Tamoxifen-resistant MCF-7 Cells Research Articles

Identification of the Significant Genes Regulated by Estrogen Receptor in Estrogen Receptor-Positive Breast Cancer and Their Expression Pattern Changes When Tamoxifen or Fulvestrant Resistance Occurs.

Breast cancer is the most frequent malignant tumor in women, and the estrogen receptor (ER) plays a vital role in the vast majority of breast cancers. The purpose of the present study was to identify the significant genes regulated by ER in ER-positive breast cancer and to explore their expression pattern changes when tamoxifen or fulvestrant resistance occurs. For this purpose, the gene expression profiles GSE11324, GSE27473, and GSE5840 from the Gene Expression Omnibus database were used, which contain gene expression data from MCF7 cells treated with estrogen, MCF7 cells with silencing of ER, and tamoxifen- and fulvestrant-resistant MCF7 cells treated with estrogen (17β-estradiol), respectively. Differentially expressed genes (DEGs) between the treatment group and negative control were identified and subjected to pathway enrichment and protein–protein interaction (PPI) analyses. There were 230 DEGs in common among the three datasets, including 160 genes positively regulated by ER and 70 genes negatively regulated by ER. DEGs mainly showed enrichment for pathways in cancer, progesterone-mediated oocyte maturation, RNA transport, glycerophospholipid metabolism, oocyte meiosis, platelet activation, and so on. PPI network and modular analysis selected three significant clusters containing 19 genes. A total of 44 genes were involved in Kyoto Encyclopedia of Gene and Genome pathway results or PPI modular analysis, and 16 of them were found to correlate with relapse-free survival in patients with ER+/human epidermal growth factor receptor 2-negative breast cancer who had undergone endocrine therapies only. Some of the genes’ expression patterns were different among wild-type, tamoxifen-resistant, and fulvestrant-resistant MCF7 cells such as DDX18, ANAPC7, MAD2L1, RSL1D1, and CALCR, etc., indicating different resistance mechanisms and potential prognostic markers or therapeutic targets for fulvestrant- or tamoxifen-resistant breast cancer.

Production of Plant-Derived Oleuropein Aglycone by a Combined Membrane Process and Evaluation of Its Breast Anticancer Properties.

Natural products and herbal therapies represent a thriving field of research, but methods for the production of plant-derived compounds with a significative biological activity by synthetic methods are required. Conventional commercial production by chemical synthesis or solvent extraction is not yet sustainable and economical because toxic solvents are used, the process involves many steps, and there is generally a low amount of the product produced, which is often mixed with other or similar by-products. For this reason, alternative, sustainable, greener, and more efficient processes are required. Membrane processes are recognized worldwide as green technologies since they promote waste minimization, material diversity, efficient separation, energy saving, process intensification, and integration. This article describes the production, characterization, and utilization of bioactive compounds derived from renewable waste material (olive leaves) as drug candidates in breast cancer (BC) treatment. In particular, an integrated membrane process [composed by a membrane bioreactor (MBR) and a membrane emulsification (ME) system] was developed to produce a purified non-commercially available phytotherapic compound: the oleuropein aglycone (OLA). This method achieves a 93% conversion of the substrate (oleuropein) and enables the extraction of the compound of interest with 90% efficiency in sustainable conditions. The bioderived compound exercised pro-apoptotic and antiproliferative activities against MDA-MB-231 and Tamoxifen-resistant MCF-7 (MCF-7/TR) cells, suggesting it as a potential agent for the treatment of breast cancer including hormonal resistance therapies.

Adenylate Kinase 4 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an m6A-Based Epitranscriptomic Mechanism

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and this methylation constitutes an important regulatory mechanism for the stability and translational efficiency of mRNA. In this study, we found that the protein levels of adenylate kinase 4 (AK4) and m6A writer METTL3 are significantly higher in tamoxifen-resistant (TamR) MCF-7 cells than in parental cells. The TamR MCF-7 cells also exhibit increased methylation at multiple m6A consensus motif sites in the 5' untranslated region (5' UTR) of AK4 mRNA, and genetic depletion of METTL3 in TamR MCF-7 cells led to a diminished AK4 protein level and attenuated resistance to tamoxifen. In addition, we observed augmented levels of reactive oxygen species (ROS) and p38 activity in TamR MCF-7 cells, and both are diminished upon genetic depletion of AK4. Reciprocally, overexpression of AK4 in MCF-7 cells stimulates ROS and p38 phosphorylation levels, and it suppresses mitochondrial apoptosis. Moreover, scavenging of intracellular ROS leads to reduced p38 activity and re-sensitizes TamR MCF-7 cells to tamoxifen. Thus, our results uncover a novel m6A-mediated epitranscriptomic mechanism for the regulation of AK4, illustrate the cellular pathways through which increased AK4 expression contributes to tamoxifen resistance, and reveal AK4 as a potential therapeutic target for overcoming tamoxifen resistance.

Identification of potential gene associated with berberine in overcoming tamoxifen resistance by functional network analysis

Previously, berberine enhanced the sensitivity of MCF-7-resistant breast cancer cells toward tamoxifen; however, its molecular mechanism still remains unclear. The purpose of this study is to identify the potential targets and molecular mechanism of berberine in overcoming breast cancer resistance toward tamoxifen by using a bioinformatics approach for functional network analysis. The microarray data of tamoxifen-resistant and berberine-treated MCF-7 cells were obtained from GSE67916 and GSE85871, which resulted in differentially expressed genes (DEGs). The analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment by using The Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that several DEGs participated in the erbB tyrosine kinase signaling pathway. The analysis of protein–protein interaction network and hub genes selection by using STRING and Cytoscape identified the top 10 genes with the highest degree score. The analysis of genetic alterations by using cBioportal demonstrated genetic alterations of six potential target genes including PRKCA, EGFR, ERBB4, AREG, ESR1, and STAT1. Moreover, importantly, the erbB signaling is a potential target for overcoming breast cancer resistance toward tamoxifen. Further studies are required to validate the results of this study.

CXCL10 mediates breast cancer tamoxifen resistance and promotes estrogen-dependent and independent proliferation

Although 70% of estrogen receptor (ER)-positive breast cancer patients can benefit from tamoxifen therapy, the rapid development of tamoxifen resistance hampers the treatment advantage. In this investigation, we found that the serum level of CXCL10 in breast cancer patients was positively correlated with tumor size and ER status. Furthermore, GSE22220 dataset analysis demonstrated that CXCL10 expression in the tumor was correlated with tumor grade and lymphatic metastasis status, and Kaplan–Meier analysis indicated that patients with high CXCL10 expression had a poor prognosis. Estrogen-deprived MCF7 cells were transfected with CXCL10 luciferase reporter plasmid and treated with 10 nM estrogen. Luciferase reporter assay confirmed that CXCL10 was regulated by estrogen. CXCL10 promoted the proliferation of both parental MCF7 cells and tamoxifen-resistant (TamR) MCF7 cells through the AKT pathway, while CXCL10 inhibition restored the sensitivity of TamR cells to tamoxifen. All of these data indicate that CXCL10 could be utilized as a biomarker to predict the prognosis of breast cancer and as a therapeutic target to treat tamoxifen resistant cases.

IGFBP-1 Expression Promotes Tamoxifen Resistance in Breast Cancer Cells via Erk Pathway Activation.

Insulin-like growth factor (IGF) system plays a significant role in many cellular processes, including proliferation, and survival. In estrogen receptor positive breast cancer, the level of circulating IGF-1 is positively associated with the incidence and at least 50% of cases have elevated IGF-1R signaling. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. Unfortunately, tamoxifen resistance is a frequent occurrence in patients receiving treatment and the molecular mechanisms that underlie tamoxifen resistance not adequately defined. It has recently been reported that the inhibition of IGF-1R activation and the proliferation of breast cancer cells upon tamoxifen treatment is mediated by the accumulation of extracellular insulin-like growth factor binding protein 1 (IGFBP-1). Elevated IGFBP-1 expression was observed in tamoxifen-resistant (TamR) MCF-7 and T-47D cells lines suggesting that the tamoxifen-resistant state is associated with IGFBP-1 accumulation. MCF-7 and T-47D breast cancer cells stably transfected with and IGFBP-1 expression vector were generated (MCF7-BP1 and T47D-BP1) to determine the impact of breast cancer cell culture in the presence of increased IGFBP-1 expression. In these cells, the expression of IGF-1R was significantly reduced compared to controls and was similar to our observations in tamoxifen-resistant MCF-7 and T-47D cells. Also similar to TamR breast cancer cells, MCF7-BP1 and T47D-BP1 were resistant to tamoxifen treatment, had elevated epidermal growth factor receptor (EGFR) expression, increased phospho-EGFR (pEGFR), and phospho-Erk (pErk). Furthermore, tamoxifen sensitivity was restored in the MCF7-BP1 and T47D-BP1 upon inhibition of Erk phosphorylation. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 inhibited pErk accumulation and increased tamoxifen sensitivity. Taken together, these data support the conclusion that IGFBP-1 is a key component of the development of tamoxifen resistance in breast cancer cells.

Dual in vitro invasion/migration suppressing and tamoxifen response modulating effects of a recombinant anti-ALCAM scFv on breast cancer cells.

It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.

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 P6-03-18: The accumulation of IGFBP-1 results in EGFR/MAPK activation and drives the development of tamoxifen resistance in breast cancer cells

Abstract Insulin-like growth factor (IGF) system plays a significantly role in cellular processes, including proliferation, survival, and mitogenesis. The IGF system consists of two soluble ligands, IGF-1 and IGF-2; two transmembrane receptors, IGF-1R and IGF-2R; and six high affinity IGF binding proteins, IGFBPs 1-6. Evidence shows that the IGF system is involved in breast cancer. For example, at least 50% of breast cancer tumors have elevated IGF-1R signaling; the level of circulating IGF-1 is found positively associated with the incident of estrogen receptor positive breast cancer. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. However, tamoxifen resistance also occurs, and its exact mechanism is not well understood. Recently, we discovered that the accumulation of IGFBP-1 was stimulated by tamoxifen treatment, which subsequently prevented the activation of IGF-1R and inhibited the proliferation in MCF-7 cells. As a result, we hypothesized that the accumulation of IGFBP-1 due to the long-term tamoxifen exposure would result in tamoxifen resistance. In order to mimic the accumulation of IGFBP-1 after tamoxifen treatment, we employed MCF-7 and T-47D breast cancer cells to generate stable cell lines with IGFBP-1 overexpression. We found that the expression of IGF-1R was largely reduced in both MCF7-BP1 and T47D-BP1 cells, which was also observed in tamoxifen resistant MCF-7 and T-47D cells. Furthermore, we demonstrated that both MCF7-BP1 and T47D-BP1 were not responsive to tamoxifen, and epidermal growth factor receptor (EGFR) signaling pathway was significantly upregulated, as shown by higher levels of EGFR, phospho-EGFR (pEGFR), and phospho-Erk (pErk). Also, we found that the inhibition of Erk phosphorylation by MEK1 inhibitor PD98059 sensitized the stable cells to tamoxifen, showing that elevated pErk was protective against tamoxifen. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 resulted in significantly lower level of pErk and increased sensitivity to tamoxifen, further showing the role of IGFBP1 accumulation in the establishment of tamoxifen resistance and the activation of EGFR/MAPK pathway. Citation Format: Yan Zheng. The accumulation of IGFBP-1 results in EGFR/MAPK activation and drives the development of tamoxifen resistance in breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-18.

The LncRNA HOTAIRM1 Promotes Tamoxifen Resistance by Mediating HOXA1 Expression in ER+ Breast Cancer Cells.

Breast cancer is one of the most commonly diagnosed cancers in women worldwide. Approximately 40% of patients with breast cancer acquire endocrine resistance following therapy with tamoxifen. Many explanations for the development of endocrine resistance have been put forward, one of them being the dysregulation of long non-coding RNAs (lncRNAs). The lncRNA HOTAIRM1, known to be involved in myelopoiesis as well as transcriptional regulation of the HOXA genes in embryonic stem cells, is also expressed in breast cancer cells. This study explored the molecular mechanisms of HOTAIRM1 involved in acquired tamoxifen resistance. We showed that HOTAIRM1 and HOXA1 are concurrently up-regulated in tamoxifen-resistant MCF7 (TAMR) cells. Knockdown of HOTAIRM1 down-regulated HOXA1 expression and restored sensitivity to tamoxifen. In addition, the knockdown of HOXA1 showed similar effects, suggesting that the HOTAIRM1/HOXA1 axis regulates tamoxifen resistance. Furthermore, we showed that HOTAIRM1 directly interacts with EZH2 and prevents the PRC2 complex from binding and depositing H3K27me3 on the putative promoter of HOXA1. Together, our findings suggest that HOXA1 and its neighboring lncRNA, HOTAIRM1, might serve as potential therapeutic targets for ER+ breast cancer patients who have acquired tamoxifen resistance.

Downregulation of 15-hydroxyprostaglandin dehydrogenase during acquired tamoxifen resistance and association with poor prognosis in ERα-positive breast cancer.

Aim:Tamoxifen (TAM) resistance remains a clinical issue in breast cancer. The authors previously reported that 15-hydroxyprostaglandin dehydrogenase (HPGD) was significantly downregulated in tamoxifen-resistant (TAMr) breast cancer cell lines. Here, the authors investigated the relationship between HPGD expression, TAM resistance and prediction of outcome in breast cancer.Methods:HPGD overexpression and silencing studies were performed in isogenic TAMr and parental human breast cancer cell lines to establish the impact of HPGD expression on TAM resistance. HPGD expression and clinical outcome relationships were explored using immunohistochemistry and in silico analysis.Results:Restoration of HPGD expression and activity sensitised TAMr MCF-7 cells to TAM and 17β-oestradiol, whilst HPGD silencing in parental MCF-7 cells reduced TAM sensitivity. TAMr cells released more prostaglandin E2 (PGE2) than controls, which was reduced in TAMr cells stably transfected with HPGD. Exogenous PGE2 signalled through the EP4 receptor to reduce breast cancer cell sensitivity to TAM. Decreased HPGD expression was associated with decreased overall survival in ERα-positive breast cancer patients.Conclusions:HPGD downregulation in breast cancer is associated with reduced response to TAM therapy via PGE2-EP4 signalling and decreases patient survival. The data offer a potential target to develop combination therapies that may overcome acquired tamoxifen resistance.

LncRNA CYTOR promotes tamoxifen resistance inbreast cancer cells via sponging miR‑125a‑5p.

Development of resistance to endocrine therapy, such as tamoxifen, remains a tricky clinical problem during the treatment of breast cancer. Accumulating evidence suggested that dysregulation of long noncoding (lnc_RNAs contributes to the development of tamoxifen resistance. In the current study, via screening, cytoskeleton regulator RNA (CYTOR) was identified as the most significantly elevated lncRNA in the established tamoxifen resistant MCF7 cell lines (MCF7/TAM1 and MCF7/TAM2) compared with the parental MCF7 cells (MCF7-P). The CCK-8 assay indicated that silencing of CYTOR increased the sensitivity of MCF7/TAM1 and MCF7/TAM2 to tamoxifen treatment. Using bioinformatic analysis, it was predicted that microRNA (miR)-125a-5p might bind to CYTOR and the expression of miR-125a-5p was negatively correlated with CYTOR in the tumor tissues of breast cancer. In addition, RT-qPCR and dual luciferase assays validated that CYTOR directly repressed miR-125a-5p expression in breast cancer cells. Through regulation of miR-125a-5p, CYTOR elevated serum response factor (SRF) expression and activated Hippo and mitogen associated protein kinase signaling pathways to promote breast cancer cell survival upon tamoxifen treatment. In the collected tumor tissues of breast cancer in the present study, high expression of CYTOR was detected in tissues from patients with no response to tamoxifen compared with those from patients who were not treated with tamoxifen. A positive correlation between CYTOR and SRF mRNA expression was observed in tissues collected from patients with breast cancer. In conclusion, the results of the present study demonstrated a pivotal role of CYTOR in mediating tamoxifen resistance in breast cancer.

Involvement of the P2X7 receptor in the migration and metastasis of tamoxifen-resistant breast cancer: effects on small extracellular vesicles production

Tamoxifen (TAM) is the standard anti-hormonal therapy for estrogen receptor-positive breast cancer. However, long-term TAM therapy can make acquisition of TAM resistance and there are still no solutions to treat TAM-resistant breast cancer. In this study, we found that protein and mRNA expression of the P2X purinoreceptor 7 (P2X7) was higher in tamoxifen resistant MCF-7 (TAMR-MCF-7) cells than in control MCF-7 cells. P2X7 inhibition potently inhibited the migration of TAMR-MCF-7 cells and the liver metastasis burden of TAMR-MCF-7 cells in the spleen-liver metastasis experiment. However, the P2X7 antagonist did not affect protein expression of matrix metalloproteinase (MMP)-2, MMP-9, and epithelial-mesenchymal transition markers. Here our data indicate a link between small extracellular vesicles (sEV) and P2X7, and suggest a new mechanism of metastasis in TAM-resistant breast cancer cells through P2X7 receptors. The migration of TAMR-MCF-7 cells was increased in a concentration-dependent manner by purified sEV treatment. The number of secreted sEVs and the protein levels of CD63 in TAMR-MCF-7 cells were decreased by the P2X7 antagonist, showing that P2X7 influences the production of sEV. Our results suggest that inhibiting the P2X7 could be considered for metastasis prevention in TAM-resistant cancer patients.

Abstract 3893: Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice

Abstract Background: Melatonin-tamoxifen drug conjugate linked with a 5-carbon chain (C5) exhibited uterus protection in female mice, which did not occur when the same doses of unlinked melatonin and tamoxifen were co-administered. Anti-cancer actions of C5 and 4 others, with varying carbon lengths (C2, C4, C9, C15), were further tested in phenotypically diverse breast cancer (BC) cell lines—MCF-7(ER+/PR+), MMC(HER2+) and triple negatives(MDA-MB-231 and BT-549) in terms of cell viability and migration. In all BC lines, C4 and C5 exhibited superior potency and efficacy compared to C2, C9, C15 and unlinked melatonin and tamoxifen or 4-OH-tamoxifen controls. In this study, C4 and C5 were further tested for their anti-cancer effects in tamoxifen-resistant MCF-7 cells(TamR-MCF-7); their pharmacokinetic parameters were assessed in vitro and in vivo; and the mechanisms underlying C4- and C5-mediated anti-cancer actions were also determined by use of inhibitors against MEK1/2, MEK5 or PI3 kinase and western blot analysis. Results: C4 and C5 exhibited similar potency (TamR-MCF-7 IC50 = 4.22µM and 7.21µM; MCF-7 IC50 = 3.62nM and 440mM) and efficacy (TamR-MCF-7 = 83% and 81% inhibition; MCF-7= 71% and 90% inhibition) to inhibit TamR-MCF-7 cell viability as they did in MCF-7 cells. Regarding metabolism, C4 and C5 displayed similar CYP-mediated pharmacokinetic profiles as tamoxifen in both mouse and human liver microsomes where 40% loss of tamoxifen, C4 or C5 occurred by 10 min in mouse vs. 20 min in human. In vivo, the Tmax for C5 (oral) was 24h compared to 0.5h for C4 (oral) resulting in lower Cmax (8.9 ng/mL) and AUC0-24 (1.6 hr.ng/mL) for C5 compared to C4 (Cmax =102.6 ng/mL and AUC0-24 =143.7 hr.ng/mL) and lower oral bioavailability (0.11%) for C5 compared to C4 (4.52%). Regarding mechanisms, the effect of C4- or C5-mediated protein modulation was “context-specific” dependent upon BC phenotype (i.e., ER+, HER2+ or ER-/PR-/HER2-); the number of carbons linking melatonin to tamoxifen (i.e., 4 carbons for C4 or 5 carbons for C5); and the concentration (i.e., 1nM or 10µM) of C4 or C5 tested. For MCF-7, MMC and MDA- MB-231 cells, inhibition of MEK1/2, MEK5 and PI3K enhanced but did not block C4’s or C5’s anti-cancer actions; while for BT-549 cells, Bix02189 blocked C5’s effects. Also, in MCF-7 cells, pERK1/2, NF-κB and Runx2 were significantly modulated by C4 or C5; for MMCs, C4 and C5 modulated NF-κB and β1-integrin expression; for MDA-MB-231, MEK1/2 and MEK5 played a more central role possibly through co-modulation of NF-κB; and for BT-549, C5-mediated effects on pERK5 or PI3K-dependent regulation of pERK1/2 may underlie its anti-cancer actions. Conclusions: Melatonin-tamoxifen drug conjugates (C4 and C5) may be superior to tamoxifen therapy alone in a variety of cancers, including TamR BC. Citation Format: Mahmud Hasan, Thomas D. Wright, Saugat Adhikari, Mohamed A. Marzouk, Alaina Pericoloso, Kelsey Murgas, Miranda Burgman, Benton P. Miller, Ulrike Holzgrabe, Darius P. Zlotos, Robert E. Stratford, Paula A. Witt-Enderby. Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3893.

Abstract SY20-02: The SIRT3-MnSOD-Ac axis in carcinogenesis and tumor resistance

Abstract ABSTRACT The molecular mechanism by which acetylation regulates manganese superoxide dismutase (MnSOD) activity, and its oncogenic and/or physiological significance, is unclear. Here we show that expression of the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) transformed wild-type primary mouse embryonic fibroblasts (pMEFs) when co-infected with either c-Myc or Ras, as measured by in vitro transformation assays and xenograft formation. Immortalized MnSOD-/- mouse embryonic fibroblasts (MEFs) infected with lenti-MnSODK68Q also exhibited an in vitro transformation permissive phenotype. In addition, MCF7 and T47D cells expressing MnSODK68Q demonstrated increased in vitro and xenograft oncogenic properties. These findings collectively identify MnSODK68Q as a tumor promoter. MCF7, T47D, and MnSOD-/- cells expressing MnSODK68Q displayed a monomeric form of MnSOD, distinct from the well-established homotetramer, and MnSOD-K68-Ac functions as a peroxidase, determined using a bacterial system for site-specific incorporation of N-(ε)-acetyl-l-lysine. Finally, tamoxifen resistant MCF7 and T47D cells, and a subgroup of human breast malignancies, displayed a MnSOD-K68-Ac signature. Citation Format: David R. Gius, Yueming Zhu. The SIRT3-MnSOD-Ac axis in carcinogenesis and tumor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY20-02.

Pharmacological, Mechanistic, and Pharmacokinetic Assessment of Novel Melatonin-Tamoxifen Drug Conjugates as Breast Cancer Drugs.

Tamoxifen is used to prevent and treat estrogen receptor-positive (ER+) breast cancer (BC); however, its chronic use can increase uterine cancer risk and induce tamoxifen resistance. Novel melatonin-tamoxifen drug conjugates may be promising to treat BC and may help offset the adverse effects of tamoxifen usage alone due to the presence of melatonin. We synthesized and screened five drug conjugates (C2, C4, C5, C9, and C15 linked) for their effects on BC cell (MCF-7, tamoxifen-resistant MCF-7, mouse mammary carcinoma, MDA-MB-231, and BT-549) viability, migration, and binding affinity to melatonin receptor 1 (MT1R) and estrogen receptor 1 (ESR1). C4 and C5 demonstrated the most favorable pharmacological characteristics with respect to binding profiles (affinity for ESR1 and MT1R) and their potency/efficacy to inhibit BC cell viability and migration in four phenotypically diverse invasive ductal BC cell lines. C4 and C5 were further assessed for their actions against tamoxifen-resistant MCF-7 cells and a patient-derived xenograft triple-negative BC cell line (TU-BcX-4IC) and for their mechanisms of action using selective mitogen-activated protein kinase kinase MEK1/2, MEK5, and phosphoinositide 3-kinase (PI3K) inhibitors. C4 and C5 inhibited tamoxifen-resistant MCF-7 cells with equal potency (IC50 = 4-8 μM) and efficacy (∼90% inhibition of viability and migration) but demonstrated increased potency (IC50 = 80-211 μM) and efficacy (∼140% inhibition) to inhibit migration versus cell viability (IC50 = 181-304 mM; efficacy ∼80% inhibition) in TU-BcX-4IC cells. Unique pharmacokinetic profiles were observed, with C4 having greater bioavailability than C5. Further assessment of C4 and C5 demonstrates that they create novel pharmacophores within each BC cell that is context specific and involves MEK1/2/pERK1/2, MEK5/pERK5, PI3K, and nuclear factor κB. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.

Oestrogen Non-Genomic Signalling is Activated in Tamoxifen-Resistant Breast Cancer.

Endocrine therapies targeting oestrogen signalling have significantly improved breast cancer management. However, their efficacy is limited by intrinsic and acquired resistance to treatment, which remains a major challenge for oestrogen receptor α (ERα)-positive tumours. Though many studies using in vitro models of endocrine resistance have identified putative actors of resistance, no consensus has been reached. We demonstrated previously that oestrogen non-genomic signalling, characterized by the formation of the ERα/Src/PI3K complex, is activated in aggressive breast cancers (BC). We wondered herein whether the activation of this pathway is also involved in resistance to endocrine therapies. We studied the interactions between ERα and Src or PI3K by proximity ligation assay (PLA) in in-vitro and in-vivo endocrine therapy-resistant breast cancer models. We reveal an increase in ERα/Src and ERα/PI3K interactions in patient-derived xenografts (PDXs) with acquired resistance to tamoxifen, as well as in tamoxifen-resistant MCF-7 cells compared to parental counterparts. Moreover, no interactions were observed in breast cancer cells resistant to other endocrine therapies. Finally, the use of a peptide inhibiting the ERα–Src interaction partially restored tamoxifen sensitivity in resistant cells, suggesting that such components could constitute promising targets to circumvent resistance to tamoxifen in BC.

CircRNA_0025202 Regulates Tamoxifen Sensitivity and Tumor Progression via Regulating the miR-182-5p/FOXO3a Axis in Breast Cancer.

Tamoxifen is the most commonly used endocrine therapy for patients with hormone receptor (HR)-positive breast cancer. Despite its initial therapeutic efficacy, many patients eventually develop drug resistance, which remains a serious clinical challenge. To investigate roles of circular RNAs (circRNAs) in tamoxifen resistance, a tamoxifen-resistant MCF-7 cell line was established and screened for its circRNA expression profile by RNA sequencing. hsa_circ_0025202, a circRNA that was significantly downregulated, was selected for further investigation. Using a large cohort of clinical specimens, we found that hsa_circ_0025202 exhibited low expression in cancer tissues and was negatively correlated with lymphatic metastasis and histological grade. Gain- and loss-of-function assays indicated that hsa_circ_0025202 could inhibit cell proliferation, colony formation, and migration and increase cell apoptosis and sensitivity to tamoxifen. Bioinformatics and luciferase reporter assays verified that hsa_circ_0025202 could act as a miRNA spongefor miR-182-5p and further regulate the expression and activity of FOXO3a. Functional studies revealed thattumorinhibition and tamoxifen sensitization effects of hsa_circ_0025202 were achieved via the miR-182-5p/FOXO3a axis. Moreover, invivo experiments confirmed that hsa_circ_0025202 could suppress tumor growth and enhance tamoxifen efficacy. Taken together, hsa_circ_0025202 served an anti-oncogenic role in HR-positive breast cancer, and it could be exploited as a novel marker for tamoxifen-resistant breast cancer.

Downregulation of CYB5D2 is associated with breast cancer progression

We report here that CYB5D2 is associated with tumor suppression function in breast cancer (BC). CYB5D2 expression was significantly reduced in tamoxifen resistant MCF7 cells and in MCF7 cell-derived xenografts treated with TAM. CYB5D2 overexpression induced apoptosis in MCF7 cells; CYB5D2 knockdown enhanced MCF7 cell proliferation. Using the TCGA and Curtis datasets within the Oncomine database, CYB5D2 mRNA expression was downregulated in primary BCs vs breast tissues and HER2-positive or triple negative BCs vs estrogen receptor (ER)-positive BCs. Using the TCGA and Metabric datasets (n = 817 and n = 2509) within cBioPortal, 660 and 4891 differentially expressed genes (DEGs) in relation to CYB5D2 were identified. These DEGs were enriched in pathways governing cell cycle progression, progesterone-derived oocyte maturation, oocyte-meiosis, estrogen-mediated S-phase entry, and DNA metabolism. CYB5D2 downregulation decreased overall survival (OS, p = 0.0408). A CYB5D2-derived 21-gene signature was constructed and robustly correlated with OS shortening (p = 5.72e-12), and independently predicted BC deaths (HR = 1.28; 95% CI 1.08–1.52; p = 0.004) once adjusting for known clinical factors. CYB5D2 reductions displayed relationship with mutations in PIK3CA, GATA3, MAP3K1, CDH1, TP53 and RB1. Impressively, 85% (560/659) of TP53 mutations occurred in the 21-gene signature-positive BC. Collectively, we provide the first evidence that CYB5D2 is a candidate tumor suppressor of BC.

Overexpression miR-24-3p repressed Bim expression to confer tamoxifen resistance in breast cancer.

Endocrine therapy resistance represents a major challenge to the successful treatment of patients with breast cancer. The development of tamoxifen resistance commonly occurrs during the treatment of patients with breast cancer whereas its underlying mechanisms remain elusive. Here, we found that miR-24-3p regulated tamoxifen sensitivity in breast cancer cells. Forced overexpression of miR-24-3p augmented tamoxifen-induced cell viability inhibition in breast cancer cells, while knockdown of miR-24-3p partially attenuated the cytotoxicity effect of tamoxifen. Moreover, we discovered Bim as a target gene of miR-24-3p in breast cancer cells by RNA immunoprecipitation, quantitative reverse transcription polymerase chain reaction, Western blot, and dual luciferase reporter assay. In our established tamoxifen resistant MCF7 cell line (MCF7/TAM), there was a significant elevation of miR-24-3p and decrease of BIM expression compared with parental MCF7 cells. In addition, the inhibition of miR-24-3p could reverse the tamoxifen resistance of MCF7/TAM cells by the induction of cell apoptosis. Silencing of Bim expression blocked miR-24-3p inhibitor-induced elevation of tamoxifen sensitivity of MCF7/TAM cells. Using tumor tissues from patients with breast cancer, we also found that the expression of miR-24-3p was negatively correlated with Bim mRNA expression. Collectively, our study highlighted the pivotal role of miR-24-3p overexpression in mediating the development of tamoxifen resistance in breast cancer and suggested miR-24-3p might be a predictor or target for patients with breast cancer.