Estrogen Receptor Protein Expression Research Articles

TP53-specific mutations serve as a potential biomarker for homologous recombination deficiency in breast cancer: a clinical next-generation sequencing study.

TP53 mutations and homologous recombination deficiency (HRD) occur frequently in breast cancer. However, the characteristics of TP53 pathogenic mutations in breast cancer patients with/without HRD are not clear. Clinical next-generation sequencing (NGS) of both tumor and paired blood DNA from 119 breast cancer patients (BRCA-119 cohort) was performed with a 520-gene panel. Mutations, tumor mutation burden (TMB), and genomic HRD scores were assessed from NGS data. NGS data from 47 breast cancer patients in the HRD test cohort were analyzed for further verification. All TP53 pathogenic mutations in patients had somatic origin, which was associated with the protein expression of estrogen receptor and progestogen receptor. Compared to patients without TP53 pathologic mutations, patients with TP53 pathologic mutations had higher levels of HRD scores and different genomic alterations. The frequency of TP53 pathologic mutation was higher in the HRD-high group (HRD score≥42) relative to that in the HRD-low group (HRD score<42). TP53 has different mutational characteristics between the HRD-low and HRD-high groups. TP53-specific mutation subgroups had diverse genomic features and TMB. Notably, TP53 pathogenic mutations predicted the HRD status of breast cancer patients with an area under the curve (AUC) of 0.61. TP53-specific mutations, namely HRD-low mutation, HRD-high mutation, and HRD common mutation, predicted the HRD status of breast cancer patients with AUC values of 0.32, 0.72, and 0.58, respectively. Interestingly, TP53 HRD-high mutation and HRD common mutation combinations showed the highest AUC values (0.80) in predicting HRD status. TP53-specific mutation combinations predict the HRD status of patients, indicating that TP53 pathogenic mutations could serve as a potential biomarker for poly-ADP-ribose polymerase (PARP) inhibitors in breast cancer patients .

Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor β (Esr2/ERβ) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERβ and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERβ and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERβ and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERβ and GPER in cumulus cells.

Abstract 3440: Targeting the mutant estrogen receptor in metastatic breast cancer

Abstract Introduction: Estrogen receptor gene (ESR1) drives approximately 70-80% of breast cancer cases, which are primarily treated with endocrine therapies. Mutations in the ESR1 exclusively occur in the ligand binding domain (LBD) and are significantly enriched following endocrine therapy. These mutations retain the estrogen receptor (ER) expression thus conferring ligand-independent growth, which is a paramount contributor to endocrine therapy resistant disease. Worse progression-free survival is consistently associated with ESR1 mutation status; therefore, there is an unmet need for therapies against these mutations. To this end, we recently developed TX-245, a bis-benzamide molecule that hampers the function of the two most common ESR1 mutations - Y537S and D538G. Methods: TX-245 was evaluated in vitro using MTT cell proliferation assay and colony formation assay carried out in multiple ER+ breast cancer cells harboring wild type (WT) ESR1 or mutant (MT) ESR1. Approaches such as tumor spheroid cultures were also implemented to study the effect of TX-245 compared to selected SERDs/SERMs via CellTiter-Glo 3D viability assay. Mechanistic insights were obtained performing TR-FRET assay, fluorescence polarization assay, RNA-seq, ChIP-seq, western blot, and qRT-PCR. The therapeutic efficacy in vivo was evaluated using cell xenografts, patient derived xenografts (PDX), patient derived explants (PDE), and metastatic mouse models of MT-ER breast cancer. Results: TX-245 demonstrated greater anti-proliferative dose-dependent activity in models harboring WT and MT ESR1, including those resistant to fulvestrant and tamoxifen, when compared to other SERDs/SERMs. RNAseq data suggests that TX-245 dramatically alters the transcription of ERα-regulated genes, with repression of canonical estradiol-upregulated and induction of estradiol-repressed genes. TX-245 was able to antagonize ER-chromatin interaction and ER-transcriptional signaling in breast cancer cells driven by ESR1 mutation. TX-245 treatment downregulated ER protein expression and decreased expression of ER targeted genes (i.e., GREB and PGR) in multiple ER+ cell lines. TX-245 treatment as a single agent or in combination with palbociclib resulted in tumor regression in xenografts expressing WT, Y537S or D538G ESR1 mutation. PDX and PDE models consistently exhibited substantial tumor growth inhibition as well as decreased proliferation (Ki67 staining). Metastases studies using intracardiac and intratibial injection demonstrated that TX-245 inhibited the progression of established metastatic niches in mice injected with Y537S or D538G ER+ breast cancer cells. Conclusion: Our results indicate that TX-245 inhibits cell proliferation in cell lines that express WT or mutant ESR1, induce ER degradation, suppress ER-mediated signaling and reduce tumor burden in mouse models. These studies highlight the utility of TX-245 in targeting mutations in the ER-LBD. Citation Format: Karla Parra, Suryavathi Viswanadhapalli, Tanner Reese, Xihui Liu, JunHao Liu, Zexuan Liu, Tae-Kyung Lee, Chia Yuan Chen, Kara Kassees, Jung-Mo Ahn, Ratna Vadlamudi, Ganesh Raj, Carlos Roggero. Targeting the mutant estrogen receptor in metastatic 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 3440.

ERK-estrogen receptor α signaling plays a role in the process of bone marrow mesenchymal stem cell-derived exosomes protecting against ovariectomy-induced bone loss

BackgroundExosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) are considered as candidates for osteoporosis (OP) therapy. Estrogen is critical in the maintenance of bone homeostasis. However, the role of estrogen and/or its receptor in BMSC-Exos treatment of OP, as well as its methods of regulation during this process remain unclear.MethodsBMSCs were cultured and characterized. Ultracentrifugation was performed to collect BMSC-Exos. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to identify BMSC-Exos. We examined the effects of BMSC-Exos on the proliferation, osteogenic differentiation, mineralization, and cell cycle distribution of MG-63 cells. The protein expression of estrogen receptor α (ERα) and the phosphorylation of ERK were investigated through western blotting. We determined the effects of BMSC-Exos on the prevention of bone loss in female rats. The female Sprague–Dawley rats were divided into three groups: the sham group, ovariectomized (OVX) group, and the OVX + BMSC-Exos group. Bilateral ovariectomy was performed in the OVX and OVX + BMSC-Exos groups, while a similar volume of adipose tissue around the ovary was removed in the sham group. The rats in OVX group and OVX + BMSC-Exos group were given PBS or BMSC-Exos after 2 weeks of surgery. Micro-CT scanning and histological staining were used to evaluate the in vivo effects of BMSC-Exos.ResultsBMSC-Exos significantly enhanced the proliferation, alkaline phosphatase activity, and the Alizarin red S staining in MG-63 cells. The results of cell cycle distribution demonstrated that BMSC-Exos increased the proportion of cells in the G2 + S phase and decreased the proportion of cells in the G1 phase. Moreover, PD98059, an inhibitor of ERK, inhibited both the activation of ERK and the expression of ERα, which were promoted by administration of BMSC-Exos. Micro-CT scan showed that in the OVX + BMSC-Exos group, bone mineral density, bone volume/tissue volume fraction, trabecular number were significantly upregulated. Additionally, the microstructure of the trabecular bone was preserved in the OVX + BMSC-Exos group compared to that in the OVX group.ConclusionBMSC-Exos showed an osteogenic-promoting effect both in vitro and in vivo, in which ERK-ERα signaling might play an important role.

High-Fat Diet Promotes Adipogenesis in Offspring Female Rats Induced by Perinatal Exposure to 4-Nonylphenol.

Both high-fat diet (HFD) and 4-nonylphenol (4-NP) could affect fat formation in adipose tissue individually. We investigated whether HFD promote abnormal adipose tissue formation caused by early exposure to 4-NP in life and preliminarily explore the possible mechanisms involved. The first-generation rats were treated with HFD on postnatal day after pregnant rats exposure to 5 ug/kg/day 4-NP. Then, the second generation rats started to only receive normal diet without 4-NP or HFD. We analyzed organ coefficient and histopathology of fat tissues, biochemical index, and gene level involved in lipid metabolism in female offspring rats. HFD and 4-NP interaction synergistically increased birth weight, body weight, and organ coefficients of adipose tissue in offspring female rats. HFD accelerately aggravated abnormal lipid metabolism and increased the adipocyte mean areas around the uterus of the offspring female rats induced by prenatal exposure to 4-NP. HFD also facilitate the regulation of gene expression involved lipid metabolism in offspring female rats induced by perinatal exposure to 4-NP, even passed on to the second generation of female rats. Moreover, HFD and 4-NP interaction synergistically declined the gene and protein expression of estrogen receptor (ER) in the adipose tissue of second-generation female rats. HFD and 4-NP synergistically regulate the expression of lipid metabolism genes in adipose tissue of F2 female rats and promote adipose tissue generation, leading to obesity in offspring rats, which is closely related to low expression of ER. Therefore, ER genes and proteins may be involved in the synergistic effect of HFD and 4-NP.

S-07-3: THE TESTOSTERONE-ANDROGEN RECEPTOR PATHWAY IS ASSOCIATED WITH CAROTID ATHEROSCLEROTIC PLAQUE INSTABILITY IN MEN WITH SEVERE CAROTID ATHEROSCLEROSIS

Objective: Carotid atherosclerotic plaques can be stable or unstable; the latter more likely to rupture resulting in strokes. Sex differences exist in carotid plaque morphology and composition: carotid plaques of men tend to be more unstable than of women, who tend to have more fibrous and stable plaques. Yet, women experience greater morbidity and mortality rates post-stroke. Given that sex hormones exert pleiotropic effects on the cell types involved in the atherosclerotic process, and given that estradiol is largely atheroprotective in women, we hypothesize that sex hormones and their receptors may affect plaque instability. Design and Methods: In this cross-sectional study, we measured sex hormones in the circulation and their receptors in the plaque of older men and postmenopausal women undergoing a carotid endarterectomy (n = 160). Using liquid chromatography mass spectrometry, circulating estradiol, testosterone, dehydroepiandrosterone (DHEA), and androstenedione were measured in blood samples. Plaques collected post-operatively were classified into 4 groups (n = 40/group): women with stable, women with unstable plaques, men with stable, and men with unstable plaques. Protein expression of estrogen receptor α (ER-α), estrogen receptor β (ER-β), G protein-coupled estrogen receptor (GPER), and androgen receptor (AR) was quantified using immunohistochemistry and western blot analysis. Plaque sex hormone receptor mRNA expression was assessed using qRT-PCR. Results: Men with unstable plaques demonstrated greater circulating concentrations of testosterone and androstenedione compared to men with stable plaques (P = 0.02 and P = 0.047, respectively). Unstable plaques in men demonstrated significantly less AR and ER-α mRNA expression compared to stable plaques from men (P = 0.0003 and P = 0.042, respectively) and compared to unstable plaques from women (P = 0.0088 and P = 0.002). Yet, AR, ER-α, ER-β, and GPER protein expression was significantly greater in unstable plaques from men compared to stable plaques from men (P = 0.039, P = 0.006, P &lt; 0.0001, and P = 0.0002, respectively) and compared to unstable plaques from women (P = 0.005, P = 0.041, P = 0.028, and P = 0.002, respectively). Expression was observed in macrophages, foam cells, endothelial cells, and smooth muscle cells. Moreover, unstable plaques from men demonstrated greater features of plaque instability compared to unstable plaques from women, including hemorrhage, large lipid core, abundance of inflammatory cells, and fibrous cap infiltration by inflammatory cells (P &lt; 0.05 for all). Conclusion: Our preliminary findings indicate a possible association between androgens and the androgen receptor pathway and plaque instability. With further investigations, our ongoing work will unravel the underlying mechanisms, and may ultimately lead to hormone-specific therapies aimed at stabilizing plaques and reducing the incidence of stroke for men and women.

Association of genetic ancestry with HER2, GRB7 AND estrogen receptor expression among Colombian women with breast cancer.

Our previous study reported higher mRNA levels of the human epidermal growth factor receptor 2 (HER2)-amplicon genes ERBB2 and GRB7 in estrogen receptor (ER)-positive breast cancer patients with relatively high Indigenous American (IA) ancestry from Colombia. Even though the protein expression of HER2 and GRB7 is highly correlated, they may also express independently, an event that could change the patients' prognosis. In this study, we aimed to explore the differences in ER, HER2 and GRB7 protein expression according to genetic ancestry, to further assess the clinical implications of this association. We estimated genetic ancestry from non-tumoral breast tissue DNA and assessed tumoral protein expression of ER, HER2, and GRB7 by immunohistochemistry in a cohort of Colombian patients from different health institutions. We used binomial and multinomial logistic regression models to test the association between genetic ancestry and protein expression. Kaplan-Meier and log-rank tests were used to evaluate the effect of HER2/GRB7 co-expression on patients' survival. Our results show that patients with higher IA ancestry have higher odds of having HER2+/GRB7- breast tumors, compared to the HER2-/GRB7- subtype, and this association seems to be stronger among ER-positive tumors (ER+/HER2+/GRB7-: OR=3.04, 95% CI, 1.47-6.37, p<0.05). However, in the multivariate model this association was attenuated (OR=1.80, 95% CI, 0.72-4.44, p=0.19). On the other hand, it was observed that having a higher European ancestry patients presented lower odds of ER+/HER2+/GRB7- breast tumors, this association remained significant in the multivariate model (OR=0.36, 95% CI, 0.13 - 0.93, p= 0.0395). The survival analysis according to HER2/GRB7 co-expression did not show statistically significant differences in the overall survival and recurrence-free survival. Our results suggest that Colombian patients with higher IA ancestry and a lower European fraction have higher odds of ER+/HER2+/GRB7- tumors compared to ER+/HER2-/GRB7- disease. However, this association does not seem to be associated with patients' overall or recurrence-free survival.

Betulinic acid alleviates zearalenone-induced uterine injury in mice

Zearalenone (ZEA) is a mycotoxin with estrogen-like biological activity, which widely present in feed and raw materials, with strong reproductive system toxicity and a major threat to animal reproduction. Betulinic acid (BA) is a natural plant compound with antioxidant, anti-inflammatory and other pharmacological activities. However, the mechanism of ZEA-induced uterine injury and the protective effect of BA have not been reported. Our results show that ZEA could cause uterine histopathological damage and cellular ultrastructural damage, affecting the secretion of sex hormones, such as estradiol (E2) and progesterone (P4), and increase the mRNA and protein expression of estrogen receptor α (ERα). ZEA could inhibit the activities of catalase (CAT) and superoxide dismutase (SOD), increase the production of malondialdehyde (MDA) and reactive oxygen species (ROS), and cause uterine oxidative stress. Furthermore, ZEA affected the homeostasis of uterine cell proliferation and death by regulating the expression of proliferating cell nuclear antigen (PCNA) and activating the mitochondrial apoptotic pathway. ZEA-induced uterine injury might be related to the activation of p38/ERK MAPK signaling pathway. However, the regulatory effect of ZEA on the uterus was reversed after BA treatment. In conclusion, the uterus is an important target organ attacked by ZEA, and BA showed a good therapeutic effect.

Calycosin suppresses colorectal cancer progression by targeting ERβ, upregulating PTEN, and inhibiting PI3K/Akt signal pathway.

High intake of phytoestrogen has been reported to be associated with the prevention of colorectal cancer (CRC). Calycosin belongs to the phytoestrogen that has been shown to suppress CRC cells in our previous study. However, its anticancer activity and molecular mechanisms have not been elucidated. In this study, we analyzed the effect of calycosin on the viability and apoptosis of human CRC HCT116 and SW480 cells via MTT assay, flow cytometry assay, and caspase-3/7 activity assay. The protein expressions of estrogen receptor β (ERβ), PTEN, and PI3K/Akt signal pathways were determined by Western blot analysis. And then, the alterations of biological behavior in CRC cells transfected with ERβ siRNA were analyzed. Mouse xenograft models were further performed to detect the antitumor effect in vivo.The results show that calycosin reduces CRC cell viability, induces cell apoptosis, and suppresses xenograft tumor growth. The protein expressions of ERβ and PTEN are significantly upregulated following calycosin treatment, whereas p-AKT/AKT ratio and Bcl-2 level are downregulated. Suppressing ERβ with siRNA partially attenuates the reduction in viability and apoptosis induced by calycosin. Our results indicate that calycosin shows inhibitory effects on CRC cells, which might be obtained by targeting ERβ, upregulating PTEN, and inhibiting the PI3K/Akt signal pathway.

Anti-inflammatory and hemostatic effects of total extract, saponins, and flavonoids of Clinopodium chinense in female rats with abnormal uterine bleeding and mechanism

This study aims to explore the anti-inflammatory and hemostatic effects of the total extract of Clinopodium chinense(TEC), total saponins of C. chinense(TSC), and total flavonoids of C. chinense(TFC) in female rats with abnormal uterine bleeding(AUB), and the possible mechanism. Mifepristone(i.g., 12.4 mg·kg~(-1)) and misoprostol(i.g., 130 μg·kg~(-1)) were used to induce AUB in SD female rats conceiving on the same day. Then the AUB rats were randomized into model group, TEC group, TSC group, TFC group, Yimucao Granules(LG) group, and estradiol valerate(EV) group, with 8 rats in each group. Another 8 non-pregnant female rats were selected as normal group. During the experiment, each group was given the corresponding drug by gavage once a day for 7 days. After the administration, blood and uterine tissue were collected. The uterine bleeding volume was measured by ultraviolet spectrophotometry and the pathological changes of endometrium were observed based on hematoxylin-eosin(HE) staining. In addition, the microvessel density of endometrium was determined by immunohistochemistry, and the content of thromboxane B2(TXB2), 6-keto-PGF_(1α), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in plasma and levels of lutenizing hormone(LH), follicle stimulating hormone(FSH), estradiol(E_2), and progesterone in serum were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA and protein expression of estrogenreceptor α(ERα), progesterone receptor(PR), matrix metalloproteinase(MMP)-2, MMP-9, and vascular endothelial growth factor(VEGF) in uterine tissue was determined by Western blot. Compared with the model group, TEC, TSC, and TFC can reduce uterine bleeding volume, alleviate the pathological damage of endometrium, and increase the microvessel density in endometrium. Moreover, TEC and TSC can significantly raise plasma TXB2 level and ratio of TXB2 to 6-keto-PGF_(1α), and TEC and TFC can significantly reduce the levels of IL-6 and TNF-α. In addition, TEC significantly elevated serum progesterone level and TFC significantly increased serum levels of E_2, FSH, and LH. TSC can significantly raise serum progesterone and FSH levels. In addition, TEC can significantly down-regulate the protein expression of PR, MMP-2, and VEGF and TSC significantly reduced the expression of MMP-9. TFC significantly decreased the expression of PR, MMP-9, and VEGF, and up-regulated the expression of ERα. In conclusion, TEC, TSC, and TFC all show therapeutic effects on AUB, particularly TEC. TSC exerts the effects by enhancing the coagulation function and promoting endometrial repair, and TFC by regulating estrogen levels and reducing inflammatory response. This study reveals the mechanism of C. chinense against AUB and also explains the holistic characteristics of Chinese medicine.

Abstract P2-02-03: Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC)

Abstract Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC)BackgroundThe presence of CTCs has been shown to be a poor prognostic factor in patients with MBC. Studies in prostate cancer have shown patients with distinct morphological subtypes of CTCs have worse outcomes than high CTC burden alone and could have treatment implications. In this study, we report a comprehensive analysis of CTC phenotypes in MBC and their impact on survival.MethodsA total of 148 blood samples from 90 patients with MBC whose disease was progressing on therapy were enrolled for CTC analysis. Blood was sent overnight to Epic Sciences, processed onto glass slides and biobanked. Replicate slides were stained with antibodies targeting the androgen receptor (AR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2) in addition to cytokeratin, CD45, and DAPI. Total CTCs were quantified per sample from approximately 3 mL of blood. A threshold defining positive AR, ER, and HER2 protein expression was set using cultured cancer cell lines expressing the individual proteins. CTC morphology subtypes (heterogeneity [Scher et al Cancer Res 2017], small cell/neuroendocrine-like [SC/NE; Brown et al CCR 2021] and chromosomal instability [CIN+; Schonhoft et al Cancer Res 2020]) were quantified as previously described. High heterogeneity samples (Het+) were defined as having a Shannon Index (SI; a measure of morphologic diversity) of ≥1. CIN positivity was defined as having ≥0.7 CIN+ CTC/mL. Association with these CTC subtypes and survival were evaluated with a univariate COX proportional hazard model. ResultsOver 90% of samples and patients had CTCs (Table 1). The percentage of patients with the CTC subtypes evaluated are below. Table 1. Percentage of blood samples with CTCs and CTC subtypes in MBC patients, by MBC subtype The presence of CTCs was associated with worse survival, with a median survival of 15.3 months (mos) vs 24.0 mos with no CTCs (p=0.028). The presence of AR+ or HER2+ CTCs was not associated with survival. Analysis of ER is ongoing and will be presented. High morphologic heterogeneity or the presence of the SC/NE or CIN+ CTC subtypes, predicted for a worse survival outcome (Table 2). Table 2. Overall survival of patients with and without distinct CTC subtypes CTC morphological subtypes did not correlate with each other, except CTC heterogeneity by SI which correlated weakly with SC/NE and CIN+. HER2+ CTCs strongly correlated with a higher SI while AR+ CTCs did not. AR and HER2 expression did not associate with the CIN+ or SC/NE subtypes. ConclusionsDigital pathology analysis of CTCs identifies distinct CTC morphological subtypes that predict for worse prognosis in MBC patients. To our knowledge, this is the first report of SC/NE CTCs in MBC patients and that MBC patients with SC/NE and CIN+ CTCs have worse outcomes. Longitudinal characterization of CTC subtypes across the MBC continuum could potentially identify effective therapies against these more lethal cancers. Citation Format: Joyce A O'Shaughnessy, Maren K Levin, Esther San Roman Rodriguez, James Lu, Joshua T Jones, Amanda KL Anderson, Alisa Tubbs, Joseph D Schonhoft, Rick Wenstrup. Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC) [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 P2-02-03.

Evaluation of bovine uterine gland functions in 2D and 3D culture system.

In ruminants, uterine glands play key roles in the establishment of pregnancy by secreting various factors into the uterine lumen. Although a three-dimensional (3D) culture system has been used for investigating cellular functions in vitro, the detailed functions of uterine gland have not been fully elucidated. In this study, we examined the benefits of 3D culture system to examine the innate functions of bovine uterine glands. Isolated bovine uterine glands were cultured on Matrigel (2D) or in Matrigel (3D), respectively, and the mRNA levels of secreted proteins (SERPINA14, MEP1B, APOA1, ARSA, CTGF, and SPP1) were measured in isolated and cultured uterine glands. The protein expression of estrogen receptor β (ERβ) and progesterone receptor (PR) and the establishment of apico-basal polarity were examined. In isolated uterine glands, the mRNA levels of secreted proteins changed during the estrous cycle. Although uterine glands cultured in both 2D and 3D expressed ERβ and PR, progesterone did not affect SERPINA14 mRNA expression. The expression of APOA1 mRNA in 2D cultured uterine glands did not respond to estrogen and progesterone. Additionally, the mRNA levels of secreted proteins in the 3D culture system were significantly higher than those in the 2D culture system, which might be attributed to the different cellular morphology between them. The locations of ZO-1 and β-catenin in 2D cultured uterine glands were disordered compared with 3D cultured uterine glands. These results showed that the hormonal responsiveness of secreted factor expression and cellular morphology were different between 2D and 3D cultured bovine uterine glands.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Androgen and Estrogen Receptor Activation Impacts Cardiac Function in a PCOS-Prone Rodent Model

Abstract Introduction: The global risk and incidence of cardiovascular disease (CVD) is increased in polycystic ovary syndrome (PCOS). Early risk factors and subclinical CVD includes atherogenic dyslipidemia, obesity, insulin resistance, blood pressure, atherosclerosis and impaired cardiac function. Androgen exposure is associated with onset of adiposity, impaired insulin-glucose and lipid metabolism, and cardiac dysfunction. The mechanisms of increased risk of CVD and cardiac dysfunction in PCOS related to hyperandrogenemia, AR and estrogen receptor (ER) activation remain unclear. Aim: The aim of this study was to investigate the effect of androgen treatment on cardiac AR and ER activation, fatty acid metabolism and cardiac function in a PCOS-prone rodent model. Methods: A PCOS-prone rodent model at 6 wks of age with obesity, apoB-remnant lipemia and insulin resistance, and controls were treated with testosterone for 12 weeks. Cardiac function was assessed using transthoracic doppler echocardiography (M-Mode 2D-imaging), lipogenic, AR, ER and other metabolic gene and protein expression were assessed using RTPCR and SDS-PAGE western blot. Results: PCOS-prone animals exhibited left ventricular (LV) hypertrophy, with increased LV mass to body weight (551.6 ± 38.85 mg vs 999 ± 96.17 mg, p&amp;lt;0.05), LV posterior wall diastolic diameter and LV internal diastolic diameter compared to controls. Isovolumetric relaxation time (IVRT) was prolonged (15.91 ± 1.591 msec vs 23.75 ± 0.722 msec, p&amp;lt;0.05). Mild systolic dysfunction was evidenced by increased isovolumetric contraction time (IVCT; 22.5 ± 1.348 msec vs 28.96 ± 1.248 msec, p&amp;lt;0.05) and decreased % ejection fraction and % fractional shortening in PCOS-prone compared to controls. T treatment increased LV mass, IVCT and IVRT in controls but did not exacerbate cardiac function in PCOS-prone animals. T treatment increased cardiac protein expression of PPAR-⍺ in PCOS-prone and controls, and T increased ACC in controls. AR protein expression tended to be reduced, and ER-⍺ was reduced in both T treated control and PCOS-prone animals. Conclusions: The PCOS-prone rodent model demonstrates early cardiac LV hypertrophy and diastolic-systolic dysfunction and T treatment alters fatty acid metabolism, and AR and ER activation are associated with altered cardiac morphology and function in the PCOS-prone and control conditions.

Anti-inflammatory activity of the Tongmai Yangxin pill in the treatment of coronary heart disease is associated with estrogen receptor and NF-κB signaling pathway

Ethnopharmacological relevanceThe Tongmai Yangxin Pill (TMYX) is a patented traditional Chinese medicine originating from two classic prescriptions, Zhigancao Decoction and Shenmai Yin, which composed of 11 Chinese medicinal herbs: Rehmannia glutinosa (Gaertn.) DC., Spatholobus suberectus Dunn, Ophiopogon japonicus (Thunb.) Ker Gawl., Glycyrrhiza uralensis Fisch., Polygonum multiflorum Thunb., Equus asinus L., Schisandra chinensis (Turcz.) Baill., Codonopsis pilosula (Franch.) Nannf., Chinemys reevesii (Gray), Ziziphus jujuba Mill. and Cinnamomum cassia (L.) J.Presl (Committee of the Pharmacopoeia of PR China, 2015). TMYX has marketed in China for the treatment of chest pain, palpitation, angina, irregular heartbeat and coronary heart disease (CHD) for several decades. Previous studies have confirmed that TMYX can treat CHD by reducing inflammation, but the underlying pharmacological mechanism remains unclear. Aim of the studyThis study aimed to declare the underlying pharmacological mechanism of anti-inflammatory activity of TMYX in the treatment of CHD via clinical trial, microarray study, bioinformatics analysis and the vitro assays. Materials and methodsEight CHD patients’ serum biochemical indices including coagulation function, lipid metabolism, endothelial injury, metalloprotease, adhesion molecule, inflammatory mediator and homocysteine were measured to investigate the reduction of CHD risk by TMYX oral administration (40 pills/time, 2 times/day) for eight weeks. The expression profile chips and Ingenuity Pathway Analysis (IPA) were assessed to reveal the global transcriptional response and predict related functions, diseases and canonical pathways. The in vitro anti-inflammatory actions of TMYX were evaluated using oxidized low-density lipoprotein (100 μg/mL) induced murine RAW264.7 macrophage with an ethanol extract from TMYX (EETMYX) (25–100 μg/mL). ResultsTMYX treatment showed reduced levels of apolipoprotein B, endothelin 1, nuclear factor κB (NF-κB) and homocysteine in CHD patients. In contrast, the treatment increased the ratio of apolipoprotein A/apolipoprotein B. EETMYX restored cell morphology and suppressed the lipid deposition of the induced foam cells. EETMYX exerted anti-inflammatory effects by raising the mRNA and protein expression of Estrogen receptor 1 (ESR1), blocking the reduction of IκBa level and the phosphorylation of IKKα/β, IκBα and NF-κB p65, accompanied by inhibiting MCP-1, TNF-α and IL-6 production, which were consistent with bioinformatics predictions. ConclusionTMYX treatment improved the biochemical indices in CHD patients. EETMYX effectively attenuated macrophage foam cell formation and exhibited anti-inflammatory activity is associated with regulating ESR1 and NF-κB signaling pathway activity.

Abstract GS4-09: Correlative studies of the breast cancer index (HOXB13/IL17BR) and ER, PR, AR, AR/ER ratio and Ki67 for prediction of extended endocrine benefit: A trans-aTTom study

Abstract Background: Several biomarkers such as estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and Ki67 have been implicated in the pathogenesis and/or as prognostic biomarkers of breast cancer, and are utilized to determine treatment. Given the heterogeneity of response to endocrine therapy, however, predictive biomarkers are critical to better individualize patient care. Previous results from the Trans-aTTom study demonstrated that the Breast Cancer Index HOXB13/IL17BR [BCI (H/I)] biomarker significantly predicts extended endocrine benefit from 10 vs 5y of tamoxifen. In this correlative study, the predictive activity of BCI (H/I) was compared with ER, PR, AR and Ki67 protein expression in node positive patients treated in the aTTom trial. Methods: Patients with available tumor tissue and biomarker analyses were included. ER, PR, AR and Ki67 were centrally assessed by immunohistochemistry (IHC) utilizing tissue microarrays. BCI gene expression analysis by RT-PCR was performed blinded to clinical outcome. Multivariate Cox models adjusting for age, tumor size, tumor grade and HER2 status were used to assess the significance of the interaction between treatment and each biomarker as continuous variables. 17-year risk of recurrence, as a function of each continuous biomarker, was estimated from Cox models in each of the 2 treatment arms. Results: Analysis of 789 HR+, N+ patients showed a weak negative correlation between BCI (H/I) and ER, PR, and AR expression whereas Ki67 and the AR/ER ratio showed no correlation (ER, cor=−0.18; PR, cor=−0.25; AR, cor=−0.14; Ki67, cor=0.04; AR/ER ratio, cor=0.02). The interaction between BCI (H/I) and extended tamoxifen treatment was significant (p=0.014). In addition, analysis of risk of recurrence as a function of continuous BCI (H/I) demonstrated that the magnitude in the reduction in recurrence risk with extended tamoxifen correlated with increasing H/I levels. In contrast, interaction P values were nonsignificant (ER, p=0.829; PR, p=0.659; AR, p=0.783; Ki67, p=0.865; AR/ER ratio, p=0.835) and the magnitude of endocrine benefit did not correlate with expression levels of any of other biomarkers. Conclusion: Results from this post-hoc analysis of the Trans-aTTom study demonstrated that whereas BCI(H/I) is a significant predictive biomarker of endocrine response, analysis of ER, PR, AR, Ki67 and AR/ER expression showed no interaction with treatment, and lacked the ability to predict benefit of extended tamoxifen in HR+ early stage breast cancer. These results add to the growing body of evidence that BCI (H/I) is distinct in its ability to predict benefit from therapy and interrogates distinct tumor biology that is not captured by other traditional biomarkers. Citation Format: Dennis C Sgroi, Kai Treuner, Yi Zhang, Tammy Piper, Ranelle Salunga, Ikhlaaq Ahmed, Lucy Doos, Sarah Thornber, Karen J Taylor, Elena F Brachtel, Sarah Pirrie, Catherine A Schnabel, Daniel W Rea, John MS Bartlett. Correlative studies of the breast cancer index (HOXB13/IL17BR) and ER, PR, AR, AR/ER ratio and Ki67 for prediction of extended endocrine benefit: A trans-aTTom study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS4-09.

FGFR1 Is Associated With Tamoxifen Resistance and Poor Prognosis of ER-Positive Breast Cancers by Suppressing ER Protein Expression.

Fibroblast growth factor receptor 1 (FGFR1) is widely recognized as a key player in mammary carcinogenesis and associated with the prognosis and therapeutic response of breast cancers. With the aim of investigating the correlation between FGFR1 expression and estrogen receptor (ER) and exploring the effect of FGFR1 on endocrine therapy response and ER+ breast cancer prognosis, we examined the FGFR1 protein expression among 184 ER-positive breast cancers by the immunohistochemistry (IHC) method, analyzed the association between FGFR1 expression and disease characters using the Pearson’s chi-square test, and assessed the prognostic role of FGFR1 among breast cancers using Cox regression and Kaplan-Meier analyses. Moreover, in vitro assays were conducted to confirm the correlation between FGFR1 and ER expression and investigate the effect of FGFR1 on tamoxifen (TAM) sensitivity in ER+ breast cancer. The results showed that ER expression was negatively correlated with FGFR1 expression (P = 0.011, r = -0.221). Moreover, FGFR1 expression was one of the prognostic factors of ER-positive breast cancer (OR = 1.974, 95% CI = 1.043-3.633), and high FGFR1 expression was correlated with decreased breast cancer overall survival. In addition, knocking down FGFR1 inhibited cell proliferation and enhanced TAM sensitivity in TAM-resistant cells. In conclusion, we found that there was a significant negative correlation between FGFR1 and ER levels in ER+ breast cancers, high FGFR1 protein expression was associated with poor breast cancer prognosis, down-regulating FGFR1 could elevate ER expression and is associated with enhanced TAM sensitivity in ER+ breast cancers.

Geniposide in Gardenia jasminoides var. radicans Makino modulates blood pressure via inhibiting WNK pathway mediated by the estrogen receptors.

To investigate the effects of geniposide in an iridoid found in Gardenia jasminoides var. radicans Makino (GJRM) in spontaneous hypertensive rat (SHR) and explore the possible mechanisms. In this study, we detected the content of geniposide in GJRM by high-performance liquid chromatography (HPLC). Then, we used acute diuretic experiments to determine whether geniposide has diuretic effect. Moreover, we carried out experiments on SHR to further study the mechanism of hypertension, while real-time PCR, Western blot and immunohistochemistry were used for the experiments in vivo test. Hypotonic model was used for in vitro test. Our data showed that the content of geniposide in the extract of GJRM is 27.54%. Meanwhile, 50mg/kg geniposide showed the strongest effect on promoting urine volume. Further study indicated that the extract of GJRM and geniposide could significantly reduce blood pressure and promote the excretion of urine and Na+ in SHR. In addition, geniposide significantly inhibited the activation of the with-no-lysine kinase (WNK) signalling pathway and significantly increases the protein expressions of estrogen receptor α (ERα), estrogen receptor β (ERβ) and G protein-coupled receptor 30 (GPR30) in SHR. In hypotonic model, geniposide significantly inhibits the phosphorylation of NKCC and NCC and could be antagonistic to estrogen receptor antagonists. Collectively, we would suggest that geniposide may potentially be utilized as an adjunct to existing thiazide and thiazide-like diuretics to control hypertension, mainly through inhibiting the activation of the WNK signalling pathway mediated by the estrogen receptor.

ESR1 genetic alterations and their association with clinicopathologic characteristics in advanced breast cancer: a single academic institution experience

Estrogen receptor (ER) alpha, a ligand-dependent nuclear transcription factor encoded by the ESR1 gene, is expressed in 70% of breast carcinomas (BCs) and is used as a target for endocrine-based therapies. However, some patients develop resistance to endocrine-based therapies due to ESR1 mutation, which leads to constitutive activation in the absence of ligand. We retrospectively analyzed 223 clinically advanced BCs using the FoundationOne CDX assay and found 13.9% (31/223) of cases had ESR1 genetic alterations (26 mutations and 5 amplifications). All ESR1 mutations occurred within the ligand binding domain, with the most prevalent being Y537S (42.3%) and D538G (38.5%), and all ESR1-mutated cases had a history of aromatase inhibitor use. No significant difference in clinicopathologic features was identified between ESR1-mutated and ESR1-amplified cases except higher frequency of HER2 positivity and TP53 mutations in ESR1-amplified cases. The prevalence of ESR1 mutations in ER-positive BCs was 19.1% (26/136). In comparison to ESR1-nonmutated ER-positive cases, ESR1-mutated cases demonstrated significantly higher percentage of tumor cells with ER and progesterone receptor expression, an increased tendency for overall distant metastasis and liver metastasis, higher frequency of FGF3/4/19 mutations, lower frequency of TP53 mutation, but no difference in overall survival and metastatic/recurrent intervals. In conclusion, our findings suggest that development of ESR1 mutations are selected for under the influence of estrogen deprivation, and a positive correlation between ESR1 mutations and ER protein expression may exist.

Efficacy of Anti-Estrogen Therapy in Estrogen Receptor Positive High-Grade Serous Ovarian Carcinoma: A Systematic Review

Therapy targeting the estrogen receptor (ER) pathway is being explored as a treatment option in ovarian carcinoma. However, studies on the efficacy of anti-estrogen therapy include a broad range of histological subtypes and/or do not select patients based on ER status. This systematic review provides an analysis of literature on the clinical benefit rate (CBR) of anti-estrogen therapy in ER positive high-grade serous carcinomas (HGSC) and on the correlation between ER expression by immunohistochemistry and clinical response. We did not find studies with populations consisting solely of ER positive HGSC. However, we included six studies reporting on 407 evaluable patients of whom 376 were HGSC (92%) and 302 were confirmed ER positive (80%). Anti-estrogen therapy resulted in a CBR of 27-65% and an overall response rate of 0-16%. No correlation was found between ER expression and clinical response. Therefore, ER protein expression alone is not a specific predictor of response. This may result from the incorrect assumption that ER expression equals ER pathway activity, since in the absence of ER activating mutations, ER pathway activity depends on availability of the estradiol ligand. In order to apply effective ER targeted therapy, it is important to develop better predictors to identify (non)-responders.