Characteristics And Clinical Significance Of TLR4 Positive Breast Cancer Stem-like Cells

Breast cancer is the most common malignant tumor of female clinically,ranking the third in all malignant tumor occurrence rate,the main tumor pathogenesis causing female die.Correlation statistics show the morbidity of breast cancer present an increasi ng trend year by year.Continuously research for breast cancer and the improvement of the treatment of breast cancer make some improvement for the therapeutic effect of breast cancer.The WHO divided breast cancer into different molecular subtypes according to the histology feature,the expression of protein immunohistochemical feature,genotype feature and the target spot of clinical therapy.Among these subtype,luminal type(including A and B)is the most main type of breast cancer,occupy about 70%.Paients with triple negative breast cancer were the poorest prognosis group.Toll-like receptors(TLRs)is an important pattern recognition receptor of biological immunity reaction.It activates immune system and promoting the release of inflammatory medium by recognizing the pathogen associated molecular pattern(PAMP)and some endogenous ligand,and trigger proper immune reaction.Toll-like receptors expressed highly in immune cell and also expressed in some normal epithelial cell in some tissues,for example,the epithelial of stomach and alveolar.Recently research shows high expression of Toll-like receptors in tumor cells reveal bad prognosis of malignant tumor.We screen the TCGA database and find TLR4 is obviously high expression in follow-up visit death case of breast cancer,indicating TLR4 is the key prognostic indicator of breast cancer.Meanwhile,TLR4 is significantly correlated with overall surival of luminal type and triple negative type of breast cancer.For further analyzing the action mechanism of TLR4 in breast cancer,we separate the TLR4 positive breast cancer cell in vitro(luminal type breast cancer cell line MCF-7,triple negative type breast cancer cell line MDA-MB-231),and the results showing TLR4 positive MCF-7 cell relate with the stemness of breast cancer and TLR4 positive MDA-MB-231 cell showed strong feature of EMT.Meanwhile,we detect the expression of TLR4 endogenous ligand HMGB1 in breast cancer tissues,hoping to propose new test index and therapeutic schedule for diagnosis an d treat for breast cancer by joint detection and analysis of HMGB1 and TLR4.Achaet scute-like 2(ASCL2),a basic helix-loop-helix(bHLH)transcription factor,is involved in the development and progression of several human cancers.Scientific researches have discovered that the high expression of ASCL2 in tumor tissues could promote the proliferation and invasion of tumor cells,and correlated to poorer prognosis of patients.Mechanically,ASCL2 was regulated by the Wnt signaling pathway,and could inhibit the expression of mi R-200 s in colorectal cancer.Meanwhile,ASCL2 could combine with chemokine receptor 4(CXCR4)and promote the invasion and metastasis of cancer cells.Furthermore,ASCL2 was identified a relativeness with the clonal cancer stem cell.However,the research between the expression of ASCL2 in breast cancer and clinical pathology of breast cancer has not been launched.In the present study,we applied the immunohistochemical staining to investigate the expression pattern of ASCL2 in breast cancer and the prognostic value for the patients.With the development of gene screening technologies and genomics,many genes related to breast cancer had been founded.The gene tesing outcomes related to breast cancer genes amplification,mutation and abnormal expression,have a important significance for early diagnose,prognosis judgement and clinical methods choices.BRCA1 and BRCA2 had been identified a significant relation to the susceptibility of breast cancer.Yang et al founded the evidence of correlation between the gene polymorphism of TLR4 gene and the prognosis of breast cancer patients.We collected the blood samples of 24 breast cancer patients.We further screened gene polymorphism of TLR4 gene on the level of germline,and analysis the relations between gene screeing outcomes and breast cancer susceptibility.Main results and conclusion as follows:1.The expression of TLR4 is outstanding enhancement in death case of breast cancer and is outstanding related with the index of of breast cancer stem cells.(1)TCGA database analysis results shows: the expression ratio(mean)of breast cancer death group and disease free survival(DFS)group are: 0.5131 and 0.0163,statistical analysis are t=3.441 df=258,p=0.0007,these two groups have highly sig nificant difference;(2)TCGA database analysis show the expression of TLR4 has significant difference(P<0.05)between death group and disease free survival(DFS)group of luminal type breast cancer;(3)Database analysis show the expression of TLR4 has significant difference(P<0.05)between death group and disease free survival(DFS)group of triple negative breast cancer;(4)Further analysis show: The r2 value of the expression of TLR4 and the expression of ALDH1A1 are 0.8316(P<0.0001)in luminal type breast cancer death cases;The r2 value of the expression of TLR4 and the expression of ALDH1A1 are 0.478(P<0.0001)in triple negative breast cancer death cases.2.Sorting TLR4 positive cell possess feature of CSCs(1)Sorted TLR4+MCF-7 cell(luminal type)making ALDH1 detecting,the results show: the rate of ALDH1 positive cell is about 76.0% in TLR4+TLR4 positive cell of MCF-7cells;the rate of ALDH1 positive cell is about 2.30% in TLR4-MCF-7 cells;(2)Detecting breast cancer cell line MDA-MB-231(triple negative breast cancer),the rate of TLR4 positive cell is about 69.4% of ALDH1 cell;(3)The stemness transcriptional factors OCT4,SOX2,Nanog of separated TLR4 positive breast cancer cell are all express higher among TLR4+MCF-7 cell,which also have increasing ball capacity;(4)The results of fluorescent quantitative PCR show the EMT related factors Vimentin 、β-catenin express higher in TLR4 positive triple negative breast cancer cells(MDA-MB-231);(5)Immunohistochemistry double-labeled results of human breast cancer: the expression of TLR4 and ALDH1A1 are co-localization in breast cancer cell;3.HMGB1 increase the resistance of luminal type breast cancer cell to tamoxifen therapy through activating TLR4 high expression.(1)The breast cancer cell MCF-7 dealt with Tamoxifen,TLR4 and stemness transcriptional factors OCT4,SOX2,Nanog express higher and present as dose dependent;(2)CCK8 detects cytotoxicity test show: TLR4 positive MCF-7 cell resisting tamoxifen therapy is stronger than TLR4 negative MCF-7 cell;(3)with the activating of HMGB1,the resistance of TLR4 positive MCF-7 cells increase significantly.4.HMGB1 increase invasion and metastasis of TNBC through activating TLR4 high expression.(1)Transwell test shows that the invasion ability of breast cancer cell MDA-MB-231 significantly increase with the ectogenic activator LPS;(2)the invasion ability of breast cancer cell MDA-MB-231 significantly increase with the endogenic activator HMGB1;(3)After adding the antagonist TAK-242 of TLR4,the going through breast cancer cell significantly weaken;(4)After the ligand HMGB1 of TLR4 is antagonism,the invasion ability of breast cancer cell MDA-MB-231 significantly weaken: after dealt with GLY(HMGB1 antagonist),Transwell test shows the going through breast cancer cell get significant less.5.The united detection of TLR4 and HMGB1 is useful marker in breast cancer.(1)In TLR4 high expression groups,the lymphatic metastasis of breast cancer significant risk in human breast cancer sample,but the total lifetime and disease free survival lifetime has no significant difference(the value of P is 0.751 and 0.162)between high expression and low expression groups of breast cancer patient;detecting no significant clinical meaning in luminal type breast cancer patient and triple negative breast cancer TLR4 high expression patient;(2)The expression of HMGB1 protein(the endogenous ligand of TLR4)mainly locate in cell nucleus and cytoplasm,the high expression rate of HMGB1 in cell nucleus is 75.4%(144/191),and the high expression rate in cytoplasm is 50.3%(96/191);(3)The tumor size becomes larger of HMGB1 high expression in cytoplasm,Ki-67 express higher(P<0.05),however,the HMGB1 high expression group in cytoplasm has no features of malignant tumor;(4)The immunohistochemistry double-labeled test results of TLR4 and HMGB1 in human breast cancer sample show that the expression of TLR4 was associated with the expression of HMGB1 in nucleus of BRCs.TLR4 high breast cancer cells have low level of HMGB1 in nucleus;(5)The combined detection results of human breast cancer sample show: the lymphatic metastasis,the size of tumor,clinical stages of TLR4-HMGB1 positive breast cancer all have significant meaning(P<0.05),and the total survival rate and without relapse survival rate of TLR4-HMGB1 positive groups are all lower than TLR4-HMGB1 negative groups(P<0.05).6.The expression of ASCL2 has a positive correlation to expression of TLR4,the breast cancer patients with high ASCL2 expression have a worse prognos is.(1)IHC tested the expression of ASCL2 in breast cancer tissuses,the scores of expression of ASCL2 is positive with the scores of TLR4.(2)According to the IHC scores,79(41.4%)specimens were defined as ASCL2 high group,and 112(58.6%)specimens were defined as ASCL2 low group.Moreover,the expression level of ASCL2 in cancerous tissue was significantly higher than that in the adjacent tissue.Among the cell line of breast cancer,the ASCL2 are located at the cell nucleus and cytoplasm of breast can cer cell.(3)Correlation between ASCL2 expression and clinicopathological features.The expression of ASCL2 was positively correlated with lymph node metastasis(p=0.026),tumor size(p=0.027)and Ki67 level(p=0.016).Accordingly,the ratio of ASCL2 high cases was significantly higher in patients with lymph node metastasis,tumor size large than 5 cm and high level of Ki67 than those without lymph node metastasis,or with tumor size less than 5 cm,low level of Ki67,respectively(Fig).However,no significant correlation was observed between ASCL2 expression and age,clinical stage,as well as molecular subtypes(all p>0.05).(4)The ASCL2 is a independent marker of prognosis or relapse of breast cancer patient.Kaplan-Meier analysis indicates the high expression of ASCL2 is the influence factor of overall survival(OS)and disease-free survival(DFS)of breast cancer patient.Multi-factor Cox regression analysis indicates the expression level of ASCL2 is the independent factor influencing the prognosis(HR=1.865,P=0.001)and relapse(HR=1.278,P=0.055)of breast cancer patient.7.TLR4 is a potential susceptibility gene of breast cancer.(1)Among 24 samples of breast cancer patients experienced TLR4 gene screening,1 patient was founded heterozygous mutation at the Chr9:120474941(H179N).(2)The patient with TLR4 gene mutation showed resistant to the Tamoxifen.