Estrogen Receptor Alpha and ESR1 Mutations in Breast Cancer.

The estrogen receptor alpha (ERα) is a nuclear transcription factor that is expressed in more than 70% of all breast cancers. Key genes involved in proliferation and tumor progression are transcriptionally regulated by ERα making it an important therapeutic target. Indeed, the first class of targeted treatments in cancer are endocrine treatments that target ERα either by competitive inhibition, reduced ligand production or receptor degradation. Despite the efficacy of these drugs, resistance to endocrine treatment remains a key clinical challenge. Only about 50% of patients treated with endocrine treatment in early-stage disease will benefit from adjuvant endocrine treatment and nearly all patients treated in the metastatic setting will develop disease progression while on endocrine treatment. Multiple mechanisms of resistance to endocrine treatment have been identified in pre-clinical models and clinical samples. These include both intrinsic (de novo) mechanisms and adaptive, acquired mechanisms. Over the past few years, gain-of-function missense mutations of ESR1, the gene encoding ERα, have been unveiled and identified as the most common genomic mechanism of acquired resistance to endocrine treatments. These mutations are clustered in a "hot spot" region within the ligand binding domain and engender constitutive, ligand-independent activity. Clinical studies evaluating these ESR1 mutations in metastatic ERα positive breast cancer demonstrate decreased overall survival which also highlights their prognostic role. In this chapter, we will provide a detailed review of structural and biophysical characteristics, functional consequences and clinical implications of the ESR1 mutations. We will also discuss potential therapeutic strategies to overcome treatment resistance in the context of ESR1 mutations and implications for future treatment selection.

Estrogen-regulated feedback loop limits the efficacy of estrogen receptor-targeted breast cancer therapy.

Endocrine therapy resistance invariably develops in advanced estrogen receptor-positive (ER+) breast cancer, but the underlying mechanisms are largely unknown. We have identified C-terminal SRC kinase (CSK) as a critical node in a previously unappreciated negative feedback loop that limits the efficacy of current ER-targeted therapies. Estrogen directly drives CSK expression in ER+ breast cancer. At low CSK levels, as is the case in patients with ER+ breast cancer resistant to endocrine therapy and with the poorest outcomes, the p21 protein-activated kinase 2 (PAK2) becomes activated and drives estrogen-independent growth. PAK2 overexpression is also associated with endocrine therapy resistance and worse clinical outcome, and the combination of a PAK2 inhibitor with an ER antagonist synergistically suppressed breast tumor growth. Clinical approaches to endocrine therapy-resistant breast cancer must overcome the loss of this estrogen-induced negative feedback loop that normally constrains the growth of ER+ tumors.

Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis.

INTRODUCTION: Real-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes. METHODS: CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher's Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes. RESULTS: In total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients. CONCLUSION: This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.

Identification of an Imidazopyridine-based Compound as an Oral Selective Estrogen Receptor Degrader for Breast Cancer Therapy.

The pro-oncogenic activities of estrogen receptor alpha (ERα) drive breast cancer pathogenesis. Endocrine therapies that impair the production of estrogen or the action of the ERα are therefore used to prevent primary disease metastasis. Although recent successes with ERα degraders have been reported, there is still the need to develop further ERα antagonists with additional properties for breast cancer therapy. We have previously described a benzothiazole compound A4B17 that inhibits the proliferation of androgen receptor-positive prostate cancer cells by disrupting the interaction of the cochaperone BAG1 with the AR. A4B17 was also found to inhibit the proliferation of estrogen receptor-positive (ER+) breast cancer cells. Using a scaffold hopping approach, we report here a group of small molecules with imidazopyridine scaffolds that are more potent and efficacious than A4B17. The prototype molecule X15695 efficiently degraded ERα and attenuated estrogen-mediated target gene expression as well as transactivation by the AR. X15695 also disrupted key cellular protein-protein interactions such as BAG1-mortalin (GRP75) interaction as well as wild-type p53-mortalin or mutant p53-BAG2 interactions. These activities together reactivated p53 and resulted in cell-cycle block and the induction of apoptosis. When administered orally to in vivo tumor xenograft models, X15695 potently inhibited the growth of breast tumor cells but less efficiently the growth of prostate tumor cells. We therefore identify X15695 as an oral selective ER degrader and propose further development of this compound for therapy of ER+ breast cancers. Significance: An imidazopyridine that selectively degrades ERα and is orally bioavailable has been identified for the development of ER+ breast cancer therapeutics. This compound also activates wild-type p53 and disrupts the gain-of-function tumorigenic activity of mutant p53, resulting in cell-cycle arrest and the induction of apoptosis.

Auricular Acupuncture During Chemotherapy Infusion in Breast Cancer Patients: A Feasibility Study.

Introduction: Breast cancer patients undergoing chemotherapy experience multiple distressing symptoms. The authors investigated the feasibility and potential benefits of auricular acupuncture during chemotherapy infusion in this population. Materials and Methods: Women with stage I-III breast cancer undergoing chemotherapy were enrolled and followed for three chemotherapy cycles. During the first cycle of chemotherapy that participants received after study enrollment, they were provided with educational materials. During the second and third cycles of chemotherapy after enrollment, they received auricular acupuncture. The primary outcome was feasibility, assessed by recruitment, retention, and completion of assessments. Secondary outcomes included symptom burden (Edmonton Symptom Assessment System-Revised Version) and anxiety (State-Trait Anxiety Inventory-State), assessed at four timepoints for each cycle: day 1, pre-education/acupuncture (T1); day 1, post-education/acupuncture (T2); day 2 (T3); and day 5 (T4). Nausea and vomiting (Multinational Association of Supportive Care in Cancer [MASCC] Antiemesis Tool) were assessed on days 2 and 5. Paired t test was used to compare patient-reported outcomes during cycle 1 (education) versus an average of outcomes during cycles 2 and 3 (acupuncture). Results: Twenty-six patients were enrolled, of which 24 completed all acupuncture sessions and 22 completed all outcome assessments. In cycles 2 and 3 versus cycle 1, participants experienced significant reductions in symptom burden (change from T1 to T4: -7.9 ± 13.6, p = 0.02), anxiety (change from T1 to T2: -3.3 ± 6.5, p = 0.02), and nausea severity on day 2 (-1.3 ± 2.6, p = 0.04). Conclusions: The delivery of auricular acupuncture during chemotherapy infusion was feasible and associated with reduction of symptom burden, anxiety, and nausea in breast cancer patients. Larger-scale clinical studies are needed to confirm these findings. Clinical Trial Registration number: NCT03170648.

Prognostic and Biologic Significance of ERBB2-Low Expression in Early-Stage Breast Cancer.

Importance: It is unclear whether ERBB2-low breast cancer should be considered an individual biologic subtype distinct from ERBB2-0 breast cancer. Objective: To investigate whether low ERBB2 expression is associated with distinct clinicopathologic characteristics and prognosis among patients with hormone receptor (HR)-positive and triple-negative breast cancer (TNBC). Design, Setting, and Participants: This cohort study was conducted using data from a prospectively maintained institutional database on all consecutive patients with breast cancer undergoing surgery between January 2016 and March 2021 at Dana-Farber Brigham Cancer Center. The study included 5235 patients with stage I through III, ERBB2-negative invasive breast cancer. Tumors were classified as ERBB2-low if they had an ERBB2 immunohistochemical (IHC) score of 1+ or 2+ with negative in situ hybridization assay and ERBB2-0 if they had an ERBB2 IHC score of 0. Data were analyzed from September 2021 through January 2022. Exposures: Standard treatment according to institutional guidelines. Main Outcomes and Measures: Comparison of clinicopathologic characteristics and disease outcomes (pathologic complete response rate [pCR], disease-free survival, distant disease-free survival, and overall survival) between patients with ERBB2-low and ERBB2-0 breast cancer. Results: Among 5235 patients with ERBB2-negative invasive breast cancer (5191 [99.2%] women; median [range] age at primary surgery, 59.0 [21.0-95.0] years), 2917 patients (55.7%) and 2318 patients (44.3%) had ERBB2-low and ERBB2-0 tumors, respectively. Expression of HR was significantly more common among ERBB2-low compared with ERBB2-0 tumors (2643 patients [90.6%] vs 1895 patients [81.8%]; P < .001). The rate of ERBB2-low tumors increased progressively, from 296 of 739 estrogen receptor (ER)-negative tumors (40.1%) to 31 of 67 ER-low (ie, ER 1%-9%) tumors (46.3%), 37 of 67 ER-moderate (ie, ER, 10%-49%) tumors (55.2%), 2047 of 3542 ER-high (ie, ER, 50%-95%) tumors (57.8%), and 499 of 803 ER-very high (ie, ER > 95%) tumors (62.1%) (P < .001). Among 675 patients receiving neoadjuvant chemotherapy, those with ERBB2-0 tumors experienced higher pCR rates (95 patients [26.8%] vs 53 patients [16.6%]; P = .002). However, there were no statistically significant differences in pCR rate between ERBB2-low and ERBB2-0 tumors when separately analyzing HR-positive, ER-low, HR-positive without ER-low, or TNBC tumors. In exploratory survival analysis, no differences by ERBB2-low expression in disease-free survival, distant disease-free survival, or overall survival were observed among patients with HR-positive tumors or TNBC. Conclusions and Relevance: The results of this cohort study did not support the interpretation of ERBB2-low breast cancer as a distinct biologic subtype. ERBB2-low expression was positively associated with level of ER expression, and ER-low tumors were enriched among ERBB2-0 tumors, suggesting that, given the worse prognosis of ER-low tumors, they may be associated with confounding of prognostic analyses of ERBB2-low expression.

GATA3 and MDM2 are synthetic lethal in estrogen receptor-positive breast cancers.

Synthetic lethal interactions, where the simultaneous but not individual inactivation of two genes is lethal to the cell, have been successfully exploited to treat cancer. GATA3 is frequently mutated in estrogen receptor (ER)-positive breast cancers and its deficiency defines a subset of patients with poor response to hormonal therapy and poor prognosis. However, GATA3 is not yet targetable. Here we show that GATA3 and MDM2 are synthetically lethal in ER-positive breast cancer. Depletion and pharmacological inhibition of MDM2 significantly impaired tumor growth in GATA3-deficient models in vitro, in vivo and in patient-derived organoids/xenograft (PDOs/PDX) harboring GATA3 somatic mutations. The synthetic lethality requires p53 and acts via the PI3K/Akt/mTOR pathway. Our results present MDM2 as a therapeutic target in the substantial cohort of ER-positive, GATA3-mutant breast cancer patients. With MDM2 inhibitors widely available, our findings can be rapidly translated into clinical trials to evaluate in-patient efficacy.

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.