Development Of Tamoxifen Resistance Research Articles

Mechanisms of tamoxifen resistance: insight from long non-coding RNAs.

Breast cancer(BC) is the second most prevalent tumor in the world and one of the most lethal tumors in women. Patients with estrogen receptor-positive breast cancer can obtain significant advantages from endocrine therapies including tamoxifen, aromatase inhibitors, and others. However, the development of primary or acquired drug resistance ultimately leads to discontinuation of treatment with adverse consequences for breast cancer patients, and the underlying mechanisms have not been fully elucidated. Long non-coding RNAs (lncRNAs) play pivotal roles in orchestrating fundamental biochemical and cellular processes. They exert regulatory control over various processes including epigenetics, gene transcription, post-transcriptional modifications, and translation. Additionally, they influence key biological events like cell cycle progression, cell differentiation, and development. For the past few years, the relationship between lncRNAs and endocrine resistance has gained increasing attention, leading to a surge in related studies. LncRNAs mediate tamoxifen resistance in cancer by utilizing a variety of molecular mechanisms, including enhanced estrogen receptor (ER) signaling, inhibition of apoptosis, autophagy, exosome-mediated transfer, epigenetic alterations, epithelial-to-mesenchymal transition, and acting as competitive endogenous RNAs(ceRNAs). In this comprehensive review, we systematically summarize the critical role and intricate molecular mechanisms by which lncRNAs influence the development of tamoxifen resistance in breast cancer. Furthermore, we propose the potential clinical significance of lncRNAs as innovative therapeutic targets and prognostic biomarkers for breast cancer.

The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer

BackgroundThough tamoxifen achieves success in treating estrogen receptor α (ERα)-positive breast cancer, the followed development of tamoxifen resistance is a common challenge in clinic. Signals downstream of prolactin receptor (PRLR) could synergize with ERα in breast cancer progression. However, the potential effect of targeting PRL-PRLR axis combined with tamoxifen has not been thoroughly investigated.MethodsHigh-throughput RNA-seq data obtained from TCGA, Metabric and GEO datasets were analyzed to explore PRLR expression in breast cancer cell and the association of PRLR expression with tamoxifen treatment. Exogenous or PRL overexpression cell models were employed to investigate the role of activated PRLR pathway in mediating tamoxifen insensitivity. Immunotoxin targeting PRLR (N8-PE24) was constructed with splicing-intein technique, and the efficacy of N8-PE24 against breast cancer was evaluated using in vitro and in vivo methods, including analysis of cells growth or apoptosis, 3D spheroids culture, and animal xenografts.ResultsPRLR pathway activated by PRL could significantly decrease sensitivity of ERα-positive breast cancer cells to tamoxifen. Tamoxifen treatment upregulated transcription of PRLR and could induce significant accumulation of PRLR protein in breast cancer cells by alkalizing lysosomes. Meanwhile, tamoxifen-resistant MCF7 achieved by long-term tamoxifen pressure exhibited both upregulated transcription and protein level of PRLR. Immunotoxin N8-PE24 enhanced sensitivity of breast cancer cells to tamoxifen both in vitro and in vivo. In xenograft models, N8-PE24 significantly enhanced the efficacy of tamoxifen and paclitaxel when treating PRLR-positive triple-negative breast cancer.ConclusionsPRL-PRLR axis potentially associates with tamoxifen insensitivity in ERα-positive breast cancer cells. N8-PE24 could inhibit cell growth of the breast cancers and promote drug sensitivity of PRLR-positive breast cancer cells to tamoxifen and paclitaxel. Our study provides a new perspective for targeting PRLR to treat breast cancer.

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.

Metabolic/hypoxial axis predicts tamoxifen resistance in breast cancer

We sought in our cross-sectional study to investigate the role of metabolic/hypoxial axis in the development of tamoxifen (TMX) resistance in BC patients. Quantification of plasma LncRNA Taurine upregulated-1 (TUG-1), miRNA 186-5p (miR-186), serum Sirtuin-3 (SIRT3), Peroxisome Proliferator Activator Receptor alpha (PPAR-1 α) and Hypoxia Inducible Factor-1 (HIF-1α) was done in a cohort of patients divided into TMX-sensitive and TMX-resistant candidates. Multiple logistic regression and Receiver Operating Characteristic curve were developed for significant predictors. Plasma TUG-1 and miR-186 were significantly elevated in TMX resistant patients. Serum proteins SIRT3, PPAR-1 α and HIF-1α were deficient in TMX resistant patients compared to TMX sensitive patients, respectively. miR-186 was associated with respiratory symptoms, while, HIF-1α was associated with metastases in TMX resistant patients. Strong correlations were found between all parameters. A predictive model was constructed with TUG-1 and HIF-1α to estimate TMX resistance in BC patients with 88.3% sensitivity and 91.6% specificity. Hypoxia and metabolic dysregulations play important role in the development of TMX resistance in BC patients. Correlation between hypoxia, carcinogenesis and patient’s mortality have led to more aggressive phenotypes, increased risk of metastasis and resistance to TMX.

Genome-wide analysis of the FOXA1 transcriptional regulatory network identifies super enhancer associated LncRNAs in tamoxifen resistance.

Breast cancer is the leading cause of death in female cancers, and what’s worse, tamoxifen resistance occurs in almost 30% breast cancer patients and has seriously attenuated the therapeutic effect. It is widely studied that epigenetic regulation has played important role in the development of tamoxifen resistance. FOXA1 is a pioneer transcription factor that can translate epigenetic signature into transcription regulation and also drive genome-wide enhancer reprogramming in breast cancer. However, the chromatin super enhancer landscape orchestrated by FOXA1 and the key downstream targets of the FOXA1 oncogenic network in tamoxifen resistance remain elusive. Through analyzing the FOXA1 ChIP-seq data in tamoxifen sensitive MCF7 and tamoxifen resistant MCF7/TamR cells, we show that the FOXA1 chromatin occupancy is enhanced in both the promoter and enhancer regions, and the recruitment events may be E2 dependent in both MCF7 and MCF7/TamR cells. By integratively analyzing the FOXA1 ChIP-seq data and RNA-seq data of MCF7 and MCF7/TamR cells, we find that the enhanced or reduced FOXA1 chromatin binding densities may synchronize the transcriptional activity in tamoxifen resistance. Besides, we identify 1003 super enhancer associated protein coding genes and five super enhancer associated lncRNAs (ATP1A1−AS1, CASC11, CASC15, KCTD21−AS1, LINC00885) in tamoxifen resistance. By KM survival analysis, we find that high expression level of ATP1A1−AS1 and its sense transcript ATP1A1 indicates favorable clinical outcome among the luminal endocrine treated breast cancer patients. Further coexpression analysis indicates that ATP1A1-AS1 is significantly correlated with ATP1A1, and RT-qPCR results show that they both are downregulated in MCF7/TamR cells. Our study shows that the FOXA1 transcriptional regulatory network may promote the development of tamoxifen resistance, and identifies one super enhancer associated lncRNA ATP1A1-AS1 that may work as promising biomarker or drug target in tamoxifen resistance.

MiR-489 Confines Uncontrolled Estrogen Signaling through a Negative Feedback Mechanism and Regulates Tamoxifen Resistance in Breast Cancer

Approximately 75% of diagnosed breast cancer tumors are estrogen-receptor-positive tumors and are associated with a better prognosis due to response to hormonal therapies. However, around 40% of patients relapse after hormonal therapies. Genomic analysis of gene expression profiles in primary breast cancers and tamoxifen-resistant cell lines suggested the potential role of miR-489 in the regulation of estrogen signaling and development of tamoxifen resistance. Our in vitro analysis showed that loss of miR-489 expression promoted tamoxifen resistance, while overexpression of miR-489 in tamoxifen-resistant cells restored tamoxifen sensitivity. Mechanistically, we found that miR-489 is an estrogen-regulated miRNA that negatively regulates estrogen receptor signaling by using at least the following two mechanisms: (i) modulation of the ER phosphorylation status by inhibiting MAPK and AKT kinase activities; (ii) regulation of nuclear-to-cytosol translocation of estrogen receptor α (ERα) by decreasing p38 expression and consequently ER phosphorylation. In addition, miR-489 can break the positive feed-forward loop between the estrogen-Erα axis and p38 MAPK in breast cancer cells, which is necessary for its function as a transcription factor. Overall, our study unveiled the underlying molecular mechanism by which miR-489 regulates an estrogen signaling pathway through a negative feedback loop and uncovered its role in both the development of and overcoming of tamoxifen resistance in breast cancers.

EGFR inhibition reverses epithelial‑mesenchymal transition, and decreases tamoxifen resistance via Snail and Twist downregulation in breast cancer cells.

Tamoxifen resistance remains a major obstacle in the treatment of estrogen receptor (ER)‑positive breast cancer. In recent years, the crucial role of the epithelial‑mesenchymal transition (EMT) process in the development of drug resistance in breast cancer has been underlined. However, the central molecules inducing the EMT process during the development of tamoxifen resistance remain to be elucidated. In the present study, it was demonstrated that tamoxifen‑resistant breast cancer cells underwent EMT and exhibited an enhanced cell motility and invasive behavior. The inhibition of snail family transcriptional repressor1 (Snail) and twist family BHLH transcription factor1 (Twist) reversed the EMT phenotype and decreased the tamoxifen resistance, migration and invasion of tamoxifen‑resistant breast cancer cells. In addition, it was observed that the inhibition of epidermal growth factor receptor (EGFR) reversed the EMT phenotype in tamoxifen‑resistant MCF7 (MCF‑7/TR) cells via the downregulation of Snail and Twist. Notably, the EGFR inhibitor, gefitinib, decreased tamoxifen resistance, migration and invasion through the inhibition of Snail and Twist. On the whole, the results of the present study suggest that EGFR may be a promising therapeutic target for tamoxifen‑resistant breast cancer. Moreover, it was suggested that gefitinib may serve as a potent novel therapeutic strategy for breast cancer patients, who have developed tamoxifen resistance.

Automated online monitoring of lactate and pyruvate in tamoxifen resistant MCF-7 cells using sequential-injection capillary electrophoresis with contactless conductivity detection (SI-CE-C4D) and correlation with MCT1 and MCT4 genes expression

Breast cancer is among the most common cancer types worldwide. The first and second line treatment protocols for various stages of breast cancer in females rely on tamoxifen. Until now, the development of tamoxifen resistance is not entirely understood. In this study, an automated sequential injection capillary electrophoresis with capacitively coupled contactless conductivity detector was developed for online levels monitoring of both lactate and pyruvate from the supernatant media of MCF-7 cells developing tamoxifen resistance. Changes in concentration of the two metabolites were simultaneously monitored from three model cell cultures and two control untreated cells. The electrophoretic separation was performed under reversed electroosmotic flow conditions. The system delivers high sample throughput at low sample consumption of 20 µL per analysis. The method is robust and achieved high sensitivity and resolution. Limits of detection were 8.0 and 17.0 nM and linear ranges were 0.15–5 and 0.01–1 mM with a correlation coefficient of 0.9951 and 0.9963 for lactate and pyruvate, respectively. Inter-run and intra-run accuracy were in the range of 95.37–107.02% with precision (RSD, %) of ≤9.84%. The method was completely validated and the data obtained were in agreement with results achieved using the lactate and pyruvate assay kits. The highly informative generated data revealed a significant increase in lactate and pyruvate production in the three tamoxifen resistant MCF-7 cells models compared to the two control cells starting from 5.8 hours and 6.8 hours culturing times, respectively. The increase in concentrations of both lactate and pyruvate were correlated with an increase in MCT1 and MCT4 genes expression.

Abstract P4-02-16: Investigating the role of NRIP1 as a novel marker and therapeutic target for breast cancer

Abstract Background:. Tamoxifen and fulvestrant target estrogen receptor (ER), which is overexpressed in more than 80% of breast cancer (BC) cases, and have significantly improved the survival rate. However, in most metastatic cases, resistance is inevitable. Identifying gene targets for improving the efficiency of hormone therapy, delaying the progress of resistance development, and restoring the efficacy of hormone therapy has great significance. Epidemiological studies of human populations found that early age at menarche significantly associates with the risk of BC. Remarkably, a five-year delay in menarche has been shown to correspond to a 35% reduction of BC. This suggests that identifying genes that regulate female sexual maturation (FSM) might provide a novel approach for exploring the genetic and molecular mechanisms of BC, providing clues in the search for new therapeutic targets. We previously identified and verified that nuclear receptor interacting protein 1 (Nrip1, also named Rip140) is a regulator of female sexual maturation. NRIP1, a co-regulator of nuclear receptors, interacts with several known signaling pathways for BC, such as E2F and estrogen receptor (ER). Human genome-wide association studies suggested that polymorphisms of NRIP1 are significantly associated with the risk of BC. Furthermore, elevating NRIP1 can promote epithelial cells proliferation and increase the development rate and size of the mammary gland, while depleting NRIP1 expression has the opposite effect. Importantly, clinical studies suggested that breast cancer patients with higher level of NRIP1 have a relatively poorer prognosis. Experiments and results:. 1. To investigate the role of NRIP1 in BC, we first measured NRIP1 expression in cancers. We found that NRIP1 was overexpressed in the tissues of BC and the BC cell lines. Importantly, immunohistochemistry assay showed that that NRIP1 is elevated in BC cell lines and tissues from patients with BC. Interestingly, overexpressed NRIP1 accumulated in the cytoplasm of the stromal cells in the benign tumors; but, in the malignant tumors, there was more accumulation in the nuclei of epithelia. Further studies revealed that suppressing NRIP1 could increase apoptosis of cancer cells in vitro and suppress the initiation and growth of DMBA (7,12-Dimethylbenz(a)anthracene) induced breast tumors. 2. Our previous studies have shown that deletion of NRIP1, in a variety of cell types, could suppress the activity of NFκB, which promotes the development of hormone therapy resistance; therefore, indicating that the suppression of NRIP1 may also suppress hormone therapy resistance. To test this hypothesis, we induced tamoxifen resistance in MCF7, which is estrogen receptor positive. During the induction, we found that NRIP1 expression increased gradually with the development of tamoxifen resistance. Interestingly, along with the upregulation of NRIP1 expression, we found that NRIP1 was translocated from the cytosol into the nucleus. These results suggest that not only the level of NRIP1 expression, but also the location, are related to the development of tamoxifen resistant. Furthermore, we found that suppressing the expression of NRIP1 by siRNA in tamoxifen resistant MCF7 (MCF7-TR) could significantly reduce the percentage of cells in the S phase and increase apoptosis with or without tamoxifen treatment. Conclusion:. 1. Investigating the genetic regulation of FSM may provide clues for identifying novel BC related genes. 2. NRIP1 plays an important role in BC. 3. Inhibiting NRIP1 may suppress the initiation and growth of BC. 4. NRIP1 overexpression and nuclear translocation are involved in the development of hormone therapy resistance. 5. The deletion of NRIP1 can suppress the development tamoxifen resistance in vitro. Citation Format: Yun Zhu, Fawwad Zaidi, Krishna Rao, Rong Yuan. Investigating the role of NRIP1 as a novel marker and therapeutic target for breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-16.

Multi-time scale transcriptomic analysis on the dynamic process of tamoxifen resistance development in breast cancer cell lines.

Approximately 30% of breast cancer patients develop endocrine resistance after tamoxifen therapy. There still lacks a comprehensive understanding on the mechanism of tamoxifen resistance. This study aims to explore the dynamic process of ER + breast cancer resistance to tamoxifen through the time course transcriptomic analysis. The transcriptome profiles of human breast cancer cell line MCF-7 treated with tamoxifen at different time scales were collected from LINCS, SRA and GEO databases. Differentially expressed genes (DEGs) were identified in the short-term tamoxifen treatment and tamoxifen-resistant cell lines. The time course analysis was used to explore the dynamic development of tamoxifen resistance using the transcriptome profiles of tamoxifen-cultured MCF-7 for 1-12weeks. After the short-term treatment of MCF-7 with tamoxifen for 6h or 24h, the expression level of gene PRSS23 was significantly reduced. However, its expression recovered in the resistant cell lines. The time course analysis identified 9 clusters of the DEGs based on the temporal trend of their expression levels. Gene PRSS23 belongs to cluster 2 in which the expression levels were significantly down-regulated in the first 4weeks but gradually recovered afterwards. Functional enrichment analysis of the DEGs in cluster 2 showed that they are significantly enriched in DNA replication, mismatch repair and cell cycle pathways. Their specific role in the resistance development needs to be further explored. The protein-protein interaction network analysis indicates that gene PRSS23 participates in the drug resistance by regulating multiple tamoxifen drug targets. The acquired drug resistance in ER + breast cancer is a complex and dynamic biological process. PRSS23 plays an important role in the development of resistance and is a potential target for overcoming resistance.

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.

SETD1A-SOX2 axis is involved in tamoxifen resistance in estrogen receptor α-positive breast cancer cells.

Rationale: Approximately 30-40% of estrogen receptor (ER)-positive breast cancer (BC) cases recur after tamoxifen therapy. Thus, additional studies on the mechanisms underlying tamoxifen resistance and more specific prognostic biomarkers are required. In this study, we investigated the role of the SET domain containing 1A (SETD1A), a histone H3-lysine 4 (H3K4) methyltransferase, in the development of tamoxifen resistance in BC.Methods: The relationship between tamoxifen resistance and SETD1A protein level was investigated using resistant cell lines derived from the parent BC cells. Biochemical and molecular assays, such as RNA-sequencing, reverse transcription-quantitative polymerase chain reaction, chromatin-immunoprecipitation, and protein-binding assays, were used to identify the SETD1A target gene in tamoxifen-resistant BC cells. Additionally, the role of SETD1A in cancer stem cells (CSCs) was investigated using CSCs isolated from tamoxifen-resistant BC cells. Comprehensive transcriptome analysis and immunofluorescence staining using clinical datasets and tissue microarray were performed to determine the correlation between the expression of the SETD1A-SRY-box transcription factor 2 (SOX2) pair and recurrence in tamoxifen-treated patients with BC.Results: SETD1A was expressed at higher levels in tamoxifen-resistant BC cells than in primary BC cells. Notably, SETD1A-depleted tamoxifen-resistant MCF-7 cells showed restored sensitivity to tamoxifen, whereas SETD1A overexpression in MCF-7 cells resulted in decreased sensitivity. SETD1A is recruited to the SOX2 gene via its interaction with SOX2, thereby enhancing the expression of SOX2 genes in tamoxifen-resistant BC cells. The growth of tamoxifen-resistant cells and CSCs was effectively suppressed by SETD1A knockdown. In addition, high levels of SETD1A and SOX2 were significantly correlated with a low survival rate in patients with ER-positive tamoxifen-resistant BC.Conclusion: Our findings provide the first evidence of the critical role of the SETD1A-SOX2 axis in tamoxifen-resistant BC cells, implying that SETD1A may serve as a molecular target and prognostic indicator of a therapeutic response in patients with tamoxifen-resistant BC.

Lactate and pyruvate levels correlation with lactate dehydrogenase gene expression and glucose consumption in Tamoxifen-resistant MCF-7 cells using capillary electrophoresis with contactless conductivity detection (CE-C4 D).

Breast cancer is the second leading cause of cancer death in women after lung cancer. The first-line treatment of metastatic breast cancer in premenopausal women relies on tamoxifen. The development of tamoxifen resistance is not fully understood. In this study, capillary electrophoresis with capacitively coupled contactless conductivity detector was developed to monitor the changes in lactate and pyruvate levels in supernatant media of three models of developed MCF-7 tamoxifen-resistant cells and correlate these metabolites changes with lactate dehydrogenase genes expression and glucose consumption. The electrophoretic separation was achieved under reversed electroosmotic flow conditions. The linear ranges were 0.15-5 and 0.01-1mM with a correlation coefficient of 0.9966 and 0.9971 and the limits of detection were 0.01 and 0.02μM for lactate and pyruvate, respectively. Inter- and intrarun accuracy were in the range of 96.88-105.94% with precision (CV, %) of ≤7.35%. The method was completely validated and the results were in agreement with those obtained using the lactate and glucose assay kits. The results revealed a significant increase in both lactate and pyruvate production in the three tamoxifen-resistant MCF-7 cells models compared to control cells. This increase was correlated with the increase of lactate dehydrogenase genes expression and the increase of glucose consumption.

Long non-coding RNA H19 mediates N-acetyltransferase 1 gene methylation in the development of tamoxifen resistance in breast cancer.

Long non-coding RNA (lncRNA) H19 is associated with proliferation, invasion and metastasis in numerous types of cancer. H19 lncRNA has been demonstrated to be an estrogen-inducible gene, the expression of which is significantly increased in tamoxifen (TAM)-resistant MCF-7 breast cancer cells. The aim of the present study was to investigate the role and molecular mechanism of lncRNA H19 in the development of TAM resistance. TAM-resistant MCF-7 (MCF-7R) cells were developed by the treatment of wild-type MCF-7 cells with 4-hydroxytamoxifen. Analysis of H19 expression in the cells indicated that upregulation of H19 contributed to the resistance of the MCF-7R cell line. Furthermore, when H19 was knocked down in the MCF-7R cells, the sensitivity to 4-hydroxytamoxifen was markedly restored. The results further demonstrated that N-acetyltransferase 1 (NAT1) may serve an important role in TAM-resistant cells, as NAT1 expression was notably downregulated in the MCF-7R cells but significantly elevated following the knockdown of H19. In addition, lower expression of NAT1 and higher expression of H19 were indicated to be associated with poor prognosis in patients with breast cancer treated with TAM. The results of bisulfite genomic sequencing PCR analysis indicated that the methylation rate of NAT1 in MCF-7R cells was significantly higher compared with that in MCF-7 cells, while the methylation rate of NAT1 in TAM-resistant cells transfected with small interfering RNA against H19 was significantly lower than that in the corresponding untransfected cells. Therefore, the present study suggests that the H19 gene regulates NAT1 expression in TAM-resistant cells via the mediation of NAT1 promoter methylation.

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography–tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid–ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11–2.25, 0.012–0.227, and 0.02–0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.

PI3K/AKT and MAPK1 molecular changes preceding matrix metallopeptidases overexpression during tamoxifen-resistance development are correlated to poor prognosis in breast cancer patients.

Metastasis and drug resistance remain a persistent key clinical obstacle to the success of breast cancer treatments. Recent years have seen an increased focus on understanding the factors that influence metastasis and drug resistance. In this study, the changes in MMPs gene expression were investigated together with their regulatory pathways-PI3K, MAPK and NFKβ pathways-during the process of developing tamoxifen resistance in MCF7 cell line. Gene correlation maps and Kaplan-Meier survival plots among all breast cancer patients and patients treated with tamoxifen were evaluated. MMPs gene expression was found to be up regulated in MCF7 cell line treated with tamoxifen during the development of tamoxifen resistance using two approaches. Up-regulation of gene expression of AKT1 and MAPK1 started in cells treated with 10μM tamoxifen that was followed with up-regulation of other genes in these pathways and MMPs in cells treated with 35μM tamoxifen. MMPs and genes from PI3K, MAPK and NFKβ pathways showed highly significant increase of expression at 50μM or when cells were treated sequentially six times with 35μM. Furthermore, increased genes expression was associated with aggressive pattern, clear morphological changes, higher growth rate, increased migration and adhesion potential and tamoxifen insensitivity. Breast cancer distant metastasis-free survival, and survival among tamoxifen treated patients had high expression levels of MAPK1, AKT1, TIMP2, MMP1, and MMP9 showed poor prognosis. Early changes of MAPK1, AKT1 gene expression upon tamoxifen treatment could possibly be used as an early marker of resistance and future poor prognosis.

HOXB5 Confers Tamoxifen Resistance in Breast Cancer Cells and Promotes Tumor Aggression and Progression.

ER-positive breast cancer patients commonly undergo endocrine therapy with drugs such as tamoxifen. Despite tamoxifen being a highly effective drug, long-term treatment results in resistance in one-third of the patients. Although many explanations for the development of tamoxifen resistance have been put forward, a clearly defined underlying mechanism is still lacking. The expression level of HOXB5 was evaluated between MCF7 breast cancer cells and tamoxifen-resistant MCF7 (TAMR) cells by RT-PCR. Then, the effect of HOXB5 on invasion and migration abilities as well as on cancer stemness were investigated through 3D culture and spheroid formation assay. In this study, we provide evidence that HOXB5 is up-regulated in TAMR cells. EGFR is concurrently overexpressed, and the EGFR signaling cascade is activated, resulting in migratory and invasive phenotypes in TAMR cells compared to MCF7 cells. However, HOXB5 knockdown in TAMR cells resulted in the de-activation of the EGFR signaling pathway, less aggressive phenotypes and restoration of sensitivity to tamoxifen treatment. More interestingly, TAMR cells expressed higher levels of stem cell markers, and as a result, their enhanced stemness allowed for a better formation of spheroids than MCF7 cells. When HOXB5 was overexpressed in MCF7 cells, they were able to form a larger number of spheroids as in TAMR cells. HOXB5 is one of the key factors involved in tumor aggression and progression in tamoxifen-resistant breast cancer cells.

Changes in Lactate Production, Lactate Dehydrogenase Genes Expression and DNA Methylation in Response to Tamoxifen Resistance Development in MCF-7 Cell Line.

Lactate dehydrogenase (LDH) is a key enzyme in the last step of glycolysis, playing a role in the pyruvate-to-lactate reaction. It is associated with the prognosis and metastasis of many cancers, including breast cancer. In this study, we investigated the changes in LDH gene expression and lactate concentrations in the culture media during tamoxifen resistance development in the MCF-7 cell line, and examined LDHB promoter methylation levels. An upregulation of 2.9 times of LDHB gene expression was observed around the IC50 concentration of tamoxifen in treated cells, while fluctuation in LDHA gene expression levels was found. Furthermore, morphological changes in the cell shape accompanied the changes in gene expression. Bisulfate treatment followed by sequencing of the LDHB promoter was performed to track any change in methylation levels; hypomethylation of CpG areas was found, suggesting that gene expression upregulation could be due to methylation level changes. Changes in LDHA and LDHB gene expression were correlated with the increase in lactate concentration in the culture media of treated MCF-7 cells.

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.

Curcumin attenuates lncRNA H19‑induced epithelial‑mesenchymal transition in tamoxifen‑resistant breast cancer cells.

The H19 long non-coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen-resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF-7/TAMR cell epithelial-mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19-mediated effects. Reverse transcription-quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit-8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF-7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E-cadherin and markedly increased the expression levels of mesenchymal marker N-cadherin in MCF-7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose-dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19-induced alterations in N-cadherin and E-cadherin expression levels. Curcumin also prevented H19-induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF-7/TAMR cells, suggesting that curcumin may prevent H19-associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer.