Targeting EZH2 reactivates a breast cancer subtype-specific anti-metastatic transcriptional program

Hirukawa, Alison; Smith, Harvey W.; Zuo, Dongmei; Dufour, Catherine R.; Savage, Paul; Bertos, Nicholas; Johnson, Radia Marie; Bui, Tung; Bourque, Guillaume; Basik, Mark; Giguère, Vincent; Park, Morag; Muller, William J.

doi:10.1038/s41467-018-04864-8

Cited by 68 publications

(57 citation statements)

References 58 publications

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“…In contrast to our findings, one study showed that tumors derived from EZH2 knockdown cells are significantly smaller (Gonzalez et al, 2014), and another study showed that EZH2 ablation promotes tumor growth (Wassef et al, 2015). Hirukawa et al (2018) tested the effect of EZH2 inhibition on two TNBC PDX models, and, consistent with our studies, they found that EZH2 inhibition did not significantly affect primary tumor growth in both models. However, they also showed that EZH2 inhibition did not affect metastasis.…”

Section: Discussionsupporting

confidence: 77%

“…It is important to note that the TNBC constitutes distinct molecular subtypes (Lehmann et al, 2016), and we show that high EZH2 expression in the BL-1 and M subtypes correlates with low GATA3 expression and response to EZH2 inhibition. Possibly, the TNBC subtype used by Hirukawa et al (2018) may not be responsive to EZH2 inhibition. Together, these studies highlight the context-dependent function of EZH2 in breast cancer subtypes (Comet et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of EZH2 Catalytic Activity Selectively Targets a Metastatic Subpopulation in Triple-Negative Breast Cancer

et al. 2020

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Section: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Inhibition of EZH2 Catalytic Activity Selectively Targets a Metastatic Subpopulation in Triple-Negative Breast Cancer

et al. 2020

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“…Because H3K27me3 is mainly established by the PRC2-EZH2 complex, we examined if PRC2-EZH2 repressed genes in breast cancer cells also gain H3K27me3 signal, as observed for CTR9 target genes. A recent study identified a panel of EZH2 repressed genes in a mouse breast cancer model, and FOXC1 was shown to be repressed by H3K27me3 in an EZH2-dependent manner (Hirukawa et al, 2018). Analyses of H3K27me3 ChIP-seq signal in the human analogues of the mouse genes showed that approximately half of the PRC2-repressed genes displayed a dramatic increase of H3K27me3 in CTR9 KD cells (Figure S6A).…”

Section: Resultsmentioning

confidence: 99%

“…PRC2-EZH2 repressed genes were obtained from a recent study in mouse ( Foxc1 and genes listed in Figure 4a of [Hirukawa et al, 2018]. All of the 24 mouse genes can be mapped uniquely to human genes except that Dbc1 was mapped to BRINP1 and CCAR2 , so we analyzed all of the 25 human genes.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional elongation machinery controls vulnerability of breast cancer cells to PRC2 inhibitors

Huang

et al. 2020

Preprint

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1 2 CTR9 is the scaffold subunit in Paf1c, a multifunctional complex regulating multiple steps 3 of RNA Pol II-mediated transcription. Using inducible and stable CTR9 knockdown breast 4 cancer cell lines, we discovered that the expression of a subset of KDMs, including 5 KDM6A and Jarid2, is strictly controlled by CTR9. Global analyses of histone modifications 6 revealed a significant increase of H3K27me3 upon loss of CTR9. Loss of CTR9 results in 7 a decrease of H3K4me3 and H3K36me3 in gene bodies, and elevated levels and genome-8 wide expansion of H3K27me3. Mechanistically, CTR9 depletion triggers a PRC2 subtype 9 switching from PRC2.2 to PRC2.1. As a consequence, CTR9 depletion generates 10 vulnerability that renders breast cancer cells hypersensitive to PRC2 inhibitors. Our 11 findings that CTR9 demarcates PRC2-mediated H3K27me3 levels and genomic 12 distribution, provide a unique mechanism of transition from transcriptionally active to 13 repressive chromatin states and sheds light on the biological functions of CTR9 in 14 development and cancer. 15 16 17 18 19 20 21 22 23 4 RESULTS 1 2 CTR9 regulates a subset of histone lysine demethylases (KDMs) in ER+ breast 3 cancer cells 4 Data mining of the CTR9-regulated transcriptome in CTR9 inducible knockdown (KD) 5 MCF7 cells (MCF7-tet-on-shCtr9) (Figure 1A) (Zeng and Xu, 2016) identified a subset of 6 KDM genes that mirrors the expression of CTR9, including KDM1A, KDM2B, KDM3B, 7KDM5B, KDM6A and JARID2. We found that these KDM genes were down-regulated 8 when CTR9 was knocked down by shRNA in MCF7 cells after a 7-day doxycycline (Dox) 9 treatment ( Figure 1B) regardless of whether 17β-estradiol (E2) was present or absent. 10RNAPII binding peaks at the transcription start site (TSS) of several KDM genes also 11 decreased in response to CTR9 depletion ( Figure S1A). We validated the transcriptome 12 array results by RT-qPCR. Figure 1C shows that silencing of CTR9 after a 7-day Dox 13 treatment reduced the total mRNA levels, and reduced the expression of ribosome-14 associated RNAs, which are indicative of actively transcribed mRNAs, of six KDMs. 15However, the mRNA levels of KDM4B and KDM6B did not significantly change. CTR9's 16 regulation of KDMs was also observed at the protein level. As shown in Figure 1D, the 17 protein levels of six KDMs significantly decreased in the total cell lysates and extracted 18 chromatin fractions. Consistent with their mRNA levels in response to CTR9 depletion, 19 KDM4B and KDM6B protein levels also remained unchanged. To exclude the possibility 20 that this observation was specific to MCF7, or due to off-target effects of anti-CTR9 shRNA, 21 we employed MCF7 cells as well as T47D cells, another well-established ERα+ breast 22 34 35

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“…EZH2 was the only functional protein distributed in the nucleus that was binding to LINC-PINT both in A375 and Mum2B. Tri-methylation of lysine 27 on histone 3 (H3K27me3) by the methyltransferase EZH2, as a part of PRC2, is one of the most important epigenetic mechanism of gene silencing (Hirukawa et al, 2018). The ChIRP-MS results showed that EZH2 protein was enriched by LINC-PINT probes, but not the negative control probes, as further confirmed by western blot (Figure 6B).…”

Section: Linc-pint Recruited Ezh2 To Inhibit Gene Expressionmentioning

confidence: 99%

Long Non-coding RNA LINC-PINT Suppresses Cell Proliferation and Migration of Melanoma via Recruiting EZH2

Wang

et al. 2019

Front. Cell Dev. Biol.

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Long non-coding RNAs (lncRNAs) have been identified as crucial regulators in many human cancers. Many lncRNAs show aberrant expression in cancer, and some of them play critical roles in tumor proliferation, invasion, and metastasis. However, the regulatory functions of lncRNAs in melanoma progression remain to be elucidated. We utilized the Real-time PCR methodology to determine the expression of LINC-PINT in melanoma cell lines. To evaluate the effect of LINC-PINT on tumorigenesis of melanoma, we used Cell Counting Kit-8 (CCK8) and colony formation assay. Flow cytometry assay was used to detect the function of LINC-PINT on cell cycle status. PINT-interacting proteins were identified by chromatin isolation using RNA purification (ChIRP). Microarray assay and bioinformatics analysis were used to find the potential target genes of LINC-PINT and the status of LINC-PINT target gene candidate was verified using chromatin immunoprecipitation assay (ChIP). LINC-PINT plays a role in suppressing the tumorigenicity of melanoma, which was further determined by xenograft model assay. LINC-PINT was significantly downregulated in melanoma tissues and cell lines. The overexpression of LINC-PINT in tumor cells resulted in significant tumor growth reduction and migration inhibition in A375, Mum2B and CRMM1 cells. Results based on the in vivo xenograft model were further consistent with the in vitro findings that LINC-PINT impeded growth and metastasis of melanoma cells. Microarray assay and bioinformatics analysis indicated that CDK1, CCNA2, AURKA, and PCNA were potential targets of LINC-PINT. In conclusion, LINC-PINT inhibits the tumorigenicity of melanoma through recruiting EZH2 to the promoter of its target genes, leading to H3K27 trimethylation and epigenetic silencing of target genes. LINC-PINT may serve as a novel diagnostic and therapeutic target for melanoma.

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Targeting EZH2 reactivates a breast cancer subtype-specific anti-metastatic transcriptional program

Cited by 68 publications

References 58 publications

Inhibition of EZH2 Catalytic Activity Selectively Targets a Metastatic Subpopulation in Triple-Negative Breast Cancer

Inhibition of EZH2 Catalytic Activity Selectively Targets a Metastatic Subpopulation in Triple-Negative Breast Cancer

Transcriptional elongation machinery controls vulnerability of breast cancer cells to PRC2 inhibitors

Long Non-coding RNA LINC-PINT Suppresses Cell Proliferation and Migration of Melanoma via Recruiting EZH2

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