Study On The Role And Clinical Significance Of NLRP3 Inflammasome In Breast Cancer

BackgroundBreast cancer is the most common malignancy,ranking the first malignancy in the incidence of cancer in women.It was reported that breast cancer alone accounted for 15%of all of the new cancers in women.Although the molecular techniques and treatment for breast cancer has been improved significantly in recent years,its mortality rate has not significantly decreased.Inflammasomes are intracellular multiprotein complexes,which play major roles in innate immunity by inducing inflammation in response to pathogens and danger signals.Up to now,NLRP3(NOD-like receptor family,pyrin domain containing protein 3)inflammasome is the most clarified one.After the assembly of NLRP3,ASC(apoptosis associated speck-like protein),and pro-caspase-1,more pro-caspase-1 is recruited and then self-activated.The later cuts pro-IL-1? and pro-IL-18 into active IL-1?(interleukin 1 beta,IL-1?)and IL-18(interleukin 18,IL-18).So the NLRP3 inflammasome is formed to promote the release of inflammatory cytokines resulting in inflammation.In recent years,many studies have focused on the role of NLRP3 inflammasome in innate immunity.However,recent studies indicated that inflammasomes had critical but contrasting functions during carcinogenesis,and played divergent roles in different types of cancers.Besides innate immune cells,the expression of NLRP3 inflammasome was also found on epithelial cells.Breast cancer is one of the most typical epithelia originated malignancy.However,the role of NLRP3 inflammasomes in breast cancer has not been reported before.In order to figure out whether the NLRP3 inflammasome functions in breast cancer progression,surgical excised breast cancer tissues from 171 metastatic breast cancer patients were collected in our study.Immunohistochemistry was applied to investigate the expression of NLRP3 inflammasome and its correlation with clinical parameters.In this study,we tried to figure out the association between NLRP3 inflammasomes and breast cancer progression,and to provide clue for novel therapeutic strategy against breast cancer.Materials and methods1.Patients and tissue samples171 breast ductal cancer patients underwent curative resection were recruited in this study.Clinical stages were classified according to the International Union against Cancer TNM Classification System.For the patients with metastasis,the tissues of both primary tumor and metastatic loci were taken from these patients.2.Research methods2.1 Preparation of paraffin sectionFresh breast cancer tissues were collected from the patients underwent surgical operation of breast cancer.The tissues were dehydrated,transpared,paraffin-embedded,thus the paraffin sections were constructed.Both the primary tumor foci and their corresponding metastatic lesions were included for patients with axillary lymph node metastases.2.2 ImmunohistochemistryImmunohistochemistry assay for NLRP3 inflammasome components including NLRP3,ASC,caspase-1,IL-1? were performed on the paraffin sections with specific antibodies.PR(progesterone receptor),ER(estrogen receptor)and Her-2(human epidermal growth factor receptor-2)were often used to evaluate the status of breast cancer.Immunohistochemistry staining of them was also performed.2.3 Evaluation of immunohistochemistry stainingSix non-overlapping fields were examined in a systematic random sampling pattern for each section.All sections were evaluated independently by two pathologists in double-blind conditions.Each section was observed twice for repeatability.The intensity of staining was graded and recorded with 0,1,2,3 according to the intensity of staining.This initial score was further classified as low expression(0?1)or high expression(2?3).2.4 Statistical analysis Data were analyzed with SPSS 16.0.Fisher's exact test was applied to compare categorical variables.Spearman's rank correlation test was used to evaluate correlations between variables.A two-tailed P value was used in all analyses and P<0.05 was considered statistically significant.Results1.Expression of NLRP3 inflammasome components showed positive staining and were positively correlated with one another Positive staining for NLRP3,ASC,caspase-1,and IL-1? was observed in breast cancer tissues.NLRP3 showed homogeneous cytoplasm expression in breast cancer cells.Positive expression rates of NLRP3,ASC,caspase-1 and IL-1? in primary breast cancer tissues were 51.6%,68.2%,73.0%and 53.5%,respectively.We further analyzed the expression level of NLRP3 inflammasome components.The staining of NLRP3 inflammasome components showed positive correlations between NLRP3 and caspase-1(P=0.001).The significant positive correlation was also observed between NLRP3 and IL-1?(P<0.001),and between caspase-1 and IL-1?(P=0.037).2.Decreased expression of NLRP3 and IL-1? was significantly correlated with increased metastasis rate.We further analyzed the correlation between the expression level of NLRP3 inflammasome components in primary tumor and metastasis rate of breast cancer.The results showed that the group with decreased NLRP3 expression showed higher metastasis rate compared with the high expression groups,P=0.038.There was also significant difference of IL-1? expression between these two groups.The group with lower expression of NLRP inflammasome components showed higher metastasis rate,P=0.001.3.Expression level of NLRP3 inflammasome components was significantly down-regulated in the axillary lymph node compared with that in the non-metastatic primary tumors.We further compared the expression level of NLRP3 inflammasome components in breast cancer tissues between primary tumor and metastatic loci.The results showed that expression level of NLRP3,ASC and IL-1? in metastatic lesion was decreased significantly in the axillary lymph node metastatic tumors compared with that in the primary non-metastatic cancer tissues.The P value for NLRP3,ASC and IL-1? was 0.012,0.003 and 0.001,respectively.4.The expression level of NLRP3 inflammasome components in invasive ductal carcinoma decreased significantly compared with that in ductal carcinoma in situ.In the tissues taken from patients with invasive ductal carcinoma,the expression level of NLRP3 inflammasome components in the district of invasive ductal carcinoma and that in ductal carcinoma in situ were also compared.The results showed that the expression level of NLRP3,ASC,Caspase-1 and IL-1? in invasive ductal carcinoma decreased significantly compared with that in ductal carcinoma in situ in the same patient.Typically,there was one case with both of the invasive and non-invasive partin the same paraffin section,which showed different expression level of NLRP3,ASC,Caspase-1 and IL-1? in these two parts.5.Expression of NLRP3 inflammasome components was significantly correlated with PR expression in the breast cancer tissues.PR was commonly used indicator in the evaluation of breast cancer.We further analyzed the correlation of the expression level of NLRP3 inflammasome components and PR.The results showed that there was significant positive correlation between the expression level of NLRP3 and PR(P=0.002).The expression level of ASC showed significant positive correlation with that of PR(P=0.022).The expression level of IL-1? showed significant positive correlation with that of PR(P=0.048).6.The correlation of PR expression level and clinical pathological parameters.The expression level of PR was positively correlated with that of ER,P<0.001.The expression level of PR was negatively significantly correlated with that of Her-2(P<0.001)and pathological grade(P=0.026).These results showed that the data collected in this study were consistent with the general clinical characteristics of breast cancer.Conclusion1.NLRP3 inflammasome was expressed in cytoplasma of breast cancer tissues.NLRP3 inflammasome components were positively correlated with one another.This suggested that NLRP3 inflammasome components constituted a multi-protein NLRP3 inflammasome platform and cooperated to play a role together in breast cancer.2.Decreased expression of NLRP3 inflammasome components including NLRP3 and IL-1? were significantly negatively correlated with higher metastasis rate,which indicated that breast cancer cells with loss of NLRP3 inflammasome expression was prone to metastasis.Expression level of NLRP3 inflammasome components was significantly down-regulated in the axillary lymph node metastatic tumors compared with that in the non-metastatic primary tumors.Expression level of NLRP3 inflammasome components was decreased in invasive ductal carcinoma compared with that in ductal carcinoma in situ.These data indicated that decreased expression of NLRP3 inflammasome might promote the progression and metastasis of breast cancer.3.NLRP3 inflammasome was significantly positively correlated with PR expression in the axillary lymph node metastatic tumors.These data indicated that NLRP3 inflammasome might be regulated by PR,and played a positive and beneficial role in breast cancer.Innovations and significance1.This is the first research to detect the expressions of NLRP3 inflammasome in human breast cancer tissues by immunohistochemistry.The results showed that NLRP3 inflammasome was expressed in breast cancer tissues.Expression levels of NLRP3 inflammasome components were positively correlated with one another.This suggested that NLRP3 inflammasome components constituted a multi-protein NLRP3 inflammasome platform and cooperated to play together in breast cancer.2.This is the first study reporting loss of NLRP3 inflammasome components expression contributes to breast cancer progression,especially metastasis in human breast cancer cases.Expression of NLRP3 inflammasome components was significantly decreased in both the metastatic axillary lymph node and invasive ductal cancer.These data indicated that the loss of NLRP3 inflammasomes might promote the metastasis of breast cancer.3.For the first time,the correlation of NLRP3 inflammasome expression and PR expression was reported.PR expression was significantly positively correlated with NLRP3 inflammasome in the axillary lymph node metastatic tumors.These data indicated a novel regulatory mechanism of NLRP3 by PR and also showed that NLRP3 might play a positive and beneficial role in breast cancer.4.For the first time,human breast cancer tissues were collected for the study of the correlation between NLRP3 and the progression of breast cancer.It could reflect the pathology in human body more realistically than that in animal tests.More persuasive conclusion could be obtained.In conclusion,we analyzed the expression levels of NLRP3 inflammasomecomponents and its correlation with clinical parameters of breast cancer.Our data indicated that loss of NLRP3 inflammasome expression was correlated with more metastasis in breast cancer patients.This indicated that NLRP3 might become a novel biological marker for the prediction of breast cancer progression and metastasis.Our data showed that NLRP3 inflammasome played a protective role in breast cancer,which suggested that NLRP3 inflammasome might be a novel treatment target.This provided a novel.potential immuno-theraputic strategy for breast cancer by regulating NLRP3 inflammasome.BackgroundNLRP3(Nod-like receptor family,pyrin domains-containing protein 3,NLRP3)is the typical representative member of the NLR family,and it is also the most clarified one.NLRP3,sensing the danger signal,can recruit apoptosis associated speck-like protein(ASC)and cysteine aspirate protease 1 precursor(pro-caspase-1)to form a multi-protein complex----the NLRP3 inflammasome.Activated caspase-1 can cleave the precursor of IL-1? and IL-18 proteins into their mature form and promote their releasing.Caspase-1 and IL-1? are key factors causing a variety of immune inflammatory response.NLRP3 is mainly expressed on macrophages,epithelia and dendritic cells.NLRP3 protein is encoded by NLRP3 gene.Up to now,about 60 single nucleotide polymorphisms of NLRP3 gene were found.Some polymorphisms of the gene are associated with the activation of inflammasomes leading to the abnormal production of IL-1? and IL-I8,which can cause abnormal inflammatory response.Cancer is one of the major public health problems worldwide and is one of the leading causes of death in the United States.In China its morbidity and mortality have been increasing,making cancer the leading cause of death since 2010.Genetic background,environmental factors and autoimmunity and other factors are involved in carcinogenesis.Among all the factors genetic factors accounts for 60%of them.Epidemiological studies have shown that the incidence of cancer is closely related to genetic factors.The carcinogenesis of many cancers is associated with persistent infection,such as helicobacter pylori infection 'and gastric cancer,human papillomavirus infection and cervical cancer.Up to date,a lot of single nucleotide polymorphisms had been found to be associated with cancer susceptibility.Some studies showed that NLRP3 polymorphisms were related to carcinogenesis,but there were conflicting conclusions.In order to figure out the relationship between the NLRP3 single nucleotide polymorphisms and cancer susceptibility,we performed this meta-analysis.Objective1.To investigate the association of NLRP3 gene rs35829419 single nucleotide polymorphism with the susceptibility to cancer.2.To investigate the association of NLRP3 gene rs10754558 single nucleotide polymorphism with the susceptibility to cancer.3.To investigate the association of NLRP3 gene rs10733113 single nucleotide polymorphism with the susceptibility to cancer.Methods1.Search strategyTo identify relevant published studies,a comprehensive literature search was conducted using the following computerized bibliographic databases:PubMed,EMBase,Elsevier science direct,Web of science,Wiley library.2.Literature screening and quality evaluationArticles about NLRP3 gene rs35829419,rs10754558 and rs10733113 single nucleotide polymorphisms and cancer susceptibilities were collected.Unqualified studies were excluded.The criteria which Thakkinstian[8]had reported were employed to assess the methodological quality of the included trials.3.Data extractionCollected information included the first author,publication year,country,ethnicity,diseases type,source of controls,confirmation of diagnosis,and demographic variables of subjects,genotype frequencies,HWE(Hardy-Weinberg equilibrium,HWE)test,and single nucleotide polymorphism detection method.4.Statistical analysisReview Manager 5.0 statistical software was applied for statistical analysis.The effect sizes representing the unadjusted OR for the presence of the target gene single nucleotide polymorphism in patients and controls were calculated.Z test was conducted to assess the significance of the overall effect.Pooled OR were generated for comparisons using the allelic model(X allele vs Y allele),codominant model(XX vs YY?XX vs XY?XY vs YY),dominant model(XX +XY vs YY),recessive model(XX vs XY + YY).The heterogeneity of all the studies was evaluated with Cochran's Q-statistic and the I2 test.A random effects model was used in the cases of significant heterogeneity;otherwise,a fixed effect model was applied.Sensitivity analysis was performed to evaluate whether removal of any single study could influence the overall outcomes.Review Manager 5.0 and a funnel plot were applied to assess publication bias.Results1.Included studiesThis meta-analysis included a total of 7 papers(13 studies).There were 7 studies relevant to rs35829419 polymorphism,4 studies relevant to rs10754558 polymorphism,2 studies relevant to rs10733113 polymorphism.2.Included studies13 case control studies published during 2012 to 2017 were included.There were a total of 2245 cancer patients and 4672 controls.3.Meta-analysis of NLRP3 rs35829419 single nucleotide polymorphismThe pooled result suggested NLRP3 gene rs35829419 single nucleotide polymorphism had no statistical significant correlation with the occurrence of cancer(P>0.05).After kicking out the data which didn't comply with the law of Hardy-Weinberg equilibrium,the pooled result still suggested that NLRP3 rs35829419 gene polymorphism had no statistical significant correlation with the occurrence of cancer(P>0.05).After kicking out the study which cause significant heterogeneity,the result still showed that NLRP3 rs35829419 gene polymorphism.had no statistical significant correlation with the susceptibility to cancer(P>0.05).4.Meta-analysis of NLRP3 gene rs10754558 single nucleotide polymorphism No obvious heterogeneity was found among studies.The fixed effect model was used.The pooled result suggested that NLRP3 gene rs10754558 single nucleotide polymorphism had no statistical significant correlation with the occurrence of cancer(P>0.05).5.Meta-analysis of NLRP3 gene rs10733113 single nucleotide polymorphism The pooled result suggested that NLRP3 gene rs10733113 polymorphism showed a significant association in codominant genotype model(GA vs AA),OR=0.51,95%CI=0.27-0.97,P=0.04.The cases with genotype AA were more susceptible to cancer than those with genotype GA.In regard to other models,no statistical significant correlation was observed between these polymorphisms and cancer risks.Conclusion1.Human NLRP3 gene rs35829419 single nucleotide polymorphism has no significant association with the susceptibility to cancer in the following models:allelic model(A vs C),codominant model including(AA vs CC),(AA vs AC)and(AC vs CC),dominant model(AA vs AC+CC),recessive model(AA+AC vs CC).2.Human NLRP3 gene rs10754558 single nucleotide polymorphism has no significant association with the susceptibility to cancer in the following model:allelic model(C vs G),codominant model including(CC vs GG),(CC vs CG)and(CG vs GG),dominant model(CC vs CG+GG),recessive model(CC+CG vs GG).3.Human NLRP3 gene rs10733113 genotype AA had higher cancer risks than genotype GA.There was no significant association with the susceptibility to cancer in other models:allelic model(G vs A),codominant model(GG vs AA)and(GG vs GA),dominant model(GG+GA vs AA),recessive model(GG vs GA+AA).More studied are needed to confirm the conclusion.4.Further studies with larger sample sizes and high quality are needed to confirm these results.