Toll‐like receptors (TLRs): An old family of immune receptors with a new face in cancer pathogenesis

Abstract In the dark path of tumorigenesis, the more carefully the cancer biology is studied, the more brilliant answers could be given to the countless questions about its orchestrating derivers. The identification of the correlation between Toll‐like receptors (TLRs) and different processes involved in carcinogenesis was one of the single points of blinding light highlighting the interconnection between the immune system and cancer. TLRs are a wide family of single‐pass membrane‐spanning receptors that have developed through the evolution to recognize the structurally conserved molecules derived from microorganisms or damaged cells. But this is not everything about these receptors as they could orchestrate several downstream signalling pathways leading to the formation or suppression of cancer cells. The present review is tempted to provide a concise schematic about the biology and the characters of TLRs and also summarize the major findings of the regulatory role of TLRs and their associated signalling in the pathogenesis of human cancers.

other roles in the cells. The results of molecular investigations shed light on the ability of TLRs in propagating specific signalling in the cells which regulates the balance between pro-and anti-apoptotic target genes. 2 Having established these regulatory functions, a new chapter has opened about the TLRs, introducing them as important regulators of tumorigenesis.

| A G LIMPS E INTO THE B I OLOGY OF TLR s , FROM THEIR B I OG ENE S IS TO THE DEP TH OF THEIR FUN C TI ON S
From the immunological point of view, the characteristics of these molecules have been well-studied in different reports to unveil the structure, signalling and functions of TLRs in cells. The results of highresolution X-ray crystallography analysis have revealed that TLRs deviate considerably from the canonical LRR structure and thereby they could interact with a wide range of ligands in a highly divergent fashion. This different structure gives TLRs the ability to be activated in a different manner from other LRRs, resulting in the involvement of these receptors in other biological processes rather than only regulating innate immunity. 3 In the following part of this article, we take a look at the biogenesis of TLRs to become more aware of their unique biology. Then, a brief explanation would be provided about their structure, their probable ligands and downstream signalling pathways. As the propagation of the TLR cascade may result in the regulation of diverse intracellular functions, we dedicate the last part of this section to explain the biological functions of the molecules so that we can learn more deeply about the functions of TLRs.

| A glance at TLR biogenesis and localization
All the TLRs are synthesized from their mRNAs into functional configurations in the endoplasmic reticulum (ER) and are translocated to the Golgi complex followed by trafficking to either the plasma membrane or endosomes. The localization of TLRs is a complicated process, which is controlled by a group of ER-associated proteins.
Thus far, ten functional TLRs have been identified in humans according to their subcellular localization. Although TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are expressed on the cell surface and migrate to phagosomes after activation, the expression site of TLR3, TLR7, TLR8 and TLR9 is in intracellular compartments, in particular the endosomes and the endoplasmic reticulum. Among all, the subcellular localization of TLR4 is unique as this TLR could be found either at the plasma membrane or at endosomal vesicles. 4 A member of the ER-resident HSP90 protein family, gp96, serves as a general chaperone for the surface-expressed TLR1, TLR2, TLR4 and TLR5, and intracellular TLR7 and TLR9. Deficiency in gp96 leads to the loss of expression of TLR1-TLR5 and TLR7 or improper TLR9 protein folding. 5 Unc-93 homolog B1 (UNC93B1), a multi-pass TM protein, controls the endosomal trafficking of TLRs, especially TLR3.
For endosomal TLRs, such as TLR3, TLR7 and TLR9, a multi-span transmembrane protein, UNC93B, enters the game to traffic these proteins to endosome. 4 Another ER-resident protein that regulates the trafficking of TLRs is named protein associated with TLR4 A (PRAT4 A), which on one hand guide TLR1, TLR2 and TLR4 to the cell membrane, and on the other hand propel TLR7 and TLR9 to endosomes. 6 TLRs tend to construct dimers in the presence of ligand.
Most of the TLRs appear to form homodimers; however, the story is quite different for TLR2 as it prefers to be presented as a heterodimer with either TLR1 or TLR6.

| A glance at TLRs structure
TLRs belong to type I transmembrane glycoproteins and contain three major domains, ectodomain, single-spanning transmembrane domain and cytoplasmic TLR domain. The ectodomain is oriented towards extracellular space or luminal space (depending on receptor localization) and contains multiple 7-19 leucine-rich repeats (LRRs) that harbour 24-29 amino acids. 20 Two types of motifs were considered for this domain: 'typical' ('T') motifs (LxxLxLxxNxLxxLxxxxF/ LxxLxx) and 'bacterial' ('S') motifs (LxxLxLxxNx LxxLPx(x)LPxx). 20 The unique horseshoe shape of the TLRs is established by folding LRR modules into parallel β-sheets and α-helix that bend into a concave surface. 21

| A glance at TLRs ligands
Although each TLR recognizes distinct ligands, the mechanisms of TLR activation and signal transduction are somehow the same.
Several microbial ligands have been enumerated for TLRs, which their lists are summarized in Table 1. Microbial ligands are not the only stimulators of TLRs, and recently, a group of other proteins, referred to as alarmin, have been identified. They could propagate TLR signalling in the cells, especially through activating TLR2 and TLR4.
The list of these endogenous ligands is also summarized in Table 1.
According to the surveillance model proposed by Johnson et al, another sort of ligands that are responsible for TLR activation is known as the degradation products of endogenous macromolecules, such as heparan sulphate and polysaccharide fragments of hyaluronan. 23 The signals that are mediated by the degenerated macromolecules mostly trigger TLRs-mediated tissue repair processes in addition to initiating protective inflammatory responses. 24

| TLR signalling
Given the importance and the key roles of TLRs in regulation of immune responses, it is not surprising that their emitted signals are a succession of complex events in the cells that lead to activation or suppression of a wide range of downstream signalling axes. Generally speaking, the TLR signalling pathway could be mediated through either MYD88-dependent or MYD88-independent manner. The TLR signalling has been well reviewed in an article provided by Luo et al 22

Innate immune responses
When it comes to TLRs, the first thing that comes to mind is its bona fide action in response to specific molecules derived from bacteria and viruses. 24 In the perspective of innate immunity, which TLRs

Adaptive immune responses
TLRs are indeed the main players of the innate immunity system; however, their central role in regulation of host protective adaptive immune responses should not be underestimated. It has been suggested that TLRs could stimulate both T cell-and B cell-mediated immune responses upon exposure to adjuvants containing microbial lysates or products. 11 Moreover, TLRs could stimulate professional antigen-presenting cells (APCs) and have a role in processing and presentation of microbial antigens, up-regulation of co-stimulatory molecules, T cell activation and suppression of regulatory T cells. 12 TLR-mediated production of IL-12 dictates differentiation of activated T cells into T helper 1 (Th1) cells. TLRs are also crucial for activation and maturation of the B cell responses during infection and vaccination. Last but not least, through both T cell-dependent and T cell-independent pathways, TLRs regulate B cell proliferation, immunoglobulin isotype class switching and somatic hypermutation. 13

| Non-canonical functions of TLRs
Apart from participation in the regulation of immune responses, which is well-described in the previous reports, TLRs have some

| TLR s IN C AN CER : DOUB LE-EDG ED SWORDS
The first evidence supporting the involvement of TLRs in the tumorigenesis event has stemmed from a report indicated that the risk of cancer development is significantly higher in organs that are directly or indirectly exposed to bacterial TLR ligands. This finding is also reflected in the low incidence of cancer development in germ-free animals. 18 Although there remains much to learn about the involvement of TLRs in tumorigenesis, a question has occupied the mind of the researchers: How TLRs ___ that are responsible for the regulation of immune responses against unknown antigens, such as cancer cells ___ could play such a controversial mechanism of action? The answer to this question may be in the amplitude and length of receptor activation. Although chronic low-grade TLR activation favours a tumour-promoting pro-inflammatory state, high-dose TLR activation induces antitumour response. 19

| The pro-tumour activity of TLRs
The idea behind the involvement of TLRs and their related signalling in the formation of human cancers has originated from the considerable number of studies reported the abnormal expression of TLRs on tumour cells, where they may influence tumour growth and immune responses. 25 The discovery of these receptors in tumour cells has heralded a renaissance in the interconnection between innate immunity and tumour biology.  32 Other examples of simultaneous expression of TLRs in cancer cells could be found in ovarian cancer cell lines which displayed overexpressed TLR2-TLR5, 33 and in lung cancer with overexpressed TLR2-TLR4 and TLR9. 34 Moreover, overexpression of TLR4, TLR5 and TLR9 not only has been reported in gastric cancer cell lines but also was observed in the metaplastic and dysplastic gastric epithelial cells of patients with Helicobacter pylori gastritis. 35 One of the human cancers which displayed the overexpression of multiple TLRs is melanoma, in which up-regulation of TLR2-TLR4 is coupled with metastasis and tumour progression. 36 The association between the overexpression of TLRs and human cancers is summarized in Table 2.

| Overexpression of TLRs in human cancers
Another critical complication that is associated with grouped TLRs expression is the regulation of drug-resistance phenotype, which eventually lead to the poor outcome of patients and even increase the risk of cancer recurrence. Co-expression of TLR1, TLR7 and TLR9 in myeloma cells has shown to be involved in induction of drug resistance. 37 Moreover, the up-regulation of TLR3, TLR4 and TLR9 expressions could increase the probability of biochemical recurrence and cancer metastasis in prostate and breast cancer, respectively. 38 TLR4 and TLR9 could also orchestrate a signal that helps cancer cells to bypass the immune responses by increasing the expression of immunosuppressive cytokines and anti-apoptosis proteins. It has been also indicated that the co-stimulation of TLR7 and TLR8 could induce  Increases cell proliferation and survival through the NK-κB and MAPK pathways Increases pro-inflammatory factors like COX-2 and prostaglandin through the STAT3 pathway Anti-apoptotic effects by inhibition of caspase 3, 6, 7 and 9 transcription Increases tumour invasion and metastasis, especially macrovascular invasion Enhance cell proliferation and inflammation 94,95 Oesophageal Cancer TLR4 TLR3, TLR4, TLR7, TLR9 Immune escape and inflammation by up-regulation of IL-8 and COX-2 Tumour metastasis via up-regulation of p38 and selectin Enhance inflammation, invasion (especially lymph node metastasis) and proliferation 96 Adrenocortical carcinoma TLR2, TLR4 Induction of inflammation via up-regulation of TNF-α, IL-6 and IL-8 97

TLR-mediated inflammation
In 2000, Hanahan and Weinberg proposed a model to define 'six hallmarks' that a tumour requires for maintaining its survival. 40 However, as the conventional knowledge about the pathogenesis of cancer was progressing, inflammation was also added as the seventh feature to the list. 41 There are a considerable number of studies with the focus on the correlation between cancer development and the previous history of microbial infection, injury, inflammation and tissue repair. Although it is early to hazard a conjecture for the mechanism through which inflammation may develop a malignancy, the possible candidate could be TLRs that engage in a unique cross-talk with the NF-κB signalling axis. Through regulating more than a hundred pro-inflammatory genes, NF-κB is a master of inflammation regulator. 42

TLR-mediated anti-apoptotic effects
As mentioned earlier, the TLR network has a tight cross-talk with the NF-κB axis through either MYD88-dependent or MYD8independent manner. 44 As such, TLR-induced NF-κB activation inhibits apoptosis and promotes tumour cell survival in colon cancer, liver cancer, stomach cancer and lung cancer. 39 In a recent study, we also examined the combination of TAK-242 and four well-known chemotherapeutic agents: paclitaxel, cisplatin, doxorubicin and arsenic trioxide in breast and ovarian cancer cells.
Interestingly, we realized that the inhibition of TLR4 boosted the cell cytotoxicity of all drugs, which indicates the fact TLR4 would confer chemo-resistance to a broad range of anti-cancer agents. 51,52 F I G U R E 2 The mechanisms through which TLRs orchestrate pro-tumour responses. TLR stimulation activates the NF-κB cascade, which in turn increases the transcription of inflammatory cytokines including IFN-α, IL-1 and IL-6. Activation of the NF-κB axis not only promotes anti-apoptotic factors (Bcl-XL, Bcl-2, survivin) but also up-regulates MMPs which are responsible for the degradation of extracellular matrix. Although TLR4 activation leads to the production of MMP2 and β1-integrin overexpression, TLR9 stimulation enhances MMP13 level in breast cancer. As represented, increasing of PG-E2 and COX-2 by TLR2, TLR4 and TLR9 and phosphorylation of EGFR by TLR4 are the most important mechanisms in TLR-mediated angiogenesis. Stimulation of TLRs in the tumour microenvironment can provoke tumour cells or tumour-infiltrating cells to produce suppressor cytokines and chemokines which not only suppress immune cells but also attract more cells to tumour microenvironment-such as MDSC, TAMs and CAFs-to fulfil tumour needs. Finally, TLR downstream signalling can make tumour cells and tumour-infiltrating cells to switch their metabolic pathway from oxidative phosphorylation to glycolysis

TLR-mediated invasion and metastasis
Having established the role of TLR-associated signalling pathways in multiple steps of tumorigenesis, intense interest has been at-

Additional mechanisms through which TLRs exert antitumour effects
Converting tumour-supporting macrophages to tumour suppressors that produce inflammatory cytokines and promote M1 polarization is another suggested mechanism that has been attributed to the anti-tumour activity of poly I:C-activated TLR3. This response is mediated by TNF-α through a MyD88-independent pathway. 83 TLR9 agonists can also exert antitumour effects through suppression of angiogenesis. It is likely that the production of IFNs, such as IFNalpha, also plays a key role in both anti-angiogenetic and tumoursuppressive effects of TLRs. 84

| CON CLUS I ON AND FUTURE PER S PEC TIVE
In many cases, cancer cells mimic many characteristics of immune cells. So, under the mask of these cells, neoplastic cells communicate and modulate the immune system for their own survival and growth.
Despite several reports shedding light on the association between TLRs and the incidence of tumorigenesis, a considerable number of questions remained unanswered about the precise mechanisms of these receptors in cancer development. Given this, further exploration and more precise understanding concerning the role of TLRs in the maintenance of cancer cells are required to increase the current knowledge about their participation in tumour biology. These findings could also shed more light on the molecular basis of innate immunity, tumorigenesis and cancer biology, which all together could start a new chapter in the modern understating of cancer and its treatment strategies.

ACK N OWLED G EM ENTS
The authors would like to express their gratitude to Shahid Beheshti University of Medical Sciences (Tehran, Iran) for supporting this study.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no data sets were generated or analysed during the current study.