Tamoxifen-resistant Cell Lines Research Articles

Abstract PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [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 PO3-28-12.

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.

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.

Discovery of a Novel Class of PROTACs as Potent and Selective Estrogen Receptor α Degraders to Overcome Endocrine-Resistant Breast Cancer In Vitro and In Vivo.

The estrogen receptor (ER) is a well-established target for endocrine therapies of ER-positive breast cancer (ER+ BC), but endocrine resistance limits the efficacy of clinical drugs. Using proteolysis targeting chimera (PROTAC) technology to degrade ERα may be an effective alternative to endocrine therapies. Herein, we disclose a novel series of potent and selective ERα PROTACs based on an oxabicycloheptane sulfonamide (OBHSA) scaffold, with no associated ERβ degradation. These PROTACs showed significant antiproliferation and ERα degradation activities against a broad spectrum of ER+ BC cells including tamoxifen-resistant and ERα mutant cell lines. Genomics analysis confirmed that these PROTACs inhibited the nascent RNA synthesis of ERα target genes and impaired genome-wide ERα binding. Compound ZD12 exhibited excellent antitumor potency and ERα degradation activity in both tamoxifen-sensitive and -resistant BC mice models, which are superior to fulvestrant. This study demonstrates the potential of these PROTACs as novel drug candidates for endocrine-resistant BC treatment.

Network-informed discovery of multidrug combinations for ERα+/HER2-/PI3Kα-mutant breast cancer

Breast cancer is a persistent threat to women worldwide. A large proportion of breast cancers are dependent on the estrogen receptor α (ERα) for tumor progression. Therefore, targeting ERα with antagonists, such as tamoxifen, or estrogen deprivation by aromatase inhibitors remain standard therapies for ERα + breast cancer. The clinical benefits of monotherapy are often counterbalanced by off-target toxicity and development of resistance. Combinations of more than two drugs might be of great therapeutic value to prevent resistance, and to reduce doses, and hence, decrease toxicity. We mined data from the literature and public repositories to construct a network of potential drug targets for synergistic multidrug combinations. With 9 drugs, we performed a phenotypic combinatorial screen with ERα + breast cancer cell lines. We identified two optimized low-dose combinations of 3 and 4 drugs of high therapeutic relevance to the frequent ERα + /HER2-/PI3Kα-mutant subtype of breast cancer. The 3-drug combination targets ERα in combination with PI3Kα and cyclin-dependent kinase inhibitor 1 (p21). In addition, the 4-drug combination contains an inhibitor for poly (ADP-ribose) polymerase 1 (PARP1), which showed benefits in long-term treatments. Moreover, we validated the efficacy of the combinations in tamoxifen-resistant cell lines, patient-derived organoids, and xenograft experiments. Thus, we propose multidrug combinations that have the potential to overcome the standard issues of current monotherapies.

Abstract P4-01-24: Targeting receptor tyrosine kinases in overcoming tamoxifen resistance and dormancy in invasive lobular cancer

Abstract Objective: The molecular hallmark of invasive lobular cancer (ILC) is the loss of E-cadherin, resulting in the unique morphology, low mitotic index and unusual metastatic spread to ovaries, gut, and peritoneum. Patients with ILC face delayed, and higher stage at diagnosis, worse disease-free and overall survival1. The most frustrating challenge is the delayed recurrence, likely due to resistance and induction of dormancy2. Despite these unique features, ILC is lumped with hormone receptor-positive invasive ductal cancers (IDC); consequently, management strategies are based on data from IDC. Hence there is an unmet need to address the unique challenges of ILC. We generated Tamoxifen-resistant (TAMR) ILC cell lines with the aim to study the distinct characteristics, dormancy, differentially activated pathways, and response to drugs targeting such pathways in the resistant cells. Methods: We used MDA-MB-134-VI and SUM44-PE cells to study the effect of tamoxifen in combination with multi-targeted receptor tyrosine kinase inhibitor, Lenvatinib. TAMR cells from both MB-134 and SUM44PE cells were generated by growing them in increasing concentrations of 4-hydroxy tamoxifen (4-HT) up to 500nM, for 6 months. Total RNA from parental and TAMR cells were subjected to RNA-seq. Whole cell extracts were analyzed for specific phospho proteins by Western Blot analysis. Growth kinetics and effect of drugs on cell viability was measured using MTT assay (Roche). Cell migration was measured using Transwell migration assay. Novel engineered 2D and 3D cultures were used to study dormancy and the effect of the drug combination. Orthotopic tumor induction in NSG mice is ongoing to determine the effect of drug combination in vivo. Results: The TAMR cell lines show distinct morphological features, small but significant increase in growth rate (p=0.05) and remarkably higher migration (MB-134TAMR:11.5-fold, p< 0.005 and SUM44TAMR:>100-fold, p< 0.005). The IC50 for tamoxifen increased from 8.1M (MB-134) to 16.8M (MB-134TAMR) and from 11.3 M (SUM44) to 26.6 M (SUM44TAMR). RNA seq analysis revealed enrichment of PI3K-AKT, MAPK, cAMP, Rap1 signaling pathways, ECM-receptor interaction, Focal adhesion, and steroid hormone biosynthesis pathways in the TAMR cells. Upon stimulation by endothelial growth factor, the MB-134TAMR but not the parental cells showed dose dependent increase in phospho-AKT and phospho-MAPK levels. Using novel 2D/3D cultures, we show differential morphologies between IDC (MCF7) cells, MB-134TAMR, and the parental cells. MCF7 and MB-134TAMR cells are more adherent to fibronectin (p< 0.0001), whereas the parental ILC cells are more adherent to COL3A1 (p< 0.0001), a feature observed in dormancy3. Further, the administration of 5 M 4-HT induced a cessation of growth in the parental cell line, with a sub-population of apparent dormant cells. The combination of Lenvatinib (5 M) and 4-HT (7.5 M) synergistically inhibited both the parental cell lines by ~60%. Lenvatinib (5 M) and 4-HT (15 M) inhibited the MB-134TAMR and SUM44TAMR cell lines synergistically by 65% and 45% respectively. In vivo, the growth of MB-134 induced tumors were completely suppressed when 5mg sustained release pellet of TAM citrate was injected in the subscapular region of tumor bearing mice (tumor @100cm3). The results of the combination using in vivo models and 2D/3D cultures will be reported. Conclusion and significance: Lenvatinib with tamoxifen is active and synergistic against parental and TAMR ILC cell lines. Our novel 2D/3D cultures show distinct pattern of growth for ILC cells and a possible induction of a dormant subpopulation upon 4-HT treatment. Overcoming resistance to conventional therapies and preventing induction of dormancy will improve long-term outcomes of patients with ILC. 1. PMID:33641217 2. PMID: 34388695 3. PMID: 35121989 Citation Format: Bhuvaneswari Ramaswamy, Nikhil Pramod, Anagh Kulkarni, Xilal Y. Rima, Eduardo Reátegui, Eswar Shankar, Sarmila Majumder. Targeting receptor tyrosine kinases in overcoming tamoxifen resistance and dormancy in invasive lobular 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 P4-01-24.

Abstract P5-13-06: Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth

Abstract Background: We had identified somatic mutations of the NAD(P) Dependent Steroid Dehydrogenase-Like (NSDHL) gene from breast tumors of patients with distant metastasis using whole-exome sequencing. This study aimed to investigate the function of NSDHL mutants in breast cancer. Methods: ER-positive and tamoxifen-resistant human breast cancer cell line (ZR75-1) and mouse macrophage cell line (RAW264,7) were used. GFP-tagged NSDHL mutants were generated by subcloning the mutant NDHL into a lentiviral vector yielding an in-frame fusion of 3′ to GFP. ZR75-1 cells expressing the GFP-tagged NSDHL mutants were selected by puromycin with subsequent FACS analysis. The following experiments were performed: western blot, immunofluorescence staining, qRT-PCR, CellTiter-Glo assay, cell cycle assay, transwell migration assay, wound healing assay, tumor spheroid formation assay, and total cellular cholesterol measurement. An orthotopic breast tumor mouse model by injection of ZR75.1 cells into mammary gland fat pad was used in vivo. Results: Four novel NSDHL somatic mutations were discovered in the breast tumor tissues of patients. While total cholesterol levels in NSDHL mutant-transduced cells did not significantly increase, they were notably higher in wild-type NSDHL-transduced cells than in parent ZR75-1 cells. When compared to the parent and wild-type NSDHL-transduced cells, there was a minor increase in growth rate and a large increase in migratory capacities in NSDHL mutant-transduced cells (P< 0.05). The size of tumor spheroids is significantly larger in NSDHL mutant-transduced cells than in wild-type NSDHL-transduced cells. Both EGFR expression and epithelial to mesenchymal transition (EMT) markers were higher in NSDHL mutant-transduced cells than in parent and wild-type NSDHL-transduced cells. The NSDHL mutant-transduced cells’ conditioned media promoted the migratory ability and M2 polarization of RAW264.7 cells. In an orthotopic xenograft mouse model, NSDHL mutant-transduced cells were shown to promote tumor growth better than parent and wild-type NSDHL-transduced cells, suggesting that somatic mutation of the NSDHL contributes to the malignant progression of breast cancer cells. Conclusion: The present data indicate that breast cancer cells harboring somatic mutants of the NSDHL gene seem to display more aggressive behavior by gaining biological worse phenotype both in vitro and in vivo. More investigation is required into the mechanisms behind the function and accumulation of mutant NSDHL proteins to speed the development of therapies for patients with breast cancer harboring mutant NSDHL. Citation Format: Moonjou Baek, Hoe Suk Kim, So-Hyun Yoon, Seungyeon Ryu, Sangeun Lee, Kyoungsook Park, Han-Byoel Lee, Wonshik Han. Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth [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 P5-13-06.

Abstract P3-11-02: CST1 Interaction with RAB1B Modulates Tamoxifen resistance of Breast Cancer by Regulating Autophagy

Abstract Background: Breast cancer is a prominent cause of cancer-related death among women worldwide. Approximately,70% of breast cancers (BC) are estrogen and progestogen receptor-positive (HR+) at diagnosis. Adjuvant endocrine therapy is a pivotal component of treatment for this type of patient. Tamoxifen (TAM), a widely recommended drug in endocrine therapy, is confronted with a high risk of recurrence after 5 years of treatment among breast cancer patients, which shows big clinical demands. Therefore, it is urgent to find new targets for tamoxifen resistance therapy. Recently, our team found that enhanced expression of Cystain SN (CST1) was detected in tissues that had undergone tamoxifen treatment appearing to recurrence and metastasis in ER-positive breast cancer patients. However, the molecular mechanisms of modulating tamoxifen resistance remain unknown. Therefore, our team suspected that there might be a correlation between the abnormal expression of CST1 and tamoxifen resistance. To investigate whether CST1 is involved in tamoxifen resistance and its molecular mechanism, we conducted further research. It is critical to elucidate the molecular mechanisms of tamoxifen resistance and to explore a novel effective therapeutic target. Method: Enhanced expression of CST1 was confirmed by immunohistochemistry in breast cancer tissue samples and by quantitative real-time PCR in Tamoxifen-resistant cancer cell lines MCF-7R/T47DR. To further investigate the molecular function of CST1, we knockdown its expression in MCF-7R/T47DR cells by transfecting with the lentiviral particles of shCST1 and corresponding negative control (Shanghai Genechem Co., Ltd.) and overexpressed CST1 in MCF-7/T47D cells using ove-GST-CST1, ove-HA-RAB1B plasmids. The effects of CST1 on cell viability and proliferation in breast cancer cells were detected by cell counting kit-8 and colony formation assays, respectively. The interaction between CST1 and RAB1B was validated by Co-immunoprecipitation and Western blot. Autophagosomes were found using transmission electron microscopy (TEM) along with Western blotting to detect the transformation of LC3-I to LC3-II protein (i.e., the LC3-II/LC3-I ratio) and P62 protein levels. Results: We found that aberrant expression of Cystatin SN (CST1) was identified in ER-positive breast cancer (BC) cells (Figure 1) and which is crucial in contributing to Tamoxifen resistance (Figure 2). The direct promotion of autophagy by RAPA can faciliate cells to endocrine resistance. And knockdown of CST1 not only suppressed autophagy in Tamoxifen-resistant cells but also attenuated the resistance, which determined the correlation of CST1 with autophagy in BC (Figure 3). Rab1b (Ras-related protein Rab-1B), a membrane protein essential for autophagosome formation, was verified to have interaction with CST1 through Coimmunoprecipitation (CO-IP) experiments (Figure 4). Meanwhile, RAB1B expression was also reduced by CST1 knockdown in the resistant cells. The interaction between CST1 and RAB1B induces autophagy which decreases Tamoxifen’s therapeutic efficacy. Furthermore, rescue experiments suggested that CST1 knockdown–induced sensitization in the efficacy of TAM Therapy and decreased autophagy could be restored via RAB1B overexpression.Conclusions: These results indicate that CST1 interacts with RAB1B to facilitate BC resistant to TAM through regulation of autophagy and CST1 might be a potential therapeutic target to overcome endocrine therapy resistance. Figure 1. Enhanced expression of CST1 in Tamoxifen-resistance breast cancer Figure 1. Enhanced expression of CST1 in Tamoxifen-resistance breast cancer Figure 2. CST1 expression is positively correlated with autophagy in ER-positive breast cancer Figure 2. CST1 expression is positively correlated with autophagy in ER-positive breast cancer Figure 3. CST1 promotes cells resistant to tamoxifen therapy by activating auophagy Figure 3. CST1 promotes cells resistant to tamoxifen therapy by activating auophagy Figure 4. CST1 interacts with RAB1B in cell autophagy Figure 4. CST1 interacts with RAB1B in cell autophagy Citation Format: li liu. CST1 Interaction with RAB1B Modulates Tamoxifen resistance of Breast Cancer by Regulating Autophagy [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 P3-11-02.

Super-enhancer-controlled positive feedback loop BRD4/ERα-RET-ERα promotes ERα-positive breast cancer.

Estrogen and estrogen receptor alpha (ERα)-induced gene transcription is tightly associated with ERα-positive breast carcinogenesis. ERα-occupied enhancers, particularly super-enhancers, have been suggested to play a vital role in regulating such transcriptional events. However, the landscape of ERα-occupied super-enhancers (ERSEs) as well as key ERα-induced target genes associated with ERSEs remain to be fully characterized. Here, we defined the landscape of ERSEs in ERα-positive breast cancer cell lines, and demonstrated that bromodomain protein BRD4 is a master regulator of the transcriptional activation of ERSEs and cognate ERα target genes. RET, a member of the tyrosine protein kinase family of proteins, was identified to be a key ERα target gene of BRD4-regulated ERSEs, which, in turn, is vital for ERα-induced gene transcriptional activation and malignant phenotypes through activating the RAS/RAF/MEK2/ERK/p90RSK/ERα phosphorylation cascade. Combination therapy with BRD4 and RET inhibitors exhibited additive effects on suppressing ERα-positive breast cancer both in vitro and in vivo, comparable with that of standard endocrine therapy tamoxifen. Furthermore, combination therapy re-sensitized a tamoxifen-resistant ERα-positive breast cancer cell line to tamoxifen treatment. Taken together, our data uncovered the critical role of a super-enhancer-associated positive feedback loop constituting BRD4/ERα–RET–ERα in ERα-positive breast cancer, and suggested that targeting components in this loop would provide a new therapeutic avenue for treating ERα-positive breast cancer in the clinic.

Role of β1-integrin in promoting cell motility and tamoxifen resistance of human breast cancer MCF-7 cells.

The mechanism of acquired resistance of tamoxifen in endocrine therapy of breast cancer is not fully understood. In this study, we investigated the genomic changes in acquired tamoxifen-resistant cell lines. Tamoxifen-resistantsubclones (MCF-7R) derived from parent MCF-7 cells, which is an ER(+) breast cancer cell line, cultured with 4-hydrotamoxifen more than 6 months were used to obtain genomic alterations. Cell growth, microarray, and quantitative real-time PCR (q-RTPCR) assays were conducted. Additionally, the ITGB1 function was investigated in MCF-7R cells and MCF-7R ITGB1-silenced subclones using MTT and Transwell assays. Online pathway analysis was performed to assess the genetic characteristics of tamoxifen resistance. The gene expression profile of the tamoxifen-resistant cell line was considerably changed compared to the tamoxifen-sensitive cell line. Of 4102 genes with altered expressions, 1986 genes were upregulated, whereas 2116 were downregulated. The ITGB1 expression in MCF-7R cells was higher than that in MCF-7 cells. Interestingly, ITGB1 silencing partially rescued the sensitivity of MCF-7R cells to tamoxifen and reduced their motility. The activation of the β1-integrin signaling pathway was probably responsible for this phenomenon. Our data confirm the presence of alterations in the genes of tamoxifen-resistance breast cancer cells. ITGB1 probably partially contributes to tamoxifen resistance and cell motility via the β1-integrin signaling pathway. Thus, ITGB1 may be a potential target for the improvement of anti-hormone therapy reaction in ER(+) breast cancer patients.

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.

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.

Single-Cell Image-Based Analysis Reveals Chromatin Changes during the Acquisition of Tamoxifen Drug Resistance.

Cancer drug resistance is the leading cause of cancer related deaths. The development of drug resistance can be partially contributed to tumor heterogeneity and epigenetic plasticity. However, the detailed molecular mechanism underlying epigenetic modulated drug resistance remains elusive. In this work, we systematically analyzed epigenetic changes in tamoxifen (Tam) responsive and resistant breast cancer cell line MCF7, and adopted a data-driven approach to identify key epigenetic features distinguishing between these two cell types. Significantly, we revealed that DNA methylation and H3K9me3 marks that constitute the heterochromatin are distinctively different between Tam-resistant and -responsive cells. We then performed time-lapse imaging of 5mC and H3K9me3 features using engineered probes. After Tam treatment, we observed a slow transition of MCF7 cells from a drug-responsive to -resistant population based on DNA methylation features. A similar trend was not observed using H3K9me3 probes. Collectively, our results suggest that DNA methylation changes partake in the establishment of Tam-resistant breast cancer cell lines. Instead of global changes in the DNA methylation level, the distribution of DNA methylation features inside the nucleus can be one of the drivers that facilitates the establishment of a drug resistant phenotype in MCF7.

Metformin reverses tamoxifen resistance through the lncRNA GAS5-medicated mTOR pathway in breast cancer

BackgroundMetformin (Met) has antitumor effects on various cancers, but it is still unclear whether it exerts a reversible effect on endocrine resistance in breast cancer (BC) patients. In the present survey, metformin’s effects on tamoxifen-resistant MCF cells (The cell line was established at the Michigan Cancer Foundation, hence the name MCF cell) were evaluated and the molecular mechanism was explored.MethodsWe constructed a tamoxifen-resistant BC cell line MCF-7R, then applied Cell Counting Kit-8 (CCK-8), flow cytometry, and EdU assessments to determine the growth and apoptosis of MCF-7R cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the expression level of lncRNA GAS5 and the mTOR-associated proteins. Overexpression and downregulation of lncRNA GAS5 were carried out to explore the molecular mechanism of Met.ResultsThe combined effect of Met and OHT (4-hydroxytamoxifen, main active metabolite of tamoxifen) was to remarkably reduce the growth and increase the apoptosis of MCF-7R cells compared with OHT alone. The levels of the mTOR, p-mTOR and p-P70S6K proteins in MCF-7R cells decreased after Met treatment, but the p-AMPK2 and PTEN proteins significantly increased. The overexpression of lncRNA GAS5 in MCF-7R cells inhibited cell growth with decreased mTOR, p-mTOR, PCNA, and Bcl-2 proteins. Metformin had no significant effect on MCF-7R cells with lncRNA GAS5 knockdown but contrarily activated p-mTOR.ConclusionsMetformin can inhibit overactivation of the mTOR signaling pathway through upregulating lncRNA GAS5 expression, thereby inhibiting the growth and inducing the apoptosis of BC cells, providing a new clinical treatment for BC.

Abstract P5-05-10: Inhibition of FASN as a potential treatment of advanced therapy-resistant breast cancer

Abstract INTRODUCTION: The majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. METHODS: The effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen-resistant and fulvestrant-resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. RESULTS: TVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant and fulvestrant-resistant cells compared to the parental cells. Additionally, TVB-3166 significantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered. Citation Format: Henriette U Balinda. Inhibition of FASN as a potential treatment of advanced therapy-resistant 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 P5-05-10.

Fascin enhances the vulnerability of breast cancer to erastin-induced ferroptosis

Ferroptosis, which is characterized by intracellular iron accumulation and lipid peroxidation, is a newly described form of regulated cell death that may play a key role in tumour suppression. In the present study, we investigated the expression profiles and biological effects of fascin actin-bundling protein 1 (Fascin, gene name FSCN1) in breast cancer. In addition, bioinformatics analysis of the TCGA cancer database and gain- and loss-of-function studies showed that Fascin enhances sensitivity to erastin-induced ferroptosis. Mechanistically, Fascin directly interacts with cysteine/glutamate transporter (xCT, gene name SLC7A11) and decreases its stability via the ubiquitin-mediated proteasome degradation pathway. Furthermore, we observed that Fascin is substantially upregulated in tamoxifen-resistant breast cancer cell lines, and drug-resistant cells were also more vulnerable to erastin-induced ferroptosis. Taken together, our findings reveal a previously unidentified role of Fascin in ferroptosis by regulating xCT. Thus, ferroptosis activation in breast cancer with high Fascin level may serve as a potential treatment.

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.

Ionizing radiation downregulates estradiol synthesis via endoplasmic reticulum stress and inhibits the proliferation of estrogen receptor-positive breast cancer cells

Breast cancer is a major threat to women’s health and estrogen receptor-positive (ER+) breast cancer exhibits the highest incidence among these cancers. As the primary estrogen, estradiol strongly promotes cellular proliferation and radiotherapy, as a standard treatment, exerts an excellent therapeutic effect on ER+ breast cancer. Therefore, we herein wished to explore the mechanism(s) underlying the inhibitory effects of radiation on the proliferation of ER+ breast cancer cells. We used the ER+ breast cancer cell lines MCF7 and T47D, and their complementary tamoxifen-resistant cell lines in our study. The aforementioned cells were irradiated at different doses of X-rays with or without exogenous estradiol. CCK8 and clone-formation assays were used to detect cellular proliferation, enzyme-linked immunosorbent assay (ELISA) to determine estradiol secretion, western immunoblotting analysis and quantitative real-time PCR to evaluate the expression of proteins, and immunofluorescence to track endoplasmic reticulum stress-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays to explore the protein expression in tumors using immunohistochemistry. We found that ionizing radiation significantly reduced the phosphorylation of estrogen receptors and the secretion of estradiol by ER+ breast cancer cells. CYP19A (aromatase) is an enzyme located in the endoplasmic reticulum, which plays a critical role in estradiol synthesis (aromatization), and we further demonstrated that ionizing radiation could induce endoplasmic reticulum stress with or without exogenous estradiol supplementation, and that it downregulated the expression of CYP19A through ER-phagy. In addition, ionizing radiation also promoted lysosomal degradation of CYP19A, reduced estradiol synthesis, and inhibited the proliferation of tamoxifen-resistant ER+ breast cancer cells. We concluded that ionizing radiation downregulated the expression of CYP19A and reduced estradiol synthesis by inducing endoplasmic reticulum stress in ER+ breast cancer cells, thereby ultimately inhibiting cellular proliferation.

Inhibition of Metabolism as a Therapeutic Option for Tamoxifen-Resistant Breast Cancer Cells

Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of aerobic oxidation alone. Glutamine is an essential amino acid in the human body. Glutaminolysis and glycolysis are crucial for cancer cell survival. In the therapy of estrogen receptor α (ERα)-positive breast cancer (BC), the focus lies on hormone sensitivity targeting therapy with selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (4-OHT), although this therapy is partially limited by the development of resistance. Therefore, further targets for therapy improvement of ERα-positive BC with secondary 4-OHT resistance are needed. Hence, increased glucose requirement and upregulated glutaminolysis in BC cells could be used. We have established sublines of ERα-positive MCF7 and T47D BC cells, which were developed to be resistant to 4-OHT. Further, glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) and glutaminase inhibitor CB-839 were analyzed. Co-treatments using 4-OHT and CB-839, 2-DG and CB-839, or 4-OHT, 2-DG and CB-839, respectively, showed significantly stronger inhibitory effects on viability compared to single treatments. It could be shown that tamoxifen-resistant BC cell lines, compared to the non-resistant cell lines, exhibited a stronger reducing effect on cell viability under co-treatments. In addition, the tamoxifen-resistant BC cell lines showed increased expression of proto-oncogene c-Myc compared to the parental cell lines. This could be reduced depending on the treatment. Suppression of c-Myc expression using specific siRNA completely abolished resistance to 4OH-tamoxifen. In summary, our data suggest that combined treatments affecting the metabolism of BC are suitable depending on the cellularity and resistance status. In addition, the anti-metabolic treatments affected the expression of the proto-oncogene c-Myc, a key player in the regulation of cancer cell metabolism.

Inhibition of STAT3 enhances sensitivity to tamoxifen in tamoxifen-resistant breast cancer cells

BackgroundThe mechanisms of endocrine resistance are complex, and deregulation of several oncogenic signalling pathways has been proposed. We aimed to investigate the role of the EGFR and Src-mediated STAT3 signalling pathway in tamoxifen-resistant breast cancer cells.MethodsThe ER-positive luminal breast cancer cell lines, MCF-7 and T47D, were used. We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. Cell viability was determined using an MTT assay, and protein expression levels were determined using western blot. Cell cycle and annexin V staining were analysed using flow cytometry.ResultsTamR cells showed decreased expression of estrogen receptor and increased expression of EGFR. TamR cells showed an acceleration of the G1 to S phase transition. The protein expression levels of phosphorylated Src, EGFR (Y845), and STAT3 was increased in TamR cells, while phosphorylated Akt was decreased. The expression of p-STAT3 was enhanced according to exposure time of tamoxifen in T47D cells, suggesting that activation of STAT3 can cause tamoxifen resistance in ER-positive breast cancer cells. Both dasatinib (Src inhibitor) and stattic (STAT3 inhibitor) inhibited cell proliferation and induced apoptosis in TamR cells. However, stattic showed a much stronger effect than dasatinib. Knockdown of STAT3 expression by siRNA had no effect on sensitivity to tamoxifen in MCF-7 cells, while that enhanced sensitivity to tamoxifen in TamR cells. There was not a significant synergistic effect of dasatinib and stattic on cell survival. TamR cells have low nuclear p21(Cip1) expression compared to MCF-7 cells and inhibition of STAT3 increased the expression of nuclear p21(Cip1) in TamR cells.ConclusionsThe EGFR and Src-mediated STAT3 signalling pathway is activated in TamR cells, and inhibition of STAT3 may be a potential target in tamoxifen-resistant breast cancer. An increase in nuclear p21(Cip1) may be a key step in STAT3 inhibitor-induced cell death in TamR cells.