You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Impact of mutations in Toll-like receptor pathway genes on esophageal carcinogenesis}
Article:
Abstract:
Esophageal adenocarcinoma( EAC) develops in an inflammatory microenvironment with reduced microbial diversity, but mechanisms for these influences remain poorly characterized. We hypothesized that mutations targeting the Toll-like receptor( TLR) pathway could disrupt innate immune signaling and promote a microenvironment that favors tumorigenesis. Through interrogating whole genome sequencing data from 171 EAC patients, we showed that non-synonymous mutations collectively affect the TLR pathway in 25/171( 14. 6%, PathScan p = 8. 7x10-5) tumors. TLR mutant cases were associated with more proximal tumors and metastatic disease, indicating possible clinical significance of these mutations. Only rare mutations were identified in adjacent Barrett’s esophagus samples. We validated our findings in an external EAC dataset with non-synonymous TLR pathway mutations in 33/149( 22. 1%, PathScan p = 0. 05) tumors, and in other solid tumor types exposed to microbiomes in the COSMIC database( 10, 318 samples), including uterine endometrioid carcinoma( 188/320, 58. 8%), cutaneous melanoma( 377/988, 38. 2%), colorectal adenocarcinoma( 402/1519, 26. 5%), and stomach adenocarcinoma( 151/579, 26. 1%). TLR4 was the most frequently mutated gene with eleven mutations in 10/171( 5. 8%) of EAC tumors. The TLR4 mutants E439G, S570I, F703C and R787H were confirmed to have impaired reactivity to bacterial lipopolysaccharide with marked reductions in signaling by luciferase reporter assays. Overall, our findings show that TLR pathway genes are recurrently mutated in EAC, and TLR4 mutations have decreased responsiveness to bacterial lipopolysaccharide and may play a role in disease pathogenesis in a subset of patients.
Introduction:
Esophageal adenocarcinoma( EAC) is increasing in incidence and has poor survival outcomes. The main risk factor for EAC is Barrett’s esophagus, a pre-malignant glandular epithelium that develops in the setting of gastro-esophageal reflux disease. Over time exposure to refluxed acid and bile in the lower esophagus leads to chronic inflammation, increased cell turnover, production of reactive oxygen species and DNA damage[1, 2]. The combination of exposure to noxious substances and defects in DNA damage repair enable cancer cells to accumulate mutations, evidenced by characteristic mutational signatures[3, 4]. Next generation sequencing studies have shown that accumulation of somatic mutations occurs along the metaplasia–dysplasia–carcinoma sequence in EAC[5, 6] and potentially impair important cell functions. However, few genes are mutated in greater than 10% of cases, underlining the heterogeneous nature of point mutations and small indels in this type of cancer. As a result it is difficult to pinpoint the genes that are relevant to tumorigenesis using traditional approaches based on the frequency of mutations in a single gene. It has been proposed that groups of genes involved in similar processes or pathways could have a cumulative effect. An example is the SWI/SNF nucleosome remodeling complex( ARID1A, SMARCA4 and ARID2), for which gene members are mutated collectively in 20% of EAC tumors[6]. Computational tools such as PathScan[7] have been developed to identify cellular pathways that are targeted by somatic mutations above the background mutation rate. Epidemiologic evidence has linked the rising incidence of EAC with the eradication of Helicobacter pylori[8, 9], and studies have suggested that the esophageal microbiota are altered in Barrett’s esophagus[10–12] and EAC with decreased microbial diversity[13]. The Toll-like receptor( TLR) signaling pathway is a key component of the innate immune system and one of the main ways in which tumor cells interact with the microbiota. TLRs are pattern recognition receptors that bind unique molecular components of the microbiota and generate a pro-inflammatory innate immune reaction through nuclear factor kappa B( NF-κB)[14]. TLR signaling exerts an effect on epithelial cell function in the gastrointestinal tract, including stimulating repair of damaged enterocytes( lipopolysaccharide), enhancing cell proliferation, and triggering secretion of antimicrobial peptides( lipopolysaccharide and flagellin)[15]. Alongside TLRs, inflammasomes also contribute to inflammation through recognition of pathogen-associated molecular patterns. NLRP6 is a member of the NOD-like receptor family that plays a role in regulating inflammation and epithelial cell repair in the intestine and has been implicated in colorectal carcinogenesis[16, 17]. Genome-wide association studies[18, 19] and next-generation sequencing studies[5, 6] have identified TLR4 gene mutations in solid tumors including EAC. We hypothesized that somatic mutations may collectively target the TLR signaling pathway in EAC and alter inflammatory signaling. We aimed to interrogate TLR pathway mutations and expression in a cohort of EAC and Barrett’s esophagus patients with clinical outcome data. We then investigated whether TLR4 mutations in EAC affect downstream inflammatory signaling using an in vitro model system. Finally, we aimed to determine the broader relevance of TLR pathway mutations in other cancers using TCGA data and the COSMIC database.
Discussion:
Our results suggest that somatic mutations may collectively target the TLR signaling pathway in EAC( 25% of cases) and other solid tumor types. TLR mutant cases were associated with more proximal tumors and metastatic disease, indicating possible clinical significance of these mutations. TLR4 was the most frequently mutated TLR gene in 5% of cases in the combined EAC cohorts. Only two TLR pathway mutations( TLR9 and TRAF6) were detected in the adjacent Barrett’s samples in the ICGC cohort. TLR4 mutations have been previously reported in high grade dysplasia[5], which implies that this mutation may be acquired later during disease progression. Although TLR4 is mutated in less than ten percent of EAC cases, it is one of the top twenty most frequently mutated genes in both the ICGC and TCGA cohorts. The most frequently mutated gene in this disease is TP53, which is mutated in approximately 70% of tumors[6]. A limited number of candidate driver genes have been identified that are mutated in greater than ten percent of EAC tumors, including CDKN2A, SMAD4, ARID1A, MYO18B and DOCK2[5, 6]. In addition to EAC, TLR pathway mutations were frequently observed in solid tumors arising in body sites exposed to microbiota[21] including the oral and gastrointestinal tract( colorectal adenocarcinoma, stomach adenocarcinoma and head and neck squamous cell carcinoma), skin( melanoma), urogenital tract( endometrial carcinoma) and respiratory tract( lung adenocarcinoma and squamous cell carcinoma). Regrettably, it was not feasible to analyze our whole genome sequencing data using pathogen discovery software such as PathSeq[25], because the concentration of microbial DNA is so low relative to human DNA in samples derived from esophageal tumor tissue and the results were unreliable with high levels of contaminants. This is a major limitation of analyzing low microbial biomass tissue samples with a WGS approach. Our recent work examining the microbiome in esophageal tumors and Barrett’s tissue samples used 16S rRNA gene amplicon sequencing and a microbial extraction protocol with rigorous reagent controls to maximize the microbial DNA yield and account for contaminants[13]. However, only one of the tumors from that study had a TLR4 mutation so it was not possible to further correlate the findings with our current study, and additional research is needed to investigate the link between TLR mutations and the microbiome in EAC. Seven TLR4 mutants in our study showed decreased signaling in response to weak agonists, and four were hypo-responsive to strong agonists. This suggests that the mutations differentially affected signaling with weak versus strong agonists, which may be relevant to the different microbial antigens present in the tumor environment. The model system was also a contributing factor; for instance E439G was hyporesponsive to LPS in HEK293 cells but not EAC cell lines. In EAC cell lines, addition of the second mutation F703C was required to significantly reduce TLR4 signaling activity. Further, loss of heterozygosity events overlapped with four TLR4 mutations, R787H, E603D, T193K, and L498V. Of these, R787H showed decreased signaling in response to strong and weak agonists, L498V showed decreased signaling in response to weak agonist only, and E603D and T193K showed no significant change. It is conceivable that loss of heterozygosity could potentially compound the effect of TLR4 missense mutations and further decrease signaling. A possible mechanism is that defective TLR4 signaling may negatively impact epithelial cell repair, which is in part dependent on TLR4 stimulation, and potentially enable microbes to breach the epithelial barrier in the tumor microenvironment. Hold et al. found that the single nucleotide polymorphism c. 896A>G( p. D299G) was associated with an increased risk of gastric cancer in two patient populations[19], and cells with this mutation have been shown to be hypo-responsive to lipopolysaccharide[26, 27]. However, Hold et al. found no significant increased risk for EAC or esophageal squamous cell carcinoma with this germline polymorphism, and the D299G polymorphism was not identified in either of the ICGC or TCGA cohorts. The relationship between TLR4 signaling and tumorigenesis is complex and involves both innate and adaptive immunity, with evidence showing that TLR4 signaling can enhance or suppress cancer development, depending on the model system. For example, in the setting of chemically induced colitis TLR4 deficient mice were protected from colon carcinogenesis[28], while villin-TLR4 mice overexpressing TLR4 in the intestinal epithelium were highly susceptible to cancer when treated with dextran sodium sulphate and azoxymethane[29]. Conversely, overexpressing TLR4 using the CD4-TLR4 transgene in the intestinal epithelium of APCMin/+ mice that are genetically susceptible to colon carcinoma reduced tumorigenesis by increasing apoptosis[30]. Additionally, analysis of TCGA expression data suggested that NLRP6 is upregulated in TLR mutant tumors, which may reflect cross-talk between the TLR pathway and NOD-like receptor signaling pathway. NLRP6 regulates inflammasome-dependent innate immune signaling and has been shown to inhibit TLR2 and TLR4-dependent activation of NF-κB and MAP-kinase signaling pathways[31]. Further understanding of how altered TLR signaling may contribute to the inflammatory tumor microenvironment in Barrett’s carcinogenesis could be helpful in cancer prevention strategies.
Esophageal adenocarcinoma ( EAC ) is a deadly human cancer that develops in the lower esophagus , which is exposed to reflux of acidic stomach contents and has reduced microbial diversity . Next generation sequencing studies have shown that accumulation of somatic mutations occurs along the metaplasia–dysplasia–carcinoma sequence in EAC , and it has been proposed that groups of genes involved in similar processes or pathways could have a cumulative effect . The Toll-like receptor ( TLR ) signalling pathway is critical for host-microbe interaction , and therefore mutations in these pathways could be important in the pathogenesis of EAC . Here we show that TLR pathway genes are recurrently mutated in 15% of EAC tumours and other solid tumour types exposed to microbial communities , including uterine endometrioid carcinoma ( 59% ) , cutaneous melanoma ( 38% ) , colorectal adenocarcinoma ( 27% ) , and stomach adenocarcinoma ( 26% ) . TLR mutant cases were associated with more proximal tumours and metastatic disease , indicating possible clinical significance of these mutations . A better understanding of how altered TLR signalling contributes to the inflammatory tumour microenvironment in EAC could help inform cancer prevention strategies .