BTF3 Promotes Prostate Cancer Progression Through Maintenance Of Cancer Stem-like Phenotype

Prostate cancer(PCa)is a common male urinary system tumor in our country.The incidence of PCa is rapidly increasing due to the change of life style,the aging of the population and the promotion of serum prostate specific antigen(PSA)screening.Although the level of diagnosis and treatment of PCa has increased in recent years,tumor metastasis and hormone resistance are still the thorny problems in the treatment of PCa,which is the main reason leading to treatment failure and death.Carcinogenesis and development of cancer is a complex network of mutual regulation and control of various factors.Identification of driving genes with important regulatory roles in this network is an important means in cancer research.Understanding the biological role and expression regulation of these driving genes in cancer may provide important clue for revealing the malignant progression mechanisms of PCa.More importantly,the abnormal expression of these driving genes is not only an important basis for the prognosis of PCa stratification,but also can be considered as a potential therapeutic target.Our team focused on finding key genes that drive the development and progression of PCa.Similar to the conclusions of most of the research groups,the previous study of our group suggests that the stem cell-like phenotype of prostate cancer is an important basis for the malignant progression of PCa.Stem cell correlated gene may be a driving gene for the malignant progression of PCa.CSCs are cells with self-renewal capacity and capable of producing heterogeneous cancer cells.CSCs account for a very small proportion of the tumors but have an important role in the survival,proliferation,metastasis and recurrence of the tumors.In the mean time,compared to normal tissues,tumor tissues exhibts dedifferentiation and stem cell-like phenotype.CSCs and stem cell-like phenotype are the important foundation of carcinogenesis and development.It has been demonstrated that CSCs and stem cell-like phenotypep play an important regulatory role in the metastasis of PCa and hormone resistance.However,the relevant research is still a minority.BTF3,consisting of 206 amino acids,is a dual-identity protein.On one hand,BTF3 belongs to basic transcription factors family,which can bind to RNA polymerase? and coordinate gene transcription;on the other hand,BTF3 belongs to the nascent polypeptide associated complex(NAC)family,which can prevent false fold regulation of protein in the process of synthesis and degradation.Recent immunohistochemical studies have shown that,compared with normal tissues,BTF3 over-expressed in embryonic stem cells and many cancer tissues including PCa,breast cancer,pancreatic cancer and so on.However,few researches studied the role of BTF3 in carcinogenesis and development.The mechanism of BTF3 functions is still unknown.Part ? BTF3 Sustains Cancer Stem-like Phenotype of Prostate Cancer via Stabilization of BMI1The treatment of PCa is one of the major problems and challenges.It is urgent to explore the complex molecular network that promotes PCa progress.Cancer stem cells and stem-like phenotype are important basis for malignant progression and metastasis of PCa.The research about molecular mechanism of tumor stem-like phenotype provides a theoretical basis for prognosis evaluation and treatment of PCa.Considering that BTF3 may be the signature gene of embryonic stem cells(ESCs),we explore whether BTF3 plays an important role in the maintenance of PCa stem-like phenotype,whether it is a driving gene for the development and progression of PCa,whether it regulates the progression of PCa,and to clarify the mechanism of BTF3 function.In this study,we used PCa cell lines to study the multi-faceted function and molecular mechanism.The results were as follows:1.Embryonic stem cell related gene BTF3 has prognostic value in PCaUsing bioinformatics database analysis,combined with TCGA and GTEx databases,we obtained genes related to prognosis and recurrence of PCa,including SOX4 and CUL4B in our previous studies.By using GSEA and GO functional annotations,stem cell signature was found to be enriched in cases with poor prognosis.Survival analysis of TCGA and MSKCC databases indicated that PCa patients with high expression of embryonic stem cells signature had poor prognosis.The genes of ECSs signature were reviewed and analyzed,and BTF3 was selected for further study.By immunohistochemical assay of 315 cases of PCa patients,the expression of BTF3 was positively correlated with Gleason score,tumor grade and metastasis.There was no significant correlation with castration resistant prostate cancer(CRPC)and a positive correlation with clinical recurrence.The expression of BTF3 was increased in neuroendocrine prostate cancer(NEPC)in combination with the database.BTF3 is associated with poor prognosis of prostate cancer.2.BTF3 expresses in stem cell enriched population of prostatic tissues and PCaWe used bioinformatics analysis and immunofluorescence techniques to detect the expression of BTF3 in prostate tissue.Public database analysis showed that the ESCs signature containing BTF3 has a guiding significance for the prognosis of PCa.By immunofluorescence staining of mouse prostate,it was found that BTF3 was expressed in the proximal region and the basal cell layer.Public database analysis suggested that BTF3 is upregulated precursor stem cells located in prostate basal cells.Comprehensive analysis of multiple prostate data sets,BTF3 expression was significantly up-regulated in PCa tissue.3.BTF3 is an activator of stem-like properties in PCaSphere formation assay,clonogenic,immunofluorescence assay,flow cytometry,bioinformatics analysis and immunohistochemistry were used to explore the effect of BTF3 on PCa stem-like phenotype.Conpared with control group,sphere formation experiments showed that low expression of BTF3 reduced sphere formation of PCa cells;clonogenic assay showed that low expression of BTF3 reduced the rate of sphere formation of PCa cells,and high expression of BTF3 increased the rate of sphere formation in PCa cells;immunofluorescence staining showed that BTF3 expression was upregulated in holoclones;flow cytometry showed that the expression of BTF3 affects the CD 133+ cell population.GSEA suggested that multiple stem cell-related databases were enriched in samples with high BTF3 expression.Database analysis,qPCR and immunohistochemical staining of PCa tissues showed that BTF3 was positively correlated with tumor stem cell markers(CD 133,CD44,NANOG,SOX2).4.BTF3 regulates growth and invasion through modeling stem-like characteristics of PCa cellsThe tumor formation experiment of subcutaneous nude mice confirmed that stable interference with BTF3 expression delayed tumor growth,and reduced tumor cell invasion.MTS and EDU results showed that compared with the control group,low expression of BTF3 inhibited the proliferation of PCa cells,and high expression of BTF3 promoted the proliferation of PCa cells.The results of transwell assay showed that,compared with control group,the low expression of BTF3 inhibited the migration and invasion of PCa cells,and the high expression of BTF3 promoted the migration and invasion ability of PCa cells.Clonal assay showed that the low expression of BTF3 decreased the colony formation of PCa cells,as well as the percentage of holoclones.The high expression of BTF3 increased the colony forming ability and percentage of holoclones of PCa cells.5.Bioinformatics analysis of BTF3 downstream genesGSEA analysis suggested that BTF3 was correlated with SHH signaling pathway,mTOR signaling pathway,PRC1,PRC2 and MYC proteins.The results of mass spectrometry showed that BTF3 could interact with BMI1,scaffold proteins IQGAP1,HSP90,AHNAK,various E3 ubiquitin ligases,etc.Among them,IQGAP1 and AHNAK are oncoproteins that promote tumor cell invasion and migration,and HSP90 is a tumor stem cell promoting protein.Of note,the intersection of GSEA enrichment analysis and mass spectrometry analysis is the BMI1 gene.6.BMI1 is a directly target of BTF3 in PCaGSEA analysis,Western Blot verification,immunofluorescence staining,immunohistochemically staining of PCa tissue and mouse subcutaneous xenograft were performed to determine BMI1 as a downstream gene of BTF3.Bioinformatics analysis of genome-wide microarray with BTF3-knockdown or BMI1-knockdown revealed that the downstream genes of BTF3 and BMI1 are overlapped;nucleoprotein separation and extraction indicated that BTF3 promote BMI1 nucleation and promote its transcriptional regulation effect.In vitro experiments showed that interference with BMI1 partially reversed the stem cell-like phenotype of PCa caused by overexpression of BTF3.7.BTF3 stabilizes BMI1 proteinsCHX stimulation of PCa cell suggesting that knockdown BTF3 reduce the stability of BMI1 protein,over-expression of BTF3 improve the stability of BMI1 protein.Proteasome inhibiter MG132 could block the regulation of BTF3 on BMI1 protein expression.After BTF3 was over-expressed,the level of ubiquitination of BMI1 was decreased.Whereas BTF3 knockdown increased the ubiquitination level of BMI1 protein.8.BTF3 physically interacts with BMI1The results of GST-pull down indicated that BTF3 could bind to BMI1 protein,and Co-IP confirmed that BTF3 binds to BMI1 protein.Co-IP assay with truncated BMI1 protein indicated that BTF3 bound primarily at the C-terminal 160-326aa of BMI1.Binding of BMI1 to BTF3 significantly increased the binding of HSP90 protein to BMI1,and also increased the binding of BMI1 to CD44.Collectively,BTF3 plays critical role in driving PCa progression by promoting cancer stem-like character of PCa cells.BTF3 interacts with BMI1 stabilizing BMI1 protein from ubiquitination and degradation.Our finding enriched the knowledge of molecular mechanism underlying PCa progression.It provides a new direction for revealing the complex molecular network of the occurrence and development of PCa.Part ? Hypoxia Promotes Stem-like Phenotype Through Modulation of BTF3 ExpressionAbnormal expression of genes in tumors could be the result of multiple factors,including gene amplification and mutation,tumor microenvironment and tumor interaction are common causes.Hypoxic microenvironment is a common feature of many solid tumors.In hypoxia environment,the stem cell-like phenotype of tumor cells increases,which in turn promotes the proliferation and migration of tumor cells.To analyze the effect and mechanism of hypoxic environment on PCa cells,and to explore the mechanism of overexpression of BTF3 in PCa,it is helpful to understand the role of PCa tumor microenvironment in PCa progression.In this study,the public database analysis and various molecular biology experiments were used to explore the molecular mechanism.The results were as follows:1.Hypoxia significantly up-regulates BTF3 expressionIn order to explore the mechanism of BTF3 upregulation in PCa,we performed bioinformation analysis,androgen deprivation and hypoxia simulation.The results showed that upregulation of BTF3 in PCa was not associated with tumor burden of PCa,and was mildly associated with androgen deprivation.Culture in a low-oxygen environment with three-gas incubator,or CoC12 mimics hypoxic environment culture significantly up-regulate the expression of BTF3 in PCa cells.2.Hypoxia promotes up-regulation of BTF3 expression by regulating miR-15aPromoter prediction result showed that no binding site of HIF in promoter region of BTF3.Multiple databases were used to analyze miRNAs whose target gene is BTF3,and 6 miRNAs were obtained:miR-15a,miR-15b,miR-16.miR-186,miR-195,miR-361.293T cells were co-transfected with wild/mutant BTF3 3'UTR plasmid and miR-15a mimics.The luciferase activity of wild-type construct of BTF3 3'UTR was inhibited by miR-15a mimics,while that of mutant construct of BTF3 3'UTR showed no significant change.RT-qPCR results showed that the expression of miR-15a decreased in hypoxia.Western-blot data showed that miR-15a mimics reduced BTF3 expression under hypoxic conditions,and transfection of miR-15a inhibitor increased BTF3 expression.3.BTF3 is involved in hypoxia-mediated stem-like phenotypeHypoxia lead to a stem cell-like phenotype of tumor cells,which is manifested by increased expression of stem cell markers(CD133,CD44,NANOG,SOX2).Inhibition of BTF3 expression in hypoxic PCa cells partially reverses the up-regulation of stem cell markers(CD 133,CD44,NANOG,SOX2)caused by hypoxia.In summary,in PCa,the hypoxic microenvironment can positively regulate BTF3 expression by inhibiting miR-15a expression.BTF3 mediates the stem cell-like phenotype of PCa induced by hypoxia microenvironment.Part ? Expression and survival analysis of BTF3 in pan-cancerTumors derived from different organs may share common genome signatures,while tumors from the same site may have distinct genomic characteristic.In order to explore the general rule of BTF3 expression in tumors,we analysis several dataset containing pan-cancer arrays.The results were as follows:1.Expression analysis of BTF3 between normal tissues and tumor tissuesAccording to the combined TPM data of TCGA and GTEx,GENT data and ONCOMINE data analysis,BTF3 expressed in most organs of normal human body.Increased expression in tumor types such as nervous system tumors and testicular cancer,and decreased expression in tumors derived from female reproductive systems.2.Survival analysis of BTF3 in different tumorsAccording to the GEPIA database,high expression of BTF3 indicates poor prognosis in 8 types of tumors,and better prognosis in 5 types of tumors.In summary,the role of BTF3 in different types of tumors is tissue-specific.Seeking their intrinsic links can help reveal the mechanism of BTF3 in tumors.