Abstract
Esophageal adenocarcinoma (AC) develops through Barrett’s esophagus (BE) and columnar dysplasia, preceded by gastro-esophageal reflux disease (GERD). Incidence of esophageal squamous cell carcinoma (SCC) is increased with tobacco smoking and alcohol abuse. Toll-like receptors (TLRs) can act as prognostic factors and potential therapeutic targets of esophageal cancer. TLRs, an important family of pattern recognition receptors, allow immune cells to recognize pathogens triggering inflammation. TLR-signaling pathway activates signaling-elements, regulating inflammatory response, possibly correlating to carcinogenesis. In the normal esophagus, TLRs recognize molecular patterns on microorganisms and inflammatory response produced by tissue-damage. TLR3, TLR4, TLR5, and TLR9 are expressed at increasing levels from GERD to AC. TLR4 is a mediator of proliferation in AC, while TRL1 and TLR4 over-expression in AC is related to poor prognosis and metastasis. Additionally, TLR3, TLR4, and TLR9 expression in SCC has been associated with lymphatic metastasis, whereas increased expression of TLR7 and TLR9 has been also associated with advanced disease. It seems that TLR expression can indicate esophageal metaplasia, dysplasia, and cancer. Herein, we aimed to present all available data regarding the relation of TLRs and esophageal cancer. They may represent significant and valuable diagnostic or prognostic factors for esophageal cancer.
Esophageal cancer (EC) is the seventh most common malignancy and the sixth leading cause of cancer-related mortality worldwide (1). EC remains a global health concern; despite multimodal therapy, the 5-year survival is 45% for local disease, 20% for locally advanced disease, and 5% for metastatic disease (2). Poor long-term results underline the mandate for exploiting both surgical and systemic therapies in these patients.
Squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the esophagus are the two predominant histologic types of EC, with greatly varying racial and geographical distribution worldwide (3). While SCC is the most common histologic type in the Eastern world, AC has become the leading histologic subtype in the Western world. This can be attributed to increased rates of obesity, gastro-esophageal reflux (GERD), and Barrett’s esophagus (BE) (4).
Toll like receptor (TLR) signaling is a major component of the innate immune system (5) and has been proven to be one of the main pathways of interaction amongst tumor cells and microbiota (6). It has been shown that TLRs are triggered by microbial elements, such as lipopolysaccharide, flagellin, DNA, and RNA, as well as endogenous ligands, including heat-shock proteins and DNA (7).
Previous studies have shown that TLRs 1-10 are expressed in the normal esophagus. In BE and esophageal AC, only TLRs 1-6 and TLR9 have been studied and correlated with malignant changes in the esophageal columnar epithelium (5, 7, 8). In contrast, in esophageal SCC, TLR3, TLR4, TLR7, and TLR9 have been associated with aggressive disease properties and metastatic potential (9).
The most important prognostic determinant, both for esophageal AC and SCC is the WHO TNM-classification as presented in the current 8th edition (10). Another key predictor of prognosis is the grade of differentiation, based on histopathologic examination (11).
The aim of this study was to examine the potential links between innate immune system, TLRs, and the development of EC.
Toll-like Receptors
Toll-like receptors are key components of the innate immune system; they can identify pathogen-related molecular patterns and generate an inflammatory response to all externally assaulting microbiota (5). Up to date, 13 TLRs have been acknowledged in mammals; each of these receptors has a unique antigen-recognizing domain (12). It is known that TLR4 identifies bacterial lipopolysaccharide (LPS) found in Gram-negative bacteria, whereas TLR5 recognizes flagellin and TLR9 subfamily members (TLR7, TLR8 and TLR9) recognize microbial DNA and RNA (12).
Toll-like receptors can be either extracellular, such as TLR1, TLR2, TLR4, TLR5, and TLR6, or intracellular such as TLR3, TLR7, TLR8, and TLR9. Extracellular TLRs can be located at the plasma membrane where they recognize macromolecules exposed on the surface of pathogens. In contrast, intracellular TLRs are found in endosomes or lysosomes; they can detect viral and bacterial nucleic acids, playing an important role in host immune responses (13-16). Recently, the ability of TLRs to induce the immune response has been exploited for the treatment of cancer (17). Dysfunction of TLRs has been correlated with inflammation-associated tumorigenesis (carcinogenesis), along the gastrointestinal tract (18, 19), and e EC amongst others. Specifically, expression of TLR3, TLR4, TLR5, and TLR9 has been suggested as a potential mediator of the progression from reflux disorders to AC (20). Following that, increased expression levels of TLR2, TLR4, TLR7, and TLR9 have been observed in SCC (21).
Toll-like Receptors in the Normal Esophagus
It has been known that TLRs 1, 2, 4 and 6 are expressed in both normal and metaplastic squamous esophageal epithelium (8). TLRs 1, 2, and 6 form heterodimers recognizing different kinds of lipopeptides, which are components of the bacterial cell wall, whereas TLR4 recognizes bacterial membrane LPS.
Epithelial cells recognize luminal pathogens via TLRs and activate immune cells and consequently inflammation (20). Bacterial infection can affect tumorigenesis through alterations in cytokine and chemokine expression. These changes stimulate inflammation, angiogenesis, and potential metastatic properties (22).
Low expression of TLRs has been correlated with normal epithelial cells, whereas increased levels have been observed in high-grade dysplastic epithelium. Moreover, TLR3, 4, 5, and 9 have been suggested as potential mediators of the progression from reflux disorders to EC (19).
It is well known, that TLRs are pattern recognition receptors, integral to innate immunity. The signaling components – mitogen-activated protein kinases (MAPKs), play a major role in the conversion of external stimuli to a cellular response (23).
Esophageal Cancer
EC is one of the leading causes of cancer-related mortality. There are two main histological subtypes of EC: esophageal AC (which is correlated to obesity, gastro-esophageal reflux disease, and BE) and esophageal SCC (which is related to alcohol and tobacco consumption, and the ingestion of high temperature beverages) (24).
GERD and BE in the lower esophagus and esophago-gastric junction result in esophageal adenocarcinoma characterized by intestinal metaplasia and dysplasia, where the normal stratified squamous esophageal epithelium is replaced by a columnar intestinal-like one, whereas exposure to alcohol abuse and tobacco smoking result in esophageal squamous cell carcinoma characterized by dysplasia of the squamous epithelial cells of the upper and middle esophagus (25).
Esophageal AC and esophageal SCC are among the indicators of the lowest 5-year survival rate worldwide. Poor prognosis is related to late diagnosis in many cases; lymph node or/and distant metastases are common in advanced disease (26). Despite the advances in clinical treatment, prognosis of EC remains poor due to its relapse and metastatic characteristics. As a result, prognostic factors are essential to improve the classic-risk classification of EC (9). It is well-known, that the natural behavior of esophageal AC and SCC develops through the metaplasia-dysplasia sequence, due to exposure to external factors and the addition of genetic alterations (27). Chronic infections and inflammation can initiate cancer growth via cytokines and chemokines, which play substantial roles in promoting angiogenesis and metastasis, the most important factors resulting to cancer development and growth (28).
Esophageal Adenocarcinoma
It is well studied and established that esophageal AC develops through the metaplasia-dysplasia-carcinoma sequence (7). Thereafter, normal, or inflammatory esophageal (and gastro-esophageal) stratified columnar epithelium is thought to transform to simple columnar epithelium (BE) or metaplastic columnar epithelium. Continuous exposure to gastric acid reflux and to its altered microbiome is the decisive factor (29, 30).
Clinical studies have shown that in the normal esophageal epithelium (cell lines), as well as in BE, stimulation of TLR4 with LPS results in NF-κB activation, increased secretion of IL-8 and induction of inflammatory response (7). Similarly, ex vivo LPS stimulation of TLR4 in BE epithelial cell lines, increased cyclo-oxygenase 2 (COX-2) activity and inflammatory response (31). Moreover, TLR4 activation has been shown to induce proliferation in AC (TLR4–MyD88– TRAF6–NF-κB signaling pathway) (5). As a result, TLR4 may be a target for immunotherapy in esophageal AC (5).
Furthermore, high expression of TLR5 has been observed during the progression from metaplasia-dysplasia to AC (32). This is a potential differentiating factor among columnar dysplasia and BE. Subsequently, increased expression of TLR5 has been observed in AC (5), without correlation to clinical and oncological outcomes. Furthermore, increased expression of TLR9 has been shown in esophageal AC. However, expression of TLR9 was associated with advanced disease, poor differentiation, and metastasis in AC (33), in comparison to TLR5. In contrast, TLR2 expression showed a tendency to increase from normal esophageal epithelium to AC. However, TLR6 levels were highest in high-grade dysplasia (5).
Expression of TLRs is cytoplasmic. However, TLR1 and TLR4 have shown nuclear staining in esophageal adenocarcinoma (8). Nuclear expression of TLR1 and TLR4 has been observed in patients with distant metastasis, while nuclear expression of TLR4 has also been correlated with advanced tumor (T) status (8), according to TNM classification (34).
Esophageal SCC
SCC of the esophagus develops in the squamous esophageal epithelium via dysplasia, in response to exogenous factors, such as tobacco smoking, alcohol abuse, and ingestion of high temperature beverages (24). When compared to the normal esophagus, a variety of TLRs, including TLR3, TLR4, TLR7, and TLR9 showed increased expression in SCC (8).
High TLR3, TLR4, and TLR9 expression in stromal cells of esophageal SCC has been associated with lymphatic metastasis, whereas increased expression of TLR7 and TLR9 has also been observed in more advanced disease (25, 35). However, TLR9 expression in fibroblastoid cells of SCC tumors has been correlated with lower tumor (T) status and smaller incidence of lymph node metastasis at diagnosis (8). Increased TLR3 in EC cells has been associated with high probability of lymph node metastasis, relating the expression of TLR3 to tumor aggressiveness and carcinogenesis (9). Increased expression levels of TLR4 in SCC have been associated with the depth of invasion (T-tumor status), while TLR7 and TLR9 levels have been correlated to the grade of differentiation in esophageal SCC (9). Over-expression of TLR9 has been observed in esophageal squamous dysplasia as well as in esophageal SCC, indicating its potential role in esophageal carcinogenesis (35).
Stimulation of TLR4 with LPS is well known to increase migration and adhesive properties of esophageal SCC cells, via selectin and p38-MAPK (9). In contrast, stimulation of TLR7 triggered NF-κB, increased expression of the antiapoptotic protein Bcl-2, tumor cell survival, and chemoresistance (9).
These findings, indicate that increased TLR expression may interfere with normal TLR function and signaling pathways, and therefore are potentially useful markers of the malignant transformation of esophageal epithelial cells. As a result, EC cells activated by over-expression of TLR may release cytokines and chemokines, stimulating immune cells to release additional inflammatory factors. This course can lead to immune tolerance, disease progression, and dissemination of the tumor (9).
Discussion
EC is one of the most lethal malignancies worldwide, with a 5-year prognosis of approximately 25% across all stages. SCC is the most prevalent histological type of EC in the East, whereas esophageal AC predominates in most of the West, (1). Poor prognosis of EC is mainly associated with its late diagnosis, a result of the absence of specific clinical symptoms in early tumor stages.
Despite its incidence and mortality rate, knowledge on its biology is still scarce. Thus, deeper understanding of the mechanisms involved in its genesis and/or progression may be useful in identifying potential markers for the diagnosis, prognosis, and potential therapeutic strategies (25).
TLRs are pattern recognition receptors, integral to innate immune system. They act as receptors for various ligands, including bacterial cell wall components, RNA, flagellin and bacterial DNA (36). The esophageal epithelium encounters bacteria from the oral cavity, and in the case of GERD, also from the stomach or maybe the duodenum. TLRs recognize bacteria-derived molecular patterns and trigger a pre-inflammatory reaction of the epithelium (7).
The role of TLRs in EC cancer has not been determined. However, there is strong evidence supporting that TLRs play a major role in pre-inflammatory, inflammatory, and pro-carcinomatous systemic response as presented by Kauppila et al. in 2014 (7); increased expression of many TLRs has been correlated to EC and a poor prognosis, firstly described in 2010 by Ioannou et al. (16) for esophageal cancer, (5, 7-9, 12, 13, 33, 35, 37).
In addition, it must be mentioned that the microbiome of the distal esophagus and gastro-esophageal junction undergoes significant changes under inflammatory conditions, such as esophagitis and BE. (29, 30). Furthermore, TLRs over-expression, specifically TLR4, TLR5, and TLR9 in EC, supports the hypothesis of bacterial involvement in the tumorigenesis of EC. These findings further support that TLRs may be important mediators of the metaplasia-dysplasia-esophageal carcinogenesis (38, 5).
In addition, TLR3, TLR4, and TLR9 have been shown to be activated from endogenous ligands derived from diseased or destroyed host-cells (38, 39). It has been shown that cellular damage by alcohol, tobacco smoking, and gastric acids from GERD, combined, is responsible for the loss of esophageal epithelial-cells wall integrity. Specifically, TLR3 and TLR9 can identify elements from diseased cells (39). As a result, an inflammatory response is induced via activation of interleukins (IL-8) and NF-kB. This inflammation status allows the entrance of bacteria through the esophageal epithelium, resulting in the loss of the host-microbiome barrier; this can further lead to increased activation of TLR2, TLR4, TLR5, and TLR9 from bacterial components (20, 22). Changes in the microbiome and inflammation of the esophageal epithelium can cause a vicious cycle of cellular strain, which can play a key role in esophageal metaplasia and tumorigenesis (5, 29, 30).
Moreover, TLRs expression is increased both in esophageal AC and SCC. This suggests that EC cells are triggered by bacteria- and host-derived molecules. Dismal prognosis in high TLR-expressing tumors could then be an indicator of increased levels of tumor–stroma connection, providing strong evidence for advance disease and poor prognosis (7).
According to Sheydihin et al. TLR3, TLR4, TLR7, and TLR9 expression is essential to the biological pathogenesis of EC (9). Furthermore, diverse phenotypes of TLR expression in esophageal stromal cells can lead to different results (depth of invasion, lymph node metastasis, and other) and as potentially prognostic and therapeutic factors in EC, these markers should be thoroughly examined.
Conclusion
EC is a highly lethal malignancy worldwide. Despite the advances in surgical and systemic therapies, prognosis of EC remains poor. This is a result of late diagnosis, due to lack of specific symptoms in early disease. TLRs comprise an important family of pattern recognition receptors that allow immune cells to recognize pathogens and trigger inflammation. The TLR signaling pathway activates several different signaling elements, which regulate the inflammatory response and possibly correlate to carcinogenesis. Herein, we examined the relation of toll-like receptors with the carcinogenesis of EC, and their potential role as diagnostic, prognostic, or therapeutic factors. Toll-like receptors are expressed in the normal esophagus as well as during the metaplasia-dysplasia-cancer sequence. This is indicative of the inflammation caused by the exposure to microbiome changes and irritable factors, and the dissemination of EC in later stages. These findings suggest their remarkable ability to act as diagnostic or prognostic factors as well as their potential value in EC-targeted therapy.
Footnotes
Authors’ Contributions
SD contributed to study conception and design, the acquisition of data and drafting the manuscript. Ακ contributed to drafting the manuscript and the analysis and interpretation of data. MB, DS and SS contributed to the analysis and interpretation of data and drafting the manuscript. TL and AC contributed to study conception and design, the acquisition of data, and the drafting and critical revision of the manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
All Authors have no conflicts of interest to declare in relation to this study.
- Received March 10, 2022.
- Revision received April 15, 2022.
- Accepted April 18, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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