment Rapamycin has been shown to display a number of useful anticancer cell properties including inhibition of cancer cell proliferation and induction of apoptosis (Hartford, 2007; Zhang, 2007). Rapamycin can also inhibit invasion and metastasis of tumour cells (Abraham, 2007). One study focused on Rapamycins ability to reverse the apoptotic resistance that can occur following TLR4 stimulation with LPS. Sun et al showed that Rapamycin reverses TLR4 ligation induced apoptotic resistance in colon cancer cells (Sun, 2008). Interestingly this same study showed that Rapamycin inhibits anti-apoptotic protein bcl-xL expression and activation of Akt and NF-κB pathways following LPS treatment of cells and this was shown to reverse apoptosis when Akt and NF- κB were inhibited. Paclitaxel has been described as a potential ligand to TLR4 (Asselin, 2001). Kelly et al demonstrated that TLR4 signalling promotes tumour growth and paclitaxel chemoresistance in ovarian cancer cells (Kelly, 2006). This behaviour was mediated by MyD88

immune surveillance and resistance to chemotherapy and radiotherapy (He, 2007).

various chemotherapy drugs.

treatment.

expression.

chemotherapeutic agent. Wadler et al combined 5-FU with INF-α and achieved a good response in colon cancer cells however this combination was not effective in clinical trials (Wadler, 1990). Early clinical trials involving a TLR3 agonist as a single adjuvant therapy in colorectal cancer showed mixed results. Lacour et al showed that Poly A:U alone as an adjuvant therapy in colorectal cancer patients who had undergone a resection of the primary tumour was unable to improve the overall survival of patients after five years compared to a placebo (Lacour, 1992). However, when used as a combination therapy there have been very positive results in a clinical trial to demonstrate a positive role for Poly A:U in cancer treatment. After a 14 year median follow-up Laplanche et al reported the results of a trial which combined Poly A:U with loco-regional radiotherapy versus chemotherapy with cyclophosphamide, methotrexate and fluorouracil (CMF) in women with operable breast cancer. They reported that the Poly A:U combination group had a significantly improved disease free survival (p=0.03) and significantly reduced the incidence of metastasis when compared to the CMF group. This trial did not however differentiate the role that Poly A:U and radiotherapy had as single therapies (Laplanche, 2000). Several studies have reported that caspase 3 is up-regulated by TLR3 agonist activity which would suggest a role for caspase 3 in cancer cell apoptosis induced by TLR3 agonists (Salaun, 2006; Khvalevsky, 2007). A recent study by Conforti et al demonstrated the synergistic effects between vaccines, chemotherapy and poly A:U (Conforti, 2010). A vaccine (OVA plus CpG) was administered prior to the combination of oxaliplatin and poly A:U and this significantly reduced tumour growth and prolonged survival. Interestingly these results were not demonstrated in nude and TRIF knockout mice.

#### **3.3 Toll-like receptor-4**

TLR4 was first discovered in 1998. Lipopolysaccharide (LPS) is a cell wall protein in gram negative bacteria and works as a ligand for TLR4. The recognition of LPS by TLR4 requires other proteins including LPS binding protein, CD14 and MD2. In resting cells TLR4 is located in the Golgi apparatus. The translocation of TLR4 from the Golgi apparatus to the plasma membrane and the binding of LPS is dependent on MD2 (Nagai, 2002; Shimazu, 1999). TLR4 signals through two pathways: the MyD88-dependant pathway and the MyD88-independant pathway. In the MyD88-dependant pathway, MyD88 binds to the Toll-IL-1 receptor domain of the TLR4 receptor and activates IL-1 receptor associated kinase (IRAK). IRAK in turn phosphorylates TRAF6, which in turn activates a MAP-3-kinase called TAK1, and TAK1 phosphorylates and activates the IκK complex. IκK then liberates NFκB. Killeen et al showed that bacterial endotoxins directly promote tumour cell adhesion and invasion through up-regulation of urokinase plasminogen activator and urokinase plasminogen activator receptor through TLR4 dependant activation of NFKB (Killeen 2009). TLR4 can also signal through the MyD88-independant pathway to stimulate the production of Interferon-B. Wang et al showed that TLR4/MyD88 over-expression was frequently detected in colo-rectal cancer with liver metastasis and TLR4/MyD88 levels were significantly higher in these patients (Wang, 2010). Although there have been a number of studies investigating the role of TLR4 in colo-rectal cancer, the exact impact of TLR4 signalling in comparison to TLR3 signalling in colon cancer cells has yet to be established.

TLR4 signalling has been shown to promote resistance of cancer cells to apoptosis following introduction of a TLR4 ligand in colon cancer cells (Cianchi, 2010). TLR4 signalling appears to promote the development of colitis associated cancer by mechanisms including enhanced

chemotherapeutic agent. Wadler et al combined 5-FU with INF-α and achieved a good response in colon cancer cells however this combination was not effective in clinical trials (Wadler, 1990). Early clinical trials involving a TLR3 agonist as a single adjuvant therapy in colorectal cancer showed mixed results. Lacour et al showed that Poly A:U alone as an adjuvant therapy in colorectal cancer patients who had undergone a resection of the primary tumour was unable to improve the overall survival of patients after five years compared to a placebo (Lacour, 1992). However, when used as a combination therapy there have been very positive results in a clinical trial to demonstrate a positive role for Poly A:U in cancer treatment. After a 14 year median follow-up Laplanche et al reported the results of a trial which combined Poly A:U with loco-regional radiotherapy versus chemotherapy with cyclophosphamide, methotrexate and fluorouracil (CMF) in women with operable breast cancer. They reported that the Poly A:U combination group had a significantly improved disease free survival (p=0.03) and significantly reduced the incidence of metastasis when compared to the CMF group. This trial did not however differentiate the role that Poly A:U and radiotherapy had as single therapies (Laplanche, 2000). Several studies have reported that caspase 3 is up-regulated by TLR3 agonist activity which would suggest a role for caspase 3 in cancer cell apoptosis induced by TLR3 agonists (Salaun, 2006; Khvalevsky, 2007). A recent study by Conforti et al demonstrated the synergistic effects between vaccines, chemotherapy and poly A:U (Conforti, 2010). A vaccine (OVA plus CpG) was administered prior to the combination of oxaliplatin and poly A:U and this significantly reduced tumour growth and prolonged survival. Interestingly these results were not

TLR4 was first discovered in 1998. Lipopolysaccharide (LPS) is a cell wall protein in gram negative bacteria and works as a ligand for TLR4. The recognition of LPS by TLR4 requires other proteins including LPS binding protein, CD14 and MD2. In resting cells TLR4 is located in the Golgi apparatus. The translocation of TLR4 from the Golgi apparatus to the plasma membrane and the binding of LPS is dependent on MD2 (Nagai, 2002; Shimazu, 1999). TLR4 signals through two pathways: the MyD88-dependant pathway and the MyD88-independant pathway. In the MyD88-dependant pathway, MyD88 binds to the Toll-IL-1 receptor domain of the TLR4 receptor and activates IL-1 receptor associated kinase (IRAK). IRAK in turn phosphorylates TRAF6, which in turn activates a MAP-3-kinase called TAK1, and TAK1 phosphorylates and activates the IκK complex. IκK then liberates NFκB. Killeen et al showed that bacterial endotoxins directly promote tumour cell adhesion and invasion through up-regulation of urokinase plasminogen activator and urokinase plasminogen activator receptor through TLR4 dependant activation of NFKB (Killeen 2009). TLR4 can also signal through the MyD88-independant pathway to stimulate the production of Interferon-B. Wang et al showed that TLR4/MyD88 over-expression was frequently detected in colo-rectal cancer with liver metastasis and TLR4/MyD88 levels were significantly higher in these patients (Wang, 2010). Although there have been a number of studies investigating the role of TLR4 in colo-rectal cancer, the exact impact of TLR4 signalling in comparison to TLR3 signalling in colon cancer cells has yet to be established. TLR4 signalling has been shown to promote resistance of cancer cells to apoptosis following introduction of a TLR4 ligand in colon cancer cells (Cianchi, 2010). TLR4 signalling appears to promote the development of colitis associated cancer by mechanisms including enhanced

demonstrated in nude and TRIF knockout mice.

**3.3 Toll-like receptor-4** 

Cox-2 expression and increased EGFR signalling (Fukata, 2007). Recent experimental studies have shown that TLR4 induced inflammatory factors may cause tumour cell escape from immune surveillance and resistance to chemotherapy and radiotherapy (He, 2007).

One proposed mechanism by which tumour cells resist treatment involves an impairment of the immune response by the inflammatory microenvironment at the tumour site (Coussens, 2002). TLR4 upregulation has been shown to increase the secretion of immunosuppressive cytokines TGF-β, VEGF and pro-angiogenic chemokine IL-8 in human lung cancer cells and induces resistance of human lung cancer cells to TNF-α and TRAIL induced apoptosis (He, 2007). The same study demonstrated secretion of VEGF and IL-8 is p38MAPK dependent and the ERK1/2 and JNK1/2 proteins are not activated. NF-κB activation contributes to apoptosis resistance of human lung cancer cells after LPS stimulation and LPS-induced apoptosis is dependent on NF-κB activation in human lung cancer cells. NF-κB is an antiapoptotic transcriptional factor induced by cellular components such as LPS, radiation and various chemotherapy drugs.

Contrasting evidence for the role of TLR4 in chemotherapy and radiotherapy resistance was reported by Apetoh et al who demonstrated reduced tumour growth and prolonged survival in immunocompetent wild type mice when treated with chemotherapeutic agents such as oxaliplatin and doxorubicin but this effect was significantly less in TLR4-/- mice and nu/nu mice (Apetoh, 2007). These findings were also demonstrated in TRIF-/- mice which behaved like the wild type mice but Myd88-/- mice behaved like TLR-/- mice. This suggests a certain dependence of tumour treatment resistance on the MyD88 dependent pathway in TLR4 signalling. These results show that when TLR4 itself is knocked out this will lead to a reduction in tumour growth when the tumour is exposed to an appropriate chemotherapeutic agent or radiotherapy. However, when TLR4 is up regulated in the presence of LPS, this results in resistance to chemotherapy and arguably radiotherapy treatment.

Various agents have been suggested as having a role in reversing TLR4 induced tumour apoptotic resistance. Rapamycin is one such agent. Rapamycin is a macrolide antifungal agent and is a potent immunosuppressive medication that is used as an anti-inflammatory and immunosuppressive drug for the treatment of autoimmune diseases such as Systemic lupus erythematosus (SLE) and transplantation rejection (Fernandez, 2006; Hackstein, 2003). In terms of cancer treatment Rapamycin has been shown to display a number of useful anticancer cell properties including inhibition of cancer cell proliferation and induction of apoptosis (Hartford, 2007; Zhang, 2007). Rapamycin can also inhibit invasion and metastasis of tumour cells (Abraham, 2007). One study focused on Rapamycins ability to reverse the apoptotic resistance that can occur following TLR4 stimulation with LPS. Sun et al showed that Rapamycin reverses TLR4 ligation induced apoptotic resistance in colon cancer cells (Sun, 2008). Interestingly this same study showed that Rapamycin inhibits anti-apoptotic protein bcl-xL expression and activation of Akt and NF-κB pathways following LPS treatment of cells and this was shown to reverse apoptosis when Akt and NF- κB were inhibited. Paclitaxel has been described as a potential ligand to TLR4 (Asselin, 2001). Kelly et al demonstrated that TLR4 signalling promotes tumour growth and paclitaxel chemoresistance in ovarian cancer cells (Kelly, 2006). This behaviour was mediated by MyD88 expression.

The Role of Inflammation in Cancer 399

The interaction between cancer cells and the innate and adaptive immune system is complex. As described previously, pro-inflammatory cytokines promote tumour growth; however the immune system has a means of restricting cancer development through immuno-surveillance and immuno-editing. The pro-tumorigenic effect of the inflammatory immune response appears generally to be a consequence of the innate immune system,

Cancer immunosurveillance is a theory formulated in 1957 by Burnet and Thomas, who proposed that lymphocytes act as sentinels in recognising and eliminating continuously arising transformed cells (Dunn, 2002; Burnet, 1957). Cancer immunosurveillance appears to be an important host protection process that inhibits carcinogenesis and maintains regular cellular homeostasis (Kim 2007). It has been shown that when mice are deficient in genes that encode for cells integral to the adaptive immune response such as T cells, B cells and natural killer T (NKT) cells, tumour incidence and tumour growth are increased (Shankaran, 2001). With the aid of additional studies, natural killer cells and T cells now appear to be the dominant cells involved in tumour immuno-surveillance. Interestingly, interferon-γ (IFN-γ) is produced by both NKT and T cells. Furthermore, IFN-γ is the most influential cytokine released by these cells. Recent studies have now shown that mice deficient in IFN-γ are more susceptible to spontaneous carcinogenesis (Shankaran, 2001). The mechanism involved in the anticancer protective effect of the immune system is complex. Generally, it is theorised that cells of the immune system recognise the presence of a growing tumour which has undergone stromal remodelling, causing local tissue damage. This is followed by the induction of inflammatory signals which recruits natural killer cells, NKT cells and macrophages to the tumour site. During this phase, the infiltrating lymphocytes such as the natural killer cells and NKT cells are stimulated to produce IFN-γ. Newly synthesised IFN-γ then induces tumour death as well as promoting the production of chemokines which play an important role in promoting tumour death by blocking the

Similarly, IL-4 is released in large quantities by NKT cells upon activation and is a key regulator in the adaptive immunity. Therapeutic attempts to recruit the immune system to curtail cancer growth have now become a keen area of interest. Although, attempts have met with limited success thus far, this is the basis of vaccine guided anti-cancer therapy

It is clear that elements of the immune system can restrict tumour growth. However, as discussed previously pro-inflammatory cytokine release initiated by the immune system can also promote tumour growth. Therefore, the immune system appears to have the effect of a double edged sword on cancer growth. If we had full understanding and control of these

Recently vaccines have been developed to take advantage of the role played by the immune system in cancer. The discovery of tumour associated antigens (TAA) expressed by colorectal carcinoma as well as recent advances in tumour immunology are providing new focus to develop biologically targeted immunotherapeutic strategies. It has been reported that immuno-deficient animals as well as humans, e.g. transplant patients, are at greater risk of developing malignancy (Penn, 2000). It has also been reported that cytotoxic T

which is a rapidly progressing area of research (Dranoff, 2004).

pathways, this could potentially lead to successful cures for cancer.

whereas the adaptive immune system exerts anti-tumorigenic effects (Karin, 2005).

**5. Immuno-surveillance** 

formation of new blood vessels.

**5.1 Vaccines** 
