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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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MYC is upregulated in the eveR setting in a BRD4-dependent mannerA. Chromatin immunoprecipitation (ChIP) analyses of MCF7 Parental or MCF7-eveR. (left) ChIP experiments were performed with an antibody specific to BRD4 (Bethyl Labs). or IgG control. Bars represent the average % input as measured by real-time PCR analyses using primers specific for the MYC gene from four independent chromatin immunoprecipitations. Bars represent SEM. Student's t-test was performed to calculated statistical significance. **p-value=0.0082. (right) H3K27me3 histone ChIPs, % input was calculated from three independent ChIPs using an antibody specific for acetylated H3K27, and were then normalized to % input from total H3. Average fold change compared to parental is shown in the histogram with SEM. B. ChIP performed similar to (A) on MCF7 Parental cells (Par), MCF7-eveR cells (eveR) or MCF7-cells treated with 500nM JQ1 for 24h (eveR + JQ1). **Student's t-test was performed to calculated statistical significance. MCF7-eveR compared to MCF7 Parental p-value =0.0012; MCF7-eveR compared to MCF7-eveR + JQ1 p=0.0021. C. Real-time analyses in MCF7 Parental cells (Par), MCF7 cells treated with 500nM JQ1 for 24h (Par + JQ1), MCF7-eveR cells (eveR) or MCF7-eveR treated with 500nM JQ1 for 24h (eveR + JQ1) with MYC specific primers. Data is an average of three biological replicates and is represented as fold change over MCF7 Parental with SEM. MCF7-eveR compared to MCF7-Parental: ***p=8.9E=-05. MCF7 eveR compared to MCF7-eveR + JQ1: ***p=0.000341. D and E. Western blot analyses of indicated cell lines. Cells were treated with JQ1 (500nM, 24h). Cells were harvested post-treatment, lysed and extracted protein was immunoblotted for MYC. α-Vinculin was used as a loading control.
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Figure 4: MYC is upregulated in the eveR setting in a BRD4-dependent mannerA. Chromatin immunoprecipitation (ChIP) analyses of MCF7 Parental or MCF7-eveR. (left) ChIP experiments were performed with an antibody specific to BRD4 (Bethyl Labs). or IgG control. Bars represent the average % input as measured by real-time PCR analyses using primers specific for the MYC gene from four independent chromatin immunoprecipitations. Bars represent SEM. Student's t-test was performed to calculated statistical significance. **p-value=0.0082. (right) H3K27me3 histone ChIPs, % input was calculated from three independent ChIPs using an antibody specific for acetylated H3K27, and were then normalized to % input from total H3. Average fold change compared to parental is shown in the histogram with SEM. B. ChIP performed similar to (A) on MCF7 Parental cells (Par), MCF7-eveR cells (eveR) or MCF7-cells treated with 500nM JQ1 for 24h (eveR + JQ1). **Student's t-test was performed to calculated statistical significance. MCF7-eveR compared to MCF7 Parental p-value =0.0012; MCF7-eveR compared to MCF7-eveR + JQ1 p=0.0021. C. Real-time analyses in MCF7 Parental cells (Par), MCF7 cells treated with 500nM JQ1 for 24h (Par + JQ1), MCF7-eveR cells (eveR) or MCF7-eveR treated with 500nM JQ1 for 24h (eveR + JQ1) with MYC specific primers. Data is an average of three biological replicates and is represented as fold change over MCF7 Parental with SEM. MCF7-eveR compared to MCF7-Parental: ***p=8.9E=-05. MCF7 eveR compared to MCF7-eveR + JQ1: ***p=0.000341. D and E. Western blot analyses of indicated cell lines. Cells were treated with JQ1 (500nM, 24h). Cells were harvested post-treatment, lysed and extracted protein was immunoblotted for MYC. α-Vinculin was used as a loading control.

Mentions: BRD4 (bromodomain-containing protein 4) is a transcriptional regulator that recognizes and binds acetylated histones and drives transcription of many genes, including MYC. While MYC regulation by BRD4 has been extensively studied in hematological malignancies, relatively little is known about this mechanism in solid tumors, particularly in breast cancer. A recent publication outlined a role for BRD-mediated transcription of MYC in tamoxifen-resistant breast cancers [20]. As mentioned above, the LTED setting mimics patient response to aromatase inhibition and tamoxifen treatment [10]. Our observations of MYC upregulation in the eveR setting and this novel connection between BRD regulation and tamoxifen resistance prompted us to investigate BRD4 as a potential regulator of MYC in the eveR lines. In order to determine whether MYC upregulation is mediated by enhanced BRD4 association with the MYC gene we performed chromatin immunoprecipitation (ChIP) analyses. We observed that eveR lines showed increase binding of BRD4 to the MYC gene (Figure 4A, left). We also performed ChIP using antibodies specific to acetylated H3K27, a mark of active transcription. We observed an acetylated H3K27 mark on the MYC gene in the MCF7-eveR line relative to MCF7 parental (Figure 4A, right). These data are consistent with the observation that MYC is upregulated at the transcript level in the eveR setting (Figure 2). Recently, the small molecule JQ1 was shown to inhibit BRD4-dependent transcription by preventing the binding of the BRD4 bromodomain to acetylated lysine residues [21]. To further test the specific regulation of MYC by BRD4 in the eveR setting we used JQ1 in our ChIP analyses. Treatment with JQ1 resulted in abrogation of BRD4-occupancy at the MYC gene in eveR lines (Figure 4B). This experiment was repeated with a second BRD4-specific antibody and obtained similar results (Supplemental Figure 2). In concordance with this, JQ1 treatment resulted in downregulation of MYC mRNA expression in the eveR lines (Figure 4C). In fact, JQ1 treatment abolished MYC upregulation in all eveR and LTED-eveR cell lines, suggesting that BRD4 is responsible for the increased MYC expression in these settings (Figure 4D and 4E). Interestingly, fractionation of MCF7 derivatives revealed that global association of BRD4 with chromatin does not change in the eveR lines (data not shown). In addition, neither MCF7 nor ZR75 eveR derivatives showed an increase in BRD4 mRNA expression (Supplemental Figure 3).


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)

MYC is upregulated in the eveR setting in a BRD4-dependent mannerA. Chromatin immunoprecipitation (ChIP) analyses of MCF7 Parental or MCF7-eveR. (left) ChIP experiments were performed with an antibody specific to BRD4 (Bethyl Labs). or IgG control. Bars represent the average % input as measured by real-time PCR analyses using primers specific for the MYC gene from four independent chromatin immunoprecipitations. Bars represent SEM. Student's t-test was performed to calculated statistical significance. **p-value=0.0082. (right) H3K27me3 histone ChIPs, % input was calculated from three independent ChIPs using an antibody specific for acetylated H3K27, and were then normalized to % input from total H3. Average fold change compared to parental is shown in the histogram with SEM. B. ChIP performed similar to (A) on MCF7 Parental cells (Par), MCF7-eveR cells (eveR) or MCF7-cells treated with 500nM JQ1 for 24h (eveR + JQ1). **Student's t-test was performed to calculated statistical significance. MCF7-eveR compared to MCF7 Parental p-value =0.0012; MCF7-eveR compared to MCF7-eveR + JQ1 p=0.0021. C. Real-time analyses in MCF7 Parental cells (Par), MCF7 cells treated with 500nM JQ1 for 24h (Par + JQ1), MCF7-eveR cells (eveR) or MCF7-eveR treated with 500nM JQ1 for 24h (eveR + JQ1) with MYC specific primers. Data is an average of three biological replicates and is represented as fold change over MCF7 Parental with SEM. MCF7-eveR compared to MCF7-Parental: ***p=8.9E=-05. MCF7 eveR compared to MCF7-eveR + JQ1: ***p=0.000341. D and E. Western blot analyses of indicated cell lines. Cells were treated with JQ1 (500nM, 24h). Cells were harvested post-treatment, lysed and extracted protein was immunoblotted for MYC. α-Vinculin was used as a loading control.
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Figure 4: MYC is upregulated in the eveR setting in a BRD4-dependent mannerA. Chromatin immunoprecipitation (ChIP) analyses of MCF7 Parental or MCF7-eveR. (left) ChIP experiments were performed with an antibody specific to BRD4 (Bethyl Labs). or IgG control. Bars represent the average % input as measured by real-time PCR analyses using primers specific for the MYC gene from four independent chromatin immunoprecipitations. Bars represent SEM. Student's t-test was performed to calculated statistical significance. **p-value=0.0082. (right) H3K27me3 histone ChIPs, % input was calculated from three independent ChIPs using an antibody specific for acetylated H3K27, and were then normalized to % input from total H3. Average fold change compared to parental is shown in the histogram with SEM. B. ChIP performed similar to (A) on MCF7 Parental cells (Par), MCF7-eveR cells (eveR) or MCF7-cells treated with 500nM JQ1 for 24h (eveR + JQ1). **Student's t-test was performed to calculated statistical significance. MCF7-eveR compared to MCF7 Parental p-value =0.0012; MCF7-eveR compared to MCF7-eveR + JQ1 p=0.0021. C. Real-time analyses in MCF7 Parental cells (Par), MCF7 cells treated with 500nM JQ1 for 24h (Par + JQ1), MCF7-eveR cells (eveR) or MCF7-eveR treated with 500nM JQ1 for 24h (eveR + JQ1) with MYC specific primers. Data is an average of three biological replicates and is represented as fold change over MCF7 Parental with SEM. MCF7-eveR compared to MCF7-Parental: ***p=8.9E=-05. MCF7 eveR compared to MCF7-eveR + JQ1: ***p=0.000341. D and E. Western blot analyses of indicated cell lines. Cells were treated with JQ1 (500nM, 24h). Cells were harvested post-treatment, lysed and extracted protein was immunoblotted for MYC. α-Vinculin was used as a loading control.
Mentions: BRD4 (bromodomain-containing protein 4) is a transcriptional regulator that recognizes and binds acetylated histones and drives transcription of many genes, including MYC. While MYC regulation by BRD4 has been extensively studied in hematological malignancies, relatively little is known about this mechanism in solid tumors, particularly in breast cancer. A recent publication outlined a role for BRD-mediated transcription of MYC in tamoxifen-resistant breast cancers [20]. As mentioned above, the LTED setting mimics patient response to aromatase inhibition and tamoxifen treatment [10]. Our observations of MYC upregulation in the eveR setting and this novel connection between BRD regulation and tamoxifen resistance prompted us to investigate BRD4 as a potential regulator of MYC in the eveR lines. In order to determine whether MYC upregulation is mediated by enhanced BRD4 association with the MYC gene we performed chromatin immunoprecipitation (ChIP) analyses. We observed that eveR lines showed increase binding of BRD4 to the MYC gene (Figure 4A, left). We also performed ChIP using antibodies specific to acetylated H3K27, a mark of active transcription. We observed an acetylated H3K27 mark on the MYC gene in the MCF7-eveR line relative to MCF7 parental (Figure 4A, right). These data are consistent with the observation that MYC is upregulated at the transcript level in the eveR setting (Figure 2). Recently, the small molecule JQ1 was shown to inhibit BRD4-dependent transcription by preventing the binding of the BRD4 bromodomain to acetylated lysine residues [21]. To further test the specific regulation of MYC by BRD4 in the eveR setting we used JQ1 in our ChIP analyses. Treatment with JQ1 resulted in abrogation of BRD4-occupancy at the MYC gene in eveR lines (Figure 4B). This experiment was repeated with a second BRD4-specific antibody and obtained similar results (Supplemental Figure 2). In concordance with this, JQ1 treatment resulted in downregulation of MYC mRNA expression in the eveR lines (Figure 4C). In fact, JQ1 treatment abolished MYC upregulation in all eveR and LTED-eveR cell lines, suggesting that BRD4 is responsible for the increased MYC expression in these settings (Figure 4D and 4E). Interestingly, fractionation of MCF7 derivatives revealed that global association of BRD4 with chromatin does not change in the eveR lines (data not shown). In addition, neither MCF7 nor ZR75 eveR derivatives showed an increase in BRD4 mRNA expression (Supplemental Figure 3).

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