The Roles And Mechanisms Of TNFAIP8 In Acute Myeloid Leukemia Chemoresistance

BackgroundAcute myeloid leukemia(AML),a rapidly progressing hematopoietic malignancy,is the most common form of acute leukemia in adults.Nowadays,extensive use of standard therapy induces complete remission(CR)in approximately 50%to 70%of AML patients,but about 76%of patients relapse or die eventually,indicating that drug resistance has become a major obstacle to an optimistic prognosis.Therefore,it is of vital importance to explore the molecular rationale underlying drug resistance in AML.Apoptosis induction underlies the therapeutic effects of most conventional antineoplastic agents.However,an expanding body of evidence has illustrated that dysregulated expression of anti-apoptotic molecules,such as BCL-2 and MCL-1,renders malignant cells resistant to the pro-apoptotic effects of cytotoxic agents.Inhibition of these molecules has increased chemosensitivity in AML,underlining the crucial role of apoptosis dysregulation in AML chemoresistance.Nevertheless,resistance to the currently available targeted inhibitors has also emerged afterwards,suggesting inter-complementary effects among anti-apoptotic molecules.Thus there arise demands for further exploration of new candidates in apoptosis regulation network in AML.Tumor necrosis factor ?-induced protein 8(TNFAIP8)is a new anti-apoptotic molecule which was first found in human head and neck squamous cell carcinoma.Recent studies have confirmed its relation to tumorigenesis,progression and invasion.The aberrant expression of TNFAIP8 has been successively validated in gastric cancer,esophageal squamous carcinoma,colon cancer and pancreatic cancer.Tumors with high TNFAIP8 expression are usually poorly differentiated,more invasive and tend to metastasize early,thus causing poor prognosis.The involvement of TNFAIP8 in chemoresistance has also been documented,albeit understudied.Current studies focused on the role of TNFAIP8 in solid tumor with a lack of research on TNFAIP8 in AML.TNFAIP8 has also been found to be highly expressed in leukemia cell lines K562 and MOLT4.Detailed roles and mechanisms of TNFAIP8 in AML remain unclear.ObjectivesPart ?In this study we sought to examine the expression of TNFAIP8 in AML and investigate the potential regulatory mechanism of TNFAIP8 expression,which may provide novel diagnostic marker for AML.Part ?In this study we sought to investigate the functional significance of TNFAIP8 in leukemia drug resistance and to explore the underlying mechanisms,which may further extend our understanding of the interaction network in AML and provide novel therapeutic targets.MethodsPart ?1.Patient samples:Bone marrow(BM)samples from 60 AML patients and 17 control donors were obtained.TNFAIP8 levels were determined in AML patient bone marrow samples and control samples using real time qPCR and western blot.2.Cell lines and cell culture:THP1,U937,K562,K562/A02,K562/G01 and HL60/ADR cells were cultured in RPMI 1640 medium,HL60 cells were cultured in IMDM medium,293T cells were cultured in DMEM medium(with 10%heat-inactivated fetal calf serum,Gibco;penicillin and streptomycin,Invitrogen;37?,5%CO2,in humidified incubator).TNFAIP8 levels were determined in AML cell lines using real time qPCR and western blot.3.Lentiviral transduction:Cells were infected with lentivirus repressing or expressing E74 like ETS transcription factor 1(ELF1)for 24 h and selected by puromycin after 96 h.ELF 1 and TNFAIP8 levels were determined using real time qPCR and western blot.4.Luciferase assay:A region of genomic DNA comprising the 1.3 kb fragment located in the 5'-flanking region of the human TNFAIP8 gene was cloned in front of the firefly luciferase gene in the vector pGL4.10.The resultant plasmid(TNFAIP8-Prom)was transfected into 293T cells and luciferase activity was measured by a luminometer to reflect TNFAIP8 promoter activity.Then we co-transfected 293T cells with ELF1 expression plasmid and TNFAIP8-Prom plasmid and luciferase activity was measured to reflect the effect of ELF1 on transcription of TNFAIP8.5.Chromatin immunoprecipitation(ChIP):Chromatin fragments derived from K562,K562/A02,HL60 and HL60/ADR cells were immunoprecipitated with ELF1 antibody.The 5'-upstream region of human TNFAIP8 gene(-1154 to-1142,TNFAIP8-promoter)was detected by PCR of genomic DNA using the primers.6.Statistical analyses:Statistical analyses were performed with SPSS statistics 25.0,Differences between 2 groups were analyzed using an unpaired Student t-test.Differences between 3 or 4 groups were analyzed using one-way ANOVA or two-way ANOVA followed by LSD test with normally distributed data.The Mann-Whitney U test was used for cases with unequal variances.P<0.05 was considered statistically significant.Part ?1.Cell lines and cell culture:K562,K562/A02 and HL60/ADR cells were cultured in RPMI 1640 medium,HL60 cells were cultured in IMDM medium(with 10%heat-inactivated fetal calf serum,Gibco;penicillin and streptomycin,Invitrogen;37?,5%CO2,in humidified incubator).2.Lentiviral transduction:Cells were infected with lentivirus repressing or expressing TNFAIP8 for 24 h and selected by puromycin after 96 h.TNFAIP8 levels were determined using real time qPCR and Western blot.3.Cell proliferation,apoptosis and IC50 testing assays:Transfected cells were seeded into culture plates and CCK8 assay was used to examine the proliferation;48h after adding chemotherapeutics,CCK8 assay and flow cytometry were performed to examine cell viability and apoptosis,respectively.4.Co-immunoprecipitation(CoIP):Co-immunoprecipitation experiments were performed to ascertain whether TNFAIP8 associates with Rac1.We transfected 293T cells and AML cells with Flag-tagged TNFAIP8 and immunoprecipitated cell lysates with anti-Flag Ab or control IgG.Subsequent western blot analysis revealed the specific co-precipitation of endogenous Racl by anti-Flag Ab.5.Rac1 activation assay:AML cells with TNFAIP8 upregulation or downregulation were serum-starved(16 h),then treated with doxorubicin(4 h,0.5?g/mL for K562 and HL60,5 ?g/mL for K562/A02 and HL60/ADR).A Rac1 activation assay biochem kit(Cytoskeleton)was used to perform PAK pull-down assays according to the manufacturer's protocol.6.RNA sequencing:Gene transcriptome profiles were detected to identify differentially expressed mRNAs between K562/A02-shTNFAIP8 and K562/A02-shNC cells,followed by the GO and pathway analysis.7.In vivo model of AML:C57BL/6 mice were intravenously injected with C1498 cells transduced with a non-targeted short hairpin RNA or TNFAIP8 shRNA.Mice were monitored daily for evidence of leukemia.On day 24,mice were euthanized and livers and spleens were measured.Leukemia infiltration into peripheral blood,bone marrow,spleen and liver was evaluated by flow cytometry detection of GFP-positive cells and hematoxylin and eosin staining.Liver,spleen and bone marrow tissues of mice were fixed(10%formaldehyde),paraffin-embedded,sectioned(4 ?m),and stained with hematoxylin and eosin.Immunohistochemical detection of TNFAIP8 and p-ERK1/2(CST)was performed on bone marrow samples.Survival was also followed and presented with a Kaplan-Meier survival plot.ResultsPart ?1.TNFAIP8 expression in primary AML samples and cell lines1.1 TNFAIP8 expression was significantly increased in AML compared to control.TNFAIP8 expression was higher in patients with newly diagnosed AML and patients with relapsed/refractory AML than in patients with complete remission,and patients with relapsed/refractory AML exhibited the highest level.1.2 TNFAIP8 is universally expressed in 7 AML cell lines compared with 293T cells.Among AML cell lines,parental sensitive AML cell lines,K562 and HL60,showed lower levels of TNFAIP8 than corresponding chemoresistant AML cell lines,K562/A02 and HL60/ADR.2.The regulatory mechanism of TNFAIP8 expression2.1 Analysis of data from CHIPBase showed that ELF1 transcription factor possesses the most potential binding sites of the TNFAIP8 promoter.2.2 Upregulation of ELF1 increased TNFAIP8 expression levels,while downregulation of ELF 1 decreased TNFAIP8 expression levels,indicating that ELF1 may promote human TNFAIP8 gene transcription.2.3 TNFAIP8-Prom plasmid which comprised the 1.3 kb fragment located in the 5'-flanking region of the human TNFAIP8 gene was transfected into 293T cells and luciferase activity was measured by a luminometer to reflect TNFAIP8 promoter activity.TNFAIP8-Prom plasmid-transfected cells had significantly higher luciferase activity compared with controls,indicating that the 1.3-kb fragment contains the functional promoter region of the human TNFAIP8 gene.Then we co-transfected 293 T cells with ELF 1 expression plasmid and TNFAIP8-Prom plasmid and found that overexpression of ELF 1 caused an increase in luciferase expression from TNFAIP8-Prom.These data support a role for ELF1 in transcriptional regulation of TNFAIP8.2.4 ChIP-qPCR analysis showed that ELF1 antibody effectively immunoprecipitated the sequence from-1154 to-1142 bp of TNFAIP8 promoter,indicating that the site from-1154 to-1142 bp of the TNFAIP8 promoter was essential for ELF 1 regulation.3.There was a positive correlation between ELF1 and TNFAIP8 expression in AML patients3.1 ELF1 was significantly elevated in AML patients compared with controls.3.2 Parental sensitive AML cell lines,K562 and HL60,showed lower levels of ELF1 than corresponding chemoresistant AML cell lines,K562/A02 and HL60/ADR.3.3 A positive correlation was observed between ELF1 and TNFAIP8 expression in AML patients.Part ?1.The effect of TNFAIP8 suppression on resistant AML cells1.1 RT-qPCR and western blot analysis verified suppression of TNFAIP8 in resistant AML cell lines transduced with TNFAIP8 shRNA.1.2 CCK8 assay showed that TNFAIP8 downregulation significantly inhibited cell growth.1.3 TNFAIP8 knockdown increased apoptosis induced by chemotherapeutics through flow cytometry analysis.1.4 CCK8 assay confirmed that TNFAIP8 knockdown reduced the IC50 of chemotherapeutics.2.The effect of TNFAIP8 overexpression on sensitive AML cells2.1 RT-qPCR and western blot analysis verified overexpression of TNFAIP8 in sensitive AML cell lines infected with TNFAIP8-expressing lentivirus.2.2 CCK8 assay showed that TNFAIP8 upregulation significantly promoted cell growth.2.3 TNFAIP8 upregulation reduced apoptosis induced by chemotherapeutics through flow cytometry analysis.2.4 CCK8 assay confirmed that TNFAIP8 upregulation increased the IC50 of chemotherapeutics.3.TNFAIP8 promotes anti-apoptotic phenotype of AML cells in an ERK-dependent manner under pressure of chemotherapeutics3.1 RNA-sequencing analysis showed that TNFAIP8 suppression in AML cells under the pressure of chemotherapeutics dramatically changed expression of genes associated with cell proliferation,apoptosis and oncogenesis.3.2 Immunoblotting analysis confirmed that TNFAIP8 knockdown suppressed phosphorylation of ERK in AML chemoresistant cells after 8-h treatment of doxorubicin,while TNFAIP8 overexpression increased ERK phosphorylation in AML chemosensitive cells.3.3 ERK inhibition reversed growth advantage of TNFAIP8-transduced cells and partially abrogated the down-regulation on the apoptotic level by overexpressing TNFAIP8,indicating that TNFAIP8 may affect proliferative capacity and chemotherapy-induced apoptosis in an ERK-dependent manner.4.TNFAIP8 promotes ERK activation by interacting with Racl,an MAPK upstream factor4.1 Co-immunoprecipitation of Flag-tagged TNFAIP8 and Rac1 was then detected in AML cells,confirming the physical interaction between TNFAIP8 and Rac1 in vitro.4.2 TNFAIP8 knockdown in AML cells reduced levels of GTP-Rac1 and vice versa,indicating that TNFAIP8 promotes Rac1 activation.4.3 Increased ERK activation in TNFAIP8-transduced cells was attenuated by Rac1 inhibitor EHOP-016,suggesting that TNFAIP8 regulates the ERK signaling pathway by interacting with Rac1.5.TNFAIP8 suppression decelerates AML development and progression in vivo5.1 In vivo model of AML:Two weeks after injection of C1498 cells,emaciation as well as tetraplegia were observed in two groups,which is indicative of disease onset and progression.Mice were sacrificed 24 days after injection and leukemia burden was evaluated.FACS analysis showed reduced circulating GFP+leukemia cells in shTNFAIP8 AML mice.Spleen and liver enlargement were significantly less in shTNFAIP8 AML mice.H&E analysis confirmed lower leukemia infiltration in the spleen,liver and bone marrow in shTNFAIP8 AML mice.5.2 The overall survival in shTNFAIP8 AML mice was longer than that in control group.5.3 Immunohistochemistry studies showed a partial lower expression of phosphorylated ERK in shTNFAIP8 AML mice,confirming that TNFAIP8 promotes AML cell proliferation by activating ERK signaling pathway.ConclusionsPart ?1.TNFAIP8 expression was upregulated in relapsed or refractory AML patients and resistant AML cell lines,indicating that TNFAIP8 may be related to AML chemoresistance.2.ELF1 promotes human TNFAIP8 gene transcription in AML by binding the site from-1154 to-1142 bp of the TNFAIP8 promoter.3.The expression of ELF1 was upregulated in AML patients and resistant AML cell lines,and was positively correlated with TNFAIP8 expression,suggesting that ELF1 regulates the abnormal expression of TNFAIP8 in AML.Part ?1.TNFAIP8 promotes cell proliferation and drug resistance,and protects cells from apoptosis induced by chemotherapeutics in AML.2.TNFAIP8 promotes ERK activation by interacting with Racl,an MAPK upstream factor and facilitates anti-apoptotic phenotype of AML cells under pressure of chemotherapeutics,which contributes to AML chemoresistance ultimately.3.TNFAIP8 suppression decelerates AML development and progression in vivo.Targeting TNFAIP8 would be a promising therapeutic strategy for AML.