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Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers.

Bihani T, Ezell SA, Ladd B, Grosskurth SE, Mazzola AM, Pietras M, Reimer C, Zinda M, Fawell S, D'Cruz CM - Oncotarget (2015)

Bottom Line: Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes.Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting.Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

View Article: PubMed Central - PubMed

Affiliation: AstraZeneca, R&D Boston, Waltham, MA.

ABSTRACT
Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

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Related in: MedlinePlus

Enrichment of MYC signatures in eveR and LTED-eveR linesA. Differential mRNA expression analyses of MCF7 parental and eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. B. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (P: Parental, eR: eveR). Data is represented as fold change over parental. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting. α-Vinculin was used as a loading control. C. Normalized Counts for 23 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in eveR cells versus Parental cells. D. Differential mRNA expression analyses of MCF7-LTED and LTED-eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. E. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (L: LTED, LeR: LTED-eveR). Data is represented as fold change over LTED controls. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. LTED-eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting α-Vinculin was used as a loading control. F. Normalized Counts for 6 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in LTED-eveR cells versus LTED cells. G. MYC protein levels measured by western blot analyses of parental and LTED derivatives treated with 500nM everolimus for the indicated timepoints (h: hours post-treatment). pS6: α-pS6 (S240/244). α-Vinculin was used as a loading control.
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Figure 2: Enrichment of MYC signatures in eveR and LTED-eveR linesA. Differential mRNA expression analyses of MCF7 parental and eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. B. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (P: Parental, eR: eveR). Data is represented as fold change over parental. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting. α-Vinculin was used as a loading control. C. Normalized Counts for 23 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in eveR cells versus Parental cells. D. Differential mRNA expression analyses of MCF7-LTED and LTED-eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. E. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (L: LTED, LeR: LTED-eveR). Data is represented as fold change over LTED controls. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. LTED-eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting α-Vinculin was used as a loading control. F. Normalized Counts for 6 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in LTED-eveR cells versus LTED cells. G. MYC protein levels measured by western blot analyses of parental and LTED derivatives treated with 500nM everolimus for the indicated timepoints (h: hours post-treatment). pS6: α-pS6 (S240/244). α-Vinculin was used as a loading control.

Mentions: To determine molecular changes that might contribute to the resistance to everolimus, we performed whole transcriptome RNA sequencing to provide a global view of altered pathways in MCF7 derivatives. One of the most robust changes observed was an increase in MYC mRNA expression in the MCF7-eveR line compared to the MCF7 parental line (Figure 2A). Using quantitative real-time PCR analyses, we validated the RNA sequencing result demonstrating that MYC mRNA is increased in all of the eveR lines (Figure 2B, top). Furthermore, increased protein expression of MYC was also seen in all eveR lines (Figure 2B, bottom). Gene Set Enrichment Analyses (GSEA) was used to identify gene signatures associated with resistance and in concordance with increased MYC expression, multiple MYC signatures were enriched (Supplemental Table 1), including those previously identified in LTED experiments [17]. Additionally, we show breast cancer specific MYC genes that are statistically differentially expressed between MCF7-eveR and MCF7-parental, suggesting the increased MYC is functional in this setting (Figure 2C) [17-19].


Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers.

Bihani T, Ezell SA, Ladd B, Grosskurth SE, Mazzola AM, Pietras M, Reimer C, Zinda M, Fawell S, D'Cruz CM - Oncotarget (2015)

Enrichment of MYC signatures in eveR and LTED-eveR linesA. Differential mRNA expression analyses of MCF7 parental and eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. B. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (P: Parental, eR: eveR). Data is represented as fold change over parental. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting. α-Vinculin was used as a loading control. C. Normalized Counts for 23 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in eveR cells versus Parental cells. D. Differential mRNA expression analyses of MCF7-LTED and LTED-eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. E. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (L: LTED, LeR: LTED-eveR). Data is represented as fold change over LTED controls. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. LTED-eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting α-Vinculin was used as a loading control. F. Normalized Counts for 6 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in LTED-eveR cells versus LTED cells. G. MYC protein levels measured by western blot analyses of parental and LTED derivatives treated with 500nM everolimus for the indicated timepoints (h: hours post-treatment). pS6: α-pS6 (S240/244). α-Vinculin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385860&req=5

Figure 2: Enrichment of MYC signatures in eveR and LTED-eveR linesA. Differential mRNA expression analyses of MCF7 parental and eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. B. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (P: Parental, eR: eveR). Data is represented as fold change over parental. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting. α-Vinculin was used as a loading control. C. Normalized Counts for 23 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in eveR cells versus Parental cells. D. Differential mRNA expression analyses of MCF7-LTED and LTED-eveR lines. Two biological replicates of each sample were sent for RNA sequencing. Graph represents the log2 TMM MYC counts. E. (top) Real-time PCR analyses measuring relative MYC mRNA expression in the indicated lines (L: LTED, LeR: LTED-eveR). Data is represented as fold change over LTED controls. Bars indicate SEM. (bottom) MYC protein expression by western blot analyses in the indicated cell lines. LTED-eveR lines were maintained in 500nM everolimus and media was replaced with fresh compound 16-24h prior to harvesting α-Vinculin was used as a loading control. F. Normalized Counts for 6 Statistically Differentially Expressed MYC Regulated Genes represented as Z-Score Log2 TMM in LTED-eveR cells versus LTED cells. G. MYC protein levels measured by western blot analyses of parental and LTED derivatives treated with 500nM everolimus for the indicated timepoints (h: hours post-treatment). pS6: α-pS6 (S240/244). α-Vinculin was used as a loading control.
Mentions: To determine molecular changes that might contribute to the resistance to everolimus, we performed whole transcriptome RNA sequencing to provide a global view of altered pathways in MCF7 derivatives. One of the most robust changes observed was an increase in MYC mRNA expression in the MCF7-eveR line compared to the MCF7 parental line (Figure 2A). Using quantitative real-time PCR analyses, we validated the RNA sequencing result demonstrating that MYC mRNA is increased in all of the eveR lines (Figure 2B, top). Furthermore, increased protein expression of MYC was also seen in all eveR lines (Figure 2B, bottom). Gene Set Enrichment Analyses (GSEA) was used to identify gene signatures associated with resistance and in concordance with increased MYC expression, multiple MYC signatures were enriched (Supplemental Table 1), including those previously identified in LTED experiments [17]. Additionally, we show breast cancer specific MYC genes that are statistically differentially expressed between MCF7-eveR and MCF7-parental, suggesting the increased MYC is functional in this setting (Figure 2C) [17-19].

Bottom Line: Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes.Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting.Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

View Article: PubMed Central - PubMed

Affiliation: AstraZeneca, R&D Boston, Waltham, MA.

ABSTRACT
Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

Show MeSH
Related in: MedlinePlus