ABTF3/HIF-1α Positive Feedback Loop Promotes Malignant Development Of Prostate Cancer By Participating In Neuroendocrine Differentiation

BackgroundProstate cancer is one of the most common malignancies of the male genitourinary system,which is the fifth leading cause of cancer-related death,and it is a highly heterogeneous cancer in clinical and pathological.Neuroendocrine prostate cancer(NEPC)is a special subtype of prostate cancer with poor prognosis.Once diagnosed,the survival period of NEPC patients is only 7 months.The de novo NEPC is generally considered rare as it accounts for approximately 1%of prostate cancer cases.Most of NEPC is induced by transcriptional reprogramming and neuroendocrine differentiation of prostate adenocarcinoma cells into neuroendocrine-like(NE-like)cells.Androgen receptor(AR)signaling plays a crucial role in development and progression of prostate cancer.Androgen-deprivation therapy(ADT)is the first-line treatment for prostate cancer.However,after 18-24 months of ADT,most patients will eventually develop resistant to ADT and relapse.Prostate cancer is prone to neuroendocrine differentiation(NED)after ADT treatment,and even evolves into NEPC.In recent years,the incidence of NEPC has further increased with the emergence of a new generation of anti-androgen receptor pathway inhibitors(ARPI).NED is a process and NE-like cell is an important intermediate state which occurs in NEPC.Currently,there is a limited understanding of the molecular mechanisms responsible for NED.Therefore,it is very urgent to explore the molecular mechanism of NED finding diagnostic markers and therapeutic targets of NEPC.Recent studies have found that multiple driving factors may be involved in NED of prostate cancer.It includes the loss of tumor suppressor genes RBI and TP53,amplification of oncogene MYCN,absence of AR,AURKA and PEG 10 mediated disorder of mitosis,epigenetic dysregulation of REST,EZH2 and SRRM4,etc.The androgen-AR axis is indispensable for the induction of NED among these driving factors.ADT is considered to be the classic stimulator of NED due to the increased probability of NED of PCa cells after ADT treatment.In addition,Epithelial-mesenchymal transition(EMT)and Prostate cancer stem cells have also been shown to participate in NED.The abnormal tumor microenvironment can also induce prostate cancer to NED,such as hypoxia and infiltrating immune cells.Basic transcription factor 3(BTF3),also known as Nascent polypeptide-associated complex subunit beta(NACB).As a member of the general transcription mechanism,BTF3 participates in transcription initiation by interacting with RNA polymerase II to form stable complex.As a multifunctional molecular chaperone,it is reported that BTF3 is also involved in the formation of new polypeptide-related complexes to prevent misfolding and aggregation of polypeptide chains.It has been reported that BTF3 can regulate the transcription of various tumor-related genes,promote the process of tumor EMT,and inhibit tumor cell apoptosis.Previous studies in our lab also found that the expression of BTF3 is up-regulated in prostate cancer tissues,which was closely related to the poor prognosis of patients.It can further maintain the stemness of prostate cancer by upregulated BMI1 which is a critical regulators of stem cell function.EMT and prostate cancer stem cells have been clearly involved in the induction of NED of prostate cancer.Therefore,we speculated that BTF3 may play an important role in NED of prostate cancer.The Gene Set Enrichment Analysis(GSEA)was performed on the previous RNAseq data of VCaP cells interfering with BTF3 in our laboratory.It was found that the hypoxia-induced gene sets were significantly downregulated in VCaP sh-BTF3 cell and hypoxia-inhibited gene sets significantly upregulated in VCaP sh-NC cell.Hypoxic microenvironment can affect the malignant progression of prostate cancer from multiple aspects,such as cell metabolism,angiogenesis,cell metastasis,EMT,maintenance of cancer stem cell characteristics and drug resistance,etc.More and more studies have confirmed that hypoxic microenvironment participates in and induces NED of prostate cancer.BTF3 is a multifunctional protein which can interact with a variety of protein as molecular chaperone.Therefore,we suspect BTF3 may be associated with hypoxia signaling pathways molecular interaction,coordination promoting NED of prostate cancer.Purposes1.To clarify whether BTF3 is involced in NED of prostate cancer.2.To explore the molecular mechanism of BTF3 involved in NED of prostate cancer.It is focused on the molecular mechanism of BTF3 affecting HIF-1α which is a key molecule of hypoxia signaling pathway,and the mechanism of abnormal overexpression of BTF3 during NED of prostate cancer.Methods1.The relationship between BTF3 expression and NED of prostate cancer1)Retrieve the data sets that are associated with NED of prostate cancer.Analyze the differental expression of BTF3 and NE marker.2)Detect the basal expression levels of BTF3 and NE markers in PC3 cells with NE phenotype and LNCaP and C4-2B cells without NE phenotype by RT-qPCR and Western blot.3)Prepare a derived cell line t-NEPC with NE phenotype.Detect the expression levels of BTF3 and NE markers in LNCaP cell and t-NEPC cell by RT-qPCR and Western blot.2.Functional study of BTF3 promoting NED of prostate cancer1)Detect the effect of BTF3 silencing on the expression of NE markers by RT-qPCR and Western blot.2)Detect the effects of BTF3 silencing on cell proliferation,migration and invasion by MTS and Transwell assay.3.The molecular mechanism study of BTF3 by activating hypoxia signaling pathway to participate in NED of prostate cancer1)Analyze the correlation between BTF3 and hypoxia signaling pathway in the malignant progression of prostate cancer by using databases.2)Establish CoCl2 hypoxia modela Detect the effects of different concentrations of CoCl2 on the proliferation of LNCaP,C4-2B,t-NEPC and PC3 cells by MTS assay.b Detect the effect of CoCl2 treatment on HIF-1α expression in LNCaP,C4-2B,t-NEPC and PC3 cells by Western blot.3)BTF3 promotes the proliferation and invasion of NE-like cells induced by hypoxiaDetect the effect of BTF3 knockdown on the proliferation and invasion of t-NEPC and PC3 cells treated with CoCl2 for 24 hours by MTS assay and Transwell assay.4)The mechanism study of BTF3 upregulating HIF-1α protein expression.a.BTF3 upregulates HIF-1α protein expressiona)Detect the effect of BTF3 overexpression/silencing on HIF-1α expression with or without CoCl2 by RT-qPCR and Western blot.b)Detect the effect of BTF3 overexpression/silencing on HIF-1α protein half-life.c)Detect the effect of BTF3 silencing on HIF-la degradation of proteasome and lysosome pathway by MG-132 and CQ assay.b.Investigate the inhibition of BTF3 on the interaction between HIF-1α-HSP70 and HIF-1α-VHL.a)Predict the potential proteins that might interact with BTF3 by online protein interaction database.b)Detect the interaction between BTF3 and HSP70,VHL by Co-IP.c)Detect the effect of BTF3 overexpression/silencing on HIF-1α-HSP70 and HIF-1α-VHL interaction by Co-IP.5)The mechanism study of BTF3 upregulating HIF-α transcriptional activity.a.Detect the effect of BTF3 silencing on the expression of HIF-1α downstream target genes PGK1,CA9 and SLC2A1 by RT-qPCR.b.Detect the effect of BTF3 overexpression/silencing on HIF-1α plasma-nuclear translocation by subcellular fractionation assay.c.Investigate the inhibition of BTF3 on the interaction between HIF-1α-HIF-1AN.a)Detect the interaction between BTF3 and HIF-1AN by Co-IP.b)Detect the effect of BTF3 overexpression/silencing on HIF-lα/HIF-1AN interaction by Co-IP.4.Hypoxia upregulates the expression of BTF3 protein mediated by HIF-1α1).Detect the effects of CoCl2 treatment on the expression of BTF3 in LNCaP,C4-2B,t-NEPC and PC3 cells by RT-qPCR and Western blot.2).Detect the effect of HIF-1α silencing on the expression of BTF3 with or without CoCl2 by RT-qPCR and Western bolt.Results1.The expression of BTF3 is upregulated in prostate cancer cells with NEDDatabase analysis showed that the expression levels of BTF3 and NE markers in PCa cells with NED were significantly up-regulated.Basal expression analysis showed that BTF3 and NE markers were up-regulated in the PC3 cell line with NE phenotype compared with the PCa cell line without NE phenotype.The expression levels of BTF3 and NE markers in NED derived cell line t-NEPC were significantly up-regulated compared with LNCaP cells.2.Knocking down BTF3 inhibits NED of prostate cancerThe expression level of NE markers was significantly decreased after knocking down of BTF3 expression in t-NEPC and PC3 cells.Meanwhile,the results of MTS and Tranwell experiments showed that the proliferation,migration and invasion ability of t-NEPC and PC3 cells were decreased compared with the control group after the knockdown of BTF3 expression.It was suggested that knock down BTF3 inhibited the proliferation,migration and invasion ability of NE-like cells,and inhibited the NED of PCa.3.BTF3 participates in NED of prostate cancer by activating hypoxia signaling pathways1)BTF3 is positively correlated with hypoxia signaling pathway in the malignant progression of prostate cancer.The results of GSEA analysis showed that the hypoxia-induced gene sets were significantly downregulated in BTF3 silencing cells and hypoxia-inhibited gene sets significantly upregulated in control cells.The dataset analysis results of Beltran et al.Nat Med 2016,GSE35988,GSE6956,Gerhauser et al.Cancer Cell 2018 and Taylor et al.Cancer Cell 2010 showed that BTF3 had a greater correlation with hypoxia fraction in NEPC(r=0.5756)than Castration-resistant prostate cancer(CRPC)(r=0.3987).In prostate cancer,the mRNA expression levels of BTF3 and HIF-1α were positively correlated.These results suggested that BTF3 and hypoxia signaling pathways were significantly positively correlated with the malignant progression of prostate cancer.It may participate in NED through mutual regulation.2)Establish CoCl2 hypoxia modelThe results of MTS showed that 50μM CoCl2 treatment had the strongest promoting proliferative effect on LNCaP,C4-2B,t-NEPC and PC3.Western blot results showed that the expression of HIF-la in t-NEPC and PC3 cells was the highest after treatment with 50μM CoCl2 for 8h.HIF-la expression was the highest in C4-2B cells after 6h treatment.HIF-1αexpression was the highest in LNCaP cells after 4h treatment.We mainly used 50μM CoCl2 treatment for 4/6/8h to establish the hypoxia model of LNCaP,C4-2B,t-NEPC and PC3 cell for subsequent experiments.3)Hypoxia promotes proliferation and invasion of NE-like cells through BTF3MTS and Transwell experiments showed that the viability and invasion ability of t-NEPC and PC3 cells stimulated with CoCl2 for 24h were significantly increased compared with the control group.The above effects could be significantly attenuated by knockdown of BTF3.4)BTF3 upregulates HIF-la expression at the post-translational levelOverexpression of BTF3 significantly increased HIF-1α protein expression in C4-2B cells and knockdown of BTF3 in PC3 cells significantly inhibited HIF-1α protein expression level rather than mRNA expression level regardless of CoCl2 treatment.The results of CHX assay showed that overexpression of BTF3 significantly prolonged HIF-1α half-life and knockdown of BTF3 significantly shortened HIF-la half-life.MG-132 and CQ experiment showed that BTF3 inhibited the proteasome degradation of HIF-1α protein.Previous GST-pull down mass spectrometry results in our laboratory showed that BTF3 and HIF-la non-oxygen dependent regulatory factor HSP70 protein interaction.PINA and IntAct intact database analysis showed that VHL,an E3 ubiquitin ligase targeting HIF-lα,is a potential BTF3 interacting molecule.Co-IP results showed that BTF3 could interact with HSP70 and VHL proteins in C4-2B and PC3 cells.Overexpression of BTF3 weakened the binding between HIF-1α and HSP70,VHL.Knockdown of BTF3 enhanced the binding between HIF-1α and HSP70,VHL.These results suggested that BTF3 inhibited HIF-1α proteasome degradation by competitive binding to HSP70 and VHL,and up-regulated HIF-lα expression at the posttranslational level.3)BTF3 upregulates the transcriptional activity of HIF-1αRT-qPCR results showed that HIF-1α target genes PGK1,CA9 and SLC2A1 mRNA expression was down-regulated after BTF3 knockdown and CoCl2 treatment in PC3 cells.The results of subcellular fractionation showed that overexpression of BTF3 increased nuclear translocation of HIF-1α and knockdown of BTF3 significantly decreased the distribution of HIF-1α nuclei.PINA and IntAct online protein interaction database analysis results showed that HIF-1AN,a transcriptional inhibitor of HIF-1α,was also a potential interacting molecule of BTF3.Co-IP results showed that BTF3 could interact with HIF-1AN in C4-2B and PC3 cells.Overexpression of BTF3 in C4-2B weakened the binding between HIF-1α and HIF-1AN.Knockdown of BTF3 in PC3 cells increased HIF-1α binding to HIF-1AN.These results suggested that BTF3 can enhance the transcriptional activity of HIF-1α by promoting HIF-1α entry into the nucleus and competitively binding HIF-1AN.4.Hypoxia upregulates the expression of BTF3 protein mediated by HIF-1αThe results of RT-qPCR and Western blot showed that the protein level of BTF3 rather than mRNA was significantly up-regulated after CoCl2 treatment in LNCaP,C4-2B,t-NEPC and PC3 cells.The upregulation of expression level was time-dependent with the extension of CoCl2 treatment time.The protein level of BTF3 was significantly down-regulated after knocking out HIF-1α in LNCaP,C4-2B,t-NEPC and PC3 cells regardless of CoCl2 treatment,while the mRNA level was not significantly changed.ConclusionThe expression of BTF3 is up-regulated in prostate cancer with NED,and participates in NED to promote the malignant progression of prostate cancer.The possible molecular mechanism is that in prostate cancer,the up-regulated BTF3 competitively binds HSP70,VHL and HIF-1AN,respectively,increasing the stability of HIF-1α protein and enhancing its transcriptional activity,leading to excessive activation of HIF-1α signaling pathway and participating in NED.The abnormal upregulation of BTF3 during NED is mediated by HIF-1α overactivation.In conclusion,a positive feedback loop is formed between BTF3/HIF-1α,which synergistically drives NED of prostate adenocarcinoma and futher promotes the malignant progression of prostate cancer.