Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and through secondary effects [RNA-seq]
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ABSTRACT: While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER’s actions, a significant number relapse. Approximately 30% of these recurrences harbor activating mutations in ER’s ligand binding domain. ER mutations have been shown to confer ligand-independent function to ER; however, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate mutant ER’s molecular effects, we developed multiple isogenic ER mutant cell lines for the two most common ER ligand binding domain mutations, Y537S and D538G. We found that these mutations induce differential expression of thousands of genes, the majority of which are specific to one or the other mutation and are not observed upon estrogen treatment of wildtype cells. The mutant-specific genes show consistent differential expression across ER mutant lines developed in other laboratories. The observed gene expression changes cannot be explained by constitutive ER activity alone, as wildtype cells with long term estrogen exposure only exhibit some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations have minor effects on ER genomic binding, with the exception of ligand independence, we observed substantial differences in chromatin accessibility due to ER mutations. Mutant ER is bound to approximately a quarter of mutant-enriched accessible regions that are enriched for other DNA binding factors including FOXA1, CTCF, and OCT1. Our findings indicate that mutant ER causes several consistent effects on gene expression both indirectly and through constant activity.
Project description:While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER’s actions, a significant number relapse. Approximately 30% of these recurrences harbor activating mutations in ER’s ligand binding domain. ER mutations have been shown to confer ligand-independent function to ER; however, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate mutant ER’s molecular effects, we developed multiple isogenic ER mutant cell lines for the two most common ER ligand binding domain mutations, Y537S and D538G. We found that these mutations induce differential expression of thousands of genes, the majority of which are specific to one or the other mutation and are not observed upon estrogen treatment of wildtype cells. The mutant-specific genes show consistent differential expression across ER mutant lines developed in other laboratories. The observed gene expression changes cannot be explained by constitutive ER activity alone, as wildtype cells with long term estrogen exposure only exhibit some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations have minor effects on ER genomic binding, with the exception of ligand independence, we observed substantial differences in chromatin accessibility due to ER mutations. Mutant ER is bound to approximately a quarter of mutant-enriched accessible regions that are enriched for other DNA binding factors including FOXA1, CTCF, and OCT1. Our findings indicate that mutant ER causes several consistent effects on gene expression both indirectly and through constant activity.
Project description:While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER’s actions, a significant number relapse. Approximately 30% of these recurrences harbor activating mutations in ER’s ligand binding domain. ER mutations have been shown to confer ligand-independent function to ER; however, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate mutant ER’s molecular effects, we developed multiple isogenic ER mutant cell lines for the two most common ER ligand binding domain mutations, Y537S and D538G. We found that these mutations induce differential expression of thousands of genes, the majority of which are specific to one or the other mutation and are not observed upon estrogen treatment of wildtype cells. The mutant-specific genes show consistent differential expression across ER mutant lines developed in other laboratories. The observed gene expression changes cannot be explained by constitutive ER activity alone, as wildtype cells with long term estrogen exposure only exhibit some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations have minor effects on ER genomic binding, with the exception of ligand independence, we observed substantial differences in chromatin accessibility due to ER mutations. Mutant ER is bound to approximately a quarter of mutant-enriched accessible regions that are enriched for other DNA binding factors including FOXA1, CTCF, and OCT1. Our findings indicate that mutant ER causes several consistent effects on gene expression both indirectly and through constant activity.
Project description:Estrogen receptor α (ER) mutations occur in up to 30% of metastatic ER-positive breast cancers. Recent data has shown that ER mutations impact the expression of thousands of genes not typically regulated by wildtype ER. While the majority of these altered genes can be explained by constant activity of mutant ER or genomic changes such as altered ER binding and chromatin accessibility, as much as 33% remain unexplained, indicating the potential for post-transcriptional effects. Here we explored the role of microRNAs in mutant ER-driven gene regulation and identified several microRNAs that are dysregulated in ER mutant cells. These differentially regulated microRNAs target a significant portion of mutant-specific genes involved in key cellular processes. When the activity of microRNAs is altered using mimics or inhibitors, significant changes are observed in gene expression and cellular proliferation related to mutant ER. An in-depth evaluation of miR-301b led us to discover an important role for PRKD3 in the proliferation of ER mutant cells. Our findings show that microRNAs contribute to mutant ER gene regulation and cellular effects in breast cancer cells.
Project description:Estrogen receptor α (ER) mutations occur in up to 30% of metastatic ER-positive breast cancers. Recent data has shown that ER mutations impact the expression of thousands of genes not typically regulated by wildtype ER. While the majority of these altered genes can be explained by constant activity of mutant ER or genomic changes such as altered ER binding and chromatin accessibility, as much as 33% remain unexplained, indicating the potential for post-transcriptional effects. Here we explored the role of microRNAs in mutant ER-driven gene regulation and identified several microRNAs that are dysregulated in ER mutant cells. These differentially regulated microRNAs target a significant portion of mutant-specific genes involved in key cellular processes. When the activity of microRNAs is altered using mimics or inhibitors, significant changes are observed in gene expression and cellular proliferation related to mutant ER. An in-depth evaluation of miR-301b led us to discover an important role for PRKD3 in the proliferation of ER mutant cells. Our findings show that microRNAs contribute to mutant ER gene regulation and cellular effects in breast cancer cells.
Project description:Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression; however, the molecular mechanisms underlying ER’s regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways; ETV4 loss impacts chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture where hollow organoids were formed. Our results show that ETV4 plays a necessary role in estrogen signaling in endometrial cancer cells.
Project description:Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression; however, the molecular mechanisms underlying ER’s regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways; ETV4 loss impacts chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture where hollow organoids were formed. Our results show that ETV4 plays a necessary role in estrogen signaling in endometrial cancer cells.
Project description:Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression; however, the molecular mechanisms underlying ER’s regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways; ETV4 loss impacts chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture where hollow organoids were formed. Our results show that ETV4 plays a necessary role in estrogen signaling in endometrial cancer cells.
Project description:The ESR1 ligand binding domain activating mutations are the most prevalent genetic mechanism of acquired endocrine resistance in metastatic hormone receptor-positive breast cancer. These mutations confer endocrine resistance that remains estrogen receptor (ER) dependent. We hypothesized that in the presence of the ER mutations, continued ER blockade with endocrine therapies that target mutant ER is essential for tumor suppression even with chemotherapy treatment. Here, we conducted comprehensive pre-clinical in vitro and in vivo experiments testing the efficacy of adding fulvestrant to fluorouracil (5FU) and the 5FU pro-drug, capecitabine, in models of wild-type (WT) and mutant ER. Our findings revealed that while this combination had an additive effect in the presence of WT-ER, in the presence of the Y537S ER mutation there was synergy. Notably, these effects were not seen with the combination of 5FU and selective estrogen receptor modulators, such as tamoxifen, or in the absence of intact P53. Likewise, in a patient derived xenograft (PDX) harboring a Y537S ER mutation the addition of fulvestrant to capecitabine potentiated tumor suppression. Moreover, multiplex immunofluorescence revealed that this effect was due to decreased cell proliferation in all cells expressing ER and was not dependent on the degree of ER expression. Taken together, these results support the clinical investigation of the combination of ER antagonists with capecitabine in patients with metastatic hormone receptor-positive breast cancer who have experienced progression on endocrine therapy and targeted therapies, particularly in the presence of an ESR1 activating mutation.
Project description:Estrogen receptor positive (ER+) breast cancers that develop resistance to therapies that target the ER are the most common cause of breast cancer death. Beyond mutations in ER, which occur in 25-30% of patients treated with aromatase inhibitors (AIs), our understanding of clinical mechanisms of resistance to ER-directed therapies remains incomplete. We identified activating HER2 mutations in metastatic biopsies from eight patients with ER+ metastatic breast cancer who had developed resistance to ER-directed agents, including AIs, tamoxifen, and fulvestrant. Examination of treatment-naïve primary tumors in five patients revealed no evidence of pre-existing mutations in four of five patients, suggesting that these mutations were acquired under the selective pressure of ER-directed therapy. These mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of acquired resistance to ER-directed therapies. In vitro analysis confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. Resistance was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, highlighting an effective treatment strategy in these patients.
Project description:HSP90 inhibitors can target many oncoproteins simultaneously, but none have made it through clinical trials due to dose-limiting toxicity and induction of heat shock response, leading to clinical resistance. We identified diptoindonesin G (dip G) as an HSP90 modulator that can promote degradation of HSP90 clients by binding to the middle domain of HSP90 without inducing heat shock response. Although dip G does not bind to the ligand binding domain (LBD) of estrogen receptor (ER), it promotes degradation of ER in breast cancer cells. We further showed that treatment of ER LBD mutantexpressing cells with dip G promoted degradation of wild type and mutant ER with similar efficacy, downregulated ER-regulated gene expression, and inhibited cell proliferation. Our data suggest that dip G circumvents some obstacles associated with HSP90 inhibition and may be developed as a new therapeutic avenue to various cancers, particularly endocrine resistant breast cancer harboring ESR1 mutations