The Role Of FoxM1-induced DNA Damage Response In Imatinib-resistant Chronic Myeloid Leukemia And It's Regulatory Mechanisms

BackgroundChronic myeloid leukemia(CML)is a malignant clonal disease derived from hematopoietic stem cells.It's clinical phases can be divided into chronic phase(CP),accelerated phase(AP)and blastic crisis(BP).The prognosis is very poor when it is in the BP.The Philadelphia chromosome(Ph)and BCR-ABL fusion gene are landmark changes in CML.The tyrosine kinase inhibitor,imatinib(IM),which is capable of targeting BCR-ABL fusion protein,is the first-line drug for CML treatment.However,the mutation and amplification of BCR-ABL gene lead to the increase of tyrosine kinase activity,which is the main cause of IM resistance in chronic myeloid leukemia,and restricts the improvement of CML seriously,so it has become a major challenge for targeted therapy of CML.Therefore,to explore the mechanism of BCR-ABL dependent IM resistance and its reversal strategy is the focus of current clinical treatment of CML.There is growing evidence that DNA damage repair is critical to the maintenance of integrity and stability of the genome,and genomic instability caused by abnormal function is closely related to the progression of CML and the development of drug resistance.Studies have shown that blocking the repair of DNA double-strand breaks can enhance the sensitivity of CML cells,Mo7e-P210 IMR and Baf3-P210 IMR,to IM.Another study reported that down-regulation of RAD51 expression by the small molecule inhibitor,IBR2,could inhibitproliferation and increased apoptosis of T315I-Ba/F3,a kind of IM drug-resistant cells,.It enhanced cells' sensitivity to IMsignificantly.So it can be seen that DNA damage repair plays an important role in IM resistance of CML cells,which may be a new mechanism of IM resistance,and its mechanism is important to overcome IM resistance.Recent studies have shown that proliferation-specific oncogene FoxMl(Forkhead box protein M1)can target DNA damage repair proteins,such as BRIP1,RAD51,BRCA2,regulating DNA damage repair,in glioblastoma,and play an important role in chemotherapeutic drugsresistance of breast cancer and other tumors.FoxMl regulates the transcription of cell cycle-specific genes,which is closely related to cell proliferation,embryonic development,tissue regeneration,DNA damage repair and tumor formation.Recent studies have shown that FoxMl is overexpressed in breast cancer and other tumors.The tumors with high expression of FoxM1 have characteristics such as,poor differentiation,high degree of malignancy,easy to distant metastasis,and poor clinical prognosis.FoxMl plays a key role in the development and progression of tumors.It affects the stability of the genome and the process of mitosisby regulating the expression of its downstream tumor-related genes,thereby promotes tumor growth,invasion and metastasis.In conclusion,FoxMl is a necessary transcription factor for tumor proliferation.It can provide new therapeutic solutions to reverse IM resistance in CMLand new target for clinical treatment by investigating the role and mechanism of FoxMl.ObjectiveThe aim of this study was to investigate the expression of FoxMl in bone marrow of patients with CML and normal controls.Furthermore we assessed the FoxMl expression and analyzed the relationship between expression of FoxMl and clinical significance.Additionally,we intended to investigate the biological effects,and reveal the effect of FoxMl on DNA damage repair as well as the mechanism of FoxM1 involved in IM drug resistance.Materials and Methods1.Investigating the expression of FoxMl in bone marrow of patients with CML,and analyzing the expression relationship and clinical significance1.1 Patients samples:Bone marrow samples from 50 CML patients and 12 normal controls were obtained after patients' consent and approval of Medical Ethical Committee(Qilu Hospital,Shandong University,China).38 patients were in IM sensitive group,and 12 were in IM resistant group.19 were newly diagnosed of all patients.Bone Marrow Mononuclear Cells(BMMCs)were isolated from bone marrow aspirates by density-gradient centrifugation using Ficoll and stored at-80 ?.1.2 Sorting CD34+ and CD34-mononuclear cells:Bone marrow ononuclear cells(BMMCs)were isolated from of CML samples and umbilical cord blood samples aspirates by density-gradient centrifugation using Ficoll.1×106 mononuclear cells were collected and stored at-80 ?.The other cells were CD34 + and CD34-cells by CD34 + magnetic bead sorting,and stored at-80 ?.1.3 Real-time RT-PCR:Total RNA was extracted from cells using TRIzol reagent and reverse transcription resperformed with PrimeScriptTM RT Master Mix.The mRNA expression of FoxM1 was evaluated in BM mononuclear cells by Real-time RT-PCR,which was performed with SYBR Green PCR Master Mix in a 10 ul reaction volume.2.Detecting the sensitivity of IM-sensitive CML cell line(K562)and its?M-resistant strain(k562/G01).2.1 Detecting the IM sensitivity of cells:Cells were treated with IM for 48h.CCK8 method2.2 an apoptosis assays by flow cytometry were used to detect cells death.2.3 Western-blot analysis:The total protein was abstracted from cells,and the protein levels of P-Tyr,P-Crkl and ?-H2AX were determined in cells as assessed by Western-blot methods.2.4.Immunofluorescence:the protein level of ?-H2AX was determined in cells as assessed by Western-blot methods.3.Studying the effect of FoxMl on the biological behavior of CML cells.3.1(1)FoxM1 lentivirus production and transduction:Lentivirus-FoxM1(LV-FoxM1)and Lentivirus-FoxM1-RNAi(LV-FoxM1-RNAi)were purchased,and the empty lentivirus were used as controls in the experiments.Cells were stably transducted with LV-FoxMl and LV-FoxM1-RNAi,and incubated with puromycin,as well as the empty lentivirus.(2)Downregulating FoxMl with inhibitors targeting FoxM1:Cells were treated with Thiostrepton(TST)and Bortezomib(BTZ)for 48 h.3.2 Real-time RT-PCR and Western-blot analysis:The mRNA and protein expression levels of FoxM1 passway,P-Tyr and P-Crkl were detected.3.3 Detecting the IM sensitivity of cells:Cells were treated with IM for 48h.CCK8 method and apoptosis assays by flow cytometry were used to detect cells death.3.4 Downregulating the expression of FoxM1 in CD34+ mononuclear cells from CML patients with TST,as well as controls.48h later,apoptosis assays by flow cytometry were used to detect cells death.4.Screening and identification of the crucial DNA damage repair gene in FoxM1 downstream,involved in IM resistance4.1 Gene expression profile chip method:K562/G01 cells transducted with LV-FoxM1-RNAi and empty lentivirus were collected to detect the differentially expressed genes between groups using gene chip.Those genes are as candidates for DNA Damage repair gene in FoxM1 downstream involved in IM resistance.4.2 Real-time RT-PCR assay:The mRNA expression levels of DNA damage repair genes in CML cells transducted with LV-FoxM1 or LV-FoxM1-RNAi,as well as the empty lentivirus were detected by RT-PCR method.The relationship between the expression of FoxMl and those candidate downstream genes was analyzed,and one of the downstream gene,which had the most significant difference,was selecte to be the target downstream gene.4.3 Western-blot assay:Total protein was abstracted from cells transducted with lentivirus.The protein level of target downstream gene was assessed by Western-blot method..5.Studying the effect of target downstream gene on the biological behavior of CML cells5.1 Target DNA damage repair gene lentivirus production and transduction:K562/G01 cells with low expression of FoxMl were up-regulated expression of the target DNA damage repair gene with plasmid transduction,and K562/G01 cells were down-regulated expression of the target DNA damage repair gene with lentivirus infection.Cells were incubated with puromycin for 2 weeks.The efficiency was verified via Real-time RT-PCR and Western-blot assays.5.2 Real-time RT-PCR and Western-blot assays:The mRNA expression levels of target DNA damage repair gene and ?-H2AX were assessed by RT-PCR;the protein expression levels of target DNA damage repair gene,?-H2AX,and P-Tyr were assessed by Western-blot assay.5.3 Detecting the IM sensitivity of cells:Cells were treated with IM for 48h.CCK8 method and apoptosis assays by flow cytometry were used to detect cells death.5.4 Immunofluorescence assay:Cells in each group were placed on the poly-lysine coated slides and fixed with paraformaldehyde.The protein expression was examined by immunofluorescence using the antibody of y-H2AX.Results1.Expression of FoxM1 in Bone Marrow Samples.1.1 The expression of FoxMl in CML patients and normal controls:Real-time RT-PCR results showed that mRNA expression of FoxMl in newly diagnosed CML patients was significantly higher than that in normal controls;FoxMl expression was associated with phase of CML disease,it's expression in AP/BC group is significantly higher than CP patients.1.2 The expression of FoxMl in stem cells:the mRNA expression of FoxM1 in CD34+ cells of CML patients was significantly higher than that in CD34' cells and CD34+ cells in normal controls.There was no significant difference in the expression of FoxM1 mRNA between CD34+ and CD34-cells in normal controls2.The IM sensitivity of K562/G01 and K562.2.1 CCK8 assay showed that the IC50 of K562/G01 to IM was 40 times of that of K562.2.2 Annexin V-FITC/PI FACS showed that the apoptotic rate of K562/G01 cells was significantly lower than that of K562 cells after the treatment with IM.2.3 Western-blot assay showed that the expressions of P-Tyr and P-Crkl in K562/G01 cells were significantly higher than those in K562 cells.3.FoxMl regulates BCR-ABL gene amplification and IM resistance.3.1 The effect of FoxMl over-expression on IM sensitivity of K562:CCK8 assay showed cell viability was increased.Annexin V FITC/PI FACS assay showed the apoptotic rate was lower.Western-blot showed the protein expressions of P-Tyr and P-Crk1 were increased.IF showed the protein expression of Y-H2AX was increased.3.2 The effect of FoxMl low-expression on IM sensitivity of K562/G01:CCK8 assay showed cell viability was decreased.Annexin V-FITC/PI FACS assay showed the apoptotic rate was higher.Western-blot showed the protein expressions of P-Tyr and P-Crk1 were decreased.IF showed the protein expression of Y-H2AX was decreased3.3 The effect of FoxMl down-regulated with TST on the sensitivity of CD34+mononuclear cells in CML patients:Annexin V-FITC/PI FACS showed a significant increasement in apoptosis.4.Identification of the crucial DNA damage repair gene in FoxMl downstream,involved in IM resistance4.1 Based on the results of gene expression profile chip,RT-PCR and Western-blot,PARP was decided to be the target DNA damage repair gene.4.2 Western-Blot showed that the expression level of PARP in K562/G01 cellswith low-expression of FoxMl was lower than that of the control group.The expression of PARP in K562 cells with over-expressed of FoxMl was higher than that in control group.4.3 The effect of down-regulation of PARP in IM-resistant cell line K562/G01:Real-time RT-PCR and Western-Blot showed that mRNA and protein expression levels of PARP were decreased;CCK8 assay showed decreased cell viability;Annexin V FITC/PI FACS showed more apoptosis.The expression of y-H2AX was increased showed by immunofluorescence and Western-blot.4.4 The effect of over-expression of PARP in K562/G01 with low FoxMl expression:Real-time RT-PCR and Western-Blot showed that mRNA and protein expression PARP were increased;CCK8 assay showed increased cell viability;Annexin V-FITC/PI FACS showed less apoptosis;.4.5 The expression of y-H2AX was decreased showed by immunofluorescence and Western-blot.Conclusion1.Our study demonstrates that FoxM1 expresses highly in CML,and the expression is Increased with progression of CML.2.Our data provide evidence that FoxM1 plays an important role in the maintenance of survival and self-renewal of stem cells.3.The expression of BCR-ABL gene is correlated with the expression of Fox Ml.The IM resistance of CML cells increases with the increasement of FoxM1 expression,and the role of FoxM1 in the regulation of BCR-ABL gene amplification and DNA damage response in IM resistance is revealed.4.PARP is the target DNA damage repair gene in FoxM1 downstream,involved in IMresistance.The IM resistance of CML cells increases with the increasement of PARP expression.Down-regulating PARP of overexpressed-FoxM1 CML cells can reless the IM resistance.FoxM1 mediate IM resistance of CML cells by regulating the expression of PARP.