| BackgroundGlioblastoma(GBM,WHO grade Ⅳ)is the most common and deadly primary intracranial malignancy in adults.Current standard treatment options include surgery with maximum safe resection,temozolomide and radiotherapy combined with adjuvant therapeutic regimens.Although some treatment benefits have been achieved in recent years,the median survival time of GBM patients is still only 14.6 months.At present,many classical cancerassociated pathways have been found to be involved in the occurrence and development of GBM,such as pathways of PI3K/AKT,NF-κB,Hippo,etc.However,the development of drugs targeting these pathways often has big challenges associated with undruggable and extensive side effects,leading to difficulties in development and few clinical applications eventually.At present,the drugs used in clinical application previously,for instance,the monoclonal antibody Bevacizumab targeting Vascular Endothelial Growth Factor(VEGF)and the inhibitor Gefitinib targeting Epidermal Growth Factor Receptor(EGFR),have been encountering the dilemma of limited efficacy.This is due to the intricate signaling pathways within GBM that have led to the interaction of different signaling pathways,and this high heterogeneity makes a single targeted therapy much less effective.In this context,many research works are focusing on exploring the molecular mechanism of signaling pathway on the regulation of GBM,aiming to develop complementary drugs targeting multiple targets or different targets,which may exert better anti-tumor effects.The Hippo signaling pathway is a growth inhibitory pathway that plays an important role in biological processes such as cell proliferation,differentiation and apoptosis.If this pathway is downregulated,it can cause uncontrolled proliferation and apoptosis inhibition.This has gained extensive research activities in recent years.The centre components of Hippo pathway are SAV1(Salvador homolog 1),MOB1(Mob kinase activator 1),MST1/2(Mammalian STE20-like protein kinase 1/2),LATS 1/2(Large tumor suppressor 1/2),YAP(Yes-associated protein,YAP)and TAZ(Transcriptional coactivator with PDZ-binding motif,and also known as WW-domain containing transcriptional regulator 1,WWTR1),etc.Among them,SAV1 and MOB1 are adaptor proteins,which can bind to MST1/2 and LATS 1/2 respectively and enhance their phosphorylation.Classical Hippo signaling is transmitted through a kinase cascade.YAP/TAZ,the main downstream effectors of Hippo,can be directly phosphorylated by LATS 1/2.Phosphorylated YAP/TAZ interact with 14-3-3 for cytoplasmic retention and undergo protein degradation.Unphosphorylated YAP/TAZ enter nuclei and interact with TEA domain(TEAD)containing family transcriptional factors to activate gene transcription for a variety of biological functions.A large number of studies have shown that abnormal regulation of Hippo pathway plays a key role in the glioma progression,including cell proliferation,apoptosis,invasion,resistance and stemness maintenance.Our group has also preliminarily explored the role of Hippo signaling pathway in GBM.At present,domestic and abroad studies are primarily focused on the intervention of Hippo pathway.However,due to the complexity of Hippo pathway network,in addition to classical upstream kinase inhibitors,upstream intervention of non-classical Hippo pathway has also been gradually discovered.Therefore,the study of new regulatory molecules targeting different key components of Hippo signaling pathway will contribute to the treatment of GBM.Gene expression involves two important processes:transcription and translation.USP39(Ubiquitin specific protease 39)containing an arginine/serine(RS)-rich domain,acts as a splicing factor.It is involved in the assembly of the spliceosomal U4/U6.U5 tri-snRNP and plays an important role in post-transcriptional regulation.The elevated expression and cancerpromoting effects of USP39 have been revealed in prostate cancer,gastric cancer,lung cancer,etc.Few in-depth studies have been conducted in glioma.Our group analyzed the expression of USP39 in human brain tissues and found that USP39 tends to be highly expressed in gliomas.All results suggested that USP39 may be related to the occurrence and development of malignant glioma.A series of luciferase reporters to assay signaling activity from 7 cancerassociated pathways revealed that USP39 significantly regulates Hippo-TAZ signaling,and the specific mechanism is worthy of further study.Ubiquitination is a common form of post-translational modification of proteins.Most of the key components of the Hippo pathway are regulated by the ubiquitin proteasome pathway.PMEPA1(Prostate transmembrane protein,androgen induced protein 1)is highly expressed in Prostate cancer,breast cancer,lung cancer,etc.Interestingly,it shows heterogeneity across different tumors.It has been reported that PMEPA1 plays a role in the degradation of androgen receptor and other proteins.PMEPA1 is a potential biomarker,which is rarely investigated in glioma.Our previous work found that PMEPA1a was the most highly expressed isoform in gliomas.Results from Co-IP(Co-immunoprecipitation)combined with LC-MS/MS showed that LATS1,the upstream kinase of Hippo pathway,was an important interaction protein of PMEPA1a.However,whether PMEPA1a modulates the degradation of LATS1 protein remains to be further clarified.In this paper,using a variety of biological techniques,the oncogenic effect of splicing factor USP39 and transmembrane protein PMEPA1a on GBM progression as well as the molecular mechanisms of regulating key elements of the Hippo pathway were revealed.These findings are helpful to provide with new strategies for studying diagnostic biomarkers and potential drug targets in GBM treatment.Part Ⅰ:RNA splicing factor USP39 promotes glioma progression by inducing TAZ mRNA maturationObjectiveTo analyze the expression levels of splicing factor USP39 in human normal brain tissue and glioma tissue samples;To reveal the cellular expression distribution of USP39 in GBM cell lines;To study the promoting effects of USP39 on GBM cell in vitro and in vivo;To screen and identify the classical cancer pathways and specific molecular mechanisms regulated by USP39.Methods1.Immunohistochemistry(IHC)and Western blot(WB)were performed to determine USP3 9 expression levels in normal brain tissues and WHO Ⅱ-Ⅳ grade gliomas.qRT-PCR and Western blot were used to analyze USP39 expression in Normal Human Astrocyte(NHA),GBM cell lines LN18,U87MG,U251,A172 and Human primary glioma cell line P3.2.Knockdown efficiency of lentiviruses expressing either two independent shRNAs(Short hairpin RNAs)targeting different sequences of USP39 were verified by qRT-PCR and Western blot.CCK-8(Cell Counting Kit-8),colony formation assay and EdU assay were utilized to detect cell proliferation level.Transwell assay was used to evalute cell invasion and migration.Orthotopic xenograft models were generated,and in vivo bioluminescence imaging system was used to monitor the growth of glioma cells in vivo;Kaplan-Meier survival curve was used to analyze survival time of tumor-bearing mice.3.A series of dual luciferase reporter gene systems(Hippo,MAPK/ERK,MAPK/JNK,NFκB,Notch,TGFβ and Wnt)were transfected to screen for the changes of USP39 knockdown on these classical cancer-associated pathways;Western blot was used to determine which core components of Hippo pathway were regulated by USP39.IHC staining was performed on mice brain tissue sections and human glioma tissues to further confirm the correlation between USP39 and Hippo pathway element TAZ.4.Specific primers were designed to distinguish and detect the splicing efficiency of TAZ mRNA;RNA-Binding protein immunoprecipitation assay(RIP)were performed to identify the interaction between USP39 protein and TAZ mRNA.5.Nuclear and cytoplasmic extracts combined with Western blot and cell immunofluorescence were used to determine the cellular localization of the TAZ protein.qRTPCR was used to detect the impact of USP39 on transcriptional levels of TAZ target genes,BIRC5,E2F1 and MYC.6.Western blot was used to detect the effects of overexpression of exogenous USP39 or TAZ after knockdown of USP39;CCK-8,Luciferase assay and qRT-PCR were utilized to determine the impacts of cell proliferation and Hippo transcription activity.The orthotopic tumor models of nude mice were established to observe the changes in vivo.Results1.Increased expression of USP39 is linked to the higher tumor grade in primary human gliomas.Compared with NHA,USP39 was significantly highly expressed in human GBM cell lines LN18,U87MG,U251,A172 and primary GBM cell line P3.2.USP39 enhances proliferation,invasion,and migration of GBM cells in vitro and in vivo.Results from CCK-8 cell,colony formation assay and EdU assay showed that knockdown of USP39 inhibited cell proliferation in vitro.Trans well assay showed knockdown of USP39 significantly weakened the abilities of invasion or migration of cells.Compared with the control group,USP39 knockdown can delay the tumorigenesis rate of GBM cells in vivo and prolong the median survival time of mice.3.TAZ is regulated by USP39 independently of classical Hippo signaling.Results from luciferase assay showed that USP39 significantly down-regulated the transcriptional activity of Hippo pathway,while other candidate pathways,including MAPK/ERK,MAPK/JNK,NFκB,and Notch,remained unchanged.Western blot confirmed that USP39 could significantly affect the protein level of TAZ,while other core components of Hippo,including LATS1,LATS2 and YAP were not affected.IHC staining results from animal experiments and human glioma tissues also confirmed that USP39 was positively correlated with TAZ.4.USP39 controls TAZ protein expression through TAZ mRNA maturation.Further results from qRT-PCR showed that USP39 knockdown significantly reduced TAZ spliced transcripts and increased unspliced transcripts,and the splicing efficiency of TAZ mRNA was significantly inhibited.RIP also confirmed that USP39 protein interacted with TAZ mRNA.5.TAZ transcriptional activity is reduced after silencing USP39.Results from Western blot analyzed nuclear and cytoplasmic proteins as well as cell immunofluorescence showed that inhibition of USP39 reduced the ratio of TAZ nuclear to TAZ total protein levels.In addition,qRT-PCR showed that knockdown of USP39 decreased the transcriptional levels of TAZ target genes,BIRC5,E2F1 and MYC.6.Ectopic expression of USP39 and TAZ restores malignant characteristics in vitro and in vivo when USP39 is lost in GBM.Results from CCK-8 showed that USP39 knockdown led to decreased cell proliferation ability,which was significantly reversed by USP39 or TAZ overexpression.Luciferase activity from a direct read-out of Hippo pathway and TAZ target genes mRNA levels were rescued in USP39 knockdown combined with additional overexpression of USP39 or TAZ group.Similar results were observed in animal experiments.Conclusion1.USP39 is highly expressed in high-grade glioma and is an important oncogene in GBM;2.USP3 9 affects downstream transcriptional activity of Hippo pathway by regulating protein level and nuclear localization of transcription coactivator TAZ,and this effect is independent of classical upstream kinase LATS 1/2.3.USP39 protein can promote the splicing efficiency of TAZ precursor mRNA;4.USP39,a novel regulator of Hippo pathway,is a potential diagnostic marker and drug target for GBM.Part Ⅱ:PMEPA1 isoform a drives progression of glioblastoma by promoting protein degradation of the Hippo pathway kinase LATS1ObjectiveTo analyze the expression levels of PMEPA1 and its transcripts in human glioma tissues and GBM cell lines and explore the influences of the main transcript PMEPA1a on the malignant biological behaviors of GBM cells;To identify the specific interacting proteins of PMEPA1a and to clarify the biological functions of the interaction between PMEPA1a and LATS1 proteins and the effects on downstream signaling in GBM.Methods1.IHC was performed to determine the expression levels of PMEPA1 protein in human glioma and normal brain tissue.qRT-PCR was used to detect the relative expression levels of the PMEPA1 transcripts in our GBM cell lines and brain tissues,using PCR primers specific for PMEPA1a,PMEPA1b,PMEPA1c,and PMEPA1d transcripts.2.Lentiviruses expressing shRNAs targeting PMEPA1a and full-length PMEPA1a were designed and synthesized.CCK-8,and Transwell assay were utilized to evaluate cell proliferation,invasion and migration.Orthotopic xenograft models were generated to track the growth of glioma cells in vivo,as well as a Kaplan-Meier survival curve to observe the survival time of tumor-bearing mice.3.Co-IP(Co-Immunoprecipitation)followed by LC-MS/MS(Liquid chromatography-tandem mass spectrometry)were performed to identify partners of PMEPA1a in GBM cells;Co-IP was used to determine the interaction between PMEPA1a and LATS1;Deletion mapping was used to explore the functional domains responsible for PMEPA1a-LATS1 complex formation.4.Western blot was used to investigate the effects of PMEPA1a on the protein levels of LATS1 and its downstream signaling pathways.Immunofluorescence,nuclear and cytoplasmic extracts followed by Western blot,Hippo luciferase reporter assay and qRT-PCR was used to verify the transcriptional activation of Hippo.5.The proteasome inhibitor MG132 and protein synthesis inhibitor CHX(Cycloheximide)were used to pretreat with GBM cells.Western blot was used to detect the effects of knockdown or overexpression of PMEPA1a on half-life of LATS1 protein.In vivo ubiquitination assay was used to analyze the total polyubiquitination levels of LATS1 protein in response to PMEPA1a.6.PMEPA1a-NEDD4 interaction deficient mutant(PMEPA1a-Y161/232A)and LATS1NEDD4 interaction deficient mutant(LATS1-Y376/559A)were constructed;Co-IP was used to explore the effect of PMEPA1a/NEDD4/LATS1 complex formation on maintaining the stability of LATS 1 protein.The growth-promoting effects of PMEPA1a-Y161/232A and PMEPA1a-LATS1 interaction deficient truncated mutant(PMEPA1a-A100-165aa)on GBM cell proliferation,invasion and migration were investigated in vitro and in vivo.7.Lentiviral constructs containing LATS1-or YAP-shRNAs and expressing full-length LATS1 or YAP-5SA mutant were established for rescue experiments.The regulation of PMEPA1a-LATS1 was verified in primary GBM#P3 cells.IHC staining was used to detect the correlation between PMEPA1a and LATS1,P-YAP(Ser127)or CYR61 expression in human glioma samples.Results1.PMEPA1 protein is highly expressed in human gliomas.Results from IHC and Western blot showed that PMEPA1 expression was up-regulated in high grade glioma compared with normal brain and low grade glioma.PMEPA1 protein levels were also increased in glioma cell lines relative to normal human astrocytes,except in the case of U87MG where PMEPA1 were nearly undetectable.Results from qRT-PCR confirmed that PMEPA1a was the most highly expressed transcript,with relative higher expression levels in GBM cells,normal brain and glioma tissues.2.PMEPA1a enhances glioma cell proliferation,migration and invasion both in vitro and in vivo.Results from CCK-8,colony formation assay and Transwell assay indicated that compared with the control group,knockdown of PMEPA1a significantly inhibited the proliferation,invasion and migration ability of cells,while the cells overexpressing PMEPA1a showed significantly enhanced cell viability,cell migration and invasion.Compared with the control group,orthotopic tumor bearing mice implanted with cells PMEPA1a knockdown showed a longer median survival time,while those implanted with cells overexpressed PMEPA1a showed the opposite results.3.PMEPA1a forms a complex with LATS1.Results from Co-IP followed by LC-MS/MS showed that LATS1 was one of the interacting proteins of PMEPA1a.Deletion mapping and Co-IP suggested that amino acids 100-165 in PMEPA1a were necessary for binding to LATS1,whereas amino acids 351-700 in LATS1 constituted the PMEP A 1 a-interacting domain.4.PMEPAla regulates downstream transcriptional activation of Hippo pathway through LATS1.Results from Western blot showed that knockdown or overexpression of PMEPAla significantly affected the activity of LATS1 and its downstream proteins.Immunofluorescence staining and Western blot of YAP showed that PMEPA1a promoted nuclear localization of YAP.Hippo luciferase reporter assay and qRT-PCR indicated that PMEPAla promoted transcriptional coactivation of downstream target genes of Hippo pathway.5.PMEPA1a enhances proteasomal degradation of LATS1.Exogenous overexpression of PMEPAla led to a decrease in the expression level of LATS1 protein,which was reversed by the use of proteasomal inhibitor MG 132.Results from CHX chase assay showed that PMEPA1a knockdown significantly extended the half-life ofLATS1 protein compared with the control group,while PMEPA1a overexpression was the opposite.In vivo ubiquitination assay also supported the above results.6.PMEPA1a enhances the interaction between NEDD4 and LATS1 protein to promote LATS1 ubiquitination.Results from Co-IPs showed that the levels of LATS1 in complex with NEDD4 increased or decreased in response to changes in PMEPA1a levels.Knockdown or overexpression of PMEPA1a will change the binding capacity between LATS1 and NEDD4 proteins accordingly.Consistent with the previous results,exogenous overexpression of PMEPA1a wild-type significantly induced a decrease in LATS1 protein and an increase in poly-ubiquitination of LATS1.This change can be reversed by NEDD4 knockdown.However,exogenous overexpression of PMEPA1a-Y161/232A mutant does not produce such a result similar to the wild-type.In addition,overexpression of PMEPA1a did not change the protein level and the ubiquitination modification level of LATS1-Y376/5 59A mutant.Compared with the control group,cells overexpressing PMEPA1 A-Y161/232A or PMEPA1a--Δ100-165aa mutant had no significant changes in biological behaviors in vitro and in vivo.7.PMEPA1a signaling is mediated via the Hippo pathway.Results suggested that compared with the control group,weakened promoting effects of GBM cells by knockdown of PMEPAla was rescued by knockdown of LATS1 or overexpression of YAP-5SA.On the contrary,PMEPA1a overexpression followed by LATS1 overexpression or YAP knockdown significantly abrogated the malignant biological behaviors of GBM cells induced by PMEPAla overexpression alone.The primary GBM cell line P3 confirmed the regulatory effect of PMEPA1a on LATS1.IHC scores revealed a negative correlation between expression of PMEPA1a and LATS1,and a positive correlation between PMEPA1a and CYR61 expression in human gliomas.Conclusion1.PMEPA1a was highly expressed in gliomas,and the expression increased with the increase of tumor grade;2.PMEPA1a plays an important role in maintaining malignant biological behaviors such as proliferation,migration and invasion of glioma cells;3.By recruiting ubiquitin ligase NEDD4,PMEPA1a leads to ubiquitination and degradation of LATS1,the upstream kinase of Hippo pathway,thereby activates downstream Hippo target genes transcription;4.The detection or drug development of PMEPA1a,a novel regulatory factor of Hippo pathway,provides new strategies for clinical diagnosis and treatment of GBM. |
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