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Oncogenesis
2018 Oct 11;710:82. doi: 10.1038/s41389-018-0092-0.
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Depletion of ZBTB38 potentiates the effects of DNA demethylating agents in cancer cells via CDKN1C mRNA up-regulation.
Marchal C
,
de Dieuleveult M
,
Saint-Ruf C
,
Guinot N
,
Ferry L
,
Olalla Saad ST
,
Lazarini M
,
Defossez PA
,
Miotto B
.
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DNA methyltransferase inhibitor (DNMTi) treatments have been used for patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and have shown promising beneficial effects in some other types of cancers. Here, we demonstrate that the transcriptional repressor ZBTB38 is a critical regulator of the cellular response to DNMTi. Treatments with 5-azacytidine, or its derivatives decitabine and zebularine, lead to down-regulation of ZBTB38 protein expression in cancer cells, in parallel with cellular damage. The depletion of ZBTB38 by RNA interference enhances the toxicity of DNMTi in cell lines from leukemia and from various solid tumor types. Further we observed that inactivation of ZBTB38 causes the up-regulation of CDKN1C mRNA, a previously described indirect target of DNMTi. We show that CDKN1C is a key actor of DNMTi toxicity in cells lacking ZBTB38. Finally, in patients with MDS a high level of CDKN1C mRNA expression before treatment correlates with a better clinical response to a drug regimen combining 5-azacytidine and histone deacetylase inhibitors. Collectively, our results suggest that the ZBTB38 protein is a target of DNMTi and that its depletion potentiates the toxicity of DNMT inhibitors in cancer cells, providing new opportunities to enhance the response to DNMT inhibitor therapies in patients with MDS and other cancers.
Fig. 1. DNA hypomethylating agents cause ZBTB38 down-regulation at protein level in solid and hematologic cancer cells.a Schematic representation of the experimental protocol. Cancer cells were treated twice with 10 µM 5-Azacytidine (Aza) or DMSO and harvested 24 h later for analysis. b Dot blot analysis of CpG methylation on preparation of genomic DNA from cells treated or not with 5-azacytidine (n = 2). c Gene expression analysis of hyper-methylated silenced genes CDH13 and DAPK1 by real-time PCR in HeLa cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells (n = 3). Expression level is expressed as the fold change between treated and control cells. d Western blot analysis of ZBTB38, MCM3, and GAPDH protein expression in HeLa cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells. e Gene expression analysis of ZBTB38 by real-time PCR in HeLa cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells (n = 3). f Western blot analysis of ZBTB38 and GAPDH protein expression in U2OS, HepG2, and HCT116 cancer cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells. g Western blot analysis of ZBTB38 and GAPDH protein expression in THP-1 and MOLM-14 AML cells treated with 5-azacytidine (Aza) compared to control (Ctrl) cells. h Western blot analysis of ZBTB38 and GAPDH protein expression in HeLa and HCT116 cancer cells treated with 5-azacytidine (Aza), 5-azacytidine plus proteasome-inhibitor MG132 (4 h) compared to control cells. i Western blot analysis of ZBTB38-ubiquitination in HCT116 cells treated with 5-azacytidine (Aza) compared to control (Ctrl). Co-immuno-precipitates of ZBTB38 were run on a SDS-page and ubiquitin-moieties detected using a specific antibody. j Western blot analysis of ZBTB38, ZBTB4, ZBTB33, GAPDH, phospho-CHK2 (P-CHK2), phospho-H2AX (P-H2AX) and phospho-ATM (P-ATM) in HeLa and HCT116 cells treated with 10 µM of either 5-azacytidine, decitabine or zebularine and in control mock-treated cells. k Western blot analysis of ZBTB38, DNMT1, phospho-H2AX (P-H2AX), H2AX, and GAPDH protein levels in different human cell lines (HeLa, U2OS, HCT116) treated with a specific siRNA against DNMT1 or a control siRNA. l Western blot analysis of ZBTB38 and GAPDH in HeLa cells treated with 10 µM of 5-azacytidine alone or co-treated with caffeine (1 mM) for 24 h
Fig. 2. Inactivation of ZBTB38 marginally impacts DNA demethylation and 5-azacytidine induced DNA demethylation.a Dot blot analysis of CpG methylation levels in HCT116 (black bar), U2OS (grey bar), and HeLa (light grey bar) cells treated with siRNA against ZBTB38 or control siRNA and further exposed to 5-azacytidine (n = 3). The data represent relative levels of the condition siRNA ZBTB38 versus siRNA control. b Dot blot analysis of CpG hydroxymethylation levels in HCT116 (black bar), U2OS (grey bar), and HeLa (light grey bar) cells treated with siRNA against ZBTB38 or control siRNA and further exposed to 5-azacytidine (n = 3). The data represent relative levels of the condition siRNA ZBTB38 versus siRNA control. c Dot blot analysis of CpG methylation and hydroxymethylation levels in THP-1 cells treated with siRNA against ZBTB38 or control siRNA and further exposed to decitabine (n = 2). The data represent relative levels of the condition siRNA ZBTB38 versus siRNA control. d RT-PCR analysis of genes modulating 5-azacytidine toxicity in HCT116, U2OS, and THP-1 cells treated with siRNA against ZBTB38 (dashed bars) and control siRNA (solid bars) (n = 3)
Fig. 4. Inactivation of ZBTB38 by RNA interference causes a strong arrest in cell proliferation upon DNMTi exposure, persistant after removal of DNMTi.a Graph depicting the growth curve of K562 cells treated with siRNAs (control and ZBTB38) and with decitabine 1 µM (DAC) or not (n = 4). ***P < 0.001. b, c Same experiment in THP-1 (b) and MOLM-14 (c). d Graph depicting the proliferative potential of THP-1, MOLM-14, and K562 cells transfected with siRNAs (control and ZBTB38) and further exposed to decitabine (DAC) or azacytine (AZA) (n = 4). ***P < 0.001. *P < 0.05. e Graph depicting the proliferative potential of THP-1 (left panel) and MOLM-14 (right panel) cells transfected with siRNAs (control and USP9X) and further exposed to decitabine 1 µM (DAC) (n = 4). ***P < 0.001
Fig. 6. High expression of CDKN1C mRNA in the bone marrow of patients with MDS coincides with a better response to 5-azacytidine plus entinostat.a RT-qPCR analysis of ZBTB38 and b
CDKN1C mRNA expression in bone marrow samples from patients with MDS, AML with myelodysplasia-related changes (AML-MRC) and de novo AML. MDS patients were grouped into lower and higher-risk categories according to the Revised International Prognostic Scoring System. c Kaplan–Meier plots of overall survival in MDS patients classed according to low and high ZBTB38 (left panel) or CDKN1C (right panel) mRNA expression levels. d Expression of ZBTB38, RBBP6, USP9X, and CDKN1C in samples from patients with MDS before azacytidine and entinostat treatment (D0) and 15 days later (D15). Responders to the therapy are highlighted in orange; non responder are presented in grey (data from ref. 1). Significant difference between responder and non-responder at Day 0 is indicated by a P-value in the graph
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