Therapy For ER-positive Breast Cancer Research Articles

Abstract 2994: MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling

Abstract Background: Estrogen receptor (ER) positive breast cancer is the most common subtype of breast cancer. Elucidation of the regulatory network in estrogen receptor pathway is critical to develop and deliver potent targeted therapy for ER-positive breast cancer. The Mir-99a-let-7c cluster host gene MIR99AHG is a long non-coding RNA involving in a variety of tumor formation and disease progression processes. However, the biological functions of MIR99AHG in ER-positive breast cancer have not been reported. Methods: The Western-blot, qRT-PCR, Immunofluorescence and fluorescence in-situ hybridization were used to identify potential target genes and regulatory pathway of estrogen receptor α (ERa), which is a key proliferative driver of ER-positive breast cancer cells. The cell proliferation assay, clone formation assay, xenograft tumor model and rescue assay were used to investigate the effect of differential expression of MIR99AHG on the proliferative capacity of ER-positive breast cancer cells MCF7 and T47D. In addition, correlation analysis was performed to verify the relation of the expression of MIR99AHG, LATS1, LATS2, CYR61, CTGF and ESR1 from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. And survival analysis was used to predict the prognostic impact of MIR99AHG in breast cancer patients. Results: MIR99AHG expression was significantly higher in ER-positive breast cancer cells MCF7 and T47D than in ER-negative breast cancer cells SKBR3, MDA-MB-231 and BT549. The high expression of MIR99AHG predicted poor prognosis in ER-positive breast cancer patients. In vitro and in vivo assays demonstrated that MIR99AHG positively regulated the proliferation of MCF7 and T47D cells. Importantly, the Hippo signaling was identified to be down-stream mediators of MIR99AHG in modulating ERα transcription. Furthermore, XMU-MP-1, which targets the Hippo signaling upstream kinase MST1/2, was able to reverse the effect of MIR99AHG on MCF7 cell proliferation. Conclusions: MIR99AHG up-regulates ERα expression and promotes proliferation in ER-positive breast cancer cells through activating Hippo signaling pathway. Targeting MIR99AHG/Hippo pathway is a potential strategy for the treatment of ER-positive breast cancer. Citation Format: Wende Wang, Danping Lin, Ziyang Lin, Yangyang Yan, Yusen Qin, Yuanke Liang, Haoyu Lin, De Zeng. MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2994.

Nanoparticle-Based Combination Therapy Enhances Fulvestrant Efficacy and Overcomes Tumor Resistance in ER-Positive Breast Cancer.

A smart nanomedicine encapsulating fulvestrant to improve its half-life, bioavailability, and tumor-targeting and coloaded with CDK4/6 inhibitor abemaciclib to block resistance is a safe and effective therapy for ER-positive breast cancer.

Abstract 3894: The role of ELF3 in acquired resistance to endocrine therapy in ER-positive breast cancer

Abstract Endocrine therapies targeting the estrogen receptor (ER) are the major treatments of ER+ breast cancer, however acquired resistance to endocrine therapy is almost unavoidable and is the main cause of death with breast cancer. The only FDA-approved selective estrogen receptor degrader (SERD) fulvestrant has been used in the second line treatment to overcome aromatase inhibitor- or tamoxifen-resistance. However, fulvestrant has poor pharmacokinetic properties, which has inspired the development of a new generation of oral SERDs including amcenestrant and giredestrant. To understand the mechanisms of acquired resistance to SERDs in ER+ breast cancer, we established ER+ breast cancer cell lines (MCF7 and T47D) resistant to fulvestrant, amcenestrant or giredestrant. We found that all of our MCF7 and T47D SERD-resistant cell lines showed cross-resistance to each of the three SERDs. We then performed multiple genome-wide profiling assays including CRISPR-KO screen, whole exome sequencing (WES), RNA-seq, ATAC-seq and H3K27ac ChIP-seq on the parental and SERD resistant cell lines. Our findings from the CRISPR-KO screens suggest that ETS transcription factor ELF3 is a potential driver of resistance to SERDs. ELF3 expression is negatively regulated by estrogen signaling. Cells that developed acquired resistance to different SERDs consistently show depletion of ER expression but also a dramatic increase of ELF3 expression, while knockdown of ELF3 in the resistant cells leads to cell growth inhibition, suggesting that ELF3 can drive cell survival in the SERD resistant cells. When comparing the open chromatin regions and active enhancer regions between SERD resistant and parental cells via ATAC-seq and H3K27ac ChIP-seq respectively, ELF3 DNA binding motif was found significantly enriched in SERD resistant cells versus parental cells further indicating that ELF3 may play an important role in the SERD resistant cells. ELF3 ChIP-seq was also performed to identify genes enriched with ELF3 binding sites in SERD resistant and parental cells. Pathway analysis of ELF3-targeted genes reveals that ELF3 may regulate cell metabolism specifically in SERD resistant cells but not in parental cells. Our study suggests that ELF3 may be a novel driver of acquired resistance to SERDs in ER+ breast cancer. Citation Format: Na Zhang, Rongbin Zheng, Myles Brown. The role of ELF3 in acquired resistance to endocrine therapy in ER-positive 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 3894.

Neoadjuvant endocrine therapy in ER-positive breast cancer: evolution, indication, and tailored treatment strategy.

In recent years, endocrine therapy (ET), an effective systemic treatment for the management of estrogen receptor (ER)-positive breast cancers, has regained interest as a neoadjuvant therapy based on evidence that ET can fulfill the aim of neoadjuvant systemic treatment for tumor shrinkage as well as elucidate important clinical information on endocrine sensitivity that enables the prognostication of patients. Moreover, neoadjuvant endocrine therapy (NET) potentially provides an opportunity for early assessment of the clinical efficacy of novel agents. Furthermore, recently reported trials have generated evidence for a more tailored approach for perioperative management of ER-positive breast cancer using clinical and molecular biomarkers, and this has provided a rationale that enables the broadening of clinical indications for NET. This review discusses the current evidence for NET, the evolution of NET trials, clinical indications, and NET-based treatment strategies.

Upregulation of an estrogen receptor-regulated gene by first generation progestins requires both the progesterone receptor and estrogen receptor alpha.

Progestins, synthetic compounds designed to mimic the activity of natural progesterone (P4), are used globally in menopausal hormone therapy. Although the older progestins medroxyprogesterone acetate (MPA) and norethisterone (NET) have been implicated in increased breast cancer risk, little is known regarding newer progestins, and no significant risk has been associated with P4. Considering that breast cancer is the leading cause of mortality in women, establishing which progestins increase breast cancer incidence and elucidating the underlying mechanisms is a global priority. We showed for the first time that the newer-generation progestin drospirenone (DRSP) is the least potent progestin in terms of proliferation of the estrogen-responsive MCF-7 BUS breast cancer cell line, while NET and P4 have similar potencies to estradiol (E2), the known driver of breast cancer cell proliferation. Notably, MPA, the progestin most frequently associated with increased breast cancer risk, was significantly more potent than E2. While all the progestogens enhanced the anchorage-independent growth of the MCF-7 BUS cell line, MPA promoted a greater number of colonies than P4, NET or DRSP. None of the progestogens inhibited E2-induced proliferation and anchorage-independent growth. We also showed that under non-estrogenic conditions, MPA and NET, unlike P4 and DRSP, increased the expression of the estrogen receptor (ER) target gene, cathepsin D, via a mechanism requiring the co-recruitment of ERα and the progesterone receptor (PR) to the promoter region. In contrast, all progestogens promoted the association of the PR and ERα on the promoter of the PR target gene, MYC, thereby increasing its expression under non-estrogenic and estrogenic conditions. These results suggest that progestins differentially regulate the way the PR and ER converge to modulate the expression of PR and ER-regulated genes. Our novel findings indicating similarities and differences between P4 and the progestins, emphasize the importance of comparatively investigating effects of individual progestins rather than grouping them as a class. Further studies are required to underpin the clinical relevance of PR/ERα crosstalk in response to different progestins in both normal and malignant breast tissue, to either confirm or refute their suitability in combination therapy for ER-positive breast cancer.

Abstract 1778: NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer

Abstract Genomic loss of in Neurofibromin 1 (NF1) is a recurrent mechanism driving metastatic progression and resistance to endocrine therapy in ER-positive breast cancer, via activation of the MAPK pathway, induction of cyclin D1 expression and modulation of estrogen receptor signaling. To identify specific mediators of tumor growth in NF1 mutant cancers, we performed a genetic knockout (KO) screen in isogenic WT and NF1 KO breast cancer cells. After employing stringent cutoff criteria, we found 28 genes that are specifically essential for NF1 KO cells but not WT cells. Among these genes, NR2F2, encoding an orphan nuclear receptor, was one of the top candidates. We validated that its loss significantly impaired the growth of multiple models of NF1 null, ER+ breast cancer. Loss of NF1 leads to the hyperactivation of Ras which sequentially activates multiple effector pathways, including the MAPK pathway and the PI3K-AKT pathway. We found that MAPK activation upon NF1 loss led to elevated NR2F2 expression levels. A negative correlation between NF1 and NR2F2 expression was further confirmed in public gene expression databases. These data suggested that induction of NR2F2 expression might be one mechanism that mediates endocrine resistance caused by NF1 loss. Consistently, we found that exogenous expression of NR2F2 could confer endocrine resistance while its knockout hypersensitized ER+ breast cancer cells to endocrine therapies and significantly attenuated NF1 loss induced resistance. RNA-seq analysis revealed that estrogen response gene sets were among the most enriched signatures upon NF1 KO, NR2F2 over-expression (OE) or NR2F2 KO. NF1 KO or NR2F2 OE decreased the expression of early estrogen response genes and impaired their E2-induced expression, most of which could be rescued by NR2F2 KO, suggesting a regulatory effect of NR2F2 on ER transcriptional activity. To further define these mechanisms, ongoing integrative analyses of NR2F2 qPlex-RIME, ER and NR2F2 ChIP-Sequencing and ATAC-Sequencing will be presented. Taken together, our studies identify NR2F2 as a MAPK driven transcript whose induction mediates endocrine resistance, and may serve as a novel therapeutic target to overcome anti-estrogen resistant breast cancer growth. Citation Format: Yanyan Cai, Peihua Zhao, Fan Wu, Hong Shao, Pedram Razavi, Guotai Xu, Maurizio Scaltriti, Sarat Chandarlapaty. NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1778.

Estrogen Receptor and the Unfolded Protein Response: Double-Edged Swords in Therapy for Estrogen Receptor-Positive Breast Cancer.

Estrogen receptor α (ERα) is a target for the treatment of ER-positive breast cancer patients. Paradoxically, it is also the initial site for estrogen (E2) to induce apoptosis in endocrine-resistant breast cancer. How ERα exhibits distinct functions, in different contexts, is the focus of numerous investigations. Compelling evidence demonstrated that unfolded protein response (UPR) is closely correlated with ER-positive breast cancer. Treatment with antiestrogens initially induces mild UPR through ERα with activation of three sensors of UPR-PRK-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6)-in the endoplasmic reticulum. Subsequently, these sensors interact with stress-associated transcription factors such as c-MYC, nuclear factor-κB (NF-κB), and hypoxia-inducible factor 1α (HIF1α), leading to acquired endocrine resistance. Paradoxically, E2 further activates sustained secondary UPR via ERα to induce apoptosis in endocrine-resistant breast cancer. Specifically, PERK plays a key role in inducing apoptosis, whereas IRE1α and ATF6 are involved in endoplasmic reticulum stress-associated degradation after E2 treatment. Furthermore, persistent activation of PERK deteriorates stress responses in mitochondria and triggers of NF-κB/tumor necrosis factor α (TNFα) axis, ultimately determining cell fate to apoptosis. The discovery of E2-induced apoptosis has clinical relevance for treatment of endocrine-resistant breast cancer. All of these findings demonstrate that ERα and associated UPR are double-edged swords in therapy for ER-positive breast cancer, depending on the duration and intensity of UPR stress. Herein, we address the mechanistic progress on how UPR leads to endocrine resistance and commits E2 to inducing apoptosis in endocrine-resistant breast cancer.

Predicting Response to Endocrine Therapy in ER-Positive Breast Cancer

Predicting Response to Endocrine Therapy in ER-Positive Breast Cancer

Dual Modulators of p53 and Cyclin D in ER Alpha Signaling by Albumin Nanovectors Bearing Zinc Chaperones for ER-positive Breast Cancer Therapy.

CDATA[The inherited mutations and underexpression of BRCA1 in sporadic breast cancers resulting in the loss or functional inactivation of BRCA1 may contribute to a high risk of breast cancer. Recent researchers have identified small molecules (BRCA1 mimetics) that fit into a BRCA1 binding pocket within Estrogen Receptor alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity, and overcome antiestrogen resistance. Studies indicate that most of the BRCA1 breast cancer cases are associated with p53 mutations. It indicates that there is a potential connection between BRCA1 and p53. Most p53 mutations are missense point mutations that occur in the DNA-binding domain. Structural studies have demonstrated that mutant p53 core domain misfolding, especially p53-R175H, is reversible. Mutant p53 reactivation with a new class of zinc metallochaperones (ZMC) restores WT p53 structure and functions by restoring Zn2+ to Zn2+ deficient mutant p53. Considering the role of WT BRCA1 and reactivation of p53 in tumor cells, our hypothesis is to target both tumor suppressor proteins by a novel biomolecule (ZMC). Since both proteins are present in the same cell and are functionally inactive, this state may be a novel efficacious therapeutic regime for breast cancer therapy. In addition, we propose to use Albumin Nanovector (ANV) formulation for target drug release.

Genistin attenuates cellular growth and promotes apoptotic cell death breast cancer cells through modulation of ERalpha signaling pathway

Estrogen receptor alpha (ERα) is a vital molecular target in ER-positive breast cancer. Genistin (GS) is one of isoflavones that can exert diverse pharmacological effects including that of anti-proliferation, anti-tumor angiogenesis, induce cell cycle arrest and apoptosis. Here, we examined the efficacy of GS as an anti-cancer agent against breast cancer cells. We observed that GS exhibited more cytotoxic activity against MCF-7 cells than MDA-MB-231cells. We found that GS caused negative regulation of ERα. It also effectively down-modulated ER nuclear translocation as well DNA binding activity in breast cancer cells. Moreover, GS effectively induced apoptosis and suppressed levels of oncogenic markers in MCF-7 cells. Interestingly, in ERα knocked-down MCF-7 cells, cell viability was found to be increased and the levels of cleaved PARP was abolished. We found completely contrasting results in ERα overexpressed MDA-MB-231 cells, where cell viability was decreased and expression level of apoptotic markers was enhanced. Our results demonstrate that GS can suppress ERα signaling and can be useful for prevention and therapy of ER-positive breast cancer.

Abstract GS6-06: A neoadjuvant trial with letrozole identifies PRR11 in the 17q23 amplicon as a mechanism of resistance to endocrine therapy in ER-positive breast cancer

Abstract Although the 17q23 amplicon has been associated with luminal B breast cancer (BC) and high risk of recurrence, a specific gene or genes in this region that would be causal to endocrine resistance have not yet been uncovered. We performed whole transcriptome analysis on RNA extracted from 58 estrogen receptor (ER)+ BCs treated with neoadjuvant letrozole for median 7.2 months. PRR11 (Proline rich 11), located in 17q23, was upregulated in non-responding tumors as defined by relapse after a median follow up of 5 years and/or a preoperative endocrine prognostic index (PEPI) ≥4. Differential gene expression analysis between tumors expressing low vs high PRR11 mRNA showed that BC signatures associated with proliferation, IGF-1 and PI3K signaling were enriched in tumors with high PRR11 expression. Rate of PRR11 amplification is 15.2% in the Metastatic Breast Cancer project, but 9.5% and 9.4% in METABRIC and The Cancer Genome Atlas (TCGA), respectively. Gene Set Enrichment Analysis revealed an enrichment of hallmark gene sets associated with proliferation in PRR11-amplified ER+ BCs in METABRIC and TCGA. Integrated analysis of gene expression with on-treatment Ki67 levels from three independent studies with operable ER+ BCs treated with neoadjuvant aromatase inhibitor (ACOSOG-Z1031, NCT00651976, Llombart-Cussac et al.) showed that PRR11 was the only gene in 17q23 with a significant correlation with a high Ki67 levels across all studies. PRR11 knockdown inhibited E2-independent growth of HCC1428 LTED (long-term estrogen deprived) and MCF7 LTED cells in culture and MCF7 xenografts. PRR11 siRNA also inhibited growth of fulvestrant-resistant and tamoxifen-resistant MCF7 cells. Conversely, PRR11 transduction induced MDA-MB-134VI cell growth under estrogen-depleted conditions. Using a PCR array with 84-cell cycle genes, we identified SKP2, CDKN1A, CCNB2, CCNA2, CKS2 and CCNB1 as genes downregulated by PRR11 knockdown. Except for SKP2 and CDKN1A, expression of all those genes was elevated in PRR11-amplifiedER+ BCs in TCGA and METABRIC. Suggesting a link to activation of PI3K signaling, we found the proline-rich motif of PRR11 associates with the SH3 domain of the p85 regulatory subunit of PI3K. We hypothesized that this association suppresses p85 homodimer formation, thus facilitating binding of PI3Kα (p110α)-p85 dimers to IRS1, retention of p110α at the plasma membrane and, hence, activation of PI3K/AKT. To test this, we co-transfected HEK293T cells with HA-p85 and FLAG-p85. Forced expression of PRR11 reduced HA-p85 and FLAG-p85 homodimers as shown by HA and FLAG pulldowns followed by FLAG and HA immunoblots, respectively. PRR11 overexpression enhanced insulin-stimulated association of IRS1 to p110α and activation of AKT. PRR11 knockdown reduced insulin/IGF-1/2-stimulated p-AKT. In METABRIC and TCGA, PRR11 amplification and PIK3CA mutations are exclusive of each other, suggesting these alterations would be functionally linked with the same pathway. Connectivity map analysis with the list of genes significantly overexpressed in ER+/PRR11-amplified BCs predicted PI3K inhibitors as perturbations that suppress such gene list. In the MGH/Sanger dataset, PRR11-amplified BC cell lines displayed significantly higher sensitivity to the pan-PI3K inhibitor pictilisib compared to cell lines without PRR11 amplification. Finally, inhibition of PI3Kα by siRNA or alpelisib abrogated E2-independent growth and insulin-stimulated growth of PRR11-transduced MDA-MB-134VI and MCF10A cells, respectively, suggesting p110α is required for the growth promoting effects of PRR11. These data suggest that 1) PRR11 is a mediator of resistance to antiestrogens via amplification of PI3K/AKT signaling, and 2) PI3Kα is a potential therapeutic target in ER+ BCs harboring PRR11 amplification. Citation Format: Kyung-min Lee, Angel Guerrero-Zotano, Ariella Hanker, Alberto Servetto, Dhivya Sudhan, Luigi Formisano, Valerie Jansen, Paula González-Ericsson, Melinda Sanders, Thomas Stricker, Lewis Cantley, Carlos Arteaga. A neoadjuvant trial with letrozole identifies PRR11 in the 17q23 amplicon as a mechanism of resistance to endocrine therapy in ER-positive breast cancer [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 GS6-06.

Abstract P3-10-09: Gene deletion of IRS1 inhibited IGF-I, insulin and estradiol stimulated MCF-7L breast cancer cell growth

Abstract Targeting of the type I insulin-like growth factor receptor (IGF-IR) has not been successful in breast cancer. Data suggest the highly homologous insulin receptor (IR) may be an alternate growth stimulatory pathway used by cancer cells. Since both receptors phosphorylate the insulin receptor substrate-1 (IRS-1) protein as an immediate consequence of ligand binding, disruption of both receptors could be accomplished by suppression of IRS-1. To test this hypothesis, we created a doxycycline-inducible IRS-1 shRNA lentiviral construct to infect hormone dependent MCF-7 breast cancer cells. IRS-1 shRNA downregulation resulted in decreased responses to IGF-I, insulin, and estradiol in monolayer and anchorage independent growth assays. Decreased IRS-1 levels also suppressed estradiol stimulated gene expression and estrogen receptor binding to DNA. Xenograft growth was also inhibited by induction of IRS-1 shRNA. These data show IRS-1 is a critical regulator of endocrine responsive breast cancer. To further study the critical effects of IRS1 in ER positive breast cancer biology, CRISPR technology was used to delete IRS1 from MCF-7L cells. We analyzed 5 CRISPR IRS1 gene edited clones. Two different commercial available IRS1 antibodies were used to examine IRS1 protein expression. Clones #16, #20 did not show any IRS1 expression, while clones #22, #33 and #38 showed truncated IRS1 expression. IGFIRβ and Insulin R levels were comparable to the parent MCF-7L cells in all the clones, while IRS2 and ERα levels were significantly lower in clone #16. IRS phosphorylation, detected by PY20 antibody, was significantly lower in clone #16. The downstream signaling measured by pAKT and pErk1,2 did not change in all clones compared to those in MCF-7L. In all the clones, ERα ser118 phosphorylation by estradiol stimulation (10 nM) and ERα downregulation by fulvestrant treatment (100 nM) were not affected by IRS1 knockout. IGF-I (5 nM), insulin (10 nM) and estradiol (10 nM) stimulated monolayer cell growth in clone #16 was completely inhibited, while other clones were partially inhibited. In contrast, growth in clone #22 was not inhibited due to extremely high IRS2 expression and compensation. In conclusion, when the IRS1 gene was deleted, multiple growth stimulatory ligands (IGF-I, insulin and estradiol) were inhibited in breast cancer cells. However, IRS-2 compensated for IRS-1 gene deletion in one of the clones we studied. Thus, targeting the IRS proteins may inhibit multiple ligands that cause growth stimulation in ER positive breast cancer therapy. Citation Format: Xihong Zhang, Douglas Yee. Gene deletion of IRS1 inhibited IGF-I, insulin and estradiol stimulated MCF-7L breast cancer cell growth [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 P3-10-09.

HDAC5-mediated deacetylation and nuclear localisation of SOX9 is critical for tamoxifen resistance in breast cancer

BackgroundTamoxifen resistance remains a significant clinical challenge for the therapy of ER-positive breast cancer. It has been reported that the upregulation of transcription factor SOX9 in ER+ recurrent cancer is sufficient for tamoxifen resistance. However, the mechanisms underlying the regulation of SOX9 remain largely unknown.MethodsThe acetylation level of SOX9 was detected by immunoprecipitation and western blotting. The expressions of HDACs and SIRTs were evaluated by qRT-PCR. Cell growth was measured by performing MTT assay. ALDH-positive breast cancer stem cells were evaluated by flow cytometry. Interaction between HDAC5 and SOX9 was determined by immunoprecipitation assay.ResultsDeacetylation is required for SOX9 nuclear translocation in tamoxifen-resistant breast cancer cells. Furthermore, HDAC5 is the key deacetylase responsible for SOX9 deacetylation and subsequent nuclear translocation. In addition, the transcription factor C-MYC directly promotes the expression of HDAC5 in tamoxifen resistant breast cancer cells. For clinical relevance, high SOX9 and HDAC5 expression are associated with lower survival rates in breast cancer patients treated with tamoxifen.ConclusionsThis study reveals that HDAC5 regulated by C-MYC is essential for SOX9 deacetylation and nuclear localisation, which is critical for tamoxifen resistance. These results indicate a potential therapy strategy for ER+ breast cancer by targeting C-MYC/HDAC5/SOX9 axis.

17β-estradiol at low concentrations attenuates the efficacy of tamoxifen in breast cancer therapy

Tamoxifen has been applied widely in the treatment of estrogen receptor (ER)-positive breast cancer. The impact of low concentrations of 17β-estradiol (E2) (a pervasive environmental pollutant) on its effectiveness was studied in vitro using an MCF-7 cell line. Cell proliferation, migration, invasion, and apoptosis were studied along with cell cycle progression, reactive oxygen species generation and mitochondrial membrane potentials repression. The signaling pathways involved were identified. Typical concentrations of E2 in the environment (10−10 to 10−8 M) were observed to promote cell growth and protect MCF-7 cells from tamoxifen's cytotoxicity. Cell migration, invasion, cell cycle progression and apoptosis all involved in reducing tamoxifen's cytotoxicity. E2 at environmental concentrations induced PI3K/Akt and MAPK/ERK signal transduction through the estrogen receptor pathways to affect cell proliferation. Taken together, the results explain how E2 in the environment may attenuate the efficacy of tamoxifen in ER-positive breast cancer therapy. They provide considerable support for E2's adverse effects on human health and cancer management.

Aberrant Regulation of RAD51 Promotes Resistance of Neoadjuvant Endocrine Therapy in ER-positive Breast Cancer

Breast cancer is one of the most common malignant cancers affecting females. Estrogen receptor (ER)-positive breast cancer is responsive to endocrine therapy. Although current therapies offer favorable prospects for improving survival, the development of resistance remains a severe problem. In this study, we explored the resistance mechanisms of ER-positive breast cancer to neoadjuvant endocrine therapy. Microarray data of GSE87411 contained 109 pairs of samples from Z1031 trial, including untreated samples and post-treated samples with neoadjuvant aromatase inhibitor (AI) therapy. The differentially expressed genes (DEGs) were obtained from two different comparisons: untreated samples versus post-treated samples with AIs, and post-treated samples sensitive versus resistant to AIs. Multiple bioinformatic methods were applied to evaluate biological function, protein-protein network and potential binding between target protein and aromatase inhibitor. Then, regulation of gene expression, DNA methylation and clinicopathological factors of breast cancer were further analyzed with TCGA data. From GSE87411 dataset, 30 overlapped DEGs were identified. Cell division was found to be the main function of overlapped DEGs by functional enrichment and gene ontology (GO) analysis. RAD51 recombinase (RAD51), a key protein of homologous recombination, was detected to interact with BReast CAncer genes 2 (BRCA2). Moreover, according to the docking simulation, RAD51 might potentially bind to AIs. Overexpressed RAD51 was associated with hypermethylation of BRCA2, resistance to AIs and poor overall survival of patients with ER-positive breast cancer. Furthermore, RAD51 was found to be a better indicator than MKI67 for predicting resistance in neoadjuvant setting. The results indicated that methylation of BRCA2 led to incomplete suppression on RAD51, which caused an increased expression of RAD51, subsequently AI-resistance and poor prognosis in ER-positive breast cancer. RAD51 could be a new candidate used as a predicative marker and therapeutic target in neoadjuvant endocrine treatment.

Abstract PD7-10: Neoadjuvant trial with letrozole identifies PRR11 in 17q21-23 amplicon as a resistance mechanism to endocrine therapy in ER-positive breast cancer

Abstract Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy relapse with metastatic disease. Previously, we identified 3 amplicons (11q11.3, 8p11.23, and 17q21-23) associated with endocrine-resistance (Giltnane et al. Sci Transl Med 2017). The 17q21-23 amplicon has been associated with highly proliferative luminal B tumors and cancers with high genomic instability. A causal role of this region in endocrine resistance is unclear. We performed whole transcriptome analysis on RNA extracted from 58 ER+ breast cancers of patients treated with letrozole for 5.4-9.2 months (median 7.2 months). PRR11 (Proline rich 11), located in 17q21-23, was significantly upregulated in non-responding tumors as defined by cancer relapse after a median follow up of 5 years and/or a preoperative endocrine prognostic index (PEPI) ≥4. Differential gene expression analysis between tumors expressing low vs high PRR11 mRNA showed that BC signatures associated with proliferation, cell cycle, IGF-1 and PI3K signaling were enriched in tumors with high PRR11 expression. In the Metastatic Breast Cancer Project and TCGA, PRR11 amplification was higher in metastatic vs. primary BCs (16.5% and 8.5%, respectively; Fisher's p=0.0088). Gene Set Enrichment Analysis of mRNA expression in METABRIC and TCGA revealed significant enrichment of hallmark gene sets associated with proliferation in PRR11 amplified ER+ BCs. Genome-scale RNAi screening in Project Achilles showed that among all genes in the 17q21-23 amplicon, PRR11 knockdown results in the 4th strongest anti-proliferative effect in MCF7 cells. PRR11 knockdown with siRNA inhibited proliferation, cell cycle progression, and RB phosphorylation in HCC1428 LTED (long-term estrogen deprived), MCF7 LTED, and fulvestrant-resistant MCF7 cells. Using a PCR array with 84-cell cycle genes, we identified SKP2, CDKN1A, CCNB2, CCNA2, CKS2 and CCNB1 as genes downregulated by PRR11 knockdown. Except for CDKN1A, expression of all those genes was elevated ER+ BCs with PRR11 gain or amplification in TCGA. PRR11 associates with the p85 regulatory subunit of PI3K via its SH3 domain. We speculated this association would suppress p85 homodimers, thus permitting binding of PI3Kα (p110α)-p85 dimers to IRS1 and, hence, activating PI3K/AKT. To test this, we co-transfected HEK293T cells with HA-p85 and FLAG-p85. Transfection of PRR11 into these HEK293T cells reduced HA-p85 and FLAG-p85 homodimers as shown by HA and FLAG pulldowns followed by FLAG and HA immunoblots, respectively. Finally, PRR11 knockdown resulted in a reduction of p110a and S473 P-AKT levels and inhibition of IGF-1/2 stimulated P-AKT. Not inconsistent with these data, PRR11 amplification and PIK3CA mutations in METABRIC and TCGA are exclusive of each other, suggesting these alterations are functionally linked with the same signaling pathway. These data support a role of PRR11 in PI3K/AKT activation that may be causal to resistance to estrogen deprivation. We propose PRR11, located in the 17q21-23 amplicon, is a potential mediator of resistance to antiestrogen therapy by amplifying PI3K/AKT signaling, suggesting PI3K may be a therapeutic target in ER+ BCs harboring PRR11 amplification. Citation Format: Lee K-M, Guerrero-Zotano A, Formisano L, Jansen V, Gonzalez Ericsson P, Arteaga C. Neoadjuvant trial with letrozole identifies PRR11 in 17q21-23 amplicon as a resistance mechanism to endocrine therapy in ER-positive 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 PD7-10.

Epigenetic biomarker screening by FLIM-FRET for combination therapy in ER+ breast cancer

Hormone-dependent gene expression involves dynamic and orchestrated regulation of epigenome leading to a cancerous state. Estrogen receptor (ER)-positive breast cancer rely on chromatin remodeling and association with epigenetic factors in inducing ER-dependent oncogenesis and thus cell over-proliferation. The mechanistic differences between epigenetic regulation and hormone signaling provide an avenue for combination therapy of ER-positive breast cancer. We hypothesized that epigenetic biomarkers within single nucleosome proximity of ER-dependent genes could serve as potential therapeutic targets. We described here a Fluorescence lifetime imaging-based Förster resonance energy transfer (FLIM-FRET) methodology for biomarker screening that could facilitate combination therapy based on our study. We screened 11 epigenetic-related markers which include oxidative forms of DNA methylation, histone modifications, and methyl-binding domain proteins. Among them, we identified H4K12acetylation (H4K12ac) and H3K27 acetylation (H3K27ac) as potential epigenetic therapeutic targets. When histone acetyltransferase inhibitor targeting H4K12ac and H3K27ac was combined with tamoxifen, an enhanced therapeutic outcome was observed against ER-positive breast cancer both in vitro and in vivo. Together, we demonstrate a single molecule approach as an effective screening tool for devising targeted epigenetic therapy.

Compensatory role of insulin-like growth factor 1 receptor in estrogen receptor signaling pathway and possible therapeutic target for hormone therapy-resistant breast cancer.

Hormone therapy targeting the estrogen receptor (ER) pathway is the most common treatment used for ER-positive breast cancer. However, some patients experience de novo or acquired resistance, which becomes a critical problem. Activation of the insulin-like growth factor (IGF) pathway allows breast cancer cells to proliferate and is associated with the ER pathway. Little is known about the role of the IGF pathway in hormone therapy and resistance; therefore, we investigated whether the inhibition of this pathway may represent a novel therapeutic target for overcoming hormone therapy resistance in ER-positive breast cancers. Crosstalk between the ER and IGF pathways was analyzed in breast cancer cell lines by inhibiting or stimulating either one or both pathways. We studied the effect of insulin-like growth factor one receptor (IGF1R) inhibition in aromatase inhibitor-resistant breast cancer cell lines and fulvestrant-resistant cell lines which were uniquely established in our laboratory. Under normal conditions, IGF signaling is controlled by ER signaling to promote cell growth. Temporary disruption of the estrogen supply results in attenuated ER signaling, and IGF-1 dramatically increased relative growth compared with normal conditions. In addition, IGF1R inhibitor strongly suppressd cell growth in hormone-resistant breast cancer cells where ER remains than cells where ER decreased or was almost lost. Our study suggests that inhibition of the IGF pathway may be an effective strategy for ER-positive breast cancer therapy, even in hormone therapy-resistant cases.

Development of Hot Melt Extruded Solid Dispersion of Tamoxifen Citrate and Resveratrol for Synergistic Effects on Breast Cancer Cells.

Primary standard therapy for ER-positive breast cancer being tamoxifen, newer delivery approach for enhancement of dissolution and therapeutic efficiency of tamoxifen through oral route could be a possible solution. In the present study, we investigated combination of tamoxifen (TAM) with resveratrol (RES) and observed that the combination is effective on MCF-7 breast cancer cells. To ensure co-delivery of the drugs, we explored the hot melt extrusion technique for simultaneously extruding two drugs together in order to enhance their bioavailability. As both are class II drugs with dissolution limited bioavailability, detailed formulation and process parameter analyses were carried out. Detailed characterization using microscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray powder diffraction (XRD) confirmed that both the drugs were molecularly dispersed in the matrix of Soluplus, CremophorRH40, and Poloxamer188, and no interactions between the ingredients were there during hot melt extrusion (HME) process. Dissolution studies confirmed that HME extrudates were able to release drug more rapidly than simple suspension formulation. Further, pharmacokinetic studies in rats were carried out for tamoxifen. Results demonstrated that extrusion significantly increased the tamoxifen oral bioavailability (p < 0.05) (Tmax = 2.00 ± 0.56h, Cmax = 3.66 ± 1.49μg/mL, AUC = 39.80 ± 16.24μgh/mL, MRT = 20.49 ± 5.71) compared to the conventional suspension of tamoxifen (Tmax = 2.00 ± 0.71h, Cmax = 2.41 ± 0.84μg/mL, AUC = 12.82 ± 3.99μgh/mL, MRT = 18.24 ± 5.95h). In vitro cytotoxicity studies of TAM, RES, and their combination (TAM-RES) were evaluated with MCF7 cells. The combination showed significantly lower IC50 compared to TAM with increasing ratio of RES which is a result of apoptosis. HME-based simultaneous extrusion of TAM and RES formulation provides a suitable formulation strategy for breast cancer treatment and establishes proof of concept for extruding multiple drugs simultaneously for other applications in future.

Reactivation of cAMP Pathway by PDE4D Inhibition Represents a Novel Druggable Axis for Overcoming Tamoxifen Resistance in ER-positive Breast Cancer.

Purpose: Tamoxifen remains an important hormonal therapy for ER-positive breast cancer; however, development of resistance is a major obstacle in clinics. Here, we aimed to identify novel mechanisms of tamoxifen resistance and provide actionable drug targets overcoming resistance.Experimental Design: Whole-transcriptome sequencing, downstream pathway analysis, and drug repositioning approaches were used to identify novel modulators [here: phosphodiesterase 4D (PDE4D)] of tamoxifen resistance. Clinical data involving tamoxifen-treated patients with ER-positive breast cancer were used to assess the impact of PDE4D in tamoxifen resistance. Tamoxifen sensitization role of PDE4D was tested in vitro and in vivo Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance.Results: PDE4D, which hydrolyzes cyclic AMP (cAMP), was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. Higher PDE4D expression predicted worse survival in tamoxifen-treated patients with breast cancer (n = 469, P = 0.0036 for DMFS; n = 561, P = 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis (n = 132, P = 0.049). Inhibition of PDE4D by either siRNAs or pharmacologic inhibitors (dipyridamole and Gebr-7b) restored tamoxifen sensitivity. Sensitization to tamoxifen is achieved via cAMP-mediated induction of unfolded protein response/ER stress pathway leading to activation of p38/JNK signaling and apoptosis. Remarkably, acetylsalicylic acid (aspirin) was predicted to be a tamoxifen sensitizer using a drug repositioning approach and was shown to reverse resistance by targeting PDE4D/cAMP/ER stress axis. Finally, combining PDE4D inhibitors and tamoxifen suppressed tumor growth better than individual groups in vivoConclusions: PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis. Clin Cancer Res; 24(8); 1987-2001. ©2018 AACR.