Gilles Buchwalter

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. Clin Cancer Res; 20(7); 1757–67. ©2014 AACR.

Regulation of MCP-1 chemokine transcription by p53

BackgroundOur 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.ResultsThe 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-α 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-α treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-α addition, directly confirming a crosstalk between p53 and MCP-1.ConclusionThese 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.

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.

TMPRSS2:ERG blocks neuroendocrine and luminal cell differentiation to maintain prostate cancer proliferation

The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains poorly understood. To address this, we compared the transcriptional effects of TMPRSS2:ERG expression in a transgenic mouse model with those of ERG knockdown in a TMPRSS2:ERG-positive PC cell line. This reveals that ERG represses the expression of a previously unreported set of androgen receptor (AR)—independent neuronal genes that are indicative of neuroendocrine (NE) cell differentiation—in addition to previously reported AR-regulated luminal genes. Cell sorting and proliferation assays performed after sustained ERG knockdown indicate that ERG drives proliferation and blocks the differentiation of prostate cells to both NE and luminal cell types. Inhibition of ERG expression in TMPRSS2:ERG-positive PC cells through blockade of AR signaling is tracked with increased NE gene expression. We also provide evidence that these NE cells are resistant to pharmacological AR inhibition and can revert to the phenotype of parental cells upon restoration of AR/ERG signaling. Our findings highlight an ERG-regulated mechanism capable of repopulating the parent tumor through the transient generation of an anti-androgen therapy-resistant cell population, suggesting that ERG may have a direct role in preventing resistance to anti-androgen therapy.

Embryonic transcription factor SOX9 drives breast cancer endocrine resistance

Significance Resistance to endocrine treatment remains a significant clinical obstacle. ESR1 mutations were found to be the mechanism of endocrine resistance in a substantial number of patients with metastatic ER-positive breast. However, these mutations are primarily linked to aromatase inhibitor resistance and are not strongly associated with tamoxifen resistance. Herein, we show that tamoxifen treatment promotes a RUNX2–ER complex, which mediates an altered ER cistrome that facilitates the up-regulation of SOX9. We show that up-regulation of SOX9, an embryonic transcription factor with key roles in metastases, is a driver of endocrine resistance in the setting of tamoxifen treatment. Our data provide putative targets for the development of new strategies to treat tamoxifen-resistant breast cancer. 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. DOI: http://dx.doi.org/10.7554/eLife.13964.001

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.

Abstract P3-05-09: Exploring bazedoxifene and palbociclib as potential therapeutic strategies for overcoming ESR1-mediated endocrine resistance

INTRODUCTION Despite effective endocrine treatments, endocrine resistance remains a major clinical challenge. We and other groups have recently detected ligand-binding domain ESR1 mutations in metastatic estrogen receptor positive ( ER+) breast cancers. Our preclinical studies showed that these mutations confer constitutive activity and relative resistance to tamoxifen and fulvestrant. In this study we sought to investigate therapeutic strategies to overcome resistance rendered by the ESR1 mutations. Since our previous studies showed relative resistance to tamoxifen or fulvestrant, we hypothesized that bazedoxifene, a high affinity third generation SERM/SERD could overcome resistance driven by the ER mutant resistance. Additionally, we hypothesized that inhibiting cyclin D1, a key ER transcriptional target gene and cell cycle regulator, is a second potential therapeutic strategy to circumvent resistance rendered by mutant ER. Therefore in this study we tested the effects of bazedoxifene, palbciclib and their combination on cell proliferation in the presence and absence of the ESR1 mutations. METHODS For this study we established doxycycline inducible MCF7 cell lines expressing the ER-LBD mutations (Y537S, Y537N and D538G) and WT-ER as control. Cell proliferation response to bazedoxifene, tamoxifen, fulvestrant, palbociclib and the bazedoxifene-palbociclib combination was evaluated. RESULTS Cells harboring mutant ER were relatively resistant to tamoxifen and fulvestrant, as expected, and remained sensitive to single agent bazedoxifene and palbociclib. The combination of bazedoxifene and palbociclib was found to be superior to the single agents and exhibits synergistic activity. CONCLUSION The combination of bazedoxifene and palbociclib inhibits mutant ER cell growth and other cell models of endocrine resistance and is a potential therapeutic combination. Citation Format: Nguyen M, Buchwalter G, Luo F, Garraway L, Brown M, Jeselsohn R. Exploring bazedoxifene and palbociclib as potential therapeutic strategies for overcoming ESR1-mediated endocrine resistance. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-09.

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

AbstractESR1 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.Genetics: Drug treatment spurs new resistance mutationsTreatment with aromatase inhibitors, a class of drugs that suppress the synthesis of estrogen, can drive the evolution of mutations in the estrogen receptor gene ESR1, leading to tumor resistance against hormone therapies. To better understand the emergence of ESR1 mutations, Rinath Jeselsohn from the Dana-Farber Cancer Institute 
in Boston, Massachusetts, USA, and coworkers tested tumor DNA contained within blood samples from 155 women with metastatic breast cancer. They found ESR1 mutations rarely in women with any molecular subtype of cancer other than estrogen receptor-positive disease. Nothing about the primary tumor predicted who would develop ESR1 mutations; however, treatment with an aromatase inhibitor was associated with mutations arising. The findings highlight the need to develop therapeutic regimens that reduce the selective pressure for ESR1 mutations and/or target these mutations directly.

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 find 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 that BZA’s selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations. Significance Bazedoxifene (BZA) is a potent orally available antiestrogen that is clinically approved for use in hormone replacement therapy (DUAVEE). We explore the efficacy of BZA to inhibit activating somatic mutants of ERα that can arise in metastatic breast cancers after prolonged exposure to aromatase inhibitors or tamoxifen therapy. Breast cancer cell line, biophysical, and structural data show that BZA disrupts helix 12 of the ERα ligand binding domain to achieve improved potency against Y537S and D538G somatic mutants compared to 4-hydroxytamoxifen.