Estrogen Receptor-positive Breast Cancer Cells Research Articles

Abstract P4-02-01: Targeting insulin receptor in estrogen receptor positive breast cancer

Abstract Obesity and metabolic dysfunction are on the rise in the United States. Hyperinsulinemia, frequently seen in these conditions, correlates with an increased risk of development and mortality from estrogen receptor (ER) positive breast cancer. Expression of components of the insulin and insulin-like growth factor (IGF) family of proteins promote cancer cell growth in vitro, link to shorter progression-free survival, and is associates with an increased risk of metastatic disease and death in breast cancer patients. Previously our lab found that development of resistance to the commonly used ER-targeting drug tamoxifen (Tam) in vitro is associated with a loss of the type I IGF receptor expression with an increased dependence on the homologous insulin receptor (IR). These data argue for targeting IR in breast cancer, but concern has been raised due to the potential of disrupting glucose homeostasis in patients with or without existing metabolic disease. To address this concern, a novel insulin-Fc fusion protein (AKS-130) was developed to lower blood glucose levels without causing clinical hypoglycemia with a potential to target tumor IR. AKS-130 was found to downregulate IR expression in models of colorectal cancer (HCT-116) and malignant melanoma (WM266.4). Further, AKS-130 suppressed xenograft growth in fed and fasted states. We examined if AKS-130 had similar effects on ER positive breast cancer cells. To test the hypothesis that resistance to endocrine therapy increases the reliance of ER positive breast cancer cells on IR signaling, we utilized the ER positive breast cancer cell lines (MCF-7L, T47D) and their Tam resistant derivatives (MCF-7L TamR, T47D TamR). Similar to in HCT-116 and WM266.4 cells, we found that AKS-130 is a modest agonist of IR in ER+ breast cancer cells. Exposure to AKS-130 led to a marked reduction in IR expression in both parental and TamR cell lines, and was associated with partially suppressed downstream signaling to Akt. Interestingly, AKS130 induced IR downregulation was inhibited by pretreatment of BMS 536924, a dual IGF1R/IR kinase inhibitor. In vivo, AKS-130 treatment by itself scaled down MCF7L parental xenograft growth as compared to vehicle group, and AKS-130 in combination with Tam trended toward extending time to a tumor size of 500mm3. In MCF7L TamR xenografts, addition of AKS-130 to Tam treatment also trended toward delaying xenograft growth, though it was not statistically significant. Importantly, prolonged treatment with AKS-130 did not lead to significant changes in weight or blood glucose. Taken together these data show that novel agents, such as AKS-130, may serve as way to disrupt IR signaling in cancer cells without affecting host glucose metabolism. Addition of AKS-130 to Tam treatment trended toward delaying xenograft growth though further studies are needed, especially animal model systems of insulin resistance. These data indicate that IR signaling may represent a target to overcome or delay endocrine therapy resistance in breast cancer. Citation Format: Jingran Cao, Lynsey M. Fettig, Kelly LaPara, Xihong Zhang, Sylaja Murikipudi, Andrea R. Delpero, Thomas M. Lancaster, Todd C. Zion, Douglas Yee. Targeting insulin receptor in estrogen receptor positive 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 P4-02-01.

Abstract PD9-07: Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer

Abstract Background: Luminal, i.e., estrogen receptor (ER)-positive breast cancer (BC) is a heterogeneous disease in terms of tumor progression, therapy response, and relapse. Additional biomarkers with a prognostic and predictive impact would facilitate advanced patient stratification and can reveal advanced therapeutic options for individual patients suffering from luminal BC subtype. First objective of this study was the hypothesis driven validation and identification of biomarkers associated with successful engraftment, augmented tumor growth, and enhanced metastasis upon xenotransplantation into HTM and non-humanized tumor mice. Second objective was the retrospective validation and correlation of aforementioned markers with clinical outcomes (disease free-survival [DFS] and overall survival [OS]) of luminal BC patients after neoadjuvant chemotherapy within the GeparTrio trial. Methods: Immunodeficient NSG mice with and without immunological humanization were transplanted with primary, ER-positive BC tissues and cells. Engraftment rates, tumor progression, and metastasis were monitored as a function of marker expression and genomic alterations, considered to be associated with tumor cell stemness and tumor initiating capacity. Functional assays were applied to demonstrate the impact of identified markers on tumor cell viability, growth and migration in-vitro. Dual color fluorescence-in-situ-hybridization to monitor mdm2 gene and the cen12 region was applied to 502 pretherapeutic ER-positive tissue specimens of BC patients treated with anthracycline/taxane based neoadjuvant chemotherapy within the GeparTrio trial. Mdm2 gene expression was analyzed in the entire luminal BC cohort as well as in luminal-A and luminal-B samples classified based on a Ki-67 cut-off of 20% (St. Gallen guideline). Associations with survival outcomes were studied by Cox regression models. Results: We observed an elevated CD44 and c-MET expression in metastatic cells compared to the primarily growing xenotransplantants. Moreover, we found mdm2 gene amplification was associated with tumor growth and pronounced metastatic potential in NSG mice. Functional assays unveiled a reduced viability, proliferation, and migration capacity of inherently mdm2 positive breast cancer cells upon mdm2 knock-down or anti-mdm2 targeting. Validation of mdm2 gain in luminal BC cohort within the GeparTrio trial revealed a significant association of mdm2 amplification with worse DFS (HR=1.80 [95%CI 1.16-2.79], log rank p=0.008) and OS (HR=1.75 [95%CI 1.00-3.05], log-rank p=0.047). This association was even stronger in luminal-A BC: DFS (HR=2.56 [95%CI 1.40-4.71], log rank p=0.002 and OS (HR=3.27 [95%CI 1.51-7.09], log-rank p=0.001). Conclusions: Mdm2 gene amplification facilitates ER-positive BC engraftment and progression in a preclinical in-vivo xenograft humanized NSG mouse model. Targeting mdm2 in-vitro reduced malignant cell propagation and growth. In addition, an increased mdm2 gene dose is strongly associated with an unfavorable outcome of luminal BC. Prospective studies are required to verify the suitability of mdm2 for advanced luminal BC stratification and therapeutic targeting. Citation Format: Anja Kathrin Wege, Valentina Vladimirova, Christine Solbach, Eva-Maria Rom-Jurek, Jens-Uwe Blohmer, Paul Jank, Bruno Sinn, Andreas Trumpp, Elisabetta Marangoni, Knut Engels, Wilko Weichert, Nicole Pfarr, Christoph Irlbeck, Bernhard Polzer, Olaf Ortmann, Marion van Mackelenbergh, Carsten Denkert, Sibylle Loibl, Gero Brockhoff. Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal 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 PD9-07.

Binding of the angiogenic/senescence inducer CCN1/CYR61 to integrin α6β1 drives endocrine resistance in breast cancer cells.

CCN1/CYR61 promotes angiogenesis, tumor growth and chemoresistance by binding to its integrin receptor αvβ3 in endothelial and breast cancer (BC) cells. CCN1 controls also tissue regeneration by engaging its integrin receptor α6β1 to induce fibroblast senescence. Here, we explored if the ability of CCN1 to drive an endocrine resistance phenotype in estrogen receptor-positive BC cells relies on interactions with either αvβ3 or α6β1. First, we took advantage of site-specific mutagenesis abolishing the CCN1 receptor-binding sites to αvβ3 and α6β1 to determine the integrin partner responsible for CCN1-driven endocrine resistance. Second, we explored a putative nuclear role of CCN1 in regulating ERα-driven transcriptional responses. Retroviral forced expression of a CCN1 derivative with a single amino acid change (D125A) that abrogates binding to αvβ3 partially phenocopied the endocrine resistance phenotype induced upon overexpression of wild-type (WT) CCN1. Forced expression of the CCN1 mutant TM, which abrogates all the T1, H1, and H2 binding sites to α6β1, failed to bypass the estrogen requirement for anchorage-independent growth or to promote resistance to tamoxifen. Wild-type CCN1 promoted estradiol-independent transcriptional activity of ERα and enhanced ERα agonist response to tamoxifen. The α6β1-binding-defective TM-CCN1 mutant lost the ERα co-activator-like behavior of WT-CCN1. Co-immunoprecipitation assays revealed a direct interaction between endogenous CCN1 and ERα, and in vitro approaches confirmed the ability of recombinant CCN1 to bind ERα. CCN1 signaling via α6β1, but not via αvβ3, drives an endocrine resistance phenotype that involves a direct binding of CCN1 to ERα to regulate its transcriptional activity in ER+ BC cells.

Research on Clinical Effectiveness of Aspirin for Treating Breast Cancer and Cell Protein Biomarkers on Aspirin Treatment in Drug-Resistant Estrogen Receptor-Positive Breast Cancer Cells

Background: Aspirin (ASA) has been reported to have an antitumor effect but the role of ASA in the prevention and treatment of breast cancer (BC) is still controversial. This study aimed to identify clinical effectiveness of ASA in the treatment of BC and explore the antitumor target proteins of ASA that may be involved in overcoming tamoxifen resistance in estrogen receptor (ER)-positive BC cells. Materials and Methods: Randomized controlled trials (RCTs) of ASA in the treatment of BC were queried from the databases, including PubMed, Web of Science, Cochrane Library, WanFang, and Chinese National Knowledge Infrastructure. According to the quality standard recommended in the Newcastle-Ottawa Scale (NOS), the outcome indexes were analyzed by RevMan 5.3 and Stata 12.0 software. Cell culture experiments were performed to explore the effect of tamoxifen combined with ASA on the proliferation of ER-positive BC cell lines MCF-7 and MCF-7/TAM. Cell cytotoxicity was determined by the 3-(4, 5-di-2-yl)-2, 5-ditetrazolium bromide (MTT) assay. A quantitative proteomic analysis was conducted between the control and experimental groups to identify differentially expressed proteins (DEPs). Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used for bioinformatic analysis of DEPs. The protein expression in patients of ER-positive BC was analyzed by immunochemistry (IHC). Results: Nine RCTs including 162,381 patients were selected for this study. The meta-analysis revealed that daily ASA use, as compared with its non-use, was associated with a decreased risk of BC death: relative risk (RR) = 0.83%, 95% CI [0.73, 0.94], Z = 2.89, P = 0.004 (P

Amorphophalli Rhizoma inhibits breast cancer growth, proliferation, migration, and invasion via the PI3K/AKT pathway

Ethnopharmacological relevanceAmorphophalli Rhizoma (APR) is widely used as an adjuvant treatment for advanced and metastatic triple-negative breast cancer (TNBC), but its effects, potential active ingredients, and mechanism of action on estrogen receptor-positive (ER+) and human epidermal growth factor receptor-positive (HER2+) breast cancer cells were not reported. Aim of the studyThe present study investigated the effects and mechanism of APR on ER+ and HER2+ breast cancer cells. Materials and methodsRotary evaporation was used to prepare different extracts of APR. Cell activity was assessed using the cell counting kit-8 (CCK-8) method. Wound healing assays were used to assess cell migration, and a cell invasion assay was performed using a Transwell chamber with Matrigel matrix. A xenograft model was used to analyze the inhibitory effects of APR on tumor growth. Bioinformatics analyses were used to explore the potential mechanism of APR in breast cancer. RT–qPCR and Western blotting were performed to reveal the molecular mechanism. ResultsThe ethyl acetate extract of APR showed the strongest tumor inhibitory effect on ER+ and HER2+ breast cancer cells compared to petroleum ether or N-butanol extracts. APR inhibited ER+ and HER2+ breast cancer cell growth, proliferation, migration, and invasion via the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. ConclusionsAPR had a significant inhibitory effect on ER+ and HER2+ breast cancer cells via the PI3K/AKT signaling pathway. Therefore, APR may be useful for preventing ER+ and HER2+ breast tumor growth, proliferation, migration, and invasion.

Understanding the Role of Estrogen Receptor Status in PRODH/POX-Dependent Apoptosis/Survival in Breast Cancer Cells.

Simple SummaryThe estrogen receptor (ER) status and the availability of agonists or antagonists of these receptors determine the processes of growth, differentiation, and proliferation of breast cancer cells. Estrogens and anti-estrogenic compounds have been shown to influence breast cancer cell survival/apoptosis via action through the mitochondrial enzyme proline dehydrogenase/proline oxidase (PRODH/POX). In this review, we highlight the molecular effects of ER stimulation/inhibition in signaling pathways.It has been suggested that activation of estrogen receptor α (ER α) stimulates cell proliferation. In contrast, estrogen receptor β (ER β) has anti-proliferative and pro-apoptotic activity. Although the role of estrogens in estrogen receptor-positive breast cancer progression has been well established, the mechanism of their effect on apoptosis is not fully understood. It has been considered that ER status of breast cancer cells and estrogen availability might determine proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that converts proline into pyrroline-5-carboxylate (P5C). During this process, ATP (adenosine triphosphate) or ROS (reactive oxygen species) are produced, facilitating cell survival or death, respectively. However, the critical factor in driving PRODH/POX-dependent functions is proline availability. The amount of this amino acid is regulated at the level of prolidase (proline releasing enzyme), collagen biosynthesis (proline utilizing process), and glutamine, glutamate, α-ketoglutarate, and ornithine metabolism. Estrogens were found to upregulate prolidase activity and collagen biosynthesis. It seems that in estrogen receptor-positive breast cancer cells, prolidase supports proline for collagen biosynthesis, limiting its availability for PRODH/POX-dependent apoptosis. Moreover, lack of free proline (known to upregulate the transcriptional activity of hypoxia-inducible factor 1, HIF-1) contributes to downregulation of HIF-1-dependent pro-survival activity. The complex regulatory mechanism also involves PRODH/POX expression and activity. It is induced transcriptionally by p53 and post-transcriptionally by AMPK (AMP-activated protein kinase), which is regulated by ERs. The review also discusses the role of interconversion of proline/glutamate/ornithine in supporting proline to PRODH/POX-dependent functions. The data suggest that PRODH/POX-induced apoptosis is dependent on ER status in breast cancer cells.

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.

Role of Orai3 in the Pathophysiology of Cancer

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two independent Ca2+ currents, the store-operated current, Icrac, involving functional interactions between the stromal interaction molecules (STIM), STIM1/STIM2, and Orai1/Orai2/Orai3, as well as the store-independent arachidonic acid (AA) (or leukotriene C4)-regulated current Iarc, which involves Orai1, Orai3 and STIM1. Overexpression of functional Orai3 has been described in different neoplastic cells and cancer tissue samples as compared to non-tumor cells or normal adjacent tissue. In these cells, Orai3 exhibits a cell-specific relevance in Ca2+ influx. In estrogen receptor-positive breast cancer cells and non-small cell lung cancer (NSCLC) cells store-operated Ca2+ entry (SOCE) is strongly dependent on Orai3 expression while in colorectal cancer and pancreatic adenocarcinoma cells Orai3 predominantly modulates SOCE. On the other hand, in prostate cancer cells Orai3 expression has been associated with the formation of Orai1/Orai3 heteromeric channels regulated by AA and reduction in SOCE, thus leading to enhanced proliferation. Orai3 overexpression is associated with supporting several cancer hallmarks, including cell cycle progression, proliferation, migration, and apoptosis resistance. This review summarizes the current knowledge concerning the functional role of Orai3 in the pathogenesis of cancer.

Synergistic Cytotoxicity between Elephantopus scaber and Tamoxifen on MCF-7-Derived Multicellular Tumor Spheroid.

Elephantopus scaber Linn, a traditional herb, exhibited anticancer properties, and it was cytotoxic against the monolayer estrogen receptor-positive breast cancer cell line, MCF-7, in the previous study. In order to determine the potential of E. scaber as a complementary medicine for breast cancer, this study aimed to evaluate the synergism between E. scaber and tamoxifen in cytotoxicity using MCF-7 in the form of 3-dimensional multicellular tumor spheroid (MCTS) cultures. MCTS represents a more reliable model for studying drug penetration as compared to monolayer cells due to its greater resemblance to solid tumor. Combination of E. scaber ethanol extract and tamoxifen, which were used in concentrations lower than their respective IC50 values, had successfully induced apoptosis on MCTS in this study. The combinatorial treatment showed >58% increase of lactate dehydrogenase release in cell media, cell cycle arrest at the S phase, and 1.3 fold increase in depolarization of mitochondrial membrane potential. The treated MCTS also experienced DNA fragmentation; this had been quantified by TUNEL-positive assay, which showed >64% increase in DNA damaged cells. Higher externalization of phospatidylserine and distorted and disintegrated spheroids stained by acridine orange/propidium iodide showed that the cell death was mainly due to apoptosis. Further exploration showed that the combinatorial treatment elevated caspases-8 and 9 activities involving both extrinsic and intrinsic pathways of apoptosis. The treatment also upregulated the expression of proapoptotic gene HSP 105 and downregulated the expression of prosurvival genes such as c-Jun, ICAM1, and VEGF. In conclusion, these results suggested that the coupling of E. scaber to low concentration of tamoxifen showed synergism in cytotoxicity and reducing drug resistance in estrogen receptor-positive breast cancer.

Palmatine, a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica, Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin.

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER+/HER2− breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC50 values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.

Targeting Estrogen Receptor-Positive Breast Microtumors with Endoxifen-Conjugated, Hypoxia-Sensitive Polymersomes.

Endoxifen is the primary active metabolite of tamoxifen, a nonsteroidal-selective estrogen receptor modulator (SERM) and widely used medication to treat estrogen receptor-positive (ER+) breast cancer. In this study, endoxifen was conjugated to the surface of polymeric nanoparticles (polymersomes) for targeted delivery of doxorubicin (DOX) to estrogen receptor-positive breast cancer cells (MCF7). Rapid cell growth and insufficient blood supply result in low oxygen concentration (hypoxia) within the solid breast tumors. The polymersomes developed here are prepared from amphiphilic copolymers of polylactic acid (PLA) and poly(ethylene glycol) (PEG) containing diazobenzene as the hypoxia-responsive linker. We prepared two nanoparticle formulations: DOX-encapsulated hypoxia-responsive polymersomes (DOX-HRPs) and endoxifen-conjugated, DOX-encapsulated hypoxia-responsive polymersomes (END-DOX-HRPs). Cellular internalization studies demonstrated eight times higher cytosolic and nuclear localization after incubating breast cancer cells with END-DOX-HRPs (targeted polymersomes) in contrast to DOX-HRPs (nontargeted polymersomes). Cytotoxicity studies on monolayer cell cultures exhibited that END-DOX-HRPs were three times more toxic to ER+ MCF7 cells than DOX-HRPs and free DOX in hypoxia. The cell viability studies on three-dimensional hypoxic cultures also demonstrated twice as much toxicity when the spheroids were treated with targeted polymersomes instead of nontargeted counterparts. This is the first report of surface-decorated polymeric nanoparticles with endoxifen ligands for targeted drug delivery to ER+ breast cancer microtumors. The newly designed endoxifen-conjugated, hypoxia-responsive polymersomes might have translational potential for ER+ breast cancer treatment.

Downregulation of RPS14 inhibits the proliferation and metastasis of estrogen receptor-positive breast cancer cells.

Ribosomal protein S14 (RPS14) is a component of the 40S ribosomal subunit and is considered to be indispensable for ribosomal biogenesis. Previously, we found that RPS14 was significantly downregulated in estrogen receptor-positive (ER+) breast cancer cells following treatment with 4-hydroxytamoxifen (4-OH-TAM). However, its role in breast cancer remains poorly understood. In the present study, we sought to demonstrate, for the first time, that RPS14 is highly expressed in ER+ breast cancer tissues and its downregulation can significantly inhibit the proliferation, cycle, and metastasis of ER+ breast cancer cells, as well as induce cell apoptosis. Quantitative RT-PCR and western blotting were used to determine the expression of target genes. Herein, lentivirus-mediated small hairpin RNA (shRNA) targeting RPS14 was designed to determine the impact of RPS14 knockdown on ER+ breast cancer cells. Further, bioinformatics analysis was used to reveal the significance of differentially expressed genes in RPS14 knockdown breast cancer cells. RPS14 was highly expressed in ER+ breast cancer tissues compared to ER- tissues. The downregulation of RPS14 in two ER+ breast cancer cell lines suppressed cell proliferation, cell cycle and metastasis, and induced apoptosis. Based on bioinformatics analysis, the expression level of several significant genes, such as ASNS, Ret, and S100A4, was altered in breast cancer cells after RPS14 downregulation. Furthermore, the BAG2 and interferon signaling pathways were identified to be significantly activated. The downregulation of RPS14 in ER+ breast cancer cells can inhibit their proliferation and metastasis.

The mechanisms involved in the resistance of estrogen receptor-positive breast cancer cells to palbociclib are multiple and change over time

PurposeCyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used for the treatment of advanced estrogen receptor (ER)-positive breast cancer. To develop a treatment strategy for cancers resistant to CDK4/6 inhibitors, here, we established palbociclib-resistant sublines and analyzed their resistance mechanisms.MethodsPalbociclib-resistant sublines were established from T47D and MCF7 cells. Sensitivity to other drugs was assessed via the WST assay. Altered expression/phosphorylation of proteins related to signal transduction and cell cycle regulation was examined using western blotting. Copy number alterations and mutations in the retinoblastoma (RB1) gene were also analyzed.ResultsAlthough an increase in CDK6 and decrease in retinoblastoma protein (Rb) expression/phosphorylation were commonly observed in the resistant sublines, changes in other cell cycle-related proteins were heterogeneous. Upon extended exposure to palbociclib, the expression/phosphorylation of these proteins became altered, and the long-term removal of palbociclib did not restore the Rb expression/phosphorylation patterns. Consistently a copy number decrease, as well as RB1 mutations were detected. Moreover, although the resistant sublines exhibited cross-resistance to abemaciclib, their response to dinaciclib was the same as that of wild-type cells. Of note, the cell line exhibiting increased mTOR phosphorylation also showed a higher sensitivity to everolimus. However, the sensitivity to chemotherapeutic agents was unchanged in palbociclib-resistant sublines.ConclusionER-positive breast cancer cells use multiple molecular mechanisms to survive in the presence of palbociclib, suggesting that targeting activated proteins may be an effective strategy to overcome resistance. Additionally, palbociclib monotherapy induces mutations and copy number alterations in the RB1 gene.

Signaling of MK2 sustains robust AP1 activity for triple negative breast cancer tumorigenesis through direct phosphorylation of JAB1

Triple negative breast cancer (TNBC) cells are generally more invasive than estrogen receptor-positive (ER + ) breast cancer cells. Consistent with the importance of activator protein 1 (AP1) transcription factors in invasion, AP1 activity is much higher in TNBC lines than ER + lines. In TNBC cells, robust AP1 activity is facilitated by both ERK and p38MAPK signaling pathways. While ERK signaling pathway regulates AP1 activity by controlling the abundance of AP1 transcription factors, p38MAPK signaling pathway does it by enhancing AP1 binding to AP1 sites without altering their abundance. Here, we show that p38MAPK regulation of AP1 activity involves both MAPKAPK2 (MK2) and JAB1, a known JUN-binding protein. MK2 not only interacts with JAB1 but also directly phosphorylates JAB1 at Ser177 in TNBC cells. Interestingly, Ser177 phosphorylation does not affect JAB1 and JUN interaction. Instead, interfering with p38MAPK signaling pathway or introducing an S to A point mutation at Ser177 of JAB1 reduces JUN recruitment to the AP1 sites in cyclin D1, urokinase plasminogen activator (uPA) and uPA receptor promoters. Moreover, knockdown of JAB1 diminishes >60% of AP1 transcriptional activity in TNBC cells. Taken together, these results indicate that MK2-mediated phosphorylation of JAB1 facilitates JUN recruitment to AP1 sites, thus augmenting AP1 activity. In line with the role of JAB1 in AP1 activity, silencing JAB1 leads to dramatic reduction in TNBC cell growth, in vitro invasion and in vivo tumor outgrowth. This study suggests that the p38MAPK-MK2 signaling pathway promotes TNBC tumorigenesis by sustaining robust AP1 activity.

Cholesterol-Induced Metabolic Reprogramming in Breast Cancer Cells Is Mediated via the ERRα Pathway.

Simple SummaryThere is increasing evidence that obesity and high circulating cholesterol levels are associated with an increased risk of recurrence and a higher mortality rate in breast cancer patients via altering the metabolic programming in breast cancer cells. However, the underlying molecular mechanism by which high cholesterol levels reprogram the metabolic pathways in breast cancer cells is not well-understood. We have previously demonstrated that cholesterol acts as an endogenous agonist of estrogen-related receptor α (ERRα), a strong regulator of cellular metabolism. The aim of the current study is to demonstrate whether cholesterol/obesity mediates its pathogenic effect in breast cancer cells via altering metabolic pathways in an ERRα-dependent manner. The findings of this study provide mechanistic insights into the link between cholesterol/obesity and metabolic reprogramming in breast cancer patients and reveal the metabolic vulnerabilities in such breast cancer patients that could be therapeutically targeted.The molecular mechanism underlying the metabolic reprogramming associated with obesity and high blood cholesterol levels is poorly understood. We previously reported that cholesterol is an endogenous ligand of the estrogen-related receptor alpha (ERRα). Using functional assays, metabolomics, and genomics, here we show that exogenous cholesterol alters the metabolic pathways in estrogen receptor-positive (ER+) and triple-negative breast cancer (TNBC) cells, and that this involves increased oxidative phosphorylation (OXPHOS) and TCA cycle intermediate levels. In addition, cholesterol augments aerobic glycolysis in TNBC cells although it remains unaltered in ER+ cells. Interestingly, cholesterol does not alter the metabolite levels of glutaminolysis, one-carbon metabolism, or the pentose phosphate pathway, but increases the NADPH levels and cellular proliferation, in both cell types. Importantly, we show that the above cholesterol-induced modulations of the metabolic pathways in breast cancer cells are mediated via ERRα. Furthermore, analysis of the ERRα metabolic gene signature of basal-like breast tumours of overweight/obese versus lean patients, using the GEO database, shows that obesity may modulate ERRα gene signature in a manner consistent with our in vitro findings with exogenous cholesterol. Given the close link between high cholesterol levels and obesity, our findings provide a mechanistic explanation for the association between cholesterol/obesity and metabolic reprogramming in breast cancer patients.

Sequential usage of imidazole followed by tamoxifen to enhance the anticancer effect of tamoxifen in estrogen receptor-positive breast cancer cell lines.

e15057 Background: In women, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide. Tamoxifen is the most commonly used drug for the endocrine treatment of breast cancer. It reduces the risk of recurrence and death from breast cancer when given to estrogen-receptor-positive breast cancer patients. It has recently been shown that imidazoles, an antifungal drug, possess anticancer potentials, and it can be a novel therapeutic in cancer treatment. However, the effects of combined treatment with imidazole and tamoxifen are unknown in estrogen receptor-positive breast cancer cell lines. Our study aimed to investigate the effects of imidazole and tamoxifen combination in estrogen receptor-positive breast cancer cell lines. Methods: MCF7 cell line, an estrogen receptor-positive breast cancer cell line, was used in this study. Following 24 hours 50 mM imidazole (molecular grade) treatment, 15µM tamoxifen was treated to MCF-7 cells by 72 hours. As control groups, following for 24-hour imidazole alone treated, only medium treated cells for 72 hours were used. MTT assay was performed for the determination of cell viability. Apoptosis was shown using acridine orange/ethidium bromide staining. The cellular morphological alterations were observed on bright-field microscopy using Giemsa staining. Cell migration status was determined using by in vitro scratch assay. Results: MTT assay results showed that tamoxifen alone treatment for 72 hours decreased cell viability by 18 percent (p < 0.001). On the other hand, cell viability is not affected by imidazole alone treatment for 24 hours compared with the control group (p > 0.05). However, it was calculated that 24 hours of imidazole followed by tamoxifen for 72 hours decreased cell viability up to 42 percent (p < 0.001). Tamoxifen alone group, compared with combined treatment with tamoxifen and imidazole, observed an increase of apoptotic cell numbers in combined treatment with tamoxifen and imidazole group, statistically significantly (p < 0.01). It was observed that cellular morphology was affected by combined treatment with tamoxifen and imidazole. Giemsa staining results showed that MCF 7 cells changed their cellular morphology, lost cell-cell contact, differentiated from parental morphology and cellular morphology, and appeared unhealthy. Parallel to these findings, a decrease in cell migration was observed in the combined-treated group compared to the tamoxifen alone group (p < 0.01). Conclusions: Our results showed that sequential usage of imidazole followed by tamoxifen has an enhanced anticancer effect of tamoxifen in estrogen receptor-positive breast cancer cell lines. These results allow us to establish a new hormonal treatment for patients with breast cancer.

CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.

Tamoxifen (TAM) is a selective estrogen receptor modulator used for breast cancer patients. Prolonged use of tamoxifen is not recommended for some patients. In this study, we aimed to identify molecular targets sensitive to TAM using a genome-wide gene deletion library screening of fission yeast heterozygous mutants. From the screening, casein kinase 1 gamma 2 (CSNK1G2), a serine-/threonine protein kinase, was the most sensitive target to TAM with a significant cytotoxicity in estrogen receptor-positive (ER+) breast cancer cells but with only a slight toxicity in the case of ER- cells. In addition, tumor sphere formation and expression of breast stem cell marker genes such as CD44/CD2 were greatly inhibited by CSNK1G2 knockdown in ER+ breast cancer cells. Consistently, CSNK1G2 altered ERα activity via phosphorylation, specifically at serine (Ser)167, as well as the regulation of estrogen-responsive element (ERE) of estrogen-responsive genes such as CTSD and GREB1. However, ERα silencing almost completely blocked CSNK1G2-induced TAM sensitivity. In ER+ breast cancer cells, combined treatment with TAM and CSNK1G2 knockdown further enhanced the TAM-mediated decrease in phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) signaling but not extracellular signal-regulated kinase (ERK) signaling. Inversely, in ER- cells treated with TAM, only ERK and PI3K signaling was altered by CSNK1G2 knockdown. The CK1 inhibitor, D4476, partly mimicked the CSNK1G2 knockdown effect in ER+ breast cancer cells, but with a broader repression ranging from PI3K/AKT/mTOR/S6K to ERK signaling. Collectively, these results suggest that CSNK1G2 plays a key role in sensitizing TAM toxicity in ER+ and ER- breast cancer cells via differently regulating PI3K/AKT/mTOR/S6K and ERK signaling.

Efficacy and reusability of mixed-phase TiO2–ZnO nanocomposites for the removal of estrogenic effects of 17β-Estradiol and 17α-Ethinylestradiol from water

Photocatalytic removal of estrogenic compounds (ECs), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2) were assessed using a TiO2–ZnO nanocomposite (NC) over a range of initial EC concentration (Co; 10 mg/L – 0.05 mg/L). Photocatalytic removal was evaluated under UV and visible irradiation using 10 mg/L NC over 240 min duration. After 240 min, analysis using GCxGC TOF MS revealed 100% transformation at Co ≤ 1 mg/L and ≥25% transformation at Co ≤ 10 mg/L under visible irradiation. Degradation was accompanied by breakdown of the fused ring structure of E2, generating smaller molecular weight by-products which were subsequently mineralized as revealed through TOC removal. With UV photocatalysis, ~30% and ~20% mineralization was attained for E2 and EE2, respectively, for Co of 10 mg/L. Under visible irradiation, ~25% and ~10% mineralization was achieved for E2 and EE2, respectively. Estrogenicity variation was estimated using the E-screen assay conducted with estrogen receptor-positive MCF-7 breast cancer cells. Complete removal of estrogenicity of ECs was confirmed after 240 min of photocatalysis under UV and visible irradiation. FTIR spectroscopy-based analysis of the NC after E2 photocatalysis revealed the presence of sorbed organics. Desorption, followed by GC × GC TOF-MS analysis revealed these organics as by-products of photocatalysis. Desorption of sorbed organics followed by recalcination at 600 °C for 1 h regenerated the active sites on the NC, enabling its efficient reuse for 3 cycles under visible irradiation without loss in activity.

Unveiling the mechanism of action behind the anti-cancer properties of cannabinoids in ER+ breast cancer cells: Impact on aromatase and steroid receptors

Breast cancer is the leading cause of cancer-related death in women worldwide. In the last years, cannabinoids have gained attention in the clinical setting and clinical trials with cannabinoid-based preparations are underway. However, contradictory anti-tumour properties have also been reported. Thus, the elucidation of the molecular mechanisms behind their anti-tumour efficacy is crucial to better understand its therapeutic potential. Considering this, our work aims to clarify the molecular mechanisms underlying the anti-cancer properties of the endocannabinoid anandamide (AEA) and of the phytocannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), in estrogen receptor-positive (ER+) breast cancer cells that overexpress aromatase (MCF-7aro). Their in vitro effects on cell proliferation, cell death and activity/expression of aromatase, ERα, ERβ and AR were investigated. Our results demonstrated that cannabinoids disrupted MCF-7aro cell cycle progression. Unlike AEA and THC that induced apoptosis, CBD triggered autophagy to promote apoptotic cell death. Interestingly, all cannabinoids reduced aromatase and ERα expression levels in cells. On the other hand, AEA and CBD not only exhibited high anti-aromatase activity but also induced up-regulation of ERβ. Therefore, all cannabinoids, albeit by different actions, target aromatase and ERs, impairing, in that way, the growth of ER+ breast cancer cells, which is dependent on estrogen signalling. As aromatase and ERs are key targets for ER+ breast cancer treatment, cannabinoids can be considered as potential and attractive therapeutic compounds for this type of cancer, being CBD the most promising one. Thus, from an in vitro perspective, this work may contribute to the growing mass of evidence of cannabinoids and cannabinoids-based medicines as potential anti-cancer drugs.

Ginsenoside-Rg2 affects cell growth via regulating ROS-mediated AMPK activation and cell cycle in MCF-7 cells

BackgroundGinsenoside-Rg2 (G-Rg2) is a protopanaxatriol-type ginsenoside isolated from ginseng. It has been found to exhibit various pharmacological effects, including antioxidant, anti-inflammatory, and anticancer effects. PurposeThis study aimed to investigate the anticancer effects of G-Rg2 on estrogen receptor-positive MCF-7 breast cancer (BC) cells, and the underlying mechanisms involving in reactive oxygen species (ROS) production. Study design/MethodsCell viability, cell cycle distribution, apoptosis, and ROS production were measured following exposure to G-Rg2. The protein expression levels of p-ERK1/2, p-Akt, PARP, p-Rb, cyclin D1, CDK6, and p-AMPK were quantified using western blot analysis. The in vivo activity of G-Rg2 was assessed in a xenograft model. Immunohistochemistry staining for p-Rb and p-AMPK was performed in tumor tissues. ResultsG-Rg2 significantly decreased cell viability but increased cell apoptosis. In MCF-7 cells, G-Rg2 increased ROS production by inhibiting ERK1/2 and Akt activation. G-Rg2-induced ROS induced G0/G1 cell cycle arrest and AMPK phosphorylation. In the xenograft model, the 5 mg/kg G-Rg2-treated group showed decreased tumor volume and weight, similar to the 5 mg/kg 4-OHT-treated group, compared to the control group. Immunohistochemistry staining showed that G-Rg2 treatment decreased Rb phosphorylation, while increasing AMPK phosphorylation in tumor tissues. ConclusionG-Rg2 has potential anticancer effects by increasing the ROS-AMPK signaling pathway and inhibiting ERK1/2 and Akt activation-mediated cell proliferation and cell cycle progression in MCF-7 BC cells.