| Background and objectiveNatural killer/T-cell lymphoma(NKTCL)is a highly aggressive non-Hodgkin’s lymphoma with rapid progression and short survival.Compared with B-cell lymphoma,the efficacy of chemotherapy for advanced-stage NKTCL is relatively poor.NKTCL is insensitive to traditional CHOP chemotherapy,and the patients’5-year overall survival rate is less than 50%,which may be attributed to P-gp protein-mediated multidrug resistance.Although chemotherapeutic regimens that are based on L-asparaginase or gemcitabine can relatively improve the overall survival(OS),approximatively 30%of patients relapse or exhibit refractory disease within a year.Therefore,drug resistance may account for these chemotherapy failures.Researchers have explored multiple biological mechanisms such as chromosomal locus deletions,tumor suppressor genes’ disorder,activation of proliferation-related signal pathways,immune checkpoint exception,and epigenetic abnormalities.However,the use of related immunotherapy and targeted therapy is limited due to these complications and individual differences.Therefore,it is urgent for the clinic to elucidate the mechanisms of chemotherapeutic resistance and improve the therapeutic efficacy.We have identified NKTCL side population(SP)cell lines,characterized with expression of surface markers of cancer stem cells(CSCs)and the drug resistant protein ATP binding cassette subfamily G member 2(ABCG2).SP cells mainly embodied a:1.Stronger chemotherapeutic resistance compared to ordinary NKTCL cells and doxorubicin-resistant NKTCL cells;2.Stem cell-like biological characteristics.SP cells are CSC precursor cells that showed similarities in experimental technology and mechanism with CSCs and that can be used to sort and identify CSCs.Recent studies have shown that CSCs are closely related to the development of hematopoietic malignancies and that epigenetic regulation may have a critical role in CSCs compared to tumor cells or other non-stem cells.N6-methyladenosine(m6A)is the most abundant modification in eukaryotic messenger RNAs(mRNAs)whose activity has been shown in almost all types of cells.The m6A modification can maintain the pluripotency of embryonic stem cells,participate in lineage differentiation of hematopoietic stem cells,and in somatic cells’reprogramming into pluripotent stem cells.It can also enrich breast CSCs and increase the leukemia CSC transformation.Therefore,the RNA m6A plays an important role in CSC’ biological functions.ALKB homolog 5(ALKB homolog 5,ALKBH5)is the second m6A demethylase that has been reported to impact hematological malignancies.ALKBH5 is highly expressed in acute myeloid leukemia(AML)and B cell lymphoma.It can maintain leukemic stem cells and promote AML progression by regulating TACC3 and receptor tyrosine kinase AXL.Most importantly,AXL is closely related to AML drug resistance.These functions suggest that ALKBH5 may contribute to the development of hematological malignancies and their associated CSCs,especially through promoting drug resistance mechanisms.This study aimed at investigating ALKBH5 expression and functions in NKTCL and SP cells.For this,immunohistochemistry(IHC)assays were conducted to determine ALKBH5 expression level in NKTCL tissues,and its correlation with NKTCL prognosis.ALKBH5 gain-or loss-of-fuction assays were used to detect its role in NKTCL SP cells.A signaling pathway which may be related to ABCG2 function in NKTCL SP cells was identified by transcriptome sequencing,immunofluorescence and pathway regulators.Dual luciferase labeling,qRT-PCR,and Western blotting were used to verify the interactional relationship between signaling pathways and ABCG2.ABCG2 gain-or loss-of-function assays were used to detect ABCG2 influence on chemotherapeutic drug sensitivity in NKTCL SP cells in vivo and vitro.Moreover,ALKBH5 gene function,molecular mechanism and effect on drug sensitivity were further evaluated using mice xenograft models.The results of this study provide novel insights into the complex drug resistance mechanism associated with m6A modification,and provide a theoretical basis for the biological functions of SP cells and their potential therapeutic targeting in NKTCL patients.Part Ⅰ.Expression and clinical significance of ALKBH5 in NK/T cell lymphomaMethodsALKBH5 expression was detected by immunohistochemical(IHC)in the patients of NKTCL and lymph node reactive hyperplasia and analyzed with the patients’ prognosis.ResultsALKBH5 expression in NKTCL tissues was significantly higher in comparison with that in lymph node reactive hyperplasia tissues(P=0.002).In NKTCL patients,ALKBH5 high expression was correlated with high Ann Arbor stage(P=0.001)and short OS(overall survival)(P=0.018)and PFS(progression free survival)(P=0.025).In NKTCL patients,ALKBH5(P=0.048)was independent OS prognostic factors.Summary1.ALKBH5 expression is higher in NKTCL tumor tissues and its expression was significantly related with the Ann Arbor stage.2.ALKBH5 is an independent OS poor prognostic factor in NKTCL patients.Part Ⅱ.Studies of ALKBH5 expression,biological function,and mechanisms in NKTCL SP cells.Methods1.The m6A content of the NKTCL cell lines(YT,SNK-6,KHYG-1)and NKTCL SP cell lines(YT-SP,SNK-6-SP,KHYG-1-SP)were detected by the microplate reader colorimetric method.2.qRT-PCR and Western blotting methods were utilized to examine ALKBH5 expression.3.Lentiviral transfection was used to generate YT-SP and SNK-6-SP ALKBH5-overexpressing cells(Lv-ALKBH5),ALKBH5-silenced(Sh-ALKBH5),and their respective control cells(Lv-NC and Sh-NC).4.Gain-or loss-of-function assays were performed to investigate ALKBH5 role in determing the biological characteristics of NKTCL SP cells,using the Cell Counting Kit-8(CCK-8)assay,EdU,Annexin V,and Transwell assays.5.To investigate the activity changes of potential signaling pathways that may be related to ABCG2 in YT-SP-Sh-ALKBH5(ALKBH5-silenced)cells,using transcriptome sequencing,immunofluorescence,qRT-PCR,dual fluorescence enzyme labeling,Western blotting,and pathway regulators.Results1.The m6A content of NKTCL SP cells were lower than that of NKTCL cells(P<0.0001).2.ALKBH5 mRNA and protein expression levels were upregulated in NKTCL SP cells compared to those in NKTCL cells(P<0.0001).3.ALKBH5 mRNA and protein expression levels were upregulated in YT-SP and SNK-6-SP cells following transduction with Lv-ALKBH5(P<0.001),and ALKBH5 mRNA and protein expression levels were downregulated following transduction with Sh-ALKBH5(P<0.01).4.In YT-SP and SNK-6-SP cells,an enhanced ALKBH5 expression can be accompanied by higher cell proliferation(P<0.01)and migration(P<0.001),and fewer apoptotic cells(P<0.01);whereas,ALKBH5 silencing can be accompanied by lower cell proliferation(P<0.05)and migration(P<0.01),and more apoptotic cells(P<0.01).5.RNA-sequence and KEGG analysis showed genes’ difference enrichment in the Wnt/β-catenin signaling pathway,and a downregulation of the transcription factor 4(TCF4)in this pathway.The immunofluorescence assay verified that the Wnt pathway proteins,β-catenin and TCF4,were downregulated in the YT-SP-ShALKBH5 group compared with those in the YT-SP-Sh-NC group(P<0.0001).Based on the results of the ABC family protein detection results,we found that ALKBH5 may regulate ABCG2 through the Wnt/β-catenin signaling pathway.Western blotting analysis showed that Wnt/β-catenin pathway inhibitors can reduce ABCG2 protein expression in YT-SP cells.The dual luciferase labeling experiment detected a binding of TCF4 to the ABCG2 promoter region and an upregulation of its expression(P=0.0012).6.In YT-SP-SH-ALKBH5 cells,Wnt/β-catenin agonist significantly upregulated the expression of TCF4 and ABCG2 and also significantly reversed the decreased cell proliferation and the increased apoptosis ability(P<0.0001).Summary1.ALKBH5 was upregulated in NKTCL SP cells compared with that in NKTCL cells.ALKBH5 decreased m6A content,increased cell proliferation and migration,and decreased apoptosis in NKTCL SP cells.2.ALKBH5 could positively regulate ABCG2 through the Wnt/β-catenin/TCF4 signaling pathway.The downregulation of TCF4 and ABCG2 protein expression levels and the changes in biological characteristics that were caused by ALKBH5 silencing could be restored by the Wnt signaling agonist.Part Ⅲ.Studies of the multidrug resistance characteristics of ABCG2,the targeted gene of ALKBH5 in NKTCL SP cellsMethods1.Detection of ABCG2 expression in SNK-6-SP cells by immunofluorescence and Western blotting.2.Lentiviral transfection was used to construct SNK-6-SP cells with ABCG2 overexpressing(Lv-ABCG2),ABCG2 silenced(Sh-ABCG2),and their respective control cells(Lv-NC and Sh-NC).3.IC50 values of NKTCL chemotherapeutic drugs(doxorubicin,cytarabine,cisplatin,gemcitabine,and L-asparaginase)in Lv-ABCG2 and Sh-ABCG2 cells were tested by the CCK-8.4.IC50 values of doxorubicin and gemcitabine in Lv-NC and Lv-ABCG2 cells were tested following treatment with in the ABCG2 inhibitor,Pelitinib.5.Gain-or loss-of-function assays were performed to investigate the the role of ABCG2 in determing the biological characteristics of NKTCL SP cells that were treated with gemcitabine.6.BALB/c nude mice,Lv-NC and Lv-ABCG2 cells were used to generate a subcutaneous xenograft tumor model for investigating ABCG2 effect on gemcitabine sensitivity.Results1.ABCG2 was significantly upregulated in SNK-6-SP cells compared with that in SNK-6 cells(P<0.001).2.ABCG2 mRNA and protein expression levels were upregulated in SNK-6-SP cells following transduction with Lv-ABCG2.ABCG2 mRNA and protein rxpression levels were downregulated in SNK-6-SP cells following transduction with Sh-ABCG2(P<0.001).3.The IC50 values of doxorubicin,cytarabine,cisplatin,and gemcitabine were significantly higher(P<0.0001)in the Lv-ABCG2 group compared with those in the Lv-NC group,but there was no difference in L-asparaginase(P=0.0822).The IC50 values of doxorubicin,cytarabine,cisplatin,and gemcitabine were significantly lower in the Sh-ABCG2 group(P<0.0001),but there was no difference in L-asparaginase(P=0.6952).4.ABCG2 inhibitor Pelitinib could reduce the IC50 values of doxorubicin and gemcitabine in SNK-6-SP cells(P<0.001).5.ABCG2 could resist gemcitabine,increase clonal ability and decrease apoptosis(P<0.001).6.ABCG2 could resist gemcitabine in the NKTCL SP transplanted tumor.Tumors’ volume and weight in the Lv-ABCG2 group were significantly increased(P<0.05).Summary1.The expression of ABCG2 was increased in the NKTCL SP cells.2.ABCG2 increased the resistance of NKTCL SP cells to doxorubicin,cytarabine,cisplatin,and gemcitabine,but had no effect on L-asparaginase.Pelitinib could inhibit ABCG2-mediated resistance.3.ABCG2 could resist the effect of gemcitabine,enhance the clonal ability of NKTCL SP cells,reduce apoptosis,and promote the formation of of NKTCL SP subcutaneous xenograft tumors.Part Ⅳ.In vivo validation of ALKBH5 function and mechanisms of action in NKTCL SP cellsMethods1.Generation of mice models for the validation of ALKBH5 function and mechanisms of action.1.1.YT-SP cells that were stably transduced with Lv-ALKBH5,Lv-NC,Sh-ALKBH5 and Sh-NC were injected into the flanks of BALB/c nude mice.1.2.The general states of the mice were monitored and the tumors’ volume and mice weights recorded.1.3.The murine tumors in the Sh-NC and Sh-ALKBH5 groups were detected by H&E staining and IHC for the NKTCL surface markers,CD56 and Perforin,the proliferation index Ki-67,ALKBH5,β-catenin and ABCG2.2.Generation of mice models to test the effect of targeting ALKBH5 on chemotherapeutic sensitivity.Gemcitabine was intraperitoneally administered to the Sh-NC and Sh-ALKBH5 mice twice a week from day nine.Results1.Tumors’ volume and weight in the Lv-ALKBH5 group of mice significantly increased,while the tumors’ volume and weight in the Sh-ALKBH5 group of mice were significantly reduced(P<0.01).H&E staining indicated the general characteristics of the malignant tumors.IHC results showed positivity for CD56 and Perforin.Ki-67,ALKBH5,β-catenin,and ABCG2 protein expression levels were significantly lower in Sh-ALKBH5 group.2.Under gemcitabine treatment,the tumors’ volume in the Sh-ALKBH5 group was significantly smaller(P<0.05).Summary1.ALKBH5 promoted NKTCL SP tumor growth in mice.2.Interference with ALKBH5 could inhibit the expression of proteins in the Wnt/β-catenin signaling pathway and ABCG2,the growth of mouse tumors,and improve the sensitivity of tumor cells to chemotherapy.Conclusions1.ALKBH5 was upregulated in tumor tissues of NKTCL patients,and was an independent OS prognostic factor.2.ALKBH5 promoted NKTCL SP cells’ proliferation and migration,inhibited apoptosis,and its expression was closely related to the malignancy of NKTCL.ALKBH5 regulated ABCG2 through the Wnt/β-catenin signaling pathway to mediate the multidrug resistance of NKTCL SP cells.3.ALKBH5 targeting could inhibit tumor growth,improve chemotherapy sensitivity,and therefore,could be used as a potential therapeutic target for NKTCL. |
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