**2. Toll-like receptors signaling pathways and their activation**

Toll-like receptors (TLRs) are transmembrane glycoprotein receptors that play a key role in the innate immune system. Usually, they are present on the cell surface, except for TLR3, TLR7, TLR8, and TLR9. These are located in the endosomal membranes of sentinel cells such as macrophages and dendritic cells that recognize structurally conserved molecules derived from microbes. TLRs are the first pattern recognition receptors (PRRs) identified and characterized in mammals [4]. They initiate key inflammatory responses, shape adaptive immunity against microbial infection, repair and regenerate tissues [5, 6]. Through their role in the immune system, TLRs are a possible tool for curing and preventing cancer. TLRs belong to type I transmembrane glycoproteins characterized by an extracellular leucine-rich domain and a cytoplasmic tail, which are primarily responsible for mediating ligand recognition and a single transmembrane helix. TLRs also contain a cytoplasmic tail domain that is homologous to the interleukin-1 receptor and is responsible for initiating various intracellular signaling cascades. These signaling cascades include activation of the nuclear factor-κB (NF-κB), which is considered a key transcription factor that promotes expression of genes involved in immune response such as cytokines and chemokines, as well as co-stimulatory and adhesion molecules [7, 8] (**Figure 1**). To date, 10 types of toll-like receptor families have been identified in humans, and each of them recognizes a specific PAMP [9–12]. Moreover, TLRs can form heterodimers such as TLR2/TLR1 and TR2/TLR6 to recognize the tri- or diacyl lipopeptides of bacteria, respectively [13, 14]. TLR2/6, along with CD36, has been found to have a role in recognizing the lipoteichoic acid (which is diacylated) of Gram-positive bacteria [15]. TLR2 can recognize the peptidoglycan of most bacterial species and fungi, while TLR4 along with CD14 recognizes the lipopolysaccharide (LPS) of Gram-negative bacteria. TLR3 can recognize the double-stranded RNA (dsRNA) that is found during the replication cycle of most viruses [15]. TLR5 recognizes bacterial flagellin [13, 14]. Furthermore, TLR7 and TLR8 can recognize the

single-stranded RNA (ssRNA) found in certain viruses and also the imidazoquinoline compounds, imiquimod and resiquimod (R-848). Correspondingly, TLR9 recognizes hypomethylated CpG motifs of bacterial double-stranded DNA (dsDNA) and DNA generated during the replication process of dsDNA viruses such as the herpes simplex virus [13, 14]. On the other hand, the PAMP recognized by TLR10 is unknown. However, TLRs can be classified into two groups based upon cellular location [16]. TLRs 1, 2, 4, 5, and 6 are found on the cell plasma membrane and can be activated by extracellular PAMPs. In contrast, TLRs 3, 7, 8, and 9 are principally found in membranes of intracellular compartments, such as endosomes and lysosomes [17]. The intracellular location of TLRs 3, 7, 8, and 9 enables them to detect nucleic acids (i.e., DNA or RNA) that have been released from viruses or bacteria and degraded within endosomes and lysosomes inside the cell [15, 16]. Thus, the inactivation of TLRs will certainly hamper immune function, leading to significant side effects for human health and well-being. The TLR signaling cascade involves (or not) the activation of the adapter molecule MyD88. Both cascades lead to the activation of NF-κB to promote transcription of pro-inflammatory cytokines, chemokines, and cationic peptides. These mediators are involved in innate and adaptive

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**Figure 1.** Toll-like receptors: localization and signaling pathways.

TLRs present in various immune cells are used to sense multiple pathogens [18, 19]. In this sense, TLR2 activation allows the activation of NF-κB and subsequent production of IL-8 and iNOS [19]. Also, it was demonstrated that the activation of TLR3 induced the production of IL-8,

immune responses.

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**Figure 1.** Toll-like receptors: localization and signaling pathways.

endogenous and exogenous danger signals by using pattern recognition molecules (DAMPs and PAMPs) and drive an adaptive immune response through their signaling pathways, which leads to NF-κB and IRF3 transactivation and induces different inflammatory cytokine genes. Over the past several years, different studies have indicated that TLR polymorphisms modify the cellular immune response and that some of these polymorphisms are associated with susceptibility to infectious and inflammatory diseases as well as cancer incidence and severity [1–3]. However, previous studies have reported that the polymorphisms on the TLR1-TLR10 cluster have been associated with increased risk of cancer. A meta-analysis also confirmed the association of TLRs with increased risk of cancer development. Our previous studies have demonstrated a strong link between TLR polymorphisms and colon as well as breast cancer in the Saudi Arabian population. Similar studies have been conducted with Korean patients with papillary thyroid cancer and their clinicopathologic features in age-matched controls using direct sequencing. The general objective of this chapter is to investigate the role of different TLR (i.e., TLR2, TLR4, and TLR6) polymorphisms and their association with cancer

**2. Toll-like receptors signaling pathways and their activation**

Toll-like receptors (TLRs) are transmembrane glycoprotein receptors that play a key role in the innate immune system. Usually, they are present on the cell surface, except for TLR3, TLR7, TLR8, and TLR9. These are located in the endosomal membranes of sentinel cells such as macrophages and dendritic cells that recognize structurally conserved molecules derived from microbes. TLRs are the first pattern recognition receptors (PRRs) identified and characterized in mammals [4]. They initiate key inflammatory responses, shape adaptive immunity against microbial infection, repair and regenerate tissues [5, 6]. Through their role in the immune system, TLRs are a possible tool for curing and preventing cancer. TLRs belong to type I transmembrane glycoproteins characterized by an extracellular leucine-rich domain and a cytoplasmic tail, which are primarily responsible for mediating ligand recognition and a single transmembrane helix. TLRs also contain a cytoplasmic tail domain that is homologous to the interleukin-1 receptor and is responsible for initiating various intracellular signaling cascades. These signaling cascades include activation of the nuclear factor-κB (NF-κB), which is considered a key transcription factor that promotes expression of genes involved in immune response such as cytokines and chemokines, as well as co-stimulatory and adhesion molecules [7, 8] (**Figure 1**). To date, 10 types of toll-like receptor families have been identified in humans, and each of them recognizes a specific PAMP [9–12]. Moreover, TLRs can form heterodimers such as TLR2/TLR1 and TR2/TLR6 to recognize the tri- or diacyl lipopeptides of bacteria, respectively [13, 14]. TLR2/6, along with CD36, has been found to have a role in recognizing the lipoteichoic acid (which is diacylated) of Gram-positive bacteria [15]. TLR2 can recognize the peptidoglycan of most bacterial species and fungi, while TLR4 along with CD14 recognizes the lipopolysaccharide (LPS) of Gram-negative bacteria. TLR3 can recognize the double-stranded RNA (dsRNA) that is found during the replication cycle of most viruses [15]. TLR5 recognizes bacterial flagellin [13, 14]. Furthermore, TLR7 and TLR8 can recognize the

development.

58 Genetic Diversity and Disease Susceptibility

single-stranded RNA (ssRNA) found in certain viruses and also the imidazoquinoline compounds, imiquimod and resiquimod (R-848). Correspondingly, TLR9 recognizes hypomethylated CpG motifs of bacterial double-stranded DNA (dsDNA) and DNA generated during the replication process of dsDNA viruses such as the herpes simplex virus [13, 14]. On the other hand, the PAMP recognized by TLR10 is unknown. However, TLRs can be classified into two groups based upon cellular location [16]. TLRs 1, 2, 4, 5, and 6 are found on the cell plasma membrane and can be activated by extracellular PAMPs. In contrast, TLRs 3, 7, 8, and 9 are principally found in membranes of intracellular compartments, such as endosomes and lysosomes [17]. The intracellular location of TLRs 3, 7, 8, and 9 enables them to detect nucleic acids (i.e., DNA or RNA) that have been released from viruses or bacteria and degraded within endosomes and lysosomes inside the cell [15, 16]. Thus, the inactivation of TLRs will certainly hamper immune function, leading to significant side effects for human health and well-being. The TLR signaling cascade involves (or not) the activation of the adapter molecule MyD88. Both cascades lead to the activation of NF-κB to promote transcription of pro-inflammatory cytokines, chemokines, and cationic peptides. These mediators are involved in innate and adaptive immune responses.

TLRs present in various immune cells are used to sense multiple pathogens [18, 19]. In this sense, TLR2 activation allows the activation of NF-κB and subsequent production of IL-8 and iNOS [19]. Also, it was demonstrated that the activation of TLR3 induced the production of IL-8, TNFα, IL-18, and type I interferon (IFNα/β) and promoted Th-1 type immune responses [20, 21]. Furthermore, other studies have demonstrated that the activation of TLR5 in human keratinocytes by its ligand, flagellin, also resulted in the production of TNFα, IL-8, and the antimicrobial peptides human β-defensins 2 and 3 (hBD2 and hBD3) [22, 23]. TLR9 activation leads to the selective production of the chemokines CXCL9 and CXCL10, which promote memory T-cell responses and the production of type I interferon [20, 21]. Miller et al. showed that TLR5 and TLR9 promote the wound healing process by production of the differentiation factor TGFα [24]. Recently, Semlali et al. reported that gingival epithelial cells in engineered human oral mucosa sense *C. albicans* infection by activating TLRs and producing antimicrobial peptides, such as HBD-2 and HBD-3 [25]. Also, Rouabhia et al. demonstrated that the mutation of the *Candida* genes *IPT1* or *ECM33* prevents TLR activation and cationic peptide (beta-defensin) expression [26]. Together, these available studies demonstrated that TLRs are expressed in the immune cells, and the deregulation of TLR expression by immune cells may promote cancer initiation and development.

**4. TLR polymorphisms and their role in cancer development**

tial functional consequences for infectious disease or cancers.

• TLR1 polymorphisms and cancer susceptibility

tric cancer [37].

• Association between TLR2 and cancer

Multiple single nucleotide polymorphisms (SNPs) have been identified in TLRs with poten-

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Several studies have linked TLR1 polymorphism to different types of cancer, including breast, colon, and gastric. Two SNPs related to TLR1 and breast cancer were previously investigated. Rs7696175 was found to be associated with increased risk of breast cancer in two populations: Chinese and of European ancestry (OR > 1) [31, 32]. Moreover, Chen et al. investigated the association of rs4833095 SNP and breast cancer in a Chinese population, and they found no association [33]. In colon cancer, rs5743618 (T1805G) on exon 4 results in I602S amino acid substitution in the junction of cytoplasmic and transmembrane TLR1 domain, affecting ligand binding [34]. However, several studies have demonstrated a significant decrease in cytokine response in rs5743618 as compared with wild type [34]. In patients with metastatic colorectal cancer (mCRC), TLR1 rs5743618 SNP was associated with a significant response to FOLFIRI plus bevacizumab chemotherapy. Based on these findings, TLR1 rs5743618 might be a predictive biomarker for the advantage of FOLFIRI plus bevacizumab response in mCRC patients [35]. According to Castano-Rodriguez and colleagues' review, rs5743618 polymorphism has also been associated with decreased risk of gastric cancer among the German population (OR < 1) [36]. Moreover, Ravishankar et al. investigated the association of rs4833095 polymorphism in TLR1, and they found that this SNP has conferred susceptibility of *Helicobacter pylori* patients to the development of gastroduodenal diseases, especially gas-

The link between TLR2 polymorphisms and cancer was specifically investigated in the context of chronic inflammation, which likely increases the risk of cancers. An association between TLR2–174 to −196 del polymorphism, 22 base-pair deletions in the promoter, has been found with breast, colon, gastric, and cervical cancer; therefore, the presence of this polymorphism might be used as a biomarker for these cancers. Theodoropoulos et al. demonstrated the association between −174 and − 196 del of TLR2 and breast cancer in Caucasian patients, and they found that this polymorphism may confer increased susceptibility to breast cancer [38]. Furthermore, Proença et al. found that −174 to −196 del are related to an elevated risk of colorectal cancer [39]. According to a review by Castano-Rodriguez and colleagues, TLR2–174 to −196 del have been associated with gastric cancer among different populations: Chinese, Brazilian, and Japanese (OR > 1). On the other hand, −174 to −196 del (Ins/Ins genotype) have been associated with cervical cancer as a protective factor among Tunisian women [40]. Among the Korean population, two TLR2 polymorphisms have been studied to investigate their association with papillary thyroid cancer (PTC)—rs3804099 and rs3804100—and it was found that the two SNPs are associated with PTC [41]. Additionally, rs3804100 and rs3804099 have been associated with increased gastric cancer risk in Brazilian and Chinese populations, respectively [36]. Slattery et al. have investigated some TLR2 SNPs for their impact on
