Estrogen Receptor Protein Levels Research Articles

An insight into the therapeutic effects of isoliquiritigenin in breast cancer.

Breast cancer ranks as the most widespread malignant condition in women, emerging as a primary contributor to mortality. The primary challenges in cancer treatments involve undesirable side effects. Therefore, exploring natural compounds as additional therapy could provide valuable insights. Isoliquiritigenin (ILN), an isoflavonoid featuring a chalcone moiety primarily sourced from Glycyrrhiza species, has garnered increasing interest in breast cancer research. This review aims to provide a comprehensive understanding of ILN's mechanisms of action in breast cancer, drawing from a range of in vitro and in vivo studies. ILN primarily acts by inhibiting angiogenesis, aromatase, inflammation, and cell proliferation, and preventing invasion and metastasis. Mechanistically, it downregulates miR-374a, phosphoinositide-3-kinase-protein kinase B/Akt, maternal embryonic leucine zipper kinase, vascular endothelial growth factor, and estrogen receptor protein levels, and causes enhancement of Wnt inhibitory factor-1, and Unc-51-like kinase 1 expression to treat breast cancer. ILN emerges as a promising natural option, offering therapeutic advantages with minimal side effects. However, it is important to note that current research on ILN is primarily limited to preclinical models, underscoring the need for further investigation to validate its potential efficacy.

Network medicine-based analysis of the hepatoprotective effects of Amomum villosum Lour. on alcoholic liver disease in rats.

Alcoholic liver disease (ALD) is characterized by high morbidity and mortality, and mainly results from prolonged and excessive alcohol use. Amomum villosum Lour. (A. villosum), a well-known traditional Chinese medicine (TCM), has hepatoprotective properties. However, its ability to combat alcohol-induced liver injury has not been fully explored. The objective of this study was to investigate the hepatoprotective effects of A. villosum in a rat model of alcohol-induced liver disease, thereby establishing a scientific foundation for the potential preventive use of A. villosum in ALD. We established a Chinese liquor (Baijiu)-induced liver injury model in rats. Hematoxylin and eosin (HE) staining, in combination with biochemical tests, was used to evaluate the protective effects of A. villosum on the liver. The integration of network medicine analysis with experimental validation was used to explore the hepatoprotective effects and potential mechanisms of A. villosum in rats. Our findings showed that A. villosum ameliorated alcohol-induced changes in body weight, liver index, hepatic steatosis, inflammation, blood lipid metabolism, and liver function in rats. Network proximity analysis was employed to identify 18 potentially active ingredients of A. villosum for ALD treatment. These potentially active ingredients in the blood were further identified using mass spectrometry (MS). Our results showed that A. villosum plays a hepatoprotective role by modulating the protein levels of estrogen receptor 1 (ESR1), anti-nuclear receptor subfamily 3 group C member 1 (NR3C1), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). In conclusion, the results of the current study suggested that A. villosum potentially exerts hepatoprotective effects on ALD in rats, possibly through regulating the protein levels of ESR1, NR3C1, IL-6, and TNF-α.

Prognostic role of HER2 expression in patients with ER-positive/HER2-negative breast cancer: Results from a population-based cancer registry study.

547 Background: Estrogen Receptor (ER)-positive (+)/Human Epidermal Growth Factor Receptor 2 (HER2)-negative (-) breast cancers (BCs) express variable protein levels of both ER and HER2, which can influence prognosis. Methods: We evaluated all invasive ER+/HER2- BCs (n = 3633) that were diagnosed and systematically collected by the Parma Province Cancer Registry, Italy, from 2004 to 2018. Tumors were classified by HER2 (IHC score of 0, 1+ or 2+ with negative FISH) and ER status (ER-low [1-9%], ER-moderate [10-79%] or ER-high [80-100%]). Comparisons of clinicopathologic characteristics and disease outcome were performed. Results: BCs with late-stage diagnosis ( P = 0.04), high histologic grade ( P < 0.0001), or high proliferative rate ( P < 0.0001) were more likely HER2 2+/FISH-. The rate of ER-high BCs did not change from 2675 of 2938 (91%) HER2 0 tumors to 508 of 560 (90.7%) HER2 1+, and 124 of 135 (91.9%) HER2 2+/FISH- tumors. Correspondingly, ER-low BCs were not enriched among HER2 0 tumors compared to the other tumors with different HER2 expression ( P = 0.6). The 5-year overall survival (OS) for HER2 2+/FISH- BCs was lower than that for HER2 0 or 1+ tumors ( P = 0.03). ER-low/moderate tumors were associated with poorer OS in comparison with ER-high BCs ( P < 0.0001). HER2 2+/FISH- status was detrimental to OS among patients (pts) with ER-high tumors ( P = 0.04), while this finding was not observed among ER-low/moderate BCs ( P = 0.21). An interaction between HER2 2+/FISH- expression and ER-high status was found for poorer OS after adjusting for prognostic variables (HR = 1.7; 95% CI: 1.1-2.9). Conclusions: The prognostic role of HER2 expression in pts with ER-positive/HER2-negative BCs seems to be restricted to ER-high tumors, while the worse prognosis of tumors with lower ER expression is not associated with HER2 status. These findings may help identify optimal patient inclusion criteria for clinical trials with novel anti-HER2 therapies in ER-positive/HER2-negative disease.

Abstract 1000: Associations of parity and body mass index with NanoString digital spatial protein profiling in early onset breast cancer

Abstract Purpose: Young women’s breast cancers (YBCs), defined herein as breast cancer (BC) diagnosed at age ≤ 40 years, are etiologically and biologically distinct from postmenopausal BCs, demonstrating a higher proportion of estrogen receptor (ER) negative subtypes and a worse prognosis. Studies have not assessed whether risk factors such as recent parity and body mass index (BMI) are linked to protein expression profiles in YBCs. Accordingly, we examined protein expression assessed with GeoMx® Digital Spatial Profiling (DSP) in 640 YBCs prepared as tissue microarrays (TMAs) in the Young Women’s BC Study (YWBCS). Methods: The YWBCS is a multi-institutional cohort of 1,297 women with incident BC diagnosed at age <40 years identified through medical record review, with risk factor data collected via questionnaires. TMAs including 1,505 cores of 640 YBCs that were collected prior to therapy and were suitable for molecular analysis were evaluated for 72 markers with NanoString GeoMx DSP. Associations of log-transformed biomarker expression with BMI and time from last live birth to BC diagnosis were carried out using linear mixed modeling approaches, accounting for multiple tumor tissue cores per individual. Results: Mean age at BC was 35.5 years (range 17-40). Tumor subtypes based on clinical immunostaining were the following: luminal B 46%; luminal A 36%; triple negative 12% and HER2+ 6%. Of 72 biomarkers tested, 53 (74%) were evaluable after QC and normalization. GeoMx ER and PR protein levels were higher in luminal A and luminal B YBCs than in HER2+ and TNBC (p=2x10-94 and 2x10-52, respectively), supporting the accuracy of the GeoMx assay. Compared to nulliparas or those with most recent birth > 3 years before BC, those who gave birth ≤ 3 years prior to BC had tumors with higher expression of PR (8x10-6) and ER (p=0.006), and lower expression of BCL-2 (p=0.006). In contrast, women with last birth ≥ 6 years prior to BC had higher expression of CD8 (p=0.006) and PD-1 (p=0.001) than nulliparas and those with more recent births. Compared to women with BMI between 18.5 and 29.9, those with higher or lower BMIs had higher expression of CD34 (p=0.0003), CD4 (p=0.0009) and CD14 (p=0.001). BCL-2 expression was higher among women with BMI < 18.5 compared to those with higher values (p=0.005). Women with BMI of 30+ had higher β2-microglobulin levels than those with lower BMIs (p=0.003). Inverse dose-response associations (with higher BMI related to lower levels) were observed for expression of ER (p=0.01), pan cytokeratin (p=0.004) and four proteins in the p13K/AKT signaling pathway (p ≤ 0.01 for each). Conclusions: Preliminarily, we identify differences in hormonal and immune markers in YBCs in relation to timing of births and BMI, suggesting that these factors may contribute to etiologic heterogeneity. Multivariate modeling is ongoing to incorporate molecular subtype and other risk factors. Citation Format: Robert A. Vierkant, Laura M. Pacheco-Spann, Stacey J. Winham, Jennifer M. Kachergus, Ji Shi, Craig Snow, Fergus J. Couch, Janet E. Olson, Chen Wang, Derek C. Radisky, Amy C. Degnim, E. Aubrey Thompson, Laura C. Collins, Kathryn J. Ruddy, Ann H. Partridge, Mark E. Sherman. Associations of parity and body mass index with NanoString digital spatial protein profiling in early onset breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1000.

Abstract P2-11-22: Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes

Abstract Background: Milk whey protein consumption has been associated with breast cancer prevention, therapy and treatment tolerance, and other studies detected these proteins or their mRNAs in breast cancer models or tissues. Our objectives are to ascertain expression of certain whey protein genes in human breast carcinoma cells isolated by Laser Capture Microdissection (LCM) and to determine if gene expression is related to risk of recurrence and overall survival in lesions with various ER/PR status. Genes selected were: LALBA (alpha-lactalbumin), LPO (lactoperoxidase), LTF (lactotransferrin), PAEP (progestogen associated endometrial protein or glycodelin) and SSP1 (secreted phosphoprotein 1 or osteopontin). This retrospective investigation is unique in that highly quantified ER and PR protein levels and ESR1 and PGR relative expression from pure populations of carcinoma cells were employed to stratify cancers into ER/PR subcategories to assess relationships of expression of candidate genes with clinical outcomes. Methods: This investigation of primary breast carcinomas from 170 postmenopausal and 77 premenopausal patients used a matchless deidentified dataset of quantified estrogen receptor (ER) and progestin receptor (PR) protein results with clinical follow-up. ER/PR protein levels, expressed as fmol/mg cytosol protein, were quantified earlier from each breast carcinoma with FDA-approved kits, either Radioligand-Binding Assay (LBA, NEN/DuPont) and/or enzyme immunoassay (EIA, Abbott Labs). Patients were treated with the standard of care at the time of diagnosis. The PixCell IleTM (ThermoFisher, Arcturus) LCM instrument was used to procure only carcinoma cells from tissue sections of de-identified, frozen biopsies, using protocols we employed earlier. Total RNA of each fixed specimen was extracted, purified and amplified to obtain relative expression levels of approximately 22,000 genes by microarray, providing an assessment of mRNA in carcinoma cells only. Relationships of relative gene expression, quantified ER/PR and other features of primary breast cancers were analyzed with clinical results using R software v4.2.0 (Vigorous Calisthenics) by Univariable Cox Regression, Kaplan Meier plots and various tests (i.e., Wilcoxon, Log Ranked, Spearman and/or Chi Square tests). Bidirectional elimination stepwise regression was also used to derive best predictors of patient outcomes. Results: Violin plots of expression distribution of each milk protein gene of 247 biopsies regardless of patient menopausal status revealed that LALBA and SSP1 were highly elevated (p < 0.001) in ER+ (n = 146) or PR+ (n = 146) breast cancer cells while PAEP was quite diminished (p < 0.001). No difference was observed in LPO expression in ER+ or PR+ cells and LTF was only elevated slightly (p < 0.05) in ER+ cells. Regardless of ER/PR status of breast cancer, LTF, LALBA, SSP1, AR and ESR1 gene expression levels were increased significantly in postmenopausal versus premenopausal patients while PAEP was decreased (p < 0.001). No difference was observed in LPO or PGR expression. Correlation matrices examined gene to gene and receptor protein to gene relationships. Using Kaplan Meier analyses of each gene alone with Log Ranked Tests of outcomes, only SSP1 expression was associated with clinical outcome, i.e., overexpression was associated with lower PFS and OS (p = < 0.05). Conclusions: Application of bidirectional elimination stepwise regression of gene expression of the five milk proteins collectively with ER/PR protein status of each carcinoma revealed that LALBA gene expression enhanced prediction of PFS in ER+ cancer while PAEP expression was associated with improved prediction of OS in ER+ carcinomas. We report that expression of LALBA and PAEP genes, largely associated with milk production in normal breast tissue, are also expressed in certain breast carcinomas and influence prediction of patient survival. Citation Format: James L. Wittliff, Michael W. Daniels. Expression of milk whey protein genes in human breast carcinoma cells isolated by LCM impacts prediction of clinical outcomes [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-22.

Abstract P2-08-22: Correlation of ER (ESR1), PR (PGR), and HER2 (ERBB2) at protein and mRNA levels in operable breast cancer

Abstract Background:. We aimed to analyze the concordance between ER (ESR1), PR (PGR), and HER2 (ERBB2) levels and breast cancer subtyping results obtained by using the immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR) methods and to assess recurrence-free survival (RFS) between subtypes as determined by the two methods. Patients and Methods:. We included a total of 441 operable breast cancer patients, consisting of the training (n = 116), testing (n = 56) and validation (n = 269) cohorts in this study. Protein levels of estrogen receptor (ER) and progesterone receptor (PR) were obtained by using the IHC and that of human epidermal growth factor receptor 2 (HER2) by the IHC/FISH methods. Expression (mRNA) levels of the corresponding genes, ESR1, PGR, and ERBB2, respectively, were determined by the RT-PCR method. We then compared the status of ER (ESR1), PR (PGR), and HER2 (ERBB2) in the training, testing and validation cohorts. We categorized all validation cases into luminal (ER/PR-positive and HER2-negative), HER2 (HER2-positive regardless of ER/PR status), and triple negative (TN: ER-, PR-, and HER2-negative) subtypes accordingly. The concordance of the two methods and RFS estimated by the Kaplan-Meier methods of the three different subtypes were revealed to evaluate the clinical performance. Results:. The concordance rates between the two methods in the validation cohort were 95.1% (Kappa = 0.74) for ER (ESR1), 91.1% (Kappa = 0.67) for PR (PGR), and 93.6% (Kappa = 0.59) for HER2 (ERBB2). Kappa statistic was 0.66 for the resulting luminal, HER2 and TN subtypes. The 5-year RFS was 83.3% for luminal vs. 73.9% for HER2 vs. 48.4% for TN subtypes as determined by the IHC (p = 0.019), and 84.3% vs. 66.3% vs. 54.4% by the RT-PCR method (p = 0.016). Conclusions:. Whereas discordance existed for some cases between the IHC and RT-PCR methods for determination of the ER (ESR1), PR (PGR), and HER2 (ERBB2) status and the resulting subtypes; both the methods showed values in RFS prognosis of the population studied. It is arguable that supplementing the conventional IHC with the molecular method for subtyping of breast cancer may have beneficial clinical implications. Citation Format: Lei Lei, Xiaojia Wang, Skye Hung-Chun Cheng. Correlation of ER (ESR1), PR (PGR), and HER2 (ERBB2) at protein and mRNA levels in operable breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-22.

Abstract 44: The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC® protein degrader, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ~ 1 nM. PROTAC® mediated ER degradation decreases the expression of classically regulated ER-target genes and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant anti-tumor activity of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (131% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also significantly reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC® protein degrader. These preclinical data supported the clinical development of ARV-471 for the treatment of patients with breast cancer. The discovery, chemical structure and initial clinical data of ARV-471 will be presented. Citation Format: Lawrence B. Snyder, John J. Flanagan, Yimin Qian, Sheryl M. Gough, Monica Andreoli, Mark Bookbinder, Gregory Cadelina, John Bradley, Emma Rousseau, Julian Chandler, Ryan Willard, Jennifer Pizzano, Craig M. Crews, Andrew P. Crew, John Houston, Marcia Dougan Moore, Ron Peck, Ian Taylor. The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 44.

3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways.

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A “seesaw” relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

Endocrine Resistance in Breast Cancer: The Role of Estrogen Receptor Stability.

Therapy of hormone receptor positive breast cancer (BCa) generally targets estrogen receptor (ER) function and signaling by reducing estrogen production or by blocking its interaction with the ER. Despite good long-term responses, resistance to treatment remains a significant issue, with approximately 40% of BCa patients developing resistance to ET. Mutations in the gene encoding ERα, ESR1, have been identified in BCa patients and are implicated as drivers of resistance and disease recurrence. Understanding the molecular consequences of these mutations on ER protein levels and its activity, which is tightly regulated, is vital. ER activity is in part controlled via its short protein half-life and therefore changes to its stability, either through mutations or alterations in pathways involved in protein stability, may play a role in therapy resistance. Understanding these connections and how ESR1 alterations could affect protein stability may identify novel biomarkers of resistance. This review explores the current reported data regarding posttranslational modifications (PTMs) of the ER and the potential impact of known resistance associated ESR1 mutations on ER regulation by affecting these PTMs in the context of ET resistance.

Abstract 1750: Combination of ZEN-3694 with CDK4/6 inhibitors reverses resistance in ER-positive breast cancer

Abstract CDK4/6 inhibitors have been shown to significantly prolong progression-free survival in patients with advanced hormone receptor (HR)-positive, HER2-negative breast cancer. In spite of the great successes with the currents lines of therapies, the patients still acquire resistance over time and, the development of additional novel therapeutic strategies is needed. The bromodomain and extra-terminal domain (BET) proteins are key epigenetic regulators that interact with acetylated lysine (AcLys) residues of histones or transcription factors, leading to the regulation of gene transcription. BET proteins have also been shown to be directly involved in the transcription of the estrogen receptor (ER) mRNA as well as cell cycle and therefore, BET inhibitors can potentially offer new strategies in the treatment of CDK4/6i- resistant and endocrine resistant ER+ breast cancer. ZEN-3694 is an orally bioavailable small molecule inhibitor of BET proteins currently being evaluated in Phase 1/2 clinical trials in metastatic castration-resistant prostate cancer (NCT02711956) and triple negative breast cancer (NCT03901469). We have shown previously that ZEN-3694 inhibits proliferation and induces apoptosis in ER+ cell lines. To assess the effects of ZEN-3694 in the combination with CDK4/6 inhibitors as a potential therapy in a CDK4/6i resistant population, we have developed a panel of ER+ cell lines resistant to palbociclib or abemaciclib by continuous stepwise exposure to increasing concentrations of CDK4/6 inhibitors. These cell lines have demonstrated cross-resistance to all three CDK4/6 inhibitors. Here, we describe that the combination treatment of ZEN-3694 with CDK4/6 inhibitors potently inhibits proliferation and induces apoptosis in all CDK4/6i resistant cell lines. The resistance to both palbociclib and abemaciclib was associated with the strong upregulation of CDK6 and CCND1protein levels in MCF7 which is consistent with their clinical mechanism of resistance. ZEN-3694 showed efficacy in all CDK4/6i-resistant models leading to downregulation of ER and CDK6 protein levels. Furthermore, our RNAseq data revealed several potential pathways involved in CDK4/6i resistance, including upregulation of cell cycle pathway and decrease of interferon signaling and demonstrated that the mechanisms of resistance to abemaciclib and palbociclib are similar but not identical. Additionally, we elucidated the mechanism of action of ZEN-3694 and in combinations with CDK4/6 inhibitors alleviating these resistance mechanisms. The pathway analysis demonstrated that the combination of ZEN-3694 with CDK4/6 inhibitors led to the strong downregulation of multiple pathways including cell cycle regulation, signaling by Rho family GTPases, STAT3, IL6 and other pathways. We conclude that ZEN-3694 has therapeutic potential in a combination with CDK4/6 inhibitors in ER+ breast cancer patients that developed resistance to endocrine therapies and/or CDK4/6 inhibitors. Citation Format: Olesya A. Kharenko, Reena G. Patel, Cyrus Calosing, Edward H. van der Horst. Combination of ZEN-3694 with CDK4/6 inhibitors reverses resistance in ER-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1750.

Exercise training attenuates cardiac inflammation and fibrosis in hypertensive ovariectomized rats.

This study investigated the effects of exercise training on cardiac inflammatory and cardiac fibrotic pathways in female spontaneously hypertensive rats (SHR), which were divided into a sham-operated sedentary hypertensive group (SHR-S), a sedentary hypertensive ovariectomized group (SHR-O), or a hypertensive ovariectomized group with treadmill exercise training (SHR-OT; 60 min/day, 5 days/wk) for 8 wk. Normotensive female Wistar-Kyoto rats (WKY) served as controls. SOD and catalase (CAT) activities were significantly increased in the SHR-OT group, when compared with the SHR-S or SHR-O groups. The protein levels of estrogen receptor (ER)-α and ER-β became decreased in the SHR-O group, when compared with the WKY or SHR-S groups, but were not changed in the SHR-OT group. The protein level of the angiotensin II type I receptor (AT1R) was increased in the SHR-S group but did not further change in the SHR-O group, whereas it was decreased in the SHR-OT group. The inflammatory-related protein levels of TNF-α, p-NF-κB, cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6, as well as the fibrotic-related protein levels of transforming growth factor-β (TGF-β), p-Smad2/3, connective tissue growth factor (CTGF), tissue-type plasminogen activator (tPA), matrix metalloproteinase (MMP)-9, and collagen I were increased in the SHR-S group and increased further in the SHR-O group, whereas they were decreased in the SHR-OT group. The coexistence of hypertension and ovariectomy additively increased cardiac inflammatory and fibrotic pathways partially through hypertension-enhanced AT1R and ovariectomy-depressed estrogen receptors. Exercise training appeared to suppress hypertensive ovariectomized heart-induced inflammatory and fibrotic pathways possibly through decreasing AT1R but not through estrogen receptors.NEW & NOTEWORTHY The coexistence of hypertension and ovariectomy appeared to increase cardiac inflammatory and fibrotic pathways likely through hypertension-enhanced angiotensin II type I receptor and ovariectomy-depressed estrogen receptors. Exercise training on a treadmill could prevent hypertensive ovariectomized heart-induced cardiac inflammation and fibrosis via an inflammatory pathway [TNF-α, p-IKK-α/β, p-NF-κB, cyclooxygenase 2 (COX-2), iNOS, and IL-6] and fibrotic pathway [transforming growth factor-β (TGF-β), p-Smad2/3, connective tissue growth factor (CTGF), tissue-type plasminogen activator (tPA), matrix metalloproteinase (MMP)-9, and collagen I] possibly through decreasing angiotensin II type I receptor but not through estrogen receptors.

Abstract P4-10-07: Tumor ER protein modulation, molecular characterization and monitoring of cfDNA in phase 1 study of LSZ102 and LSZ102 + ribociclib in patients with ER+ MBC

Abstract Background: LSZ102 is an orally bioavailable selective estrogen receptor degrader (SERD) that inhibits estrogen receptor (ER) mediated gene transcription, induces receptor degradation, and blocks ER-dependent cell growth in preclinical models. This first-in-human study of LSZ102 is evaluating LSZ102 as a single agent (SA) and in combination with the CDK4/6 inhibitor ribociclib or the PI3K inhibitor alpelisib (BYL719) in patients (pts) with locally advanced/metastatic ER-positive (ER+) breast cancer (BC). Here, we present the immunohistochemistry (IHC) and circulating tumor DNA (ctDNA) data obtained from a subset of pts treated with either LSZ102 SA or the combination of LSZ102 and ribociclib. Methods: ER IHC was performed with an antirabbit antibody (clone SP1) on FFPE samples collected at baseline and cycle 1 day 15 (C1D15), and the ER value was reported using the H-score method. A Novartis exploratory PanCancer next-generation sequencing ~600 gene panel for cell-free DNA (cfDNA) was used on plasma samples collected from pts treated with SA or the combination at baseline, on treatment (C1D1, C2D1/C3D1, C5D1, CXD1) at radiological assessments and at the end of treatment (EOT). Results: One hundred fifty-three pts with MBC (median number of prior lines of therapy in metastatic setting = 4; range, 0-10) were treated with LSZ102 SA or the combination of LSZ102 and ribociclib (data cutoff, November 30, 2018). ER protein levels, assessed by IHC H-score, are downregulated in about 75% of C1D15 samples in both SA and combination-treated pts. In this cohort of heavily pretreated pts, cfDNA sequencing was performed on approximately 400 samples from 135 pts, and the genomic landscape is described both at baseline and EOT for pts with detectable ctDNA (~77% of pts). At baseline, approximately 40% of pts had mutations in the ESR1 ligand-binding domain (LBD), and approximately 40% of pts had PIK3CA activating mutations. LBD ESR1 mutations were mainly subclonal with several pts harboring multiple mutations at baseline. The frequency of ESR1 Y537S mutation was no higher in EOT samples after LSZ102 treatment compared with baseline. Moreover, the tumor reduction of target lesions > 30% was observed in pts harboring different baseline ESR1 mutations, including Y537S, treated with either SA or the combination. Furthermore, the longitudinal cfDNA analysis showed a consistent reduction of the overall circulating tumor fraction (ctDNA fraction), from baseline for the majority of patients who achieved either partial response or stable disease with either SA or the combination. Conclusions: Consistent with being a SERD, LSZ102 treatment leads to the degradation of ER in tumor biopsies. The addition of ribociclib does not alter LSZ102-induced ER downregulation. The most common genetic alterations found in cfDNA collected from enrolled ER+ MBC patients before the study treatment were ESR1 and PIK3CA mutations. Importantly, the antitumor activity was observed in pts harboring a variety of ESR1 LBD mutations. Furthermore, enrichment of ESR1 Y537S mutations was not observed after LSZ102 treatment. Finally, the on-treatment longitudinal assessment of cfDNA measuring ctDNA fraction and mutation dynamics is maturing as a useful tool for monitoring the treatment efficacy. Citation Format: Komal Jhaveri, Dejan Juric, Sara Cresta, Yoon-Sim Yap, Francois P. Duhoux, Catherine Terret, Rachel Layman, Alejandro Balbin, Qing Sheng, Serena Liao, Adam Crystal, Giuseppe Curigliano. Tumor ER protein modulation, molecular characterization and monitoring of cfDNA in phase 1 study of LSZ102 and LSZ102 + ribociclib in patients with ER+ MBC [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-07.

Abstract P6-03-09: Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells

Abstract Background: CDK4/6 inhibitors combined with endocrine therapy (ET) are mainstay to treat metastatic estrogen receptor (ER) positive patients. Yet, almost 60% develop resistance to CDK4/6 inhibition within 2 years of initial treatment. An ongoing clinical challenge has thus been identifying biomarkers of response to predict patients that will either respond or not respond to palbociclib. Further, there is an unmet need to identify actionable targets for patients that have progressed on CDK4/6 blockade regimens. Currently, the only biomarker being used to identify patients for anti-CDK4/6 therapy is estrogen receptor (ER) by IHC. The goal of our study was thus to identify the therapeutic vulnerabilities of CDK4/6 inhibitor resistance cells and identify key markers that can longitudinally correlate with development of resistance. Methods: We have developed MCF7 and T47D resistant cells by treating them with increasing doses (1.2, 2.4, 3.6, and 4.8μΜ) of palbociclib over a 6-month period. At each dose, resistant cells were harvested to observe dose dependent changes that occur as the cells acquire resistance to palbociclib. At endpoint i.e. 4.8μM, multi-omics approach was used to compare sensitive vs. resistant cells. Results: Our multi-omics data indicates that palbociclib resistant cells show i) Upregulation of the STAT3-IL6 pathway ii) Loss of ERα and iii) Loss RB protein levels. Primarily, resistant cells adapt to palbociclib treatment by a dose dependent upregulation of IL6. Further, we observe that induction of IL-6 is an early event which also correlates with senescence-associated secretory phenotype (SASP) signature in resistant cells, predicted to be induced with CDK4/6 inhibition. Importantly, time-course studies using 1μM palbociclib indicates that IL-6 is needed in overcome SASP and promote proliferation in cells adapting and eventually developing resistance to CDK4/6 inhibition. Lastly, we subjected MCF7 and T47D parental cells, which are sensitive to endocrine therapy and palbociclib, with exogenous IL-6 and IL-6 receptor (IL-6R). Treatment with IL-6/IL-6R lead to the downregulation of ER and RB protein levels. IL-6 treated parental cells are 5-fold more resistant to tamoxifen and 7-fold more resistant to fulvestrant compared to parental cells not treated with IL-6. Additionally, dose response studies with palbociclib showed parental cells treated with IL-6 are 5-fold more resistant to palbociclib treatment compared to the cells cultured in the absence of IL-6. Conclusions: Collectively, our data suggests that resistance to palbociclib results in a cascade of events initiating with induction of senescence, leading to sustained upregulation of IL-6 which leads to the promotion of an aggressive tumor growth, accompanied by resistance to endocrine therapy. Our ongoing studies are geared towards delineating the role of each step of CDK4/6 resistance and determining if IL-6 is required to maintain CDK4/6 inhibition resistance. Our long-term goal is to correlate provide the pre-clinical rationale for using IL-6 as a predictive biomarker in patients receiving CDK4/6 inhibitor based therapies. Citation Format: Nicole M Kettner, Tuyen Bui, Xian Chen, Kelly K Hunt, Debu Tripathy, Khandan Keyomarsi. Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-09.

Estrogen signaling effects on muscle-specific immune responses through controlling the recruitment and function of macrophages and T cells

BackgroundEstrogen signaling is indispensable for muscle regeneration, yet the role of estrogen in the development of muscle inflammation, especially in the intramuscular T cell response, and the influence on the intrinsic immuno-behaviors of myofibers remain largely unknown. We investigated this issue using the mice model of cardiotoxin (CTX)-induced myoinjury, with or without estrogen level adjustment.MethodsCTX injection i.m. (tibialis anterior, TA) was performed for preparing mice myoinjury model. Injection s.c. of 17β-estradiol (E2) or estrogen receptor antagonist 4-OHT, or ovariectomy (OVX), was used to change estrogen level of animal models in vivo. Serum E2 level was evaluated by ELISA. Gene levels of estrogen receptor (ERs) and cytokines/chemokines in inflamed muscle were monitored by qPCR. Inflammatory infiltration was observed by immunofluorescence. Macrophage and T cell phenotypes were analyzed by FACS. Immunoblotting was used to assess protein levels of ERs and immunomolecules in C2C12 myotubes treated with E2 or 4-OHT, in the presence of IFN-γ.ResultsWe monitored the increased serum E2 level and the upregulated ERβ in regenerated myofibres after myotrauma. The absence of estrogen in vivo resulted in the more severe muscle inflammatory infiltration, involving the recruitment of monocyte/macrophage and CD4+ T cells, and the heightened proinflammatory (M1) macrophage. Moreover, estrogen signaling loss led to Treg cells infiltration decrease, Th1 response elevation in inflamed muscle, and the markedly expression upregulation of immunomolecules in IFN-γ-stimulated C2C12 myotubes in vitro.ConclusionOur data suggest that estrogen is a positive intervention factor for muscle inflammatory response, through its effects on controlling intramuscular infiltration and phenotypes of monocytes/macrophages, on affecting accumulation and function of Treg cells, and on suppressing Th1 response in inflamed muscle. Our findings also imply an inhibition effect of estrogen on the intrinsic immune behaviors of muscle cells.

Structure-activity relationship study of estrogen receptor down-regulators with a diphenylmethane skeleton.

Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC50 as a measure of potency, and Emax as a measure of efficacy.

Abstract P5-04-18: ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ˜ 2 nM. PROTAC-mediated ER degradation decreases the expression of classically-regulated ER-target genes (PR, GREB1, TFF) and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically-relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant tumor volume regressions of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (˜130% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC-degrader. Citation Format: Flanagan JJ, Qian Y, Gough SM, Andreoli M, Bookbinder M, Cadelina G, Bradley J, Rousseau E, Willard R, Pizzano J, Crews CM, Crew AP, Taylor I, Houston J. ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-18.

Combined effects of 17β-estradiol and exercise training on cardiac apoptosis in ovariectomized rats.

BackgroundThe purpose of this study was to investigate the combined 17β-estradiol (E2) and exercise training on cardiac pro-survival and anti-apoptotic pathways in ovariectomized rats.MethodsFifty-six female Sprague–Dawley rats were divided into a sham-operated (Sham), a bilaterally ovariectomized (OVX), an OVX treated with E2 (OVX-E2; 10μg/kg/day), and an OVX with E2 and treadmill exercise training (OVX-E2-EX; 60 min/day, 5 days/week) for 10 weeks. Following 10 weeks of exercise training, rat hearts were isolated for the evaluation of Histopathological analysis, TUNEL assay, and Western blotting.ResultsThe protein levels of estrogen receptor α (ERα), estrogen receptor β (ERβ), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-phosphatidylinositol 3-kinase (p-PI3K) (estrogen receptors/IGF-1-related survival pathway) were significantly increased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. The protein levels of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL) and phosphorylated-Bad (p-Bad) (Bcl-2 family survival pathway) were significantly increased in the OVX-E2-EX group when compared with the OVX group. Only the p-Bad was significantly increased in the OVX-E2 group when compared with the OVX group. The protein levels of truncation of Bid (t-Bid), Bcl-2-associated death promotor (Bad), Bcl-2-associated X protein (Bax), Cytochrome c, caspases-9, and caspases-3 (mitochondria-dependent apoptotic pathway), as well as the protein levels of tumor necrosis factor-α (TNF-α), Fas ligand, Fas receptors, Fas-associated death domain (FADD), activated caspase-8 and activated caspase-3 (Fas receptor–dependent apoptotic pathway) were significantly decreased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. Furthermore, when compared with the OVX-E2 group, the protein levels of ERβ, IGF-1, IGF-1R, Bcl-2 and Bcl-xL were further enhanced in the OVX-E2-EX group as well as the protein levels of Cytochrome c, Fas receptors, FADD, activated caspase-8, activated caspase-9 and activated caspase-3 were further decreased in the OVX-EX-E2 group.ConclusionsCombined E2 and exercise training exhibited a positive effect of protection on ovariectomy-induced cardiac apoptosis by enhancing ERβ-related survival pathways, which might provide a more effective therapeutic effect on cardiac protection in bilaterally oophorectomized or menopausal women than E2 treatment only.

Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics

Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics

Correlates of Breast Cancer and Indole-3-Carbinol: Medicinal Development and Strategies of Natural Products

Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor ER and ERß. Indole-3-carbinol (I3C) strongly down-regulated ER protein and transcript levels, without altering the level of ERß protein, in both cell lines. In cells transfected with the ER promoter linked to a luciferase gene reporter, I3C ablated ER promoter activity1-5. Dietary indole-3-carbinol prevents the development of estrogen-enhanced cancers including breast. Whereas estrogen increases the growth and survival of tumors, indole-3-carbinol causes growth arrest and increased apoptosis and ameliorates the effects of estrogen. In these findings best use indole-3-carbinol together with other nutrients (genistein) to achieve maximum benefits for cancer prevention. Investigator evaluated whether genistein, which is the major isoflavonoid in soy, would alter the ability of indole-3-carbinol/DIM to cause apoptosis and decrease expression driven by the estrogen receptor (ER)-alpha. Synergistic effect of indole-3-carbinol and genistein for induction of GADD expression, thus increasing apoptosis, and for decrease of expression driven by ER-alpha. Because of the synergistic effect of indole-3-carbinol and genistein, the potential exists for prophylactic or therapeutic efficacy of lower concentrations of each phytochemical when used in combination. I3C inhibits the enzyme elastase. High levels of elastase in breast cancer cells are suggestive of a poor prognosis because elastase shortens cyclin E, a cellular chemical involved in controlling the cell cycle, and the shortened version of cyclin E speeds up the cell cycle, therefore making cancer cells proliferate faster.I3C prevents the elastase shortening of cyclin E, thus halting the growth of breast cancer cells.

Histone deacetylase inhibitors alter the expression of molecular markers in breast cancer cells via microRNAs.

Histone deacetylase inhibitors(HDACis) are able to suppress breast cancer cells invitro and invivo by altering the expression of estrogen receptor(ER), progesterone receptor(PR) or human epidermal growth factor receptor2(Her2/neu). Since HDACis can alter the expression of various microRNAs (miRNAs/miRs), the present study aimed to examine the role of miRNAs in the effects of HDACis on breast cancer cells. We first examined the mRNA expression of ER, PR, and Her2/neu using RT-PCR and the protein levels of ER, PR, and Her2/neu using western blot analysis in MDA-MB-231 and BT474 cells, after trichostatin A (TSA) or vorinostat (SAHA) treatment. We then conducted miRNA expression profiling using microarrays after BT474 cells were treated with TSA or SAHA. Finally, we examined the effects of synthetic miR-762 and miR-642a-3p inhibitors on SAHA-induced downregulation of Her2/neu and SAHA-induced apoptosis and PARP cleavage in BT474 cells. The results indicated that TSA and SAHA dose‑dependently enhanced the mRNA and protein expression levels of ER and PR in MDA‑MB‑231 and BT474 cells. In addition, the mRNA expression levels of Her2/neu were reduced in MDA‑MB‑231 cells, and the mRNA and protein expression levels of Her2/neu were reduced in BT474 cells in response to SAHA and TSA. Furthermore, treatment with TSA (0.2µM) or SAHA (5.0µM) induced a marked alteration in the expression of various miRNAs in BT474 cells. Notably, when cells were cotransfected with miR‑762 and miR‑642a‑3p inhibitors, SAHA‑induced downregulation of Her2/neu was inhibited, and SAHA‑induced apoptosis and poly (ADP‑ribose) polymerase cleavage were significantly reduced in BT474 cells. These results indicated that numerous HDACi‑induced miRNAs are required to downregulate Her2/neu levels and promote apoptosis of Her2‑overexpressing breast cancer cells.