Abstract
Background: As a crucial regulator of inflammation and immune responses, the NF-κB transcription factor regulates the transcription of target genes which are closely related with cell survival, cell proliferation, apoptosis, invasion and metastasis. The toll-like receptor (TLR) family is critical in aiding pathogen recognition and the subsequent activation of innate immunity. TLR4, one of the TLR family members, is the main receptor of innate immunity and functions to identify pathogens. However, the significance of the expressions of both NF-κB and TLR-4 in the occurrence, development and prognosis of esophageal squamous cell carcinoma (ESCC) remains unclear. Methods: NF-κB and TLR-4 expressions were analyzed in tissue microarrays made up of low grade intraepithelial neoplasia (LGIN), high grade intraepithelial neoplasia (HGIN), early-stage ESCC, advanced ESCC tissue samples, as well as normal healthy esophageal mucosa. Chi-squared tests and Kaplan-Meier plots were utilized to determine the prognostic values of the NF-κB and TLR-4 expressions. Results: We discovered that NF-κB and TLR-4 expressions were progressively increasing from normal esophageal mucosa, LGIN, HGIN and were highest in early-stage ESCC. Interestingly, TLR-4 and NF-κB expressions were attenuated in advanced ESCC tissue. Suppressed expressions of TLR-4 and NF-κB correlated to increasingly malignant ESCC. Additionally, there was a positive correlation between NF-κB and TLR-4 expressions. Conclusion: TLR-4 and NF-κB are associated with the occurrence, development and malignant evolution of ESCC.
Keywords: NF-κB, toll-like receptor-4, esophageal squamous cell carcinoma, prognosis
Introduction
Esophageal cancer is ranked seventh in terms of morbidity, making up 572,000 (3.2%) out of 18,078,957 newly diagnosed cancer cases while ranking sixth in terms of overall mortality, causing 509,000 deaths (5.3%) out of all 9,555,027 cancer related deaths. Such high mortality portends that esophageal cancer is responsible for an estimated 1 in every 20 cancer deaths in 2018. The regional difference of esophageal cancer incidence is striking. The incidence of esophageal cancer is thought to be highest in Eastern Asia, in which Mongolia and China are ranked 5th worldwide [1]. In 2015, the incidence rate of esophageal cancer in China ranked third, with mortality ranking fourth [2]. The two most common histologic sub-types of esophageal cancer are squamous cell carcinoma (SCC) and adenocarcinoma (AC) both of which have been found to differ significantly in terms of their etiologies [1], geographical distribution, and pathogenesis. The Asian Esophageal Cancer Belt denotes regions where esophageal cancer incidences are the highest in the world and spans from northern China to northwestern Iran, including India and Japan [3].
The ability of innate immunity to incite inflammation in the body is a crucial defense system that confers protection against viral and bacterial infections, facilitating wound healing and maintaining tissue homeostasis. NF-κB is made up of five different master transcription factors, including RelA/p65, RelB, c-Rel, NF-κB1/p105 and NF-κB2/p100. As a master regulator, NF-κB regulates the crosstalk between inflammation and cancer in multiple aspects [4]. This molecule has the ability to form homodimers and heterodimers with specific promoter regions in the target DNA of the respective genes [5]. Tumor tissues often have elevated NF-κB activity, resulting in an accumulation of proinflammatory cytokines, contributing to a protumorigenic microenvironment. Protumorigenic cytokines such as IL1, IL17 and NFα have been documented to infiltrate the gastrointestinal mucosa, causing an elevation of NF-κB activity in patients with inflammatory bowel disease, thereby increasing the risk of colon cancer [6]. Continuous and consistent activation of the NF-κB signaling pathway results in a tumorpromoting microenvironment, leading to an increased likelihood of tumor development and progression [7].
Toll-like receptors (TLRs) are able to discern invading pathogens, triggering an innate immune response which then potentiates an adaptive immune response [8-10]. Extensive research has gone into dissecting the components of the TLR signaling pathway. Recent literature has linked cancer development and TLRs. Of all the TLRs, the TLR4 signaling pathway has been found to be involved in the inflammatory response and the progression of cancer [11]. It does this by triggering two separate cell signaling pathways-the myeloid differentiation factor 88 (MyD88)-independent pathway and the MyD88-dependent pathway. The latter pathway involves the activation of MyD88, which results in the recruitment of tumor necrosis factor-receptor associated factor 6 (TRAF6) and IL-1 receptor-associated kinase, both of which go on to stimulate the κB-inhibitor (IκB) complex (IKK) and degradation of IκBα. This sequence of events results in NF-κB activation and nuclear translocation, finally culminating in the production of pro-inflammatory cytokines (e.g., TNF-α and IL-6) [12]. Engaging TLR4 ultimately triggers the transcription of inflammatory-related genes and produces a myriad of antimicrobial inflammatory responses at the site of the inflammation or infection [12,13].
In this report, we focus on the expressions of TLR-4 and NF-κB proteins and analyze their different expressions in normal esophageal mucosa, LGIN, LGIN, early stage ESCC, and advanced ESCC, as well as their correlations with clinicopathological parameters such as gender, age, tumor size, and histologic grade in both early stage and advanced ESCC patients. Furthermore, the impact of TLR-4 and NF-κB expressions on overall patient survival are also assessed.
Materials and methods
Study cohort and tissue specimens
In our investigation, we gathered 104 paraffin-embedded samples of ESCC tissues, of which 31 cases were early-Stage ESCC and 73 cases were advanced ESCC. These samples were harvested from ESCC patients who underwent curative surgery between 2010 and 2013 in the Wuwei Tumor Hospital of Gansu, China. All ESCC cases were diagnosed clinically and histopathologically confirmed to have ESCC without distant metastasis. In addition to this, 26 cases of HGIN, 36 cases of LGIN and 33 cases of normal esophageal mucosa were included. None of the patients received chemotherapy or radiotherapy prior to their surgical resection.
All participants with or without ESCC provided written informed consent. All study protocols were approved by the Research Ethics Committee of Wuwei Tumor Hospital. The epidemiological and clinical characteristics of the ESCC patients were retrospectively documented from medical records in the Wuwei Tumor Hospital of Gansu. The following clinical and pathological parameters were noted: age, sex, site of the tumor, size of the tumor, histological grade, depth of invasion, lymph node metastasis, and survival time after definitive diagnosis. The histological grade was determined by the World Health Organization criteria [14]: histological grade I (mainly includes large, differentiated, keratinocyte-like squamous cells); histological grade II (falling between Grade I and Grade III); histological grade III (predominantly consists of basal-type cells with highly irregular mitosis). The survival time was determined in terms of number of months. The ESCC patients were stratified into two groups: early-stage ESCC (further subdivided into mucosal and submucosal carcinomas based on the depth of infiltration and absence of lymph metastasis) and advanced ESCC (microscopically subdivided into muscularis propria and adventitia carcinomas based on the depth of infiltration or the presence of lymphatic metastasis).
Tissue microarray construction
There were three pathologists (Xiang Li, Ping Fan, Haiying Li) who reviewed all the ESCC tumor H&E stained slides. We selected representative areas of each tumor sample that were exclusive of hemorrhage and necrosis regions, which were pre-marked on the corresponding paraffin blocks. 1 mm diameter cylindrical core samples were punched out using a hollow needle from the tumor samples. Each tumor, LGIN, HGIN and healthy tissue sample supplied one core biopsy each. These tissue cores were then carefully embedded onto a separate paraffin block in a pre-determined array pattern. The resultant tissue microarray was neat, complete, structurally intact with well-preserved histological target sites.
Immunohistochemical staining
4 μm consecutive sections were arranged onto slides for immunohistochemical (IHC) staining. The IHC staining was carried out by an automatic staining machine. A TLR-4 mouse monoclonal antibody (1:100, Abcam, USA) and an NF-κB rabbit anti-human polyclonal antibody (Fuzhou Maixin Biotechnology Development Company) were used to stain the sections, with PBS-staining used to produce the negative controls.
Immunohistochemical scoring
The TLR-4 and NF-κB protein expression levels were assessed with a microscopic examination of the stained tissue sections. The TLR-4 expression level included two aspects: the percentage of positive tumor cells (positive cytoplasm as well as cytomembrane) and the intensity of positive staining. The percentage of positive stained tumor cells in the marked regions was evaluated using the following standards: negative (score 0), 0-25% (score 1), 26-50% (score 2), 51-75% (score 3), and 76-100% (score 4). Positive staining intensity was assessed via the following standards: negative (score 0), borderline (score 1), weak (score 2), moderate (score 3), and strong (score 4). The NF-κB protein expression levels were estimated by determining whether there was positive staining in the nuclei. A positively-stained nuclei was taken to indicate positive NF-κB expression levels.
The overall IHC score was a product of the percentage of positive tumor cells and the staining intensity score, which ranged from 0 to 16. Both parameters were scored and determined by two independent pathologists (Xiang Li and Haiying Li) who were blinded to the samples. Any discrepancies in the scores were discussed between the two pathologists until a final consensus was reached.
Statistical analysis
SPSS software version 20.0 was used to analyze all the statistical data. A Chi-square test was used to assess the relationships between the TLR-4/NF-κB protein expressions and the clinicopathological characteristics. Duration of lifespan was defined as the duration between confirmed diagnosis and death due to any cause. Overall survival rates were determined by Kaplan-Meier curves. In all the statistical analyses, a P value < 0.05 was considered statistically significant, and all P values were two-sided.
Results
Clinical characteristics of ESCC patients
A total of 104 ESCC patients were included in this study and comprised 32 cases of early-stage ESCC and 72 cases of advanced ESCC. The gender ratio of male to female was 3.52:1. the median age was 61 years (ranging from 45 to 80 years). The median tumor size (maximum diameter) was 3.3 cm (ranging from 0.7 to 6.5 cm). There were 49 cases of histological grade I and II tumors (47.1%), and there were 55 cases of histological grade III tumors (52.9%). The patients were split into two groups based on the depth of tumor invasion: early-Stage ESCC (n = 31, 29.8%) and advanced ESCC (n = 73, 70.2%). The number of patients with lymphatic metastasis was 40 (39.4%) and without lymphatic metastasis was 64 (60.6%). All the patients were followed up from between 6 to 83 months after being diagnosed with ESCC.
TLR-4 and NF-κB expressions differ between noncancerous tissues and ESCC tissues
The IHC staining analyses of the tumor samples are shown in Figure 1. The TLR-4 protein was found to exist primarily in the cytoplasm and cytomembrane, while the NF-κB protein was primarily found in the nucleus and/or cytoplasm. Brown cytoplasm/cytomembrane immunoreactivity for the TLR-4 and brown nucleus immunoreactivity for the NF-κB was taken to indicate positive staining. The median staining score of TLR-4 expression in the tissue microarray was 8. Low TLR-4 protein expressions were determined in samples that had overall staining scores of 0-8, while those with positive staining scores of 9-16 were determined to have high expression for TLR-4 proteins. NF-κB levels were defined as either positively or negatively expressed based on wither the nucleus was colored.
Figure 1.
The location of NF-κB and TLR-4 in ESCC tissues (n = 104). The expression of TLR-4 was found to be mainly located in the cytoplasm/cytomembrane and NF-κB was found to be mainly located in the cytoplasm/nucleus and nucleus, respectively.
In this study, the expressions of TLR-4 and NF-κB in five different tissue subtypes were compared. As shown in Table 1 and Figure 2, positive NF-κB expressions were identified in 30 out of 72 TMA advanced ESCC tissues, 24 out of 32 TMA early-stage ESCC tissues, 20 out of 26 HGIN tissues, 32 out of 37 LGIN tissues, and 31 out of 34 normal esophageal squamous epithelial tissues (41.7%, 75.0%, 76.9%, 86.5% and 91.2%, respectively). High expressions of TLR-4 were detected in 28 out of 72 TMA advanced ESCC tissues, 24 out of 32 TMA early-stage ESCC tissues, 11 out of 26 HGIN tissues, 7 out of 37 LGIN tissues and 1 out of 34 normal esophageal squamous epithelial tissues (38.9%, 75.0%, 42.3%, 18.9% and 2.9%, respectively). Through the above analysis of the data, we conclude that positive expression rates of NF-κB in normal esophageal mucosa, low grade intraepithelial neoplasia (LGIN), HGIN and early-stage ESCC were progressively lower. On the other hand, TLR-4 expression in normal esophageal mucosa, low grade intraepithelial neoplasia (LGIN), HGIN and early-stage ESCC were noted to be progressively increasing. Conversely, the expression rates of both TLR-4 and NF-κB in advanced ESCC tissues were attenuated (Table 1; Figure 2).
Table 1.
Comparison of NF-κB and TLR-4 protein expression in ESCC tissues, Early-Stage ESCC tissues, HGIN tissues, LGIN tissues and normal tissues
| Group | Positive expression of NF-κB | Negative expression of NF-κB | High expression of TLR-4 | Low expression of TLR-4 |
|---|---|---|---|---|
| Advanced ESCC | 30/72 (41.7%) | 42/72 (58.3%) | 28/72 (38.9%) | 44/72 (61.1%) |
| Early-stage ESCC | 24/32 (75.0%) | 8/32 (25.0%) | 24/32 (75.0%) | 8/32 (25.0%) |
| HGIN** | 20/26 (76.9%) | 6/26 (23.1%) | 11/26 (42.3%) | 15/26 (57.7%) |
| LGIN*** | 32/37 (86.5%) | 5/37 (13.5%) | 7/37 (18.9%) | 30/37 (81.1%) |
| Normal | 31/34 (91.2%) | 3/34 (8.8%) | 1/34 (2.9%) | 33/34 (97.1%) |
| χ2 | 65.34 | 42.97 | ||
| P value* | 0.000 | 0.000 | ||
Chi square test;
High grade intraepithelial neoplasia;
Low grade intraepithelial neoplasia.
Figure 2.
Expression of TLR-4 and NF-κB in five different organizations. Positive expression rates of NF-κB in normal esophageal mucosa, low grade intraepithelial neoplasia (LGIN), HGIN and early-stage ESCC were lower and lower. And high expression rates of TLR-4 in normal esophageal mucosa, low grade intraepithelial neoplasia (LGIN), HGIN and early-stage ESCC were higher and higher. However, positive expression rates of TLR-4 and NF-κB in advanced ESCC tissue were depressed. All pictures were taken under 10× magnification.
Association of TLR-4/NF-κB expressions and clinicopathological variables of ESCC patients
The associations among TLR-4, NF-κB expression in ESCC including early-stage and advanced ESCC, with their respective clinicopathological characteristics, were determined and tabulated in Table 2. Positive NF-κB expressions correlated significantly with the female gender, histological grade I+II, early-stage ESCC and the absence of lymph node metastasis (P = 0.002, 0.010, 0.002, 0.020, respectively). On the other hand, elevated TLR-4 expressions correlated significantly with the female gender, histological grade I+II, and early-stage ESCC (P = 0.002, 0.003, 0.001). The rest of the clinicopathological variables of the ESCC patients had no significant differences with TLR-4/NF-κB expression (P > 0.05). Moreover, TLR-4 expressions were found to correlate significantly with NF-κB expressions (P = 0.000).
Table 2.
Associations between clinicopathological variables of ESCC patients and expressions of NF-κB and TLR-4 protein
| Variable | N | NF-κB expression | P value* | TLR-4 expression | P value* | ||
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| Positive | Negative | High | Low | ||||
| Gender | 0.002 | 0.002 | |||||
| Male | 81 | 35 (43.2%) | 46 (56.8%) | 34 (42.0%) | 47 (58.0%) | ||
| Female | 23 | 19 (82.6%) | 4 (17.4%) | 18 (78.3%) | 5 (21.7%) | ||
| Age | 0.449 | 0.238 | |||||
| ≤ 61 years** | 56 | 31 (55.4%) | 25 (44.6%) | 31 (55.4%) | 25 (44.6%) | ||
| > 61 years | 48 | 23 (47.9%) | 25 (52.1%) | 21 (43.8%) | 23 (56.3%) | ||
| Tumor size | 0.441 | 0.432 | |||||
| ≤ 3.3 cm*** | 54 | 30 (55.6%) | 24 (44.4%) | 29 (53.7%) | 25 (46.3%) | ||
| > 3.3 cm | 50 | 24 (48.0%) | 26 (52.0%) | 23 (46.0%) | 27 (54.0%) | ||
| Site of tumor | 0.891 | 0.896 | |||||
| Upper | 5 | 3 (5.6%) | 2 (4.0%) | 3 (5.8%) | 2 (3.8%) | ||
| Middle | 50 | 25 (50.0%) | 25 (46.3%) | 25 (48.1%) | 25 (48.1%) | ||
| Lower | 49 | 26 (48.1%) | 23 (46.0%) | 24 (46.2%) | 25 (48.1%) | ||
| Histological Grade | 0.010 | 0.003 | |||||
| Grade I+II**** | 49 | 32 (59.3%) | 17 (34.0%) | 32 (61.4%) | 17 (32.7%) | ||
| Grade III | 55 | 22 (40.7%) | 33 (66.0%) | 20 (38.5%) | 35 (67.3%) | ||
| Group | 0.002 | 0.001 | |||||
| Early-stage ESCC | 32 | 24 (44.4%) | 8 (16.0%) | 24 (46.2%) | 8 (15.4%) | ||
| Advanced ESCC | 72 | 30 (55.6%) | 42 (84.0%) | 28 (53.8%) | 44 (84.6%) | ||
| Lymph node metastasis | 0.020 | 0.107 | |||||
| Yes | 40 | 15 (27.8%) | 25 (50.0%) | 16 (30.8%) | 24 (46.2%) | ||
| No | 64 | 39 (72.2%) | 25 (50.0%) | 36 (69.2%) | 28 (53.8%) | ||
| TLR-4 expression | 0.000 | ||||||
| High | 52 | 50 (96.2%) | 2 (3.8%) | ||||
| Low | 52 | 4 (7.2%) | 48 (92.2%) | ||||
Chi square test;
61 years was the median age;
3.3 cm was the median tumor size;
Number of histological grade I was too less.
The relationship between clinicopathological characteristics, TLR-4 and NF-κB expressions, and overall survival by univariate analysis
We applied Kaplan-Meier survival curves in univariate survival analyses, with a further statistical analysis performed using log-rank tests (Table 3). Kaplan-Meier analysis revealed features such as gender, tumor size, histological grade, ESCC group and lymph node metastasis (P = 0.021, 0.007, 0.027, 0.000, 0.015) (Figure 3) had a significant impact on overall survival rate, highlighting these features as valuable prognostic factors in ESCC patients. Additionally, we discovered that patients who had both attenuated TLR-4 expressions and negative NF-κB expressions had much shorter overall survival durations in contrast to those who had elevated TLR-4 expressions and positive NF-κB expressions (Table 3; Figure 4). The average overall survival time was 63.0 months and 62.0 months in patients with high expression of TLR-4 and a positive expression of NF-κB compared with 44.2 months and 44.3 months in patients with low expression of TLR-4 and a negative expression of NF-κB, respectively (P = 0.000, 0.000).
Table 3.
Correlations among clinicopathological features, expressions of NF-κB and TLR-4 proteins and survival time of ESCC patients
| Patients features | N | Mean ± SE (month) | Median ± SE (month) | P value* |
|---|---|---|---|---|
| Gender | 0.021 | |||
| Male | 81 | 50.7±2.3 | 50.0±2.0 | |
| Female | 23 | 61.9±4.5 | 63.0±5.9 | |
| Age | 0.772 | |||
| ≤ 61 years | 56 | 52.5±3.0 | 50.0±3.2 | |
| > 61 years | 48 | 54.2±2.9 | 55.0±2.0 | |
| Tumor size | 0.007 | |||
| ≤ 3.3 cm | 54 | 59.5±2.9 | 58.0±5.2 | |
| > 3.3 cm | 50 | 46.4±2.6 | 49.0±3.7 | |
| Site of tumor | 0.899 | |||
| Upper | 5 | 53.4±10.0 | 53.0±22.2 | |
| Middle | 50 | 54.9±2.9 | 55.0±2.7 | |
| Lower | 49 | 50.4±3.1 | 49.0±4.2 | |
| Histological grade | 0.027 | |||
| Grade I+II | 49 | 58.9±3.3 | 58.0±5.8 | |
| Grade III | 55 | 49.0±2.6 | 49.0±3.8 | |
| Group | 0.000 | |||
| Early ESCC | 32 | 78.7±2.3 | - | |
| Advanced ESCC | 72 | 44.9±2.0 | 45.0±1.8 | |
| Lymph node metastasis | 0.015 | |||
| Yes | 40 | 46.7±3.1 | 48.0±3.7 | |
| No | 64 | 58.3±2.8 | 56.0±6.8 | |
| TLR-4 protein expression | 0.000 | |||
| Low | 52 | 44.2±2.8 | 44.0±3.8 | |
| High | 52 | 63.0±2.6 | 63.0±3.6 | |
| NF-κB protein expression | 0.000 | |||
| Positive | 54 | 62.0±2.6 | 63.0±3.9 | |
| Negative | 50 | 44.3±2.9 | 41.0±2.8 |
Univariate survival analysis.
Figure 3.
The relationship between gender, tumor size, histological grade, group, lymph node metastasis, ESCC group and expressions of NF-κB, TLR-4 and survival rates by Kaplan-Meier survival curve (n = 104). We found that gender, tumor size, histological grade, ESCC group and lymph node metastasis both had significant impacts on overall survival (P = 0.021, 0.007, 0.027, 0.000, 0.015).
Figure 4.
The relationship between expression of TLR-4/NF-κB and survival rates by Kaplan-Meier survival curve (n = 104). The overall survival times of patients with low expression of TLR-4 and negative expression of NF-κB were significantly shortened compared to patients with a high expression of TLR-4 and a positive expression of NF-κB (P = 0.000, 0.000).
The above results showed us that raised TLR-4 expressions and a positive expression of NF-κB were both independent prognostic factors for overall survival. Additionally, clinicopathological parameters such as gender, tumor size, histological grade, and lymph node metastasis were identified as independent prognostic factors for overall survival of ESCC patients.
Discussion
Esophageal cancer is one of the most frequently occurring, malignant alimentary canal cancers, particularly in Asian populations [15]. There has been a dramatic rise in the incidence and mortality of esophageal cancer [16,17]. ESCC is the predominant subtype of esophageal cancer in China, occurring at a high incidence (13 per 100,000) [18] and conferring an overall dismal prognosis which can be attributed to the scarcity of early and efficacious clinical detection methods in the past [19]. To this day, patients are faced with a low five-year survival rate [20] and an equally poor prognosis [21,22] despite advances in early detection, surgical techniques and chemoradiotherapy. The prognostication of ESCC clinical prognosis is highly dependent on conventional pathologic components such as tumor size and grade, as well as the presence of lymph node metastasis [23,24]. However, the relationship between inflammation and cancer in ESCC is still unknown.
Cancer initiation and progression have been shown to be associated with infection and inflammation. Examples include colon cancer and chronic inflammatory bowel diseases, Gastric cancer and chronic inflammation mediated by Helicobacter pylori infection [25]. Recent studies revealed prostate cancer to be linked to prostatitis [26]. TLRs are crucial components of the innate immune system that function to defend the host against viral or bacterial invasion. Pathogen associated molecular patterns (PAMPs) as well as endogenous molecules both activate TLRs, triggering the inflammatory cascade. Continuous activation results in the secretion of a myriad of pro-inflammatory chemokines and cytokines. TLR-facilitated IRF and NF-κB activation are thought to be crucial elements linking cancer to inflammation [27].
In this study, we gathered different tissues of patients, including 31 cases of early-stage ESCC, 73 cases of advanced ESCC, 26 cases of HGIN tissues, 36 cases of LGIN tissues, and 33 cases of normal esophageal mucosa tissues. To further explore the relationship between inflammation and ESCC development, we analyzed TLR-4 and NF-κB expressions in each collected sample. We discovered a progressively less positive NF-κB expression beginning from normal esophageal mucosa, low grade intraepithelial neoplasia (LGIN), HGIN to early-stage ESCC. Conversely, TLR-4 expressions were found to be progressively higher beginning with normal esophageal mucosa, low grade intraepithelial neoplasia (LGIN), HGIN, to early-stage ESCC. On the other hand, positive expression rates of TLR-4 and NF-κB in advanced ESCC tissues were attenuated. Moreover, high expression of TLR-4 with positive NF-κB expressions both significantly correlated with histological grade I+II. In addition, we discovered a positive correlation between NF-κB nuclear expression and TLR-4 expressions. Our results indicated that the protein of TLR-4 facilitated the transition from normal esophageal mucosa to early-stage ESCC. However, it appeared to not play a role in the progression from early-stage ESCC to advanced ESCC. Interestingly, NF-κB protein expressions remained low in all samples ranging from normal esophageal mucosa to advanced ESCC. Therefore, we hypothesize that TLR-4 and NF-κB might play different roles in the development of ESCC. It is possible that the TLR-4 protein could facilitate NF-κB protein translocation from the cytoplasm to the nucleus.
We also analyzed the correlation between TLR-4 and NF-κB expressions and ESCC patients’ overall survival time (n = 102). Our results revealed that patients with low TLR-4 and negative NF-κB expressions had markedly shorter overall survival times in contrast to those who had high TLR-4 expressions and positive NF-κB expressions. These results indicated that the TLR-4 and NF-κB expressions were independent prognosis indexes of ESCC, and they might have the potential to regulate ESCC aggressiveness. Also, we analyzed the relationship between clinical variables and ESCC patients’ overall survival times. Some prognostically significant clinical variables such as tumor size, gender, histological grade, ESCC group and lymph node metastasis were also discovered to potentially be able to predict the outcome of ESCC patients.
In summary, TLR-4 and NF-κB proteins played different roles in the occurrence and development of ESCC. Overall, high expression of TLR-4 or positive NF-κB expressions alone were associated with longer survival times. TLR-4 and NF-κB may function as potential diagnostic and therapeutic targets of ESCC.
Acknowledgements
This work was sponsored by the First Technology Projects in 2016 in Wuwei, Gansu Province, no. WW160109.
Disclosure of conflict of interest
None.
References
- 1.Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424. doi: 10.3322/caac.21492. [DOI] [PubMed] [Google Scholar]
- 2.Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66:115–32. doi: 10.3322/caac.21338. [DOI] [PubMed] [Google Scholar]
- 3.Wang C, Pu W, Zhao D, Zhou Y, Lu T, Chen S, He Z, Feng X, Wang Y, Li C, Li S, Jin L, Guo S, Wang J, Wang M. Identification of hyper-methylated tumor suppressor genes-based diagnostic panel for esophageal squamous cell carcinoma (ESCC) in a Chinese Han Population. Front Genet. 2018;9:356. doi: 10.3389/fgene.2018.00356. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol. 2002;2:725–34. doi: 10.1038/nri910. [DOI] [PubMed] [Google Scholar]
- 5.Iliopoulos D, Hirsch HA, Struhl K. An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell. 2009;139:693–706. doi: 10.1016/j.cell.2009.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Terzic J, Grivennikov S, Karin E, Karin M. Inflammation and colon cancer. Gastroenterology. 2010;138:2101–14. e5. doi: 10.1053/j.gastro.2010.01.058. [DOI] [PubMed] [Google Scholar]
- 7.Xia Y, Shen S, Verma IM. NF-kappaB, an active player in human cancers. Cancer Immunol Res. 2014;2:823–30. doi: 10.1158/2326-6066.CIR-14-0112. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol. 2001;2:675–80. doi: 10.1038/90609. [DOI] [PubMed] [Google Scholar]
- 9.Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol. 2001;1:135–45. doi: 10.1038/35100529. [DOI] [PubMed] [Google Scholar]
- 10.Nishimura M, Naito S. Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biol Pharm Bull. 2005;28:886–92. doi: 10.1248/bpb.28.886. [DOI] [PubMed] [Google Scholar]
- 11.Chen CY, Kao CL, Liu CM. The cancer prevention, anti-inflammatory and anti-oxidation of bioactive phytochemicals targeting the TLR4 signaling pathway. Int J Mol Sci. 2018;19 doi: 10.3390/ijms19092729. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol. 2014;5:461. doi: 10.3389/fimmu.2014.00461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Ou T, Lilly M, Jiang W. The pathologic role of toll-like receptor 4 in prostate cancer. Front Immunol. 2018;9:1188. doi: 10.3389/fimmu.2018.01188. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kleihues P, Sobin LH. World Health Organization classification of tumors. Cancer. 2000;88:2887. doi: 10.1002/1097-0142(20000615)88:12<2887::aid-cncr32>3.0.co;2-f. [DOI] [PubMed] [Google Scholar]
- 15.Gao H, Wang L, Cui S, Wang M. Combination of meta-analysis and graph clustering to identify prognostic markers of ESCC. Genet Mol Biol. 2012;35:530–7. doi: 10.1590/S1415-47572012000300021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Lam TK, Freedman ND, Fan JH, Qiao YL, Dawsey SM, Taylor PR, Abnet CC. Prediagnostic plasma vitamin C and risk of gastric adenocarcinoma and esophageal squamous cell carcinoma in a Chinese population. Am J Clin Nutr. 2013;98:1289–97. doi: 10.3945/ajcn.113.061267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74–108. doi: 10.3322/canjclin.55.2.74. [DOI] [PubMed] [Google Scholar]
- 18.Chen JW, Xie JD, Ling YH, Li P, Yan SM, Xi SY, Luo RZ, Yun JP, Xie D, Cai MY. The prognostic effect of perineural invasion in esophageal squamous cell carcinoma. BMC Cancer. 2014;14:313. doi: 10.1186/1471-2407-14-313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Kato K, Hida Y, Miyamoto M, Hashida H, Shinohara T, Itoh T, Okushiba S, Kondo S, Katoh H. Overexpression of caveolin-1 in esophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Cancer. 2002;94:929–933. [PubMed] [Google Scholar]
- 20.Parkin DM, Bray FI, Devesa SS. Cancer burden in the year 2000. The global picture. Eur J Cancer. 2001;37(Suppl 8):S4–66. doi: 10.1016/s0959-8049(01)00267-2. [DOI] [PubMed] [Google Scholar]
- 21.Koshy M, Esiashvilli N, Landry JC, Thomas CR Jr, Matthews RH. Multiple management modalities in esophageal cancer: combined modality management approaches. Oncologist. 2004;9:147–159. doi: 10.1634/theoncologist.9-2-147. [DOI] [PubMed] [Google Scholar]
- 22.Lin DC, Du XL, Wang MR. Protein alterations in ESCC and clinical implications: a review. Dis Esophagus. 2009;22:9–20. doi: 10.1111/j.1442-2050.2008.00845.x. [DOI] [PubMed] [Google Scholar]
- 23.Ashida A, Boku N, Aoyagi K, Sato H, Tsubosa Y, Minashi K, Muto M, Ohtsu A, Ochiai A, Yoshida T. Expression profiling of esophageal squamous cell carcinoma patients treated with definitive chemoradiotherapy: clinical implications. Int J Oncol. 2006;28:1345–1352. [PubMed] [Google Scholar]
- 24.Balkwill F, Coussens LM. Cancer: an inflammatory link. Nature. 2004;431:405–6. doi: 10.1038/431405a. [DOI] [PubMed] [Google Scholar]
- 25.Ernst PB, Takaishi H, Crowe SE. Helicobacter pylori infection as a model for gastrointestinal immunity and chronic inflammatory diseases. Dig Dis. 2001;19:104–11. doi: 10.1159/000050663. [DOI] [PubMed] [Google Scholar]
- 26.Sfanos KS, De Marzo AM. Prostate cancer and inflammation: the evidence. Histopathology. 2012;60:199–215. doi: 10.1111/j.1365-2559.2011.04033.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Hallam S, Escorcio-Correia M, Soper R, Schultheiss A, Hagemann T. Activated macrophages in the tumour microenvironment-dancing to the tune of TLR and NF-kappaB. J Pathol. 2009;219:143–52. doi: 10.1002/path.2602. [DOI] [PMC free article] [PubMed] [Google Scholar]