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DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression.

Cho MH, Park JH, Choi HJ, Park MK, Won HY, Park YJ, Lee CH, Oh SH, Song YS, Kim HS, Oh YH, Lee JY, Kong G - Nat Commun (2015)

Bottom Line: DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown.DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions.Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, Korea.

ABSTRACT
DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown. Here we identify that DOT1L cooperates with c-Myc and p300 acetyltransferase to epigenetically activate epithelial-mesenchymal transition (EMT) regulators in breast cancer progression. DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions. The upregulation of these EMT regulators by the DOT1L-c-Myc-p300 complex enhances EMT-induced breast cancer stem cell (CSC)-like properties. Furthermore, in vivo orthotopic xenograft models show that DOT1L is required for malignant transformation of breast epithelial cells and breast tumour initiation and metastasis. Clinically, DOT1L expression is associated with poorer survival and aggressiveness of breast cancers. Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

No MeSH data available.


Related in: MedlinePlus

DOT1L increases Snail, ZEB1 and ZEB2 expression to repress E-cadherinexpression and breast CSC property.(a) CDH1 mRNA levels in the indicated cell lines were analysedusing qRT–PCR. For transient knockdown of DOT1L, five differentsiRNAs against DOT1L (siDOT1L, #1–5) or siCON weretransfected into MDA-MB-231 cells (middle). MCF10A cells expressing DOT1Lwild-type (DOT1L) or DOT1L siRNA (#2)-resistant mutant (DOT1L-siMUT) were transfected with DOT1L siRNA #2 or control siRNA for48 h, and subjected to qRT–PCR for analysis ofCDH1 expression (right). *P<0.05 versuscontrols (CON, siCON, CON/siCON) by Student's t-test.(b) Effect of DOT1L on CDH1 promoter activity. Cells weretransfected with luciferase constructs of wild-type (CDH1 pro-WT) orE-box-mutant (CDH1 pro-MUT) CDH1 promoters for 24 hand the luciferase activity was measured. RLU, relative light units.*P<0.05 versus CON (Student'st-test). (c–e) Effects of DOT1L on theexpression of CDH1 transcriptional regulators were examined usingimmunoblotting (c), qRT–PCR (d) andimmunofluorescence staining (e). *P<0.05 versusCON or siCON (Student's t-test). Scale bars in e,100 μm. (f) Binding by EMT-TFs to theCDH1 promoter region was analysed using ChIP–qPCR.*P<0.05 versus CON (Student'st-test). (g,h) Effect of EMT-TFs on DOT1L-inducedcancer stemness. Cells were transfected with siRNAs against Snail, ZEB1 andZEB2, and the CSC population was measured using flow cytometry. Theknockdown of EMT-TFs was confirmed by immunoblotting (g). The FACSresults were quantified and shown as a bar graph (h). * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). Error bars ina,b,d,f,h indicate themeans±s.d. of experiments in triplicate.
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f3: DOT1L increases Snail, ZEB1 and ZEB2 expression to repress E-cadherinexpression and breast CSC property.(a) CDH1 mRNA levels in the indicated cell lines were analysedusing qRT–PCR. For transient knockdown of DOT1L, five differentsiRNAs against DOT1L (siDOT1L, #1–5) or siCON weretransfected into MDA-MB-231 cells (middle). MCF10A cells expressing DOT1Lwild-type (DOT1L) or DOT1L siRNA (#2)-resistant mutant (DOT1L-siMUT) were transfected with DOT1L siRNA #2 or control siRNA for48 h, and subjected to qRT–PCR for analysis ofCDH1 expression (right). *P<0.05 versuscontrols (CON, siCON, CON/siCON) by Student's t-test.(b) Effect of DOT1L on CDH1 promoter activity. Cells weretransfected with luciferase constructs of wild-type (CDH1 pro-WT) orE-box-mutant (CDH1 pro-MUT) CDH1 promoters for 24 hand the luciferase activity was measured. RLU, relative light units.*P<0.05 versus CON (Student'st-test). (c–e) Effects of DOT1L on theexpression of CDH1 transcriptional regulators were examined usingimmunoblotting (c), qRT–PCR (d) andimmunofluorescence staining (e). *P<0.05 versusCON or siCON (Student's t-test). Scale bars in e,100 μm. (f) Binding by EMT-TFs to theCDH1 promoter region was analysed using ChIP–qPCR.*P<0.05 versus CON (Student'st-test). (g,h) Effect of EMT-TFs on DOT1L-inducedcancer stemness. Cells were transfected with siRNAs against Snail, ZEB1 andZEB2, and the CSC population was measured using flow cytometry. Theknockdown of EMT-TFs was confirmed by immunoblotting (g). The FACSresults were quantified and shown as a bar graph (h). * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). Error bars ina,b,d,f,h indicate themeans±s.d. of experiments in triplicate.

Mentions: To further clarify the role of DOT1L in EMT and CSC, we investigated themolecular mechanism underlying the reduction by DOT1L of E-cadherin expression.On the basis of a previous finding showing DOT1L inhibitor-induced E-cadherinmRNA expression10, we confirmed that DOT1L regulates E-cadherinat the transcriptional level, as the mRNA level of the CDH1 gene(encoding E-cadherin) was decreased by DOT1L overexpression in MCF10A cells(left) and increased by siRNA-mediated DOT1L knockdown in MDA-MB-231 cells(middle; Fig. 3a). Consistently, transient DOT1L knockdownrescued the repression of CDH1 in DOT1L-overexpressing MCF10A cells, butnot in DOT1L siRNA-resistant mutant-expressing cells (Fig.3a, right). Furthermore, DOT1L repressed CDH1 promoteractivity and this effect was abolished by mutation of the E-box site within itspromoter (Fig. 3b), indicating the importance of the E-boxmotif in DOT1L regulation of CDH1. Several EMT-TFs including Snail, ZEB1and ZEB2 repress E-cadherin transcription by directly binding to the E-box motifwithin the CDH1 promoter13. Consistent with the results inDOT1L-overexpressing MCF10A cells (Fig. 2c), the proteinand mRNA levels of these EMT-TFs were positively regulated by DOT1L in bothMCF10A and MDA-MB-231 cells, as assessed by immunoblotting, immunofluorescencestaining and quantitative real-time PCR (qRT–PCR; Fig.3c–e). This effect was abolished in the siRNA-resistantDOT1L mutant-expressing MCF10A cells (Supplementary Fig. 8). By performing chromatinimmunoprecipitation–quantitative PCR (ChIP–qPCR) analysis,we further confirmed that DOT1L enhances direct binding of EMT-TFs on theCDH1 promoter (Fig. 3f). Moreover, transientknockdown of Snail, ZEB1 and ZEB2 using their siRNAs recovered E-cadherinexpression in DOT1L-overexpressing MCF10A cells (Fig. 3g).Thus, these results suggest that DOT1L represses the E-cadherin transcriptionthrough EMT-TF upregulation. Unexpectedly, DOT1L expression was also inhibitedby Snail and ZEB2 knockdown (Fig. 3g), suggesting thepossible positive feedback between these molecules. Because Snail, ZEB1 and ZEB2modulate CSC activity1121, we further explored whetherDOT1L-induced CSC-like properties is mediated by these EMT-TFs. The transientknockdown of Snail, ZEB1 and ZEB2 halted the stem cell expansion inDOT1L-overexpressing MCF10A cells (Fig. 3h). Collectively,these results indicated that DOT1L-induced EMT-TF expression is critical forboth enhancing EMT and CSC-like properties.


DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression.

Cho MH, Park JH, Choi HJ, Park MK, Won HY, Park YJ, Lee CH, Oh SH, Song YS, Kim HS, Oh YH, Lee JY, Kong G - Nat Commun (2015)

DOT1L increases Snail, ZEB1 and ZEB2 expression to repress E-cadherinexpression and breast CSC property.(a) CDH1 mRNA levels in the indicated cell lines were analysedusing qRT–PCR. For transient knockdown of DOT1L, five differentsiRNAs against DOT1L (siDOT1L, #1–5) or siCON weretransfected into MDA-MB-231 cells (middle). MCF10A cells expressing DOT1Lwild-type (DOT1L) or DOT1L siRNA (#2)-resistant mutant (DOT1L-siMUT) were transfected with DOT1L siRNA #2 or control siRNA for48 h, and subjected to qRT–PCR for analysis ofCDH1 expression (right). *P<0.05 versuscontrols (CON, siCON, CON/siCON) by Student's t-test.(b) Effect of DOT1L on CDH1 promoter activity. Cells weretransfected with luciferase constructs of wild-type (CDH1 pro-WT) orE-box-mutant (CDH1 pro-MUT) CDH1 promoters for 24 hand the luciferase activity was measured. RLU, relative light units.*P<0.05 versus CON (Student'st-test). (c–e) Effects of DOT1L on theexpression of CDH1 transcriptional regulators were examined usingimmunoblotting (c), qRT–PCR (d) andimmunofluorescence staining (e). *P<0.05 versusCON or siCON (Student's t-test). Scale bars in e,100 μm. (f) Binding by EMT-TFs to theCDH1 promoter region was analysed using ChIP–qPCR.*P<0.05 versus CON (Student'st-test). (g,h) Effect of EMT-TFs on DOT1L-inducedcancer stemness. Cells were transfected with siRNAs against Snail, ZEB1 andZEB2, and the CSC population was measured using flow cytometry. Theknockdown of EMT-TFs was confirmed by immunoblotting (g). The FACSresults were quantified and shown as a bar graph (h). * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). Error bars ina,b,d,f,h indicate themeans±s.d. of experiments in triplicate.
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f3: DOT1L increases Snail, ZEB1 and ZEB2 expression to repress E-cadherinexpression and breast CSC property.(a) CDH1 mRNA levels in the indicated cell lines were analysedusing qRT–PCR. For transient knockdown of DOT1L, five differentsiRNAs against DOT1L (siDOT1L, #1–5) or siCON weretransfected into MDA-MB-231 cells (middle). MCF10A cells expressing DOT1Lwild-type (DOT1L) or DOT1L siRNA (#2)-resistant mutant (DOT1L-siMUT) were transfected with DOT1L siRNA #2 or control siRNA for48 h, and subjected to qRT–PCR for analysis ofCDH1 expression (right). *P<0.05 versuscontrols (CON, siCON, CON/siCON) by Student's t-test.(b) Effect of DOT1L on CDH1 promoter activity. Cells weretransfected with luciferase constructs of wild-type (CDH1 pro-WT) orE-box-mutant (CDH1 pro-MUT) CDH1 promoters for 24 hand the luciferase activity was measured. RLU, relative light units.*P<0.05 versus CON (Student'st-test). (c–e) Effects of DOT1L on theexpression of CDH1 transcriptional regulators were examined usingimmunoblotting (c), qRT–PCR (d) andimmunofluorescence staining (e). *P<0.05 versusCON or siCON (Student's t-test). Scale bars in e,100 μm. (f) Binding by EMT-TFs to theCDH1 promoter region was analysed using ChIP–qPCR.*P<0.05 versus CON (Student'st-test). (g,h) Effect of EMT-TFs on DOT1L-inducedcancer stemness. Cells were transfected with siRNAs against Snail, ZEB1 andZEB2, and the CSC population was measured using flow cytometry. Theknockdown of EMT-TFs was confirmed by immunoblotting (g). The FACSresults were quantified and shown as a bar graph (h). * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). Error bars ina,b,d,f,h indicate themeans±s.d. of experiments in triplicate.
Mentions: To further clarify the role of DOT1L in EMT and CSC, we investigated themolecular mechanism underlying the reduction by DOT1L of E-cadherin expression.On the basis of a previous finding showing DOT1L inhibitor-induced E-cadherinmRNA expression10, we confirmed that DOT1L regulates E-cadherinat the transcriptional level, as the mRNA level of the CDH1 gene(encoding E-cadherin) was decreased by DOT1L overexpression in MCF10A cells(left) and increased by siRNA-mediated DOT1L knockdown in MDA-MB-231 cells(middle; Fig. 3a). Consistently, transient DOT1L knockdownrescued the repression of CDH1 in DOT1L-overexpressing MCF10A cells, butnot in DOT1L siRNA-resistant mutant-expressing cells (Fig.3a, right). Furthermore, DOT1L repressed CDH1 promoteractivity and this effect was abolished by mutation of the E-box site within itspromoter (Fig. 3b), indicating the importance of the E-boxmotif in DOT1L regulation of CDH1. Several EMT-TFs including Snail, ZEB1and ZEB2 repress E-cadherin transcription by directly binding to the E-box motifwithin the CDH1 promoter13. Consistent with the results inDOT1L-overexpressing MCF10A cells (Fig. 2c), the proteinand mRNA levels of these EMT-TFs were positively regulated by DOT1L in bothMCF10A and MDA-MB-231 cells, as assessed by immunoblotting, immunofluorescencestaining and quantitative real-time PCR (qRT–PCR; Fig.3c–e). This effect was abolished in the siRNA-resistantDOT1L mutant-expressing MCF10A cells (Supplementary Fig. 8). By performing chromatinimmunoprecipitation–quantitative PCR (ChIP–qPCR) analysis,we further confirmed that DOT1L enhances direct binding of EMT-TFs on theCDH1 promoter (Fig. 3f). Moreover, transientknockdown of Snail, ZEB1 and ZEB2 using their siRNAs recovered E-cadherinexpression in DOT1L-overexpressing MCF10A cells (Fig. 3g).Thus, these results suggest that DOT1L represses the E-cadherin transcriptionthrough EMT-TF upregulation. Unexpectedly, DOT1L expression was also inhibitedby Snail and ZEB2 knockdown (Fig. 3g), suggesting thepossible positive feedback between these molecules. Because Snail, ZEB1 and ZEB2modulate CSC activity1121, we further explored whetherDOT1L-induced CSC-like properties is mediated by these EMT-TFs. The transientknockdown of Snail, ZEB1 and ZEB2 halted the stem cell expansion inDOT1L-overexpressing MCF10A cells (Fig. 3h). Collectively,these results indicated that DOT1L-induced EMT-TF expression is critical forboth enhancing EMT and CSC-like properties.

Bottom Line: DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown.DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions.Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, Korea.

ABSTRACT
DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown. Here we identify that DOT1L cooperates with c-Myc and p300 acetyltransferase to epigenetically activate epithelial-mesenchymal transition (EMT) regulators in breast cancer progression. DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions. The upregulation of these EMT regulators by the DOT1L-c-Myc-p300 complex enhances EMT-induced breast cancer stem cell (CSC)-like properties. Furthermore, in vivo orthotopic xenograft models show that DOT1L is required for malignant transformation of breast epithelial cells and breast tumour initiation and metastasis. Clinically, DOT1L expression is associated with poorer survival and aggressiveness of breast cancers. Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

No MeSH data available.


Related in: MedlinePlus