Acquired Tamoxifen Resistance Research Articles

EZH2 PROTACs target EZH2- and FOXM1-associated oncogenic nodes, suppressing breast cancer cell growth.

Breast cancer (BC) remains the second leading cause of cancer-related mortalities in women. Resistance to hormone therapies such as tamoxifen, an estrogen receptor (ER) inhibitor, is a major hurdle in the treatment of BC. Enhancer of zeste homolog 2 (EZH2), the methyltransferase component of the Polycomb repressive complex 2 (PRC2), has been implicated in tamoxifen resistance. Evidence suggests that EZH2 often functions noncanonically, in a methyltransferase-independent manner, as a transcription coactivator through interacting with oncogenic transcription factors. Unlike methyltransferase inhibitors, proteolysis targeting chimeras (PROTAC) can suppress both activating and repressive functions of EZH2. Here, we find that EZH2 PROTACs, MS177 and MS8815, effectively inhibited the growth of BC cells, including those with acquired tamoxifen resistance, to a much greater degree when compared to methyltransferase inhibitors. Mechanistically, EZH2 associates with forkhead box M1 (FOXM1) and binds to the promoters of FOXM1 target genes. EZH2 PROTACs induce degradation of both EZH2 and FOXM1, leading to reduced expression of target genes involved in cell cycle progression and tamoxifen resistance. Together, this study supports that EZH2-targeted PROTACs represent a promising avenue of research for the future treatment of BC, including in the setting of tamoxifen resistance.

Abstract PO3-18-07: Therapeutic Potential of Combining Eltanexor with Tamoxifen to Treat Primary and Metastatic Estrogen Receptor Positive (ER+) Breast Cancers

Abstract Estrogen receptors are key pathway initiators in the aggressive development of more than two-thirds of all breast cancers. Tamoxifen (TAM) is a selective estrogen receptor modulator that disables the ER pathway and inhibits the progression of ER+ breast cancer. Unfortunately, 10-40% of patients with ER+ breast cancer who were treated with TAM experience recurrence and metastasis within 10 years. We have previously identified exportin 1 (XPO1) to be more highly expressed in breast cancers with acquired TAM resistance compared to TAM-sensitive counterparts and showed that a combination treatment of TAM with an XPO1 inhibitor, selinexor, is an effective method of tumor suppression in preclinical models. In this study we investigated the activity of eltanexor, an investigational second generation XPO1 inhibitor with minimal penetration of the blood-brain barrier, to inhibit cancer growth without leading to weight loss. Our results show that mice harboring MCF7-ESR1Y537Sxenograft tumors had significantly less (P < .05) flank tumor growth when administered a treatment regimen of TAM and eltanexor compared to control mice. In metastatic tumor models, we observed decreased metastatic burden in mice harboring MCF7-ESR1 D538G and MCF7-ESR1Y537S tumors when co-administered TAM and eltanexor. We did not observe weight changes in either model. Our findings suggest that eltanexor may be a relevant treatment for further exploration in the context of a TAM combination therapy for treatment of ER+ breast cancer. Citation Format: Vedant Jain, Myra Kamdar, Hannah McGee, Qianying Zuo, Jin Young Yoo, Yosef Landesman, Christopher Walker, Zeynep Madak-Erdogan. Therapeutic Potential of Combining Eltanexor with Tamoxifen to Treat Primary and Metastatic Estrogen Receptor Positive (ER+) Breast Cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-07.

Comparative metabolomics of MCF-7 and MCF-7/TAMR identifies potential metabolic pathways in tamoxifen resistant breast cancer cells.

Breast cancer is the most common cancer and the leading cause of cancer-related death among women. An Estrogen Receptor (ER) antagonist called tamoxifen is used as an adjuvant therapy for ER-positive breast cancers. Approximately 40% of patients develop tamoxifen resistance (TAMR) while receiving treatment. Cancer cells can rewire their metabolism to develop resistant phenotypes, and their metabolic state determines how receptive they are to chemotherapy. Metabolite extraction from human MCF-7 and MCF-7/TAMR cells was done using the methanol-methanol-water extraction method. After treating the dried samples with methoxamine hydrochloride in pyridine, the samples were derivatized with 2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, and Chlorotrimethylsilane (MSTFA + 1% TMCS). The Gas chromatography/mass spectrometry (GC-MS) raw data were processed using MSdial and Metaboanalyst for analysis. Univariate analysis revealed that 35 metabolites were elevated in TAMR cells whereas 25 metabolites were downregulated. N-acetyl-D-glucosamine, lysine, uracil, tyrosine, alanine, and o-phosphoserine were upregulated in TAMR cells, while hydroxyproline, glutamine, N-acetyl-L-aspartic acid, threonic acid, pyroglutamic acid, glutamine, o-phosphoethanolamine, oxoglutaric acid, and myoinositol were found to be downregulated. Multivariate analysis revealed a distinct separation between the two cell lines, as evidenced by their metabolite levels. The enriched pathways of deregulated metabolites included valine, leucine, and isoleucine degradation, Citric Acid Cycle, Warburg effect, Malate-Aspartate shuttle, glucose-alanine cycle, propanoate metabolism, and Phospholipid biosynthesis. This study revealed dysregulation of various metabolic processes in TAMR cells, which may be crucial in elucidating the molecular basis of the mechanisms underlying acquired tamoxifen resistance.

Abstract P4-01-38: A single arm phase II trial of Palbociclib in combination with Tamoxifen as first line therapy for metastatic hormone receptor positive breast cancer

Abstract Background: Palbociclib is a CDK4/6 inhibitor used to treat metastatic hormone receptor-positive (HR+) breast cancer (MBC) in combination with endocrine therapy. Tamoxifen is an effective treatment for HR+ MBC, with different toxicity profile compared with aromatase inhibitors (AI) and fulvestrant. Preclinical data demonstrated synergy for the combination of tamoxifen and palbociclib, being effective in a model of acquired tamoxifen resistance. Methods: We conducted a non-randomized, open-label, single-arm, multicenter, phase II trial of palbociclib in combination with tamoxifen in patients with HR+/HER2 - advanced BC, with no prior therapy for MBC. Ovarian suppression was recommended for pre-menopausal women. Primary objective was progression free survival, PFS. Secondary objectives: objective response rate, ORR (CR or PR) based on RECIST 1.1 or MDA Criteria (for patients with bone only disease); safety and tolerability (using CTCAE v4); clinical benefit rate, CBR (CR, PR or SD lasting min 24 weeks); 2-year overall survival. Correlative objectives: proteomic analysis of plasma exosomes to identify mechanisms of primary and secondary resistance to tamoxifen/palbociclib. Results: Between 6/30/2016 and 7/02/2019, we enrolled 49 patients (47 evaluable): 23 pts with de-novo metastatic disease and 24 pts with recurrent BC (12 pts were on adjuvant treatment with AI at time of recurrence and 12 pts on surveillance). As of 6/30/2022 data cut-off, 2 pts were still on treatment. This is an updated analysis with median follow-up time of 24 months (range 8-51). Median age was 60 (range 39-82). The median PFS was 19.8 months with 95% CI (8-41) for pts with de-novo MBC and 7 months (2-12) for pts with recurrent BC. The ORR was 30% overall, 39% for pts with de novo MBC, 21% for pts with recurrent BC. CBR was 64% overall, 77% for pts with de novo MBC and 50% for pts with recurrent BC. CBR was 65% for white pts and 55% for AA pts. Best response per RECIST1.1: 14 pts (34%) had PR, 18 pts (44%) had SD, 9 pts (22%) had PD. All 6 pts with bone only disease had SD. The most common drug related grade ≥ 3 AE was neutropenia (51%), transient and manageable by dose modifications, no cases of febrile neutropenia. Four patients (8%) developed thromboembolic events (grades 2, 3, 4) and discontinued treatment. One patient died while on treatment from PD. More than 800 exosome proteins were detected in the samples analyzed so far. Exosomal protein networks in pretreatment samples predicted treatment response with 89% sensitivity and 95% specificity in unsupervised clustering. Data on correlative objectives will be presented in a separate abstract. Conclusions: The combination of palbociclib and tamoxifen showed tolerable, expected safety profile. This may be an alternative approach for selected patients in first line treatment of HR+ MBC, especially those who present with de-novo metastatic disease and are intolerant to AI; although this small study indicates a lower PFS. Table 1: Baseline Demographics Citation Format: Oana C. Danciu, Kent Hoskins, Jennifer Weiss, Cristina I. Truica, Anne Blaes, Deimante Tamkus, Jatin Rana, Tandra Pavankumar, Lauren Green, Yu Gao, Menggang Yu, Qianqian Zhao, Deborah Toppmeyer, Ruth O’Regan, Kari B. Wisinski. A single arm phase II trial of Palbociclib in combination with Tamoxifen as first line therapy for metastatic hormone receptor positive breast cancer [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 P4-01-38.

Inhibition of Increased Invasiveness of Breast Cancer Cells With Acquired Tamoxifen Resistance by Suppression of CYR61.

Hormone sensitivity-targeted therapy with selective estrogen receptor modulators (SERMs), such as 4-hydroxytamoxifen (4-OHT), is the mainstay of treatment for breast cancers (BCs) that express estrogen receptor α (ERα). However, development of resistance limits this therapy approach. The question arises whether changes associated with 4-OHT resistance could be exploited therapeutically. First, 4-OHT-resistant sublines of ERα-positive breast carcinoma cell lines MCF-7 and T47D were generated. Viability was assessed by the Alamar Blue assay. Cell invasion was quantified in modified Boyden chambers with Matrigel. Changes in expression of CYR61, S100A4, and ERα were examined by RT-qPCR. Expression of CYR61 was suppressed by transient gene silencing using siRNA. Successful suppression was verified by western blot. Efficacy of 4-OHT treatment was analyzed by quantification of viability using Alamar Blue assay. Correlation of CYR61 levels in patients with luminal A BC to distant metastases-free survival was determined by Kaplan-Meier analysis. ERα-positive MCF-7 and T47D BC cells exhibit an extremely weak invasion rate. Acquired tamoxifen resistance significantly increased the invasive behavior of both tamoxifen-resistant MCF-7-TR and T47D-TR sublines. In addition, expression of CYR61 and S100A4 showed significantly increased levels, whereas expression of ERα was decreased. Suppression of CYR61 expression resulted in a significant decreased invasion rate. In addition, expression of S100A4 was reduced, whereas expression of ERα was increased. Furthermore, suppression of CYR61 resulted in re-sensitization to 4-OHT. High CYR61 levels in patients with luminal A BC resulted in reduced distant metastases-free survival. The prometastatic factor CYR61 appears to play an important role in the increased invasiveness of tamoxifen-resistant ERα-positive BC cells. Its suppression leads to a lower invasion rate. Given the few therapeutic options available for tamoxifen-resistant BC, therapy that reduces CYR61 may improve its treatability in future.

Design, synthesis, and metabolite identification of Tamoxifen esterase-activatable prodrugs

Tamoxifen (TAM) is used in treatment of hormonal dependent breast cancer, both in premenopausal and postmenopausal women. TAM is intrinsically metabolized by CYP450 enzymes to more active metabolites. Recent reports identified CYP2D6, an enzyme involved in the conversion of TAM to the more potent 4-OH-TAM, is encoded by theCYP2D6gene, which is highly polymorphic. Women with inactive alleles are poor metabolizers; in many cases they suffer acquired TAM resistance. Herein we report synthesis and biological evaluation of novel TAM analogues. The novel analogues are designed to elude CYP2D6 metabolism. Hydrolysis of the carbamate moiety on ring C is mediated via carboxylesterases. Compound 3d [E/Z Benzyl-carbamic acid4-{2-benzyl-1-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-but-1-enyl}-phenyl ester] showed GI50 = 0.09 µM on MCF-7 and GI50 = 1.84 µM on MDA-MB231 cell lines. To further validate our hypothesis, metabolites of selected novel analogues were determined in vitro under different incubation conditions. The hydroxylated analogues were obtained under non CYP2D6 dependent conditions. Compound 8d, a benzyl carbamate derivative, was the least-stable analog and showed the highest rate of metabolism among all tested analogues. Our in silico model showed the novel flexible analogues can still adopt an antiestrogenic binding profile occupying the same pocket as 4-OH-TAM.

Alteration of Trop-2 expression in breast cancer cells by clinically used therapeutic agents and acquired tamoxifen resistance

BackgroundSacituzumab govitecan is an antibody–drug conjugate that delivers SN-38, an active metabolite of irinotecan, to the target molecule, trophoblast cell-surface antigen 2 (Trop-2). It is a promising drug for triple-negative breast cancer and is anticipated to be effective for luminal breast cancer. The efficacy of the agent relies on the expression of Trop-2 rather than its intracellular function. However, conditions that alter the Trop-2 expression have not been well investigated.MethodsWe tested a range of clinically related treatments for their effect on Trop-2 expression in cultured breast cancer cell lines.ResultsThe expression level of Trop-2 differed among cell lines, independent of their subtypes, and was highly variable on treatment with kinase inhibitors, tamoxifen, irradiation, and chemotherapeutic agents including irinotecan. While inhibitors of AKT, RSK, and p38 MAPK suppressed the Trop-2 expression, tamoxifen treatment significantly increased Trop-2 expression in luminal cancer cell lines. Notably, luminal cancer cells with acquired resistance to tamoxifen also exhibited higher levels of Trop-2. We identified transcription factor EB (TFEB) as a possible mechanism underlying tamoxifen-induced elevation of Trop-2 expression. Tamoxifen triggers dephosphorylation of TFEB, an active form of TFEB, and the effect of tamoxifen on Trop-2 was prevented by depletion of TFEB. A luciferase reporter assay showed that Trop-2 induction by TFEB was dependent on a tandem E-box motif within the Trop-2 promoter region.ConclusionsOverall, these results suggest that the effectiveness of sacituzumab govitecan could be altered by concomitant treatment and that tamoxifen could be a favorable agent for combined therapy.

Relationship of micro-RNA, mRNA and eIF Expression in Tamoxifen-Adapted MCF-7 Breast Cancer Cells: Impact of miR-1972 on Gene Expression, Proliferation and Migration.

Background: Tamoxifen-adapted MCF-7-Tam cells represent an in-vitro model for acquired tamoxifen resistance, which is still a problem in clinics. We here investigated the correlation of microRNA-, mRNA- and eukaryotic initiation factors (eIFs) expression in this model. Methods: MicroRNA- and gene expression were analyzed by nCounter and qRT-PCR technology; eIFs by Western blotting. Protein translation mode was determined using a reporter gene assay. Cells were transfected with a miR-1972-mimic. Results: miR-181b-5p,-3p and miR-455-5p were up-, miR-375, and miR-1972 down-regulated and are significant in survival analysis. About 5% of the predicted target genes were significantly altered. Pathway enrichment analysis suggested a contribution of the FoxO1 pathway. The ratio of polio-IRES driven to cap-dependent protein translation shifted towards cap-dependent initiation. Protein expression of eIF2A, -4G, -4H and -6 decreased, whereas eIF3H was higher in MCF-7-Tam. Significant correlations between tamoxifen-regulated miRNAs and eIFs were found in representative breast cancer cell lines. Transfection with a miR-1972-mimic reverses tamoxifen-induced expression for a subset of genes and increased proliferation in MCF-7, but reduced proliferation in MCF-7-Tam, especially in the presence of 4OH-tamoxifen. Migration was inhibited in MCF-7-Tam cells. Translation mode remained unaffected. Conclusions: miR-1972 contributes to the orchestration of gene-expression and physiological consequences of tamoxifen adaption.

Lactate is a potential promoter of tamoxifen resistance in MCF7 cells

BackgroundTamoxifen is a widely used estrogen receptor inhibitor, whose clinical success is limited by the development of acquired resistance. This compound was also found to inhibit mitochondrial function, causing increased glycolysis and lactate production. Lactate has been widely recognized as a signaling molecule, showing the potential of modifying gene expression. These metabolic effects of tamoxifen can by hypothesized to contribute in driving drug resistance. MethodsTo test this hypothesis, we used MCF7 cells together with a tamoxifen resistant cell line (MCF7-TAM). Experiments were aimed at verifying whether enhanced lactate exposure can affect the phenotype of MCF7 cells, conferring them features mirroring those observed in the tamoxifen resistant culture. ResultsThe obtained results suggested that enhanced lactate in MCF7 cells medium can increase the expression of tafazzin (TAZ) and telomerase complex (TERC, TERT) genes, reducing the cells' attitude to undergo senescence. In long term lactate-exposed cells, signs of EGFR activation, a pathway related to acquired tamoxifen resistance, was also observed. ConclusionsThe obtained results suggested lactate as a potential promoter of tamoxifen resistance. The off-target effects of this compound could play a role in hindering its therapeutic efficacy. General significanceThe features of acquired tamoxifen resistance have been widely characterized at the molecular level; in spite of their heterogeneity, poorly responsive cells were often found to display upregulated glycolysis. Our results suggest that this metabolic asset is not simply a result of neoplastic progression, but can play an active part in driving this process.

The tamoxifen-regulated, long non-coding RNA LINC00992 affects proliferation, migration, and expression of tamoxifen resistanceassociated genes in MCF-7 breast cancer cells

Tamoxifen-adapted MCF-7 breast cancer cells (MCF-7-TAM-R) are a model for acquired tamoxifen resistance in oestrogen receptor-positive breast cancer. In this system, the expression of long-non-coding RNA LINC00992 is decreased. LINC00992 might therefore contribute to tamoxifen adaption and associated gene expres-sion changes. Here, we investigated whether a modulation of LINC00992 modifies gene expression, proliferation, and migration. Up- and down-- regulation of LINC00992 was performed using plasmid vectors and siRNA. Gene expression was measured via nCounter® and quantitative real-time polymerase chain reaction. Database analysis was performed using GEPIA2 and cBioportal. Furthermore, we performed scratch assays, colony-forming assays, and proliferation assays with MCF-7 and MCF-7-TAM-R after up-regulation of LINC00992. Up- and down-regulation of LINC00992 caused gene expression changes in 4 of the 42 tamoxifen-regulated genes tested. Especially ubiquitin D, single-minded homologue 1 (SIM1) carcinoembryonic antigen-related cell adhesion molecule 5 and the G-protein coupled oestrogen receptor 1 were affected. In tamoxifen-adapted MCF-7-TAM-R cells, LINC00992 overexpression resulted in augmented viability and proliferation and enhanced migration. Database analyses revealed that luminal breast cancers have increased expression of LINC00992 compared to Her2-type/neu- or basal type. Furthermore, higher expression of LINC00992 was associated with poor prognosis in luminal-A carcinomas. Changes in the expression of tamoxifen-regulated genes could be induced by manipulating LINC00992's abundance, suggesting that it is at least partially involved in the establishment of the tamoxifen-induced gene expression pattern. LINC00992 may also serve as a prognostic biomarker and may indicate the development of tamoxifen resistance.

Protein Aggregation Patterns Inform about Breast Cancer Response to Antiestrogens and Reveal the RNA Ligase RTCB as Mediator of Acquired Tamoxifen Resistance.

Simple SummaryAcquired resistance to antiestrogenic therapy remains the major obstacle to curing luminal subtype breast cancer. While current treatment in acquired endocrine-resistant settings includes combined therapy with receptor tyrosine kinase or cyclin-dependent kinase inhibitors, progression to incurable disease remains possible. In recent years, the antioxidant system and the protein quality control network have been associated with the enhanced resistance of breast cancer cells to hormonal therapy. In this work, we raise the hypothesis that antiestrogen treatment induces the accumulation of protein aggregates in sensitive cells, which in turn could hinder the activation of survival pathways. We present evidence concerning a novel way to identify antiestrogen response and disclose a novel protein, RTBC, that controls acquired antiestrogen resistance. This work opens a new avenue for research towards finding breast cancer prognostic markers and therapeutic targets, where the identification of proteins prone to aggregate could help to identify antiestrogen response and understand mechanisms of disease.The protein quality control network, including autophagy, the proteasome and the unfolded protein response (UPR), is triggered by stress and is overactive in acquired antiestrogen therapy resistance. We show for the first time that the aggresome load correlates with apoptosis and is increased in antiestrogen-sensitive cells compared to endocrine-resistant variants. LC-MS/MS analysis of the aggregated proteins obtained after 4OH-tamoxifen and Fulvestrant treatment identified proteins with essential function in protein quality control in antiestrogen-sensitive cells, but not in resistant variants. These include the UPR modulators RTCB and PDIA6, as well as many proteasome proteins such as PSMC2 and PSMD11. RTCB is a tRNA and XBP1 ligase and its aggregation induced by antiestrogens correlated with impaired XBP1s expression in sensitive cells. Knock down of RTCB was sufficient to restore sensitivity to tamoxifen in endocrine-resistant cells and increased the formation of aggresomes, leading to apoptotic cell death. Analysis of primary human breast cancer samples and their metastases appearing after endocrine treatment showed that RTCB is only localized to aggresomes in the primary tumors, while total aggresomes, including aggregated RTCB, were significantly reduced in the metastases. Therefore, different protein aggregation patterns may indicate loss of function of essential proteins resulting in enhanced protein aggregation that can be used to identify antiestrogen-resistant breast cancer cells and improve the response to antiestrogenic therapy.

A single-arm phase II trial of palbociclib in combination with tamoxifen as first-line therapy for metastatic hormone receptor-positive breast cancer.

1056 Background: Palbociclib is a CDK4/6 inhibitor used to treat metastatic hormone receptor-positive (HR+) breast cancer (MBC) in combination with endocrine therapy. Tamoxifen is an effective treatment for HR+ MBC, with different toxicity profile compared with aromatase inhibitors (AI) and fulvestrant. Preclinical data demonstrated synergy for the combination of tamoxifen and palbociclib, being effective in a model of acquired tamoxifen resistance. Methods: We conducted an open-label, single-arm, multicenter phase II trial of palbociclib in combination with tamoxifen in patients with HR+/HER2 - advanced BC, with no prior therapy for MBC. Ovarian suppression was recommended for pre-menopausal women, but not required. Primary objective was progression free survival. Secondary objectives: objective response rate (CR or PR) based on RECIST 1.1 or MDA Criteria (for patients with bone only disease); safety and tolerability (using CTCAE v4); clinical benefit rate (CR, PR or SD lasting min 24 weeks); 2-year overall survival. Correlative objectives: proteomic analysis of plasma exosomes to identify mechanisms of primary and secondary resistance to tamoxifen/palbociclib. Results: Between 6/30/2016 and 7/02/2019, we enrolled 49 patients (47 evaluable): 23 pts with de-novo metastatic disease and 24 pts with recurrent BC (12 pts were on adjuvant treatment with AI at time of recurrence and 12 pts on surveillance). As of 1/5/21 data cut-off, 7 pts were still on treatment. Median follow-up time was 24 months (range 8-42). Median age was 60 (range 39-82). The median PFS was 14.6 months with 95% CI (7-41) for pts with de-novo MBC and 6 months (2-12) for pts with recurrent BC. The ORR was 30% overall, 39% for pts with de novo MBC, 21% for pts with recurrent BC. CBR was 64% overall, 78% for pts with de novo MBC and 50% for pts with recurrent BC. CBR was 65% for white pts and 55% for African American pts. Best response per RECIST1.1: 14 pts (34%) had PR, 18 pts (44%) had SD, 9 pts (22%) had PD. All 6 pts with bone only disease had SD. The most common drug related grade ≥ 3 AE was neutropenia (51%), transient and manageable by dose modifications, no cases of febrile neutropenia. Four patients developed thromboembolic events (1 grade 2, 2 grade 3, 1 grade 4). One patient died while on treatment from PD. Conclusions: The combination of palbociclib and tamoxifen showed tolerable, expected safety profile. This may be an alternative approach for selected patients in first line treatment of HR+ MBC, especially those who are intolerant to AI, although this small study indicates a lower PFS. Clinical trial information: 02668666 .[Table: see text]

Overexpression of TMEM47 Induces Tamoxifen Resistance in Human Breast Cancer Cells.

Background:Tamoxifen (TAM) is the eminent first-line drug for endocrine therapy of hormone receptor positive premenopausal breast cancer and reduces the risk of recurrence by ∼50%. However, many patients developed TAM resistance and their diseases recurred. Our previous study on transcriptome profile of TAM resistant breast cancer cells revealed that the TMEM47 is one of the most significantly differentially expressed genes. The mechanism of how TMEM47 is involved in TAM resistance was not known.Methods:We constructed a mammal breast cancer cell line, in which TMEM47 was stably overexpressed (TMEM47-OE/MCF-7), to further verify the role of TMEM47 in TAM resistance. siRNA targeting TMEM47 was transfected into TAMR / MCF-7 cells by Liposome. TMEM47 expression was validated on mRNA and protein level by qRT-PCR and western blotting. We tested the cytotoxicity of TAM in the cells. Apoptosis was detected by flow cytometry.Results:Compared to the MCF7 cells, TMEM47 mRNA was significantly up regulated more than 6 folds in the TAMR/MCF7 cells and so its protein. TMEM47 expression level in TMEM47-OE/MCF-7 was similar as in the TAMR/MCF-7 cells. The 50% inhibitory concentration (IC50) value (mean ± SD) of TAM in MCF-7, TAMR/MCF-7 and TMEM47-OE/MCF-7 cells was 1.58 ± 0.19, 2.74 ± 0.24 and 3.12 ± 0.32 µγ/mL, respectively. The apoptosis rates of TAMR/MCF-7 and TMEM47-OE/MCF-7 cell lines were significantly lower than that of MCF-7 cells. After 24 and 48 hours TAM treatments, cell viability was significantly inhibitied in TMEM47 knockdown TAMR/MCF7 cells (P < 0.01). Consistant with the decreased cell viability, the apoptosis rate in TMEM47 knockdown TAMR/MCF-7 cells was significantly increased.Conclusions:Our results suggest that overexpression of TMEM47 in MCF-7 cells acquired TAM resistance to those cells, and knockdown of TMEM47 in TAMR/MCF-7 cells reversed their resistance to TAM. TMEM47 might confer TAM resistance on MCF-7 cells through the inhibition of apoptosis.

Upregulation of PI3K/AKT/PTEN pathway is correlated with glucose and glutamine metabolic dysfunction during tamoxifen resistance development in MCF-7 cells

Tamoxifen resistance is emerging as a big challenge in endocrine therapy of luminal A breast cancer patients. In this study, we aimed to determine the molecular changes of PI3K/AKT/PTEN signaling pathway during tamoxifen-resistance development using gradually increased doses of tamoxifen in one model, while fixing tamoxifen treatment dose at 35 μM for several times in the second model. An upregulation of AKT/PI3K genes was noticed at 30 μM tamoxifen concentration in cells treated with a gradual increase of tamoxifen doses. In the second model, significant upregulation of AKT1 was seen in cells treated with 35 μM tamoxifen for three times. All genes studied showed a significant increase in expression in resistant cells treated with 50 µM and 35 µM six times tamoxifen. These genes’ upregulation was accompanied by PTEN and GSK3 ß genes’ down-regulation, and it was in correlation to the changes in the metabolic rate of glucose in tamoxifen-resistant models. A significant increase in glucose consumption rate from culture media was observed in tamoxifen resistant cells with the highest consumption rate reported in the first day of culturing. Increased glucose consumption rates were also correlated with GLUL significant gene expression and non-significant change in c-MYC gene expression that may lead to increased endogenous glutamine synthesis. As a result, several molecular and metabolic changes precede acquired tamoxifen resistance could be used as resistance biomarkers or targets to reverse tamoxifen resistance.

Acquired tamoxifen resistance is surmounted by GW8510 through ribonucleotide reductase M2 downregulation-mediated autophagy induction

Tamoxifen resistance is a major roadblock in the treatment of patients with breast cancer. Ribonucleotide reductase M2 (RRM2) was found to be involved in acquired resistance of breast cancer cells (BCCs) to tamoxifen. Here, we used GW8510, which has been identified as a potential RRM2 inhibitor, to evaluate the effect of RRM2 inhibition on reversing resistance of BCCs to tamoxifen and investigate its mechanisms. We showed that RRM2 overexpression played a key role in the development of acquired tamoxifen resistance in BCCs through downregulation of autophagy level. Combination treatment with tamoxifen and GW8510 significantly inhibited survival of the tamoxifen-resistant BCCs through induction of autophagic cell death compared to either of the two drugs. Furthermore, combination of tamoxifen and GW8510 resulted in marked growth inhibition of tamoxifen-resistant BBC xenograft tumor in vivo compared to tamoxifen or GW8510 alone. In conclusion, tamoxifen in combination with GW8510 can overcome acquired tamoxifen resistance in BCCs and may be a rational therapeutic approach against breast cancer with high RRM2 expression.

Abstract P1-19-30: Safety results from a phase 2 study of Palbociclib in combination with Tamoxifen as first line therapy for metastatic hormone receptor positive breast cancer

Abstract Background: Palbociclib is a reversible, oral, small molecule inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6). Palbociclib combined with various endocrine agents showed significantly improved progression-free survival in patients with metastatic hormone receptor-positive (HR+) breast cancer (MBC). Preclinical data demonstrates synergy for the combination of tamoxifen and palbociclib and that the combination is effective in a model of acquired tamoxifen resistance. Tamoxifen is an effective treatment for HR+ MBC and presents a different toxicity profile when compared with aromatase inhibitors and fulvestrant. Combining palbociclib with tamoxifen in first line treatment of HR+ MBC offers an alternative to other endocrine combinations. Methods: This is a non-randomized, open-label, single-arm, multicenter, phase II study of palbociclib in combination with tamoxifen in patients with HR+/HER2-negative advanced BC. The primary objective is to determine the objective response rate (complete or partial response) based on RECIST 1.1 or MDA Criteria (for patients with bone only disease). Secondary objectives are: safety and tolerability, progression-free survival, clinical benefit rate, 2-year overall survival. Correlative objectives will explore alterations in circulating tumor DNA and changes in gene expression pattern at the time of progression. Safety assessments were performed at baseline, days 1 and 15 (first 2 cycles) and day 1 with each subsequent cycles. Adverse events (AEs) were graded according to NCI-CTCAE v4. Results: Forty-nine patients from 6 academic centers were enrolled in this trial at the time of this report. Baseline demographics and disease characteristics are reported in the table. Median age was 60 (range 39-82). Twenty-three (47%) patients presented with de-novo metastatic disease. Out of 26 (53%) patients who presented with recurrent metastatic disease, 13 (50%) were receiving adjuvant treatment with aromatase inhibitors at the time of recurrence and 13 (50%) were on surveillance with no active treatment. The most common drug related grade ≥ 3 AE was neutropenia (46%) that was transient and manageable by dose modifications, with no development of febrile neutropenia. The most common drug-related any grade AEs were neutropenia (68%), fatigue (43%), nausea (41%), anemia (32%), minor infections (30%), thrombocytopenia (29%), hot flashes (27%), lymphocytopenia (16%), alopecia (11%), diarrhea (11%).Two patients developed thromboembolic events (grades 2 and 4) and discontinued treatment. One patient died while on treatment. Conclusions: The combination of palbociclib and tamoxifen was well tolerated with expected toxicities. Neutropenia was the most frequently reported AE. This is one of the first trials to assess safety of the combination of palbociclib with tamoxifen in HR+ MBC and has implications for the early stage setting. Baseline Demographics and Disease CharacteristicsParameterN%SexFemale49100RaceWhite3367Black or African American1123Asian12American Indian or Alaska Native12Unknown36EthnicityHispanic or Latino36Non-Hispanic4694ECOG Performance status0336711531212Menopausal statusPost-menopausal4082Pre-menopausal918Site of metastasesBone3878Liver1326Lung816Other2245Bone only510 Citation Format: Oana C Danciu, Kent Hoskins, Cristina Truica, Anne Blaes, Deimante Tamkus, Jatin Rana, Tandra Pavankumar, Lauren Green, Li Liu, Menggang Yu, Deborah Toppmeyer, Ruth O'Regan, Kari Wisinski. Safety results from a phase 2 study of Palbociclib in combination with Tamoxifen as first line therapy for metastatic hormone receptor 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 P1-19-30.

Dynamic surveillance of tamoxifen-resistance in ER-positive breast cancer by CAIX-targeted ultrasound imaging.

Tamoxifen‐based hormone therapy is central for the treatment of estrogen receptor positive (ER+) breast cancer. However, the acquired tamoxifen resistance, typically co‐exists with hypoxia, remains a major challenge. We aimed to develop a non‐invasive, targeted ultrasound imaging approach to dynamically monitory of tamoxifen resistance. After we assessed acquired tamoxifen resistance in 235 breast cancer patients and a list of breast cancer cell lines, we developed poly(lactic‐co‐glycolic acid)‐poly(ethylene glycol)‐carbonic anhydrase IX mono antibody nanobubbles (PLGA‐PEG‐mAbCAIX NBs) to detect hypoxic breast cancer cells upon exposure of tamoxifen in nude mice. We demonstrate that carbonic anhydrase IX (CAIX) expression is associated with breast cancer local recurrence and tamoxifen resistance both in clinical and cellular models. We find that CAIX overexpression increases tamoxifen tolerance in MCF‐7 cells and predicts early tamoxifen resistance along with an oscillating pattern in intracellular ATP level in vitro. PLGA‐PEG‐mAbCAIX NBs are able to dynamically detect tamoxifen‐induced hypoxia and tamoxifen resistance in vivo. CAIX‐conjugated NBs with noninvasive ultrasound imaging is powerful for dynamically monitoring hypoxic microenvironment in ER+ breast cancer with tamoxifen resistance.

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.

The ERα-miR-575-p27 feedback loop regulates tamoxifen sensitivity in ER-positive Breast Cancer.

Background: Breast cancer is the most common malignancy, and approximately 70% of breast cancers are estrogen receptor-α (ERα) positive. The anti-estrogen tamoxifen is a highly effective and commonly used treatment for patients with ER+ breast cancer. However, 30% of breast cancer patients fail adjuvant tamoxifen therapy and most of metastatic breast cancer patients develop tamoxifen resistance. Although increasing evidence suggests that microRNA (miRNA) dysregulation influences tamoxifen sensitivity, the mechanism of the cross-talk between miRNA and ERα signaling remains unclear. miR-575 has been reported to be involved in carcinogenesis and progression, however, the role of miR-575 in breast cancer remains limited. The aim of this study was to understand the mechanism of miR-575 in breast cancer tamoxifen resistance.Method: RT-qPCR was employed to assess miR-575 expression in breast cancer tissues and cell lines. The association of miR-575 expression with overall survival in patients with breast cancer was evaluated with KM plotter. Additionally, the effects of miR-575 on breast cancer proliferation and tamoxifen sensitivity were investigated both in vitro and in vivo. Bioinformatic analyses and luciferase reporter assays were performed to validate CDKN1B and BRCA1 as direct targets of miR-31-5p. The ERα binding sites in the miR-575 promoter region was validated with ChIP and luciferase assays. ERα interactions with CDKN1B, cyclin D1 or BRCA1 were determined by IP analysis, and protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively.Results: miR-575 levels were higher in ER+ breast cancer than in ER- breast cancer and patients with high miR-575 expression had a significantly poorer outcome than those with low miR-575 expression. ERα bound the miR-575 promoter to activate its transcription, and tamoxifen treatment downregulated miR-575 expression in ER+ breast cancer. Overexpression of miR-575 decreased tamoxifen sensitivity by targeting CDKN1B and BRCA1. CDKN1B and BRCA1 were both able to antagonize ERα activity by inhibiting ERα nuclear translocation and interaction with cyclin D1. Furthermore, miR-575 expression was found to be upregulated in ER+ breast cancer cell with acquired tamoxifen resistance, whereas depletion of miR-575 partially re-sensitized these cells to tamoxifen by regulation of CDKN1B.Conclusions: Our data reveal the ERα-miR-575-CDKN1B feedback loop in ER+ breast cancer, suggesting that miR-575 can be used as a prognostic biomarker in patients with ER+ breast cancer, as well as a predictor or a promising target for tamoxifen sensitivity.

ERα36 as a Potential Therapeutic Target for Tamoxifen-Resistant Breast Cancer Cell Line Through EGFR/ERK Signaling Pathway.

BackgroundAcquired tamoxifen resistance is one of the major barriers to the successful treatment of breast cancer. Recently, overexpression of ERα36 was demonstrated to be a potential mechanism for the generation of acquired tamoxifen resistance. This study aims to evaluate the possibility of ERα36 being a therapeutic target for tamoxifen-resistant breast cancer.MethodsA tamoxifen-resistant cell subline (MCF-7/TAM) was established by culturing MCF-7 cells in medium plus 1 μM tamoxifen over 6 months. Colony-forming assay was used to determine the sensitivity of MCF-7/TAM cells to tamoxifen. Stable transfection was used to knockdown ERα36 expression in MCF-7/TAM cells. MTT assay and Transwell migration assay were used to assess the in vitro proliferation and migration, respectively. Nude mouse tumorigenicity assay was used to evaluate in vivo tumorigenicity. Western blot analysis and quantitative real-time PCR (qRT-PCR) were used to examine the expression of ERα36, ERα, EGFR and phosphorylated ERK1/2. The dual-luciferase reporter assay was used to determine the effect of ERα36 on the activity of EGFR-promotor.ResultsMCF-7/TAM cells possessed greatly increased ERα36 expression and EGFR expression and exhibited significantly increased in vitro proliferation and migration ability, as well as increased in vivo tumor growth ability, compared to parental MCF-7 cells. Knockdown of ERα36 expression inhibited in vitro proliferation and migration, as well as in vivo tumor growth ability of MCF-7/TAM cells. ERα36 regulated EGFR expression at the transcriptional level, and knockdown of ERα36 in MCF-7/TAM cells downregulated EGFR expression and then blocked EGFR/ERK signaling pathway.ConclusionKnockdown of ERα36 inhibits the growth of MCF-7/TAM cells in vitro and in vivo by blocking EGFR/ERK signaling pathway. ERα36 may be a candidate therapeutic target for the treatment of tamoxifen-resistant breast cancer.