Tamoxifen-resistant Breast Cancer Cells Research Articles

Abstract PO4-24-09: Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive breast cancer

Abstract Purpose Because more than 60% of breast cancers are hormone-receptor (HR) positive, endocrine therapy plays an important role. Tamoxifen is an antagonist of estrogen receptor-α and main treatment drug for HR-positive breast cancers. However, efficacy is not satisfactory because of the development of resistance. EGFR expression was associated with poor prognosis in HR-positive breast cancers. Loss of ER by EGFR activation induced tamoxifen resistance. And extracellular signal-regulated kinase (ERK) is another important factor for tamoxifen resistance. Activation of ERK signaling in breast cancer is not only associated with augmented tumor growth and metastasis but also with tamoxifen resistance. Therefore, the inhibition of ERK/EGFR signaling associated with tamoxifen resistance could improve the survival rate. Grape seed extract (GSE) is which contains a high content of flavonoid polyphenolic compounds. GSE is known to have various pharmacological effects, including antioxidant, anti-inflammatory, antitumor activity. In this study, we explored the activity of GSE in tamoxifen resistant breast cancer cells by inhibiting EGFR signaling pathway. Method We examined the suppressive effect of GSE on various signaling pathways. To confirm the pharmacological activity of GSE, we treated EGFR positive breast cancer cells with GSE. Levels of mRNA and protein expression were analyzed by real-time PCR and western blot. And cell proliferation and invasiveness were analyzed by the colony-forming assay and the Boyden chamber assay. Results 1. GSE down regulates EGFR and ERK in EGFR positive breast cancer cell line. The cells were treated with the indicated concentrations GSE for 48 hrs. The levels of p- and t-ERK and t-EGFR were measured through Western blotting. The levels of phosphorylated EGFR and ERK were decreased by GSE at the indicated doses for 48hrs. 2. EGF, a ligand of EGFR, upregulates the MMP-9 and GSE suppresses MMP-9. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are upregulated in most cancers and play crucial roles in modulating invasion and metastasis. EGFR with its ligand epidermal growth factor (EGF) regulates Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). After treating EGFR positive breast cancer cells with EGF and TGF, MMP-9 levels were increased. However, MMP-9 level was decreased with GSE as indicated doses for 48hrs. Conclusion Taken together, GSE inhibited the ERK/EGFR and EGF/MMP-9 signaling pathway in EGFR positive breast cancer cells. Therefore, we propose the possibility of GSE as an effective adjuvant for tamoxifen resistant breast cancer. Citation Format: Seung Pil Jung, Eun-Shin Lee, Ji Young You, Jung Hwa Chun, Hyunyou Kim. Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive breast cancer [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 PO4-24-09.

SiRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression

BackgroundBreast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets.MethodsTranscriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target.ResultsIntegrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours.ConclusionOur findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

Long non-coding RNA SOX2OT in tamoxifen-resistant breast cancer.

Hormone receptor (HR)-positive breast cancer can become aggressive after developing hormone-treatment resistance. This study elucidated the role of long non-coding RNA (lncRNA) SOX2OT in tamoxifen-resistant (TAMR) breast cancer and its potential interplay with the tumor microenvironment (TME). TAMR breast cancer cell lines TAMR-V and TAMR-H were compared with the luminal type A cell line (MCF-7). LncRNA expression was assessed via next-generation sequencing, RNA extraction, lncRNA profiling, and quantitative RT-qPCR. SOX2OT overexpression effects on cell proliferation, migration, and invasion were evaluated using various assays. SOX2OT was consistently downregulated in TAMR cell lines and TAMR breast cancer tissue. Overexpression of SOX2OT in TAMR cells increased cell proliferation and cell invasion. However, SOX2OT overexpression did not significantly alter SOX2 levels, suggesting an independent interaction within TAMR cells. Kaplan-Meier plot analysis revealed an inverse relationship between SOX2OT expression and prognosis in luminal A and B breast cancers. Our findings highlight the potential role of SOX2OT in TAMR breast cancer progression. The downregulation of SOX2OT in TAMR breast cancer indicates its involvement in resistance mechanisms. Further studies should explore the intricate interactions between SOX2OT, SOX2, and TME in breast cancer subtypes.

The SMAC mimetic GDC-0152 is a direct ABCB1-ATPase activity modulator and BIRC5 expression suppressor in cancer cells

Upregulation of the multidrug efflux pump ABCB1/MDR1 (P-gp) and the anti-apoptotic protein BIRC5/Survivin promotes multidrug resistance in various human cancers. GDC-0152 is a DIABLO/SMAC mimetic currently being tested in patients with solid tumors. However, it is still unclear whether GDC-0152 is therapeutically applicable for patients with ABCB1-overexpressing multidrug-resistant tumors, and the molecular mechanism of action of GDC-0152 in cancer cells is still incompletely understood. In this study, we found that the potency of GDC-0152 is unaffected by the expression of ABCB1 in cancer cells. Interestingly, through in silico and in vitro analysis, we discovered that GDC-0152 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multidrug efflux activity at sub-cytotoxic concentrations (i.e., 0.25×IC50 or less). Further investigation revealed that GDC-0152 also decreases BIRC5 expression, induces mitophagy, and lowers intracellular ATP levels in cancer cells at low cytotoxic concentrations (i.e., 0.5×IC50). Co-treatment with GDC-0152 restored the sensitivity to the known ABCB1 substrates, including paclitaxel, vincristine, and YM155 in ABCB1-expressing multidrug-resistant cancer cells, and it also restored the sensitivity to tamoxifen in BIRC5-overexpressing tamoxifen-resistant breast cancer cells in vitro. Moreover, co-treatment with GDC-0152 restored and potentiated the anticancer effects of paclitaxel in ABCB1 and BIRC5 co-expressing xenograft tumors in vivo. In conclusion, GDC-0152 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide essential information to physicians for designing a more patient-specific GDC-0152 clinical trial program in the future.

Small molecule inhibitor targeting the Hsp70-Bim protein–protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance

IntroductionEstrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance.MethodsA specific inhibitor of Hsp70-Bim protein–protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 μM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days.ResultsIt was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume.ConclusionsThe in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Favipiravir, an antiviral drug, in combination with tamoxifen exerts synergistic effect in tamoxifen-resistant breast cancer cells via hTERT inhibition

Tamoxifen (TAM) is one of the most successful treatments for breast cancer; however, TAM resistance continues to be a significant barrier. TAM resistance has been reported to be associated with increased expression of human telomerase reverse transcriptase (hTERT). This enzyme shares structural similarity with RNA-dependent RNA polymerase (RdRp) enzyme of RNA viruses, suggesting that RdRp inhibitors may also inhibit hTERT. Favipiravir (FAV) is an antiviral drug that inhibits RdRp of RNA viruses. Thus, we propose that FAV may also elicit an antitumor effect by suppressing hTERT. This study aimed to investigate the effect of FAV and TAM on TAM-resistant breast cancer (TAMR-1). The cell viabilities were determined. The levels of CDK1/ hTERT, in addition to regulators of hTERT-targeted signaling pathways were measured. Apoptosis, migration, and cell cycle distribution were also determined. Our data revealed that the combination of TAM and FAV suppressed cell proliferation synergistically (CI < 1) and resulted in a significant change in cell migration and apoptosis. Indeed, this was associated with reduced levels of hTERT and CDK1 and shift in the cell cycle distribution. Our findings suggest that the TAM/FAV combination exhibits synergistic effects against TAMR-1 human breast cancer cells by targeting hTERT.

USP46 enhances tamoxifen resistance in breast cancer cells by stabilizing PTBP1 to facilitate glycolysis

Tamoxifen (TAM) is the primary drug for treating estrogen receptor alpha-positive (ER+) breast cancer (BC). However, resistance to TAM can develop in some patients, limiting its therapeutic efficacy. The ubiquitin-specific protease (USP) family has been associated with the development, progression, and drug resistance of various cancers. To explore the role of USPs in TAM resistance in BC, we used qRT-PCR to compare USP expression between TAM-sensitive (MCF-7 and T47D) and TAM-resistant cells (MCF-7R and T47DR). We then modulated USP46 expression and examined its impact on cell proliferation, drug resistance (via CCK-8 and EdU experiments), glycolysis levels (using a glycolysis detection assay), protein interactions (confirmed by co-IP), and protein changes (analyzed through Western blotting). Our findings revealed that USP46 was significantly overexpressed in TAM-resistant BC cells, leading to the inhibition of the ubiquitin degradation of polypyrimidine tract-binding protein 1 (PTBP1). Overexpression of PTBP1 increased the PKM2/PKM1 ratio, promoted glycolysis, and intensified TAM resistance in BC cells. Knockdown of USP46 induced downregulation of PTBP1 protein by promoting its K48-linked ubiquitination, resulting in a decreased PKM2/PKM1 ratio, reduced glycolysis, and heightened TAM sensitivity in BC cells. In conclusion, this study highlights the critical role of the USP46/PTBP1/PKM2 axis in TAM resistance in BC. Targeted therapy against USP46 may represent a promising strategy to improve the prognosis of TAM-resistant patients.

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.

Underlying anti-cancer mechanisms of histone deacetylase (HDAC) inhibitors in tamoxifen-resistant breast cancer cells.

Breast cancer is an important women's malignancy with high cancer-related deaths worldwide. Drug resistance lowers the treatment efficacy in this malignancy. This study aimed to explore the underlying mechanisms of histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to overcome resistance to tamoxifen in breast cancer cells. Tamoxifen-resistance in MCF-7 breast cancer cells was simulated. MTT assay was used to detect the cytotoxic effects of HDAC inhibitor and PI3K inhibitor on the cancer cells. Trans-well assay was applied to evaluate the invasion and migration of the treated cancer cells. Flow cytometer assay was also applied to evaluate cell cycle phases in the treated cancer cells. Finally, expression of vascular endothelial growth factor (VEGF), E-cadherin, Vimentin, phosphorylated phosphatidylinositol kinase (p-PI3k), phosphorylated protein kinase B (p-AKT), and phosphorylated mammalian target protein of rapamycin (p-mTOR) was evaluated by western blotting. The obtained results indicated that HDAC inhibitor treatments significantly decreased viability, migration, and invasion in the cancer cells. Furthermore, the frequency of the treated cancer cells significantly increased in the S phase as well as significantly decreasing in the G2/M phase of the cell cycle. Moreover, HDAC inhibitor modified levels of VEGF, E-cadherin, Vimentin, p-PI3k, p-AKT, and p-mTOR proteins. However, HDAC inhibitor combined with PI3K inhibitor exerts more profound effects on the cancer cells as compared to HDAC inhibitor monotherapy. HDAC inhibitors inhibited the survival of breast cancer drug-resistant cells, invasion, migration, and angiogenesis by inhibiting the PI3k/Akt/mTOR signaling pathway.

FoxO3a Drives the Metabolic Reprogramming in Tamoxifen-Resistant Breast Cancer Cells Restoring Tamoxifen Sensitivity.

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy.

Role of monocarboxylate transporter I/lactate dehydrogenase B-mediated lactate recycling in tamoxifen-resistant breast cancer cells.

Although tamoxifen (TAM) is widely used in patients with estrogen receptor-positive breast cancer, the development of tamoxifen resistance is common. The previous finding suggests that the development of tamoxifen resistance is driven by epiregulin or hypoxia-inducible factor-1α-dependent glycolysis activation. Nonetheless, the mechanisms responsible for cancer cell survival and growth in a lactic acid-rich environment remain elusive. We found that the growth and survival of tamoxifen-resistant MCF-7 cells (TAMR-MCF-7) depend on glycolysis rather than oxidative phosphorylation. The levels of the glycolytic enzymes were higher in TAMR-MCF-7 cells than in parental MCF-7 cells, whereas the mitochondrial number and complex I level were decreased. Importantly, TAMR-MCF-7 cells were more resistant to low glucose and high lactate growth conditions. Isotope tracing analysis using 13C-lactate confirmed that lactate conversion to pyruvate was enhanced in TAMR-MCF-7 cells. We identified monocarboxylate transporter1 (MCT1) and lactate dehydrogenase B (LDHB) as important mediators of lactate influx and its conversion to pyruvate, respectively. Consistently, AR-C155858 (MCT1 inhibitor) inhibited the proliferation, migration, spheroid formation, and in vivo tumor growth of TAMR-MCF-7 cells. Our findings suggest that TAMR-MCF-7 cells depend on glycolysis and glutaminolysis for energy and support that targeting MCT1- and LDHB-dependent lactate recycling may be a promising strategy to treat patients with TAM-resistant breast cancer.

Inhibiting MEK1 R189 citrullination enhances the chemosensitivity of docetaxel to multiple tumour cells.

Drug resistance is still a big challenge for cancer patients. We previously demonstrated that inhibiting peptidylarginine deiminase 2 (PADI2) enzyme activity with Cl-amine increases the efficacy of docetaxel (Doc) on tamoxifen-resistant breast cancer cells with PADI2 expression. However, it is not clear whether this effect applies to other tumour cells. Here, we collected four types of tumour cells with different PADIs expression and fully evaluated the inhibitory effect of the combination of PADIs inhibitor (BB-Cla) and Doc in vitro and in vivo on tumour cell growth. Results show that inhibiting PADIs combined with Doc additively inhibits tumour cell growth across the four tumour cells. PADI2-catalysed citrullination of MEK1 Arg 189 exists in the four tumour cells, and blocking the function of MEK1 Cit189 promotes the anti-tumour effect of Doc in these tumour cells. Further analysis shows that inhibiting MEK1 Cit189 decreases the expression of cancer cell stemness factors and helps prevent cancer cell stemness maintenance. Importantly, this combined treatment can partially restore the sensitivity of chemotherapy-resistant cells to docetaxel or cisplatin in tumour cells. Thus, our study provides an experimental basis for the combined therapeutic approaches using docetaxel- and PADIs inhibitors-based strategies in tumour treatment. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Candidate Oligo Therapeutic Target, miR-330-3p, Induces Tamoxifen Resistance in Estrogen Receptor-Positive Breast Cancer Cells via HDAC4.

Tamoxifen is a drug used for treating breast cancer (BC), especially for individuals diagnosed with estrogen receptor-positive (ER+) BC. Its prolonged use could reduce the risk of recurrence and significantly lengthen the survival rate of BC patients. However, an increasing number of patients developed resistance to tamoxifen treatment, which reduced therapeutic efficiency and caused substandard prognosis. Therefore, the exploration of the molecular processes involved in tamoxifen resistance (TR) is urgently required. This investigation aimed to elucidate the relationship of microRNA-330 (miR-330-3p) with the TR of BC. There is little information on miR-330-3p's link with drug-resistant BC, although it is well known to regulate cell proliferation and apoptosis. Primarily, miR-330-3p expression in parental BC (MCF7/T47D), TR (MCF7-TR), and T47D/TR cell lines was detected by qRT-PCR. Then, the impact of miR-330-3p on the TR of BC cells was assessed by a cell proliferation assay. Lastly, dual-luciferase reporter, qRT-PCR, and western blot assessments were carried out to identify histone deacetylase 4 (HDAC4) as the potential miR-330-3p target gene. The data indicated that miRNA-330 was overexpressed in TR ER+ BC cells and its overexpression could induce TR. Furthermore, miRNA-330 could also reduce the expression of HDAC4, which is closely linked to TR, and overexpression of HDAC4 could reverse miRNA-330-induced drug resistance. In summary, miR-330-3p could induce TR of ER+ BC cells by downregulating HDAC4 expression, which might be a novel marker of TR and a possible treatment target against BC patients who are tamoxifen-resistant.

High expression of autophagy-related gene EIF4EBP1 could promote tamoxifen resistance and predict poor prognosis in breast cancer.

Breast cancer (BC) remains a public health problem. Tamoxifen (TAM) resistance has caused great difficulties for treatment of BC patients. Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) plays critical roles in the tumorigenesis and progression of BC. However, the expression and mechanism of EIF4EBP1 in determining the efficacy of TAM therapy in BC patients are still unclear. To investigate the expression and functions of EIF4EBP1 in determining the efficacy of TAM therapy in BC patients. High-throughput sequencing data of breast tumors were downloaded from the Gene Expression Omnibus database. Differential gene expression analysis identified EIF4EBP1 to be significantly upregulated in cancer tissues. Its prognostic value was analyzed. The biological function and related pathways of EIF4EBP1 was analyzed. Subsequently, the expression of EIF4EBP1 was determined by real-time reverse transcription polymerase chain reaction and western blotting. Cell Counting Kit-8 assays, colony formation assay and wound healing assay were used to understand the phenotypes of function of EIF4EBP1. EIF4EBP1 was upregulated in the TAM-resistant cells, and EIF4EBP1 was related to the prognosis of BC patients. Gene Set Enrichment Analysis showed that EIF4EBP1 might be involved in Hedgehog signaling pathways. Decreasing the expression of EIF4EBP1 could reverse TAM resistance, whereas overexpression of EIF4EBP1 promoted TAM resistance. This study indicated that EIF4EBP1 was overexpressed in the BC and TAM-resistant cell line, which increased cell proliferation, invasion, migration and TAM resistance in BC cells.

Targeting HMG-CoA synthase 2 suppresses tamoxifen-resistant breast cancer growth by augmenting mitochondrial oxidative stress-mediated cell death

AimsIn this study, we aimed to investigate previously unrecognized lipid metabolic perturbations in tamoxifen-resistant breast cancer (BC) by conducting comprehensive metabolomics and transcriptomics analysis. We identified the role of 3-hydroxy-3-methylglutary-coenzyme-A-synthase 2 (HMGCS2), a key enzyme responsible for ketogenesis, in tamoxifen-resistant BC growth. Main methodsComprehensive metabolomics (CE-TOFMS, LC-TOFMS) and transcriptiomics analysis were performed to characterize metabolic pathways in tamoxifen-resistant BC cells. The upregulation of HMGCS2 were verified thorugh immunohistochemistry (IHC) in clinical samples obtained from patients with recurrent BC. HMGCS2 inhibitor was discovered through surface plasmon resonance analysis, enzyme assay, and additional molecular docking studies. The effect of HMGCS2 suppression on tumor growth was studied thorugh BC xenograft model, and intratumoral lipid metabolites were analyzed via MALDI-TOFMS imaging. Key findingsWe revealed that the level of HMGCS2 was highly elevated in both tamoxifen-resistant T47D sublines (T47D/TR) and clinical refractory tumor specimens from patients with ER+ breast cancer, who had been treated with adjuvant tamoxifen. Suppression of HMGCS2 in T47D/TR resulted in the accumulation of mitochondrial reactive oxygen species (mtROS) and apoptotic cell death. Further, we identified alphitolic acid, a triterpenoid natural product, as a novel HMGCS2-specific inhibitor that elevated mtROS levels and drastically retarded the growth of T47D/TR in in vitro and in vivo experiments. SignificanceEnhanced ketogenesis with upregulation of HMGCS2 is a potential metabolic vulnerability of tamoxifen-resistant BC that offers a new therapeutic opportunity for treating patients with ER+ BC that are refractory to tamoxifen treatment.

Pterostilbene-isothiocyanate reduces miR-21 level by impeding Dicer-mediated processing of pre-miR-21 in 5-fluorouracil and tamoxifen-resistant human breast cancer cell lines.

Converging evidences identifies that microRNA-21 (miR-21) is responsible for drug resistance in breast cancer. This study aims to evaluate the miR-21-modulatory potential of a hybrid compound, pterostilbene-isothiocyanate (PTER-ITC), in tamoxifen-resistant MCF-7 (TR/MCF-7) and 5-fluorouracil-resistant MDA-MB 231 (5-FUR/MDA-MB 231) breast cancer cell lines, established by repeated exposure to gradually increasing the concentrations of tamoxifen and 5-fluorouracil, respectively. The outcome of this study shows that PTER-ITC effectively reduced the TR/MCF-7 (IC50: 37.21µM) and 5-FUR/MDA-MB 231 (IC50: 47.00µM) cell survival by inducing apoptosis, inhibiting cell migration, colony and spheroid formations in TR/MCF-7 cells, and invasiveness of 5-FUR/MDA-MB 231 cells. Most importantly, PTER-ITC significantly reduced the miR-21 expressions in these resistant cell lines. Moreover, the downstream tumor suppressor target gene of miR-21 such as PTEN, PDCD4, TIMP3, TPM1, and Fas L were upregulated after PTER-ITC treatment, as observed from transcriptional (RT-qPCR) and translational (immunoblotting) data. In silico and miR-immunoprecipitation (miR-IP) results showed reduced Dicer binding to pre-miR-21, after PTER-ITC treatment, indicating inhibition of miR-21 biogenesis. Collectively, the significance of this study is indicated by preliminary evidence for miR-21-modulatory effects of PTER-ITC that highlights the potential of this hybrid compound as an miR-21-targeting therapeutic agent.

CircPVT1 promotes ER-positive breast tumorigenesis and drug resistance by targeting ESR1 and MAVS.

The molecular mechanisms underlying estrogen receptor (ER)-positive breast carcinogenesis and endocrine therapy resistance remain incompletely understood. Here, we report that circPVT1, a circular RNA generated from the lncRNA PVT1, is highly expressed in ERα-positive breast cancer cell lines and tumor samples and is functionally important in promoting ERα-positive breast tumorigenesis and endocrine therapy resistance. CircPVT1 acts as a competing endogenous RNA (ceRNA) to sponge miR-181a-2-3p, promoting the expression of ESR1 and downstream ERα-target genes and breast cancer cell growth. Furthermore, circPVT1 directly interacts with MAVS protein to disrupt the RIGI-MAVS complex formation, inhibiting type I interferon (IFN) signaling pathway and anti-tumor immunity. Anti-sense oligonucleotide (ASO)-targeting circPVT1 inhibits ERα-positive breast cancer cell and tumor growth, re-sensitizing tamoxifen-resistant ERα-positive breast cancer cells to tamoxifen treatment. Taken together, our data demonstrated that circPVT1 can work through both ceRNA and protein scaffolding mechanisms to promote cancer. Thus, circPVT1 may serve as a diagnostic biomarker and therapeutic target for ERα-positive breast cancer in the clinic.

Abstract P3-07-19: Anti-depressant Amitriptyline Augments the Efficacy of Tamoxifen in ER positive breast cancer

Abstract Anti-depressant Amitriptyline Augments the Efficacy of Tamoxifen in ER positive breast cancer Pitta Venkata Prabhakar1, Arhan D Rao1, Timilsina Santosh1, Deepika Singh1, Ratna Vadlamudi1,2, Virginia Kaklamani3, Manjeet Rao1,2. 1Greehey Children’s Cancer Research Institute, University of Texas Health San Antonio, San Antonio, TX, USA. 2Mays Cancer Center, University of Texas Health San Antonio, San Antonio, TX, USA. 3Department of Medicine, UT Health, San Antonio, USA. Background: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-related deaths in women worldwide, with 1 out of 8 women being diagnosed with invasive breast cancer during their lifetime. The majority of breast cancer patients (80%) are diagnosed with Estrogen Receptor positive (ER+) breast cancer. The most commonly used treatment option for ER+ Breast cancer is endocrine therapy, such as Tamoxifen(TAM). Although survival of breast cancer patients has dramatically improved with the use of endocrine therapy, acquired resistance and debilitating side effects of treatment became major concerns. Therefore, safer treatment options that effectively suppress cancer progression, reduce treatment associated side effects, and improve efficacy of standard of care therapies are much needed. Repurposing of clinically approved or investigational drugs has become promising alternative approach for treating cancer. Therefore, we tested the repurposing potential of anti-depressant Amitriptyline for the treatment of ER+ breast cancer. Methods: The anti-cancer effect of Amitriptyline was determined in vitro using short and long-term viability, migration, and apoptosis assays. To substantiate the in vitro data, the effect of Amitriptyline on tumor growth was assessed using orthotopic xenograft model. RNA-seq analysis was performed in Vehicle and Amitriptyline treated breast cancer cells to understand the mechanism of action. Finally, to test whether amitriptyline can sensitize ER+ breast cancer cells to endocrine therapy, we performed cell viability assays after treatment with Amitriptyline and Tamoxifen. Results: Amitriptyline treatment resulted in significant reduction of short term and long term viability, as well as, migration of ER+ BC cells. Furthermore, Amitriptyline treatment significantly increased apoptosis in BC cells. Our RNA-seq analysis revealed that Amitriptyline treatment inhibited important genes involved in cancer growth and survival including E2F signaling, G2/M pathway, and DNA repair pathways. Confirming our in vitro findings, Amitriptyline treatment blocked the growth of ER+ BC growth in pre-clinical orthotopic xenograft model of breast cancer. Importantly, Amitriptyline treatment sensitized ER+ BC to TAM, showing highly synergistic effects. Amitriptyline treatment significantly improved the effects of TAM on cell viability, survival, migration, and apoptosis. Furthermore, Amitriptyline augmented the efficacy of Tamoxifen in TAM resistant BC cells. Conclusion: Our study establishes potential of Amitriptyline as a repurposable drug as safe and robust treatment option for ER+ patients, either as a monotherapy or in combination. We are poised to begin a clinical trial and test the therapeutic efficacy of Amitriptyline for treating breast cancer patients. Citation Format: Prabhakar Pitta Venkata, Arhan D. Rao, Santosh Timilsina, Deepika Singh, Ratna K. Vadlamudi, Virginia Kaklamani, Manjeet K. Rao. Anti-depressant Amitriptyline Augments the Efficacy of Tamoxifen in ER 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 P3-07-19.

Abstract P4-02-08: Towards Precision Radiation Oncology: Endocrine Therapy Resistance as a Biomarker for Radiation Resistance in ER-Positive Breast Cancer

Abstract Pre-operative endocrine therapy use in post-menopausal women with localized, ER-positive breast cancer affords comparable rates of response and breast preservation, but lower toxicity relative to chemotherapy. Pre-operative endocrine therapy exerts selective pressure on cancer cells and promote evolution and/or enrichment of pathogenic alternations such as ESR1 mutations and other cellular adaptations. How such endocrine therapy-induced adaptations alter response to radiation therapy remains poorly defined. In this study, we show that diverse mechanisms that confer endocrine therapy resistance also drive radiation resistance by reprogramming of DNA repair pathways. We also show that BRD4, a member of bromodomain and extraterminal domain (BET) family of proteins, mediates such DNA repair reprogramming. BRD4 also plays a key role in trascriptional reprogramming in ER-positive breast cancer cells that confers endocrine therapy resistance. Thus, OTX015, a BET inhibitor with a favorable safety profile in early stage clinical trials, reverses both endocrine therapy resistance and radiation resistance in ER-positive breast cancer cells and tumors. Our findings are also consistent with reports that tamoxifen-resistant breast cancer cells are resistant to DNA damaging chemotherapeutic agents such as adriamycin and cisplatin. Overall, our findings suggest that endocrine therapy resistance in the pre-operative setting serves as a biomarker for reduced response to adjuvant radiation therapy, and that pharmacological BET inhibition reverses radiation resistance in such patients. The increasing use of “window of opportunity” studies to assess response to endocrine therapy in the pre-operative setting will further facilitate personalization of radiation treatments in these patients based on their response to endocrine therapy. Thus, we provides a therapeutic rationale for personalization of radiation treatments in breast cancer patients based on their response to endocrine therapy. We also provide a molecularly targeted approach for reversing radiation resistance in such patients. Thus, our study provides a framework for advancing Precision Radiation Oncology in breast cancer patients. Citation Format: SM Nashir Udden, Asal Rahimi, Dong W. Nathan Kim, Prasanna Alluri. Towards Precision Radiation Oncology: Endocrine Therapy Resistance as a Biomarker for Radiation Resistance in ER-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 P4-02-08.

Abstract P2-26-18: Adiponectin regulates stem cell activity in tamoxifen-resistant breast cancer cells

Abstract Introduction: Breast cancer is the most diagnosed neoplasia and the second leading cause of malignancy death in women. Drug resistance is still the major challenge in the clinical management of breast cancer patients. Indeed, despite the improvements in the early diagnosis and in the therapeutic approaches, many breast cancer patients experience disease relapse due to de novo or acquired drug resistance. Growing evidence recognized a small population of breast cancer cells, named Breast Cancer Stem Cells (BCSCs), as the leading cause of tumor progression, metastasis formation and resistance against conventional therapy. BCSCs have some specific properties such as self-renewal, differentiation into different cell types, migration, tumorsphere formation, antioxidative activity, that make tumors more aggressive. Tumor microenvironment plays a crucial role in the regulation of stem cell proliferation and resistance to apoptosis through the secretion of cytokines and growth factors. Adipocytes represent the most abundant cellular component of mammary microenvironment. The excessive fat accumulation in obesity leads to the development of a dysfunctional adipose tissue, producing an unbalance in adipokines secretion. Among the secreted factors, adiponectin plays a crucial role in breast cancer development and progression. The aim of the present study was to investigate the effects of low adiponectin level (5 μl/ml), hallmark of obese status, on BCSCs activity in hormone-resistant cells. Methods: We tested the ability of MCF-7 wild type (WT) and tamoxifen-resistant (TR) to grow as mammospheres (Mammospheres Forming Efficiency, MFE), measuring the ability to maintain cell viability (self-renewal) upon serial non-adherent passages. mRNA levels of stemness, EMT and cell cycle markers were evaluated by qRT-PCR. CD44+/24- cell ratio, ALDH expression, ROS production, cell cycle, were analyzed by flow cytometry. Results: Adiponectin treatment significantly enhanced MFE and self-renewal capacity in TR-MCF-7 cells compared to MCF-7 WT cells. To identify the presence of BCSCs into mammospheres it has been evaluated the CD44+/CD24– biomarker signature. Flow cytometry revealed an enrichment of CD44, a trans-membrane glycoprotein which regulates growth signals in stem cells, in TR-MCF-7 mammospheres, whereas expression levels of the differentiation marker CD24 were decreased. The gene expression of these biomarkers was also analyzed by qRT-PCR. The increased BCSCs subpopulation in adiponectin-treated TR-MCF-7 mammospheres was also confirmed by the enhanced ALDH-expressing cells. qRT-PCR revealed that adiponectin increased the mRNA levels of stemness and EMT markers in TR-MCF-7 cells mammospheres. Interestingly, cell cycle analysis showed in adiponectin-treated TR-MCF-7 mammospheres a reduction of apoptosis. Moreover, flow cytometry analysis displayed a reduction of ROS levels, which generally are assumed as cues determining DNA damage-induced cell death, in adiponectin-treated TR-MCF-7 mammospheres. Conclusions: Our results demonstrated that low adiponectin level, as it occurs in obese breast cancer microenvironment, driving EMT, enhances stem-like features in TR-MCF-7 cells to sustain tumor progression. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin regulates stem cell activity in tamoxifen-resistant breast cancer cells [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 P2-26-18.