The Mechanism Of Chemotherapeutic Agent Inhibiting Tumor Development By Regulating The Interaction Between PML And TET2

1.BackgroundModern epigenetics refers to changes in gene expression and function that produce phenotypes that can be inherited stably without changes in the sequence of DNA.Epigenetics mainly includes DNA methylation,RNA interference,chromatin remodeling,histone modification and so on.DNA methylation is one of the earliest identified and most intensively studied epigenetic regulatory mechanisms.Numerous studies have shown that DNA methylation can cause changes in chromatin structure,DNA conformation,DNA stability and the way DNA interacts with proteins,thus controlling gene expression.In addition to gene transcriptional regulation,DNA methylation is involved in a variety of biological processes,including transposon silencing,gene imprinting,X chromosome inactivation,and the occurrence and development of cancer.TET protein catalyzes the conversion of 5-methylcytosine(5mC)to5-hydroxymethylcytosine(5hmC),an important enzyme in the process of DNA demethylation,and is important for maintaining stem cell pluripotency.Mutations in the TET gene can cause a variety of tumors,especially tumors of the hematopoietic system.Aberrant DNA methylation plays a critical role in the development and progression of cancer.Failure to demethylate and to consequently reactivate methylation-silenced genes in cancer contributes to chemotherapeutic resistance,yet the regulatory mechanisms of DNA demethylation in response to chemotherapeutic agents remain unclear.Here,we show that promyelocytic leukemia(PML)recruits ten–eleven translocation dioxygenase 2(TET2)to regulate DNA modification and cell proliferation in response to chemotherapeutic agents,contributing to insight into the mechanisms regulating DNA modification in response to chemotherapeutic agents.2.Methods2.1 Considering that DNA modification pathways may be deregulated in cancer cells,MEF cells(normal cells)were first used and DNA 5mC and 5hmC levels in MEF cells after doxorubicin treatment were examined by MS to determine the alteration of DNA modification in response to chemotherapeutic drugs.After confirming the trend of change,due to low oxygen concentration decreases in the in vivo activity of oxygen-dependent TETs^[52],we supposed that TETs might play more notable roles in head and neck squamous cell carcinoma(HNSC),which is likely to be exposed to more oxygen.Therefore,HNSC SCC-15 and SCC-25 cells were used for further study.Follow up with the addition of chemotherapeutic agents other than Doxorubicin.2.2 Determining which TET is the cause of chemotherapy-promoted 5hmC by immunoblotting and RNAi/sh RNAi.2.3 To identify the candidate functional partners of TET2 in response to chemotherapy,SILAC MS analysis of anti-TET2 coimmunoprecipitation(Co-IP)was carried out.2.4 Co-IP analysis identifies regions where TET2 and PML bind to each other.2.5 Immunofluorescence,dot blotting and MS analysis to investigate the role of PML in regulation of TET2 in response to chemotherapy.2.6 To study the effect of PML and TET2 on cell proliferation in the presence of chemotherapeutic agents in vitro and in vivo.3.Results3.1 The DNA 5mC and 5hmC levels of the treated MEF cells were assessed by MS.MS analyses indicated that the 5hmC level(5hmC/C)was increased about 1.5-fold by doxorubicin treatment.Similarly,doxorubicin enhanced 5hmC in HNSC SCC-15 and SCC-25 cells and HEK293 cells;other chemotherapeutic agents such as mitomycin C and cisplatin also increased 5hmC.3.2 The expression of TET1,TET2,or TET3 was not notably altered by doxorubicin in HEK293,SCC-15,and SCC-25 cells.Knockdown of Tet2 notably diminished doxorubicin-promoted 5hmC. We created HEK293,SCC-15,and SCC-25 cells stably expressing scramblesh RNA(control),sh TET1(#1/#2),sh TET2(#1/#2),or sh TET3(#1/#2), doxorubicin-promoted 5hmC was also decreased by the knockdown of TET2. Hence,it seemed that TET2 promotes DNA 5hmC modification in response to doxorubicin.3.3 To identify the candidate functional partners of TET2 in response to chemotherapy,SILAC MS analysis of anti-TET2 coimmunoprecipitation (Co-IP)was carried out. Plasmids encoding 65 genes were transfected into HEK293.MS analyses of the resultant cells demonstrated that PML showed the most notable ability to enhance 5hmC.We reasoned that PML was a functional partner of TET2. PML was subsequently determined to recruit TET2 to PML-NBs(DNMT3A as control)by Co-IP and immunoblotting.3.4 To determine the regions of TET2 that bind to PML,HEK293 cells weretransfected with plasmids encoding PML and each of the TET2-deletionmutants.Co-IP analyses of the resultant cells demonstrated that overexpressed TET2-F2(Cys-rich domain)showed poor interaction with overexpressed PML,whereas TET2-F1,TET2-F3,and TET2-F4 showed notable interaction with PML.Then we extended our analysis of the interaction between overexpressed PML and each of the TET2-deletion mutants in U2OS cells by immunofluorescence.Without the transfection of PML,TET2-deletion mutants were nuclear or cytoplasm diffuse.With the transfection of PML, only TET2-F2(Cys-rich domain)was not delocalized to the PML-NBs, whereas TET2-F1,TET2-F3,and TET2-F4 were colocalized with PML-NBs. To determine the regions of PML to interact with TET2,HEK293 and U2OS cells were transfected with the plasmids encoding TET2 and each of the nuclear-located PML isoforms(PML I–VI).Co-IP and immunofluorescence analysis showed that PML bound to TET2 ina PML C-terminal-dependent way.3.5 Immunofluorescence,dot blot and MS analysis showed that doxorubicin-promoted 5hmC was not stemmed from the alteration of TET protein levels.We found that doxorubicin-promoted 5hmC was decreased by the depletion of either TET2 or PML gene,but not additively by the codepletion of both genes.3.6 Enhanced expression of PML and/or TET2 WT increased the ability of doxorubicin to inhibit cell proliferation,whereas TET2 MUT showed no ability to enhance doxorubicin-promoted inhibition of cell proliferation.And si RNA-or sh RNA-mediated knockdown of PML and/or TET2 resulted in poorer response to doxorubicin in MEF,HEK293,SCC-15,and SCC-25 cells.Comparing with knock down of PML,we did not detect additive ability of PML-TET2 codepletion to decrease response to doxorubicin.SCC-15 cells stably expressing sh Scram(control scrambled sh RNA),sh PML, sh TET2,or sh PML/sh TET2 were injected sub-cutaneously into the flank of mice.Then,these grouped tumor-bearing mice were treated with vehicle or doxorubicin.Consistent with the observation in vitro,knockdown of PML and/or TET2 resulted in poorer response to doxorubicin.To further evaluate the role of PML and TET2 in vivo,we examined the clinical relevance in HNSC patients.Using data set from The Cancer Genome Atlas^[58],Kaplan– Meier plotter was built to analyze the overall survival of HNSC patients with different PML and TET2 levels.Low expression of PML and TET2 was associated with poorer overall survival in HNSC patients.4.Conclusions4.1 TET2 promotes DNA 5hmC modification in response to chemotherapeuticagents.4.2 PML physically binds to TET2 and recruits TET2 to PML nuclear bodies.4.3 PML-RARA t(15;17)mutation disrupts the ability of PML to bind to TET2.4.4 PML recruits TET2 to regulate 5hmC in response to doxorubicin.4.5 PML-TET2 regulates cell proliferation in response to doxorubicin.