ERα-negative Breast Cancer Research Articles

9304 Integration of Glucocorticoid and Mineralocorticoid Receptor Signaling in Triple-negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

7873 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

9304 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

Escalation of Actions of Endocrine Networks for Treating Triple Negative Breast Cancer with Greater Efficacy: A Narrative Review

Earlier we had reviewed the detailed etiopathogenesis, treatment of BC,besidesupdate on management of triple negative breast cancer(TNBC) along with how dysregulated cholesterol metabolism including oxysterolswere implicated in the pathophysiology of breast cancer along with use that pathway for treating it,apart from ’using tocopherols/ phytosterols derived from Pequi(Caryocar brasiliansis Camb.) for complementing chemotherapy for BC treatment/alone or as new herbal therapy for improving 5year survival, and part of syndecans in pathogenesis&detailed Selective estrogen receptors modulators(SERM) & Selective estrogen receptors degraders(SERD’s)in avoiding post menopausal fractures &for BC therapy,As per the immunohistochemical categorized estrogen/ progesterone(P4) receptors decide the endocrine plausibility of the course of treatment of breast cancer. Possessing the properties of deficiency of estrogen receptor α,ERα negative breast cancer become detached from any kinds of endocrine regimens as far as the clinical scenario is concerned in routine specificallyin case of TNBC . Nevertheless, this paradigm got challenges by the fact that triple negative breast cancers continued to possess retention of sensitivity along with susceptibility to endocrine substances. The crosstalking of hormone effects program earlier unappreaciated was over time realized in addition to carcinogenic signaling, which were escalatingly examined.Correspondingly,the previously ignored endocrine reactiveness in ERα negative breast cancers garnered interest providing innovative therapeutic approach in an ERα independent manner.Here we summarize the genomic in addition to non genomic steroid hormone effects along with endocrine correlated signals in TNBC, accompanied by the tag of hormone insensitivity. Moreover, insight is provided over non classical mechanistic modes determined in usual hormone substances for illustrating their pleiotropic actions .Further we highlighted how modulating gut microbiota(GM)might enhance these actions.

TWIST1 Drives Cytotoxic CD8+ T-Cell Exhaustion through Transcriptional Activation of CD274 (PD-L1) Expression in Breast Cancer Cells.

In breast cancer, epithelial-mesenchymal transition (EMT) is positively associated with programmed death ligand 1 (PD-L1) expression and immune escape, and TWIST1 silences ERα expression and induces EMT and cancer metastasis. However, how TWIST1 regulates PD-L1 and immune evasion is unknown. This study analyzed TWIST1 and PD-L1 expression in breast cancers, investigated the mechanism for TWIST1 to regulate PD-L1 transcription, and assessed the effects of TWIST1 and PD-L1 in cancer cells on cytotoxic CD8+ T cells. Interestingly, TWIST1 expression is correlated with high-level PD-L1 expression in ERα-negative breast cancer cells. The overexpression and knockdown of TWIST1 robustly upregulate and downregulate PD-L1 expression, respectively. TWIST1 binds to the PD-L1 promoter and recruits the TIP60 acetyltransferase complex in a BRD8-dependent manner to transcriptionally activate PD-L1 expression, which significantly accelerates the exhaustion and death of the cytotoxic CD8+ T cells. Accordingly, knockdown of TWIST1 or BRD8 or inhibition of PD-L1 significantly enhances the tumor antigen-specific CD8+ T cells to suppress the growth of breast cancer cells. These results demonstrate that TWIST1 directly induces PD-L1 expression in ERα-negative breast cancer cells to promote immune evasion. Targeting TWIST1, BRD8, and/or PD-L1 in ERα-negative breast cancer cells with TWIST1 expression may sensitize CD8+ T-cell-mediated immunotherapy.

Methylation of ESR1 promoter induced by SNAI2-DNMT3B complex promotes epithelial-mesenchymal transition and correlates with poor prognosis in ERα-positive breast cancers.

Estrogen receptor α (ERα) serves as an essential therapeutic predictor for breast cancer (BC) patients and is regulated by epigenetic modification. Abnormal methylation of cytosine phosphoric acid guanine islands in the estrogen receptor 1 (ESR1) gene promoter could silence or decrease ERα expression. In ERα-negative BC, we previously found snail family transcriptional repressor 2 (SNAI2), a zinc-finger transcriptional factor, recruited lysine-specific demethylase 1 to the promoter to transcriptionally suppress ERα expression by demethylating histone H3 lysine 4 dimethylation (H3K4me2). However, the role of SNAI2 in ERα-positive BC remains elusive. In this study, we observed a positive correlation between SNAI2 and ESR1 methylation, and SNAI2 promoted ESR1 methylation by recruiting DNA methyltransferase 3 beta (DNMT3B) rather than DNA methyltransferase 1 (DNMT1) in ERα-positive BC cells. Subsequent enrichment analysis illustrated that ESR1 methylation is strongly correlated with cell adhesion and junction. Knocking down DNMT3B could partially reverse SNAI2 overexpression-induced cell proliferation, migration, and invasion. Moreover, high DNMT3B expression predicted poor relapse-free survival and overall survival in ERα-positive BC patients. In conclusion, this study demonstrated the novel mechanisms of the ESR1 methylation mediated with the SNAI2/DNMT3B complex and enhanced awareness of ESR1 methylation's role in promoting epithelial-mesenchymal transition in BC.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Abstract P3-06-05: Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice

Abstract Although immune checkpoint blockers (ICBs) have revolutionized the treatment of many cancers, only less than 30% of patients respond to any ICB as a monotherapy. Estrogens, produced by the adipose tissue in the mammary glands, are immunomodulators in the tumor microenvironment (TME). Estrogens suppress anti-tumor immunity and activate immunosuppression through the estrogen receptor α (ERα) in the immune cells but activate anti-tumor immunity through ERβ. Genistein (GEN) is a biologically active isoflavone and is structurally similar to estradiol (E2). However, in contrast to E2, GEN binds preferentially to ERβ. Here we studied if GEN combined with ERα inhibitor fulvestrant (Fulv) alters response to anti-PD1 therapy in ERα negative, ERβ positive E0771 mouse mammary tumors. Syngeneic C57BL/6 mice were fed either a control (CON) diet or GEN supplemented diet for 5 weeks and then allografted E0771 mammary tumor cell into the 4th mammary fat pad. When tumors reached an average size of 16mm2, mice in both diet groups were further divided into 4 sub-groups treated with: 1) anti-PD1 (150µg or 100µg every 3 days for a total of 3 doses), 2) Fulv (1mg) once weekly, 3) anti-PD1+Fulv or 4) IgG control group. We found that three doses of 150µg of anti-PD1 alone effectively reduced tumor growth in the CON (p=0.0005) group and its efficacy was notably weakened in the GEN group (p=0.04).. Combination with Fulv did not improve PD1 response. These findings suggest that when using a highly effective anti-PD1 dose, GEN may reduce rather than improve the response, and anti-ERα does not add to anti-PD1’s effect in control of GEN group. A suboptimal dose (100 µg) of anti-PD1 alone did not inhibit E0771 tumor growth in mice fed CON diet, but in the GEN group the tumor inhibition by anti-PD1 alone almost reached statistical significance (p=0.057). The combination of suboptimal PD1+Fulv significantly reduced tumor growth compared with IgG (p=0.04) or Fulv (p=0.02). During GEN feeding, anti-PD1+Fulv was even more potent in inhibiting tumor growth when compared with IgG group (p=0.02) and Fulv group (p=0.007). Thus, when anti-PD1 alone is not effective, either Fulv or GEN will improve responsiveness to anti-PD1. In the suboptimal anti-PD1 dose experiment, GEN feeding increased CD8+ T cells (P=0.04) and decreased CD4+Foxp3+ cells (p=0.01) in the TME. Further, GEN reduced the proportion of immunosuppressive MDSC cells (CD3-CD11B+Ly6Ghigh6Clow) in the PD1+ Fulv group (p=0.01) in the Peyer’s patches. In vitro experiments showed that both GEN and Fulv, and especially their combination, inhibited the E0771 cells proliferation, indicating that these two treatments can also directly suppress the growth of ERα negative but ERβ positive breast cancer cells. Taken together, the data show that anti-estrogens and GEN in the diet may improve response to anti-PD1 therapy, possibly both through activation of tumor immune response and inhibition of tumor cell proliferation and induction of tumor cell death. Citation Format: Fabia de Oliveira Andrade, Vivek Verma, Ester Molina Hoyo, Karla Andrade de Oliveira, Pal Koak, Lu Jin, Leena Hilakivi-Clarke. Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice [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 P3-06-05.

Bioinformatics Identification of TUBB as Potential Prognostic Biomarker for Worse Prognosis in ERα-Positive and Better Prognosis in ERα-Negative Breast Cancer.

Tubulin β class I gene (TUBB) is highly expressed in various cancers and plays several roles in carcinogenesis. However, the prognostic value of TUBB in breast cancer remains to be investigated. GEPIA and Breast Cancer Gene-Expression Miner were used to explore TUBB expression in breast cancer patients. Kaplan–Meier Plotter was used to assess the relationship between TUBB expression and several prognostic indicators including overall, distant metastasis-free, and relapse-free survival in ERα-positive and ERα-negative breast cancer. The genes that correlate with TUBB in ERα-positive and ERα-negative breast cancer were explored and the pathways were investigated using GSCA. The correlation between TUBB and several gene markers of immune cells was explored using GEPIA. ERα-positive breast cancer patients with increased TUBB showed worse prognosis, possibly through the activation of the TSC/mTOR pathway, whereas ERα-negative breast cancer patients with increased TUBB mRNA showed better prognosis. Significant positive correlations were observed between TUBB and gene markers of immune cells in ERα-positive breast cancer patients, whereas significant negative correlations were observed in ERα-negative breast cancer patients. The analysis revealed that TUBB might be considered as a predictive biomarker for worse prognosis in ERα-positive and better prognosis in ERα-negative breast cancer.

Rewiring of the Endocrine Network in Triple-Negative Breast Cancer

The immunohistochemical definition of estrogen/progesterone receptors dictates endocrine feasibility in the treatment course of breast cancer. Characterized by the deficiency of estrogen receptor α, ERα-negative breast cancers are dissociated from any endocrine regimens in the routine clinical setting, triple-negative breast cancer in particular. However, the stereotype was challenged by triple-negative breast cancers’ retained sensitivity and vulnerability to endocrine agents. The interplay of hormone action and the carcinogenic signaling program previously underscored was gradually recognized along with the increasing investigation. In parallel, the overlooked endocrine-responsiveness in ERα-negative breast cancers attracted attention and supplied fresh insight into the therapeutic strategy in an ERα-independent manner. This review elaborates on the genomic and non-genomic steroid hormone actions and endocrine-related signals in triple-negative breast cancers attached to the hormone insensitivity label. We also shed light on the non-canonical mechanism detected in common hormone agents to showcase their pleiotropic effects.

Protein disulfide isomerase A1‑associated pathways inthedevelopment of stratified breast cancer therapies.

The oxidoreductase protein disulfide isomerase A1 (PDIA1) functions as a cofactor for many transcription factors including estrogen receptor α (ERα), nuclear factor (NF)-κB, nuclear factor erythroid 2-like 2 (NRF2) and regulates the protein stability of the tumor suppressor p53. Taking this into account we hypothesized that PDIA1, by differentially modulating the gene expression of a diverse subset of genes in the ERα-positive vs. the ERα-negative breast cancer cells, might modify dissimilar pathways in the two types of breast cancer. This hypothesis was investigated using RNA-seq data from PDIA1-silenced MCF-7 (ERα-positive) and MDA-MB-231 (ERα-negative) breast cancer cells treated with either interferon γ (IFN-γ) or etoposide (ETO), and the obtained data were further analyzed using a variety of bioinformatic tools alongside clinical relevance assessment via Kaplan-Meier patient survival curves. The results highlighted the dual role of PDIA1 in suppressing carcinogenesis in the ERα(+) breast cancer patients by negatively regulating the response to reactive oxygen species (ROS) and promoting carcinogenesis by inducing cell cycle progression. In the ERα(−) breast cancer patients, PDIA1 prevented tumor development by modulating NF-κB and p53 activity and cell migration and induced breast cancer progression through control of cytokine signaling and the immune response. The findings reported in this study shed light on the differential pathways regulating carcinogenesis in ERα(+) and ERα(−) breast cancer patients and could help identify therapeutic targets selectively effective in ERα(+) vs. ERα(−) patients.

Estrogen Receptor β Expression and Its Clinical Implication in Breast Cancers: Favorable or Unfavorable?

There are two estrogen receptor (ER) genes (ESR1/ERα and ESR2/ERβ) in humans. Of those. ERβ, the second ER isotype identified in 1996, is differentially expressed in different phenotypes and molecular subtypes of breast cancer (BCa), and is highly expressed in ERα-negative BCa and triple-negative BCa (TNBC). This review summarizes the potential clinical relevance of ERβ in BCa and the challenges associated with studies on the role of ERβ in BCa. The experimental and clinical studies evaluating clinical outcomes and associations with clinical characteristics and responses to endocrine therapy on targeting ERβ reviewed herein indicate that ERβ is a clinically important biomarker in BCa. The reviewed studies also suggest that each ERβ isoform has a distinct role in BCa subtypes and the potential of novel- targeted therapies in BCa, especially ERα-negative BCa and TNBC. However, the findings of many studies on ERβ are inconsistent, and the exact role of ERβ in BCa remains elusive; this may potentially be attributed to the complexity of ERβ isoforms, but also to the lack of standardized testing protocol. Thus, successful clinical application of ERβ requires the development of standardized, reproducible, and objective measurement methods for ERβ that can be widely and routinely applied in clinical setting.

Perfluorooctanoic acid (PFOA) as a stimulator of estrogen receptor-negative breast cancer MDA-MB-231 cell aggressiveness: Evidence for involvement of fatty acid 2-hydroxylase (FA2H) in the stimulated cell migration.

Detailed in vitro studies on the effects of perfluorooctanoic acid (PFOA) have demonstrated that activation of peroxisome proliferator-activated receptor α (PPARα) is a key process by which PFOA affects the malignancy of estrogen receptor α (ERα)-positive breast cancer cells. However, there is very little information on the PPARα-regulated genes responsible for the effects of PFOA in ERα-negative breast cancer cell malignancy. We recently demonstrated that fatty acid 2-hydroxylase (FA2H) stimulates the migration of ERα-negative human MDA-MB-231 cells, and PPARα is a key factor for the induction of FA2H in these cells. However, evidence for the relationship between PFOA exposure and PPARα-FA2H axis-driven migration has not been obtained. Here we analyzed the effects of PFOA on PPARα transcription and FA2H expression in relation to MDA-MB-231 cell migration. We found that simultaneously with stimulated migration, PFOA upregulated FA2H and activated the transcription of PPARα. FA2H-selective siRNA, but not siRNA control, clearly dampened PFOA-mediated cell migration. There is an inhibitory interaction between PPARα and PPARβ/δ (i.e., PPARβ/δ can suppress PPARα-mediated transcription) in MDA-MB-231 cells, but even in the presence of PPARβ/δ expression, PFOA appeared to free PPARα to upregulate FA2H. Collectively, our findings show that i) PFOA activates PPARα-mediated transcription, ii) PFOA stimulates migration dependent on FA2H expression, and iii) mechanistically, PFOA relieves PPARβ/δ suppression of PPARα activity to upregulate FA2H in MDA-MB-231 cells.

Expression of androgen receptor splice variant, AR-V7, in high-grade serous ovarian cancer

<h3>Objectives:</h3> Androgen receptors (AR) are expressed in up to 55% of ERα-negative breast cancers overall and up to 35% of those classified as TNBC. Similarly, AR is expressed in approximately 40% of high grade serous ovarian cancer (HGSOC). The development of AR splice variants (AR-SV), particularly AR-V7, has been shown to be a poor prognostic feature, and mechanism of androgen resistance in breast and prostate cancers. We sought to determine if AR-V7 was present in HGSOC and if there was a correlation with platinum sensitivity and/or treatment outcomes. <h3>Methods:</h3> Pair-matched chemo-naïve and platinum-treated HGSOC specimens were identified from our tissue repository. Formalin-fixed paraffin-embedded tumor (FFPE) blocks were collected and tested for expression of AR-V7. Gene fusion detection and variant detection were performed on mRNA isolated from a formalin-fixed paraffin-embedded tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required. <h3>Results:</h3> Twelve chemo-naïve specimens collected at time of diagnosis by initial biopsy (n=8) or primary debulking surgery (n=4) were pair-matched with the patients' subsequent platinum-treated specimens from either interval debulking surgery (n=6) or biopsy of recurrence (n=6). Following penultimate platinum, 50% were platinum resistant and 50% were platinum sensitive at time of second pathology. Only 19 of the 24 specimens submitted had sufficient RNA for testing, 8 chemo-naïve and 11 platinum-treated. AR-V7 variant transcript was not detected in any of the specimens submitted. <h3>Conclusions:</h3> AR-V7 was not expressed in any specimen before or after platinum based chemotherapy suggesting it may not be a useful prognostic marker in the management of HGSOC. Further investigation into gynecologic malignancies with high AR expression and treated with androgen deprivation therapy may elucidate different results.

Sesamol Epigenetically Induces Estrogen Receptor α Re-expression by Upregulating miR-370-3p in Estrogen Receptor α-Negative Breast Cancer.

Due to lack of estrogen receptor α (ERα, gene name: ESR1), ERα-negative breast carcinoma is insensitive to endocrine therapy, and restoration of ERα has become a promising strategy for ERα-negative breast cancer treatment. Sesamol, a naturally occurring phenolic compound, is usually extracted from sesame seeds. Previous investigations have unmasked its anti-oxidant and anti-inflammation properties. In this study, sesamol induced ERα functional re-expression followed by upregulation of its downstream pS2 and GREB1 genes in ERα-negative breast carcinoma. Moreover, it endowed responsiveness of ERα-negative breast carcinoma to the endocrine treatment drug 4-hydroxytamoxifen without influencing the viability of normal human umbilical vein endothelial cells. Mechanistically, sesamol induced ESR1 gene promoter demethylation by downregulating the expression of the DNA methyltransferases DNMT3A and DNMT3B, without affecting DNMT1. Moreover, the non-coding RNA miR-370-3p directly targeted DNMT3A and DNMT3B mRNA, and its expression increased upon treatment with sesamol. Artificial abrogation of miR-370-3p expression with an antagomir abolished the inhibition of DNMT3A and DNMT3B expression by sesamol, resulting in a fallback in ERα reactivation. In mice, sesamol significantly induced ERα re-expression via miR-370-3p-mediated downregulation of DNMT3A and DNMT3B. Sesamol may be a safe and effective option for clinical adjuvant therapy in patients with ERα-negative breast cancer.

Abstract 2154: Expression of androgen receptor splice variant, AR-V7, in high grade serous ovarian cancer

Abstract Objective: Androgen receptors (AR) are expressed in up to 55% of ERα-negative breast cancers overall and up to 35% of those classified as TNBC. Similarly, AR is expressed in approximately 40% of high grade serous ovarian cancer (HGSOC). The development of AR splice variants (AR-SV), particularly AR-V7, has been shown to be a poor prognostic feature, and mechanism of androgen resistance in breast and prostate cancers. We sought to determine if AR-V7 was present in HGSOC and if there was a correlation with platinum sensitivity and/or treatment outcomes. Methods: Pair-matched chemo-naïve and platinum-treated HGSOC specimens were identified from our tissue repository. Formalin-fixed paraffin-embedded tumor (FFPE) blocks were collected and tested for expression of AR-V7. Gene fusion detection and variant detection were performed on mRNA isolated from a formalin-fixed paraffin-embedded tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required. Results: Twelve chemo-naïve specimens collected at time of diagnosis by initial biopsy (n=8) or primary debulking surgery (n=4) were pair-matched with the patients' subsequent platinum-treated specimens from either interval debulking surgery (n=6) or biopsy of recurrence (n=6). Following penultimate platinum, 50% were platinum resistant and 50% were platinum sensitive at time of second pathology. Only 19 of the 24 specimens submitted had sufficient RNA for testing, 8 chemo-naïve and 11 platinum-treated. AR-V7 variant transcript was not detected in any of the specimens submitted. Conclusions: AR-V7 was not expressed in any specimen before or after platinum based chemotherapy suggesting it may not be a useful prognostic marker in the management of HGSOC. Further investigation into gynecologic malignancies with high AR expression and treated with androgen deprivation therapy may elucidate different results. Citation Format: Benjamin Wilson, Joanne Xiu, Michael Ulm, Tiffany Redfern, Ramesh Narayanan, Adam ElNaggar. Expression of androgen receptor splice variant, AR-V7, in high grade serous ovarian 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 2154.

Glucocorticoid Receptor: A Multifaceted Actor in Breast Cancer.

Breast cancer (BC) is one of the most common cancers in women worldwide. Even though the role of estrogen receptor alpha (ERα) is extensively documented in the development of breast tumors, other members of the nuclear receptor family have emerged as important players. Synthetic glucocorticoids (GCs) such as dexamethasone (dex) are commonly used in BC for their antiemetic, anti-inflammatory, as well as energy and appetite stimulating properties, and to manage the side effects of chemotherapy. However, dex triggers different effects depending on the BC subtype. The glucocorticoid receptor (GR) is also an important marker in BC, as high GR expression is correlated with a poor and good prognosis in ERα-negative and ERα-positive BCs, respectively. Indeed, though it drives the expression of pro-tumorigenic genes in ERα-negative BCs and is involved in resistance to chemotherapy and metastasis formation, dex inhibits estrogen-mediated cell proliferation in ERα-positive BCs. Recently, a new natural ligand for GR called OCDO was identified. OCDO is a cholesterol metabolite with oncogenic properties, triggering mammary cell proliferation in vitro and in vivo. In this review, we summarize recent data on GR signaling and its involvement in tumoral breast tissue, via its different ligands.

Cryptotanshinone Inhibits ERα-Dependent and -Independent BCRP Oligomer Formation to Reverse Multidrug Resistance in Breast Cancer.

Both long-term anti-estrogen therapy and estrogen receptor-negative breast cancer contribute to drug resistance, causing poor prognosis in breast cancer patients. Breast cancer resistance protein (BCRP) plays an important role in multidrug resistance. Here, we show that cryptotanshinone (CPT), an anti-estrogen compound, inhibited the oligomer formation of BCRP on the cell membrane, thus blocking its efflux function. The inhibitory effect of CPT on BCRP was dependent on the expression level of estrogen receptor α (ERα) in ERα-positive breast cancer cells. Furthermore, ERα-negative breast cancer cells with high expression of BCRP were also sensitive to CPT because CPT was able to bind to BCRP and inhibit its oligomer formation on the cell membrane, suggesting that the high level of BCRP expression is crucial for CPT to reverse drug resistance. The combination of CPT and chemotherapeutic agents displayed enhanced anticancer effects. The results suggest that CPT is a novel BCRP inhibitor via blocking the oligomer formation of BCRP on the cell membrane. CPT is able to inhibit the activity of BCRP in an ERα-dependent and -independent manner, sensitizing breast cancer cells to chemotherapy.

Molecular mechanism study of BPAF-induced proliferation of ERα-negative SKBR-3 human breast cancer cells in vitro/in vivo

Bisphenol AF (BPAF) is a known estrogen disruptor of the ERα pathway. The aim of the present study was to characterize the proliferation effects of BPAF on ERα-negative SKBR-3 breast cancer cells with mechanistic insights. BPAF at low concentrations (0.001–0.1 μM) significantly induced the proliferation of SKBR-3 cells. In a SKBR-3 tumor model in BALB/c nude mice, BPAF at 100 mg/kg body weight/day also significantly promoted the growth of SKBR-3 tumors. Low concentrations of BPAF markedly increased the expression of G protein-coupled estrogen receptor (GPER1), c-Myc, CyclinD1 and c-Fos proteins, and enhanced phosphorylation of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in SKBR-3 cells. Further, BPAF significantly upregulated mRNA levels of related target genes in SKBR-3 cells and SKBR-3 tumor tissues in nude mice. The GPER1 inhibitor G15 and phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin (WM) inhibited phosphorylation of Erk and Akt. The specific signal inhibitors also markedly decreased the expression of target genes and weakened the cell proliferation induced by low-concentration BPAF. The findings showed that GPER1 could independently regulate BPAF-induced proliferation of SKBR-3 cells without requiring ERα. These results provide mechanistic insights into the effects of BPAF regarding ERα-negative human breast cancer development.

Protein disulfide isomerase A1 regulates breast cancer cell immunorecognition in a manner dependent on redox state.

Oxidoreductase protein disulphide isomerases (PDI) are involved in the regulation of a variety of biological processes including the modulation of endoplasmic reticulum (ER) stress, unfolded protein response (UPR), ER-mitochondria communication and the balance between pro-survival and pro-death pathways. In the current study the role of the PDIA1 family member in breast carcinogenesis was investigated by measuring ROS generation, mitochondrial membrane disruption, ATP production and HLA-G protein levels on the surface of the cellular membrane in the presence or absence of PDIA1. The results showed that this enzyme exerted pro-apoptotic effects in estrogen receptor (ERα)-positive breast cancer MCF-7 and pro-survival in triple negative breast cancer (TNBC) MDA-MB-231 cells. ATP generation was upregulated in PDIA1-silenced MCF-7 cells and downregulated in PDIA1-silenced MDA-MB-231 cells in a manner dependent on the cellular redox status. Furthermore, MCF-7 and MDA-MB-231 cells in the presence of PDIA1 expressed higher surface levels of the non-classical human leukocyte antigen (HLA-G) under oxidative stress conditions. Evaluation of the METABRIC datasets showed that low PDIA1 and high HLA-G mRNA expression levels correlated with longer survival in both ERα-positive and ERα-negative stage 2 breast cancer patients. In addition, analysis of the PDIA1 vs. the HLA-G mRNA ratio in the subgroup of the living stage 2 breast cancer patients exhibiting low PDIA1 and high HLA-G mRNA levels revealed that the longer the survival time of the ratio was high PDIA1 and low HLA-G mRNA and occurred predominantly in ERα-positive breast cancer patients whereas in the same subgroup of the ERα-negative breast cancer mainly this ratio was low PDIA1 and high HLA-G mRNA. Taken together these results provide evidence supporting the view that PDIA1 is linked to several hallmarks of breast cancer pathways including the process of antigen processing and presentation and tumor immunorecognition.