The SERM/SERD bazedoxifene disrupts ESR1 helix 12 to overcome acquired hormone resistance in breast cancer cells.

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα)) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER +breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We found that BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show BZA's selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.

Unraveling the clinicopathological features driving the emergence of ESR1 mutations in metastatic breast cancer.

ESR1 mutations were recently found to be an important mechanism of endocrine resistance in ER-positive (ER + ) metastatic breast cancer. To determine the clinicopathological features driving the emergence of the ESR1 mutations we studied plasma cfDNA and detailed clinical data collected from patients with metastatic breast cancer. Droplet Digital PCR was performed for the detection of the most common ESR1 mutations and PIK3CA mutations. Among the patients with ER + /HER2- disease, ESR1 mutations were detected in 30% of the patients. There were no associations between the pathological features of the primary disease or time to distant recurrence and the emergence of ESR1 mutations in metastatic disease. The prevalence of the ESR1 mutations was significantly associated with prior treatment with an aromatase inhibitor in the adjuvant or metastatic setting. The prevalence of the ESR1 mutations was also positively associated with prior fulvestrant treatment. Conversely, the prevalence of ESR1 mutations was lower after treatment with a CDK4/6 inhibitor. There were no significant associations between specific systemic treatments and the prevalence of PIK3CA mutations. These results support the evolution of the ESR1 mutations under the selective pressure of treatment with aromatase inhibitors in the adjuvant and metastatic settings and have important implications in the optimization of adjuvant and metastatic treatment in ER + breast cancer.

Allele-Specific Chromatin Recruitment and Therapeutic Vulnerabilities of ESR1 Activating Mutations.

Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER+) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets.

Embryonic transcription factor SOX9 drives breast cancer endocrine resistance.

The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2-ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor.

The TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR's ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.

ESR1 mutations­a mechanism for acquired endocrine resistance in breast cancer.

Approximately 70% of breast cancers are oestrogen receptor α (ER) positive, and are, therefore, treated with endocrine therapies. However, about 25% of patients with primary disease and almost all patients with metastases will present with or eventually develop endocrine resistance. Despite the magnitude of this clinical challenge, the mechanisms underlying the development of resistance remain largely unknown. In the past 2 years, several studies unveiled gain-of-function mutations in ESR1, the gene encoding the ER, in approximately 20% of patients with metastatic ER-positive disease who received endocrine therapies, such as tamoxifen and aromatase inhibitors. These mutations are clustered in a 'hotspot' within the ligand-binding domain (LBD) of the ER and lead to ligand-independent ER activity that promotes tumour growth, partial resistance to endocrine therapy, and potentially enhanced metastatic capacity; thus, ER LBD mutations might account for a mechanism of acquired endocrine resistance in a substantial fraction of patients with metastatic disease. In general, the absence of detectable ESR1 mutations in patients with treatment-naive disease, and the correlation between the frequency of patients with tumours harbouring these mutations and the number of endocrine treatments received suggest that, under selective treatment pressure, clonal expansion of rare mutant clones occurs, leading to resistance. Preclinical and clinical development of rationale-based novel therapeutic strategies that inhibit these ER mutants has the potential to substantially improve treatment outcomes. We discuss the contribution of ESR1 mutations to the development of acquired resistance to endocrine therapy, and evaluate how mutated ER can be detected and targeted to overcome resistance and improve patient outcomes.

Emergence of constitutively active estrogen receptor-α mutations in pretreated advanced estrogen receptor-positive breast cancer.

PURPOSE: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. EXPERIMENTAL DESIGN: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER(+)/HER2(-)) and, as controls, 115 ER-negative (ER(-)) tumors. The ER(+) samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. RESULTS: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER(+) metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%-21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%-41%). These mutations were not detected in primary or treatment-naïve ER(+) cancer or in any stage of ER(-) disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. CONCLUSIONS: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER(+) breast cancer.

PDEF promotes luminal differentiation and acts as a survival factor for ER-positive breast cancer cells.

Breast cancer is a heterogeneous disease and can be classified based on gene expression profiles that reflect distinct epithelial subtypes. We identify prostate-derived ETS factor (PDEF) as a mediator of mammary luminal epithelial lineage-specific gene expression and as a factor required for tumorigenesis in a subset of breast cancers. PDEF levels strongly correlate with estrogen receptor (ER)-positive luminal breast cancer, and PDEF transcription is inversely regulated by ER and GATA3. Furthermore, PDEF is essential for luminal breast cancer cell survival and is required in models of endocrine resistance. These results offer insights into the function of this ETS factor that are clinically relevant and may be of therapeutic value for patients with breast cancer treated with endocrine therapy.

Digital quantification of gene expression in sequential breast cancer biopsies reveals activation of an immune response.

Advancements in molecular biology have unveiled multiple breast cancer promoting pathways and potential therapeutic targets. Large randomized clinical trials remain the ultimate means of validating therapeutic efficacy, but they require large cohorts of patients and are lengthy and costly. A useful approach is to conduct a window of opportunity study in which patients are exposed to a drug pre-surgically during the interval between the core needle biopsy and the definitive surgery. These are non-therapeutic studies and the end point is not clinical or pathological response but rather evaluation of molecular changes in the tumor specimens that can predict response. However, since the end points of the non-therapeutic studies are biologic, it is critical to first define the biologic changes that occur in the absence of treatment. In this study, we compared the molecular profiles of breast cancer tumors at the time of the diagnostic biopsy versus the definitive surgery in the absence of any intervention using the Nanostring nCounter platform. We found that while the majority of the transcripts did not vary between the two biopsies, there was evidence of activation of immune related genes in response to the first biopsy and further investigations of the immune changes after a biopsy in early breast cancer seem warranted.

Regulation of MCP-1 chemokine transcription by p53.

BACKGROUND: Our previous studies showed that the expression of the monocyte-chemoattractant protein (MCP)-1, a chemokine, which triggers the infiltration and activation of cells of the monocyte-macrophage lineage, is abrogated in human papillomavirus (HPV)-positive premalignant and malignant cells. In silico analysis of the MCP-1 upstream region proposed a putative p53 binding side about 2.5 kb upstream of the transcriptional start. The aim of this study is to monitor a physiological role of p53 in this process. RESULTS: The proposed p53 binding side could be confirmed in vitro by electrophoretic-mobility-shift assays and in vivo by chromatin immunoprecipitation. Moreover, the availability of p53 is apparently important for chemokine regulation, since TNF-alpha can induce MCP-1 only in human keratinocytes expressing the viral oncoprotein E7, but not in HPV16 E6 positive cells, where p53 becomes degraded. A general physiological role of p53 in MCP-1 regulation was further substantiated in HPV-negative cells harboring a temperature-sensitive mutant of p53 and in Li-Fraumeni cells, carrying a germ-line mutation of p53. In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-alpha treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-alpha addition, directly confirming a crosstalk between p53 and MCP-1. CONCLUSION: These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.

The ternary complex factor net is downregulated by hypoxia and regulates hypoxia-responsive genes.

Hypoxia and the Net ternary complex factor (TCF) regulate similar processes (angiogenesis, wound healing, and cellular migration) and genes (PAI-1, c-fos, erg-1, NOS-2, HO-1, and vascular endothelial growth factor genes), suggesting that they are involved in related pathways. We show here that hypoxia regulates Net differently from the other TCFs and that Net plays a role in the hypoxic response in vivo in mice and in cells. Hypoxia induces Net depletion from target promoters, nuclear export, ubiquitylation, and proteasomal degradation. Key mediators of the hypoxic response, the prolyl-4-hydroxylases containing domain proteins (PHDs), regulate Net. PHD downregulation in normoxia leads to Net degradation, and PHD overexpression delays Net downregulation by hypoxia. Net inhibition by RNA interference or mutation leads to altered regulation by hypoxia of the Net targets PAI-1, c-fos, and egr-1. We propose that hypoxia stimulates transcription of target promoters through removal of the repressor function of Net. Interestingly, the hematocrit response to a chemical inducer of hypoxia-like responses (cobalt chloride) is strongly altered in Net mutant mice. Our results show that the Net TCF is part of the biological response to hypoxia, adding a new component to an important pathological and physiological process.

The ternary complex factor Net regulates cell migration through inhibition of PAI-1 expression.

Net, Elk-1, and Sap-1 are members of the ternary complex factor (TCF) subfamily of Ets transcription factors. They form ternary complexes with serum response factor (SRF) on serum response elements of immediate early genes such as c-fos and egr-1 and mediate responses to growth factors and mitogen-activated protein kinase signaling. Although the TCFs have been extensively studied as intermediates in signaling cascades, surprisingly little is known about their different target genes and physiological functions. We report that Net homozygous mutant mouse embryonic fibroblasts have a defect in cell migration. This defect results at least in part from increased expression of plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor (serpin) that controls extracellular proteolysis and cell matrix adhesion. The defect in cell migration can be reverted by the addition of a PAI-1 blocking antibody. Net represses PAI-1 promoter activity and binds to a specific region of the promoter containing Ets binding sites in the absence of SRF. We conclude that Net is a negative regulator of PAI-1 expression and is thereby involved in cell migration.

Loss of net as repressor leads to constitutive increased c-fos transcription in cervical cancer cells.

We have investigated the expression of c-fos in cervical carcinoma cells and in somatic cell hybrids derived therefrom. In malignant cells, c-fos was constitutively expressed even after serum starvation. Dissection of the c-fos promoter showed that expression was mainly controlled by the SRE motif, which was active in malignant cells, but repressed in their non-malignant counterparts. Constitutive SRE activity was not mediated by sustained mitogen-activated protein kinase activity but because of inefficient expression of the ternary complex factor Net, which was either very low or even barely discernible. Chromatin immunoprecipitation assays revealed that Net directly binds to the SRE nucleoprotein complex in non-tumorigenic cells, but not in malignant segregants. Small interfering RNA targeted against Net resulted in enhanced c-fos transcription, clearly illustrating its repressor function. Conversely, stable ectopic expression of Net in malignant cells negatively regulated endogenous c-fos, resulting in a disappearance of the c-Fos protein from the AP-1 transcription complex. These data indicate that loss of Net and constitutive c-fos expression appear to be a key event in the transformation of cervical cancer cells.

Siah-1b is a direct transcriptional target of p53: identification of the functional p53 responsive element in the siah-1b promoter.

Siah proteins are E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina), a protein required for the R7 photoreceptor development. We have previously found that the expression of human siah-1 and its mouse homologue siah-1b are induced by p53 during apoptosis and tumor reversion. So far, no evidence that the siah-1b gene is a direct transcriptional target of p53 has been provided. In the present study we investigate this issue. Northern blot analysis with a specific probe demonstrates an increase in siah-1b transcription on activation of endogenous and inducible exogenous p53. To explore whether this effect is directly mediated by p53 we analyzed 20 kb of chromosome X DNA, containing the siah-1b locus. A p53-binding site was identified in the siah-1b promoter, located at nucleotides -2155/-2103 relative to the translational start site. This site is composed of two half-sites, conforming to the p53-binding consensus sequence but separated by a nonclassical 33-bp spacer. In luciferase assays, p53 induces a substantial increase in siah-1b promoter activity. Gel shift and DNase-I-footprinting studies, combined with mutational analysis and chromatin immunoprecipitation, indicate that p53 effectively binds the siah-1b promoter in vitro and in vivo. Thus, the siah-1b gene is a direct transcriptional target of p53.

The tumor suppressor p53 inhibits Net, an effector of Ras/extracellular signal-regulated kinase signaling.

The tumor suppressor function of p53 is linked to its ability to repress gene expression, but the mechanisms of specific gene repression are poorly understood. We report that wild-type p53 inhibits an effector of the Ras oncogene/mitogen-activated protein (MAP) kinase pathway, the transcription factor Net. Tumor-associated mutant p53s are less efficient inhibitors. p53 inhibits by preventing phosphorylation of Net by MAP kinases. Loss of p53 in vivo leads to increased Net phosphorylation in response to wound healing and UV irradiation of skin. Our results show that p53 can repress specific gene expression by inhibiting Net, a factor implicated in cell cycle entry.

Ets ternary complex transcription factors.

The three ternary complex factors (TCFs) Elk-1, Net and Sap-1 form a subfamily of the E twenty-six (Ets) domain transcription factors. Their characteristic property is the ability to form a ternary nucleoprotein complex with the serum response factor (SRF) over the serum response element (SRE) of the c-fos promoter. The molecular mechanisms that underlie the function and regulation of these factors have been extensively studied and the TCFs are a paradigm for the study of transcriptional regulation in response to extracellular signalling through the mitogen-activated protein (MAP) kinase pathway. As final effectors of multiple signalling pathways and components of protein complexes on immediate early promoters, they represent key elements in the complex and dynamic regulation of gene expression. This review summarises the molecular, structural and biochemical studies that have led to the understanding of the functional domains of the TCFs, ternary complex formation, transcriptional regulation, protein partners and target genes in cell lines. Finally, the emerging studies of the biological roles of the TCFs in vivo will be discussed.