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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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Combination effect observed with JQ1 and everolimus treatmentA and B. (left) ZR75 (top) or MCF7 (bottom) derivative cell lines were grown in three-dimensional Matrigel culture for six days in the presence of indicated compounds (JQ1; 200nM) and representative photomicrographs were taken using a 20X objective. (right) Proliferation of 3D structures was measured by CellTiterGlo six days post-treatment. An average percent of control is depicted in the histograms with standard deviation. eve: everolimus
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Figure 5: Combination effect observed with JQ1 and everolimus treatmentA and B. (left) ZR75 (top) or MCF7 (bottom) derivative cell lines were grown in three-dimensional Matrigel culture for six days in the presence of indicated compounds (JQ1; 200nM) and representative photomicrographs were taken using a 20X objective. (right) Proliferation of 3D structures was measured by CellTiterGlo six days post-treatment. An average percent of control is depicted in the histograms with standard deviation. eve: everolimus

Mentions: Given the aforementioned role of MYC as a driver of everolimus resistance, and as a target for BRD4 regulation in this setting, we hypothesized that treatment with JQ1, a small molecule inhibitor of BRD4, could be combined with everolimus to increase growth inhibition. The 3D Matrigel assay allows us to examine changes in growth while taking into consideration ECM and basement membrane interactions that more closely mimic growth in vivo [16]. Therefore, this assay was used to examine responses to combinations of everolimus and JQ1 (Figure 5). MCF7 and ZR75 derivatives were plated in Matrigel and their sensitivity to everolimus, JQ1 and the combination were examined. ZR75 Parental cells were relatively sensitive to everolimus alone at both concentrations and as previously shown (Figure 1 and 5A). However, the treatment with everolimus did not result in cell death. Rather, single cells remained intact and relatively viable in culture. Treatment with JQ1 in combination, however, resulted in an even further decrease in cell number (Figure 5A, right). This was consistent with the increased disintegration of cells depicted in the everolimus + JQ1 treatment group, suggesting the addition of JQ1 caused cell death. As expected, everolimus treatment of the ZR75-eveR line resulted in minimal sensitivity to the high concentration of everolimus, however when combined with JQ1, the lines were resensitized to everolimus and the combination resulted in greater attenuation of proliferation (Figure 5A). In fact, JQ1 resensitized the eveR lines to everolimus to a level comparable to that of the parental cells treated with everolimus alone. The ZR75-LTED lines exhibited less sensitivity to everolimus alone than the parental lines as seen previously (Figure 1 and Figure 5A). Combinations of everolimus and JQ1 in the LTED cells significantly decreased growth compared with either agent alone. Similar to parental cells, the combination also resulted in increased cell disintegration, indicative of cell death (Figure 5). Interestingly, the ZR75-LTED-eveR line was very sensitive to JQ1, even in the absence of everolimus. The combination of both drugs significantly reduced proliferation and greatly induced cell disintegration in the ZR75-LTED-eveR line (Figure 5). Similar results were seen with MCF7 cells (Figure 5B).


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)

Combination effect observed with JQ1 and everolimus treatmentA and B. (left) ZR75 (top) or MCF7 (bottom) derivative cell lines were grown in three-dimensional Matrigel culture for six days in the presence of indicated compounds (JQ1; 200nM) and representative photomicrographs were taken using a 20X objective. (right) Proliferation of 3D structures was measured by CellTiterGlo six days post-treatment. An average percent of control is depicted in the histograms with standard deviation. eve: everolimus
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Combination effect observed with JQ1 and everolimus treatmentA and B. (left) ZR75 (top) or MCF7 (bottom) derivative cell lines were grown in three-dimensional Matrigel culture for six days in the presence of indicated compounds (JQ1; 200nM) and representative photomicrographs were taken using a 20X objective. (right) Proliferation of 3D structures was measured by CellTiterGlo six days post-treatment. An average percent of control is depicted in the histograms with standard deviation. eve: everolimus
Mentions: Given the aforementioned role of MYC as a driver of everolimus resistance, and as a target for BRD4 regulation in this setting, we hypothesized that treatment with JQ1, a small molecule inhibitor of BRD4, could be combined with everolimus to increase growth inhibition. The 3D Matrigel assay allows us to examine changes in growth while taking into consideration ECM and basement membrane interactions that more closely mimic growth in vivo [16]. Therefore, this assay was used to examine responses to combinations of everolimus and JQ1 (Figure 5). MCF7 and ZR75 derivatives were plated in Matrigel and their sensitivity to everolimus, JQ1 and the combination were examined. ZR75 Parental cells were relatively sensitive to everolimus alone at both concentrations and as previously shown (Figure 1 and 5A). However, the treatment with everolimus did not result in cell death. Rather, single cells remained intact and relatively viable in culture. Treatment with JQ1 in combination, however, resulted in an even further decrease in cell number (Figure 5A, right). This was consistent with the increased disintegration of cells depicted in the everolimus + JQ1 treatment group, suggesting the addition of JQ1 caused cell death. As expected, everolimus treatment of the ZR75-eveR line resulted in minimal sensitivity to the high concentration of everolimus, however when combined with JQ1, the lines were resensitized to everolimus and the combination resulted in greater attenuation of proliferation (Figure 5A). In fact, JQ1 resensitized the eveR lines to everolimus to a level comparable to that of the parental cells treated with everolimus alone. The ZR75-LTED lines exhibited less sensitivity to everolimus alone than the parental lines as seen previously (Figure 1 and Figure 5A). Combinations of everolimus and JQ1 in the LTED cells significantly decreased growth compared with either agent alone. Similar to parental cells, the combination also resulted in increased cell disintegration, indicative of cell death (Figure 5). Interestingly, the ZR75-LTED-eveR line was very sensitive to JQ1, even in the absence of everolimus. The combination of both drugs significantly reduced proliferation and greatly induced cell disintegration in the ZR75-LTED-eveR line (Figure 5). Similar results were seen with MCF7 cells (Figure 5B).

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