Activity In Breast Cancer Cells Research Articles

A PET-Surrogate Signature for the Interrogation of the Metabolic Status of Breast Cancers.

Metabolic alterations in cancers can be exploited for diagnostic, prognostic, and therapeutic purposes. This is exemplified by 18F-fluorodeoxyglucose (FDG)-positron emission tomography (FDG-PET), an imaging tool that relies on enhanced glucose uptake by tumors for diagnosis and staging. By performing transcriptomic analysis of breast cancer (BC) samples from patients stratified by FDG-PET, a 54-gene signature (PETsign) is identified that recapitulates FDG uptake. PETsign is independently prognostic of clinical outcome in luminal BCs, the most common and heterogeneous BC molecular subtype, which requires improved stratification criteria to guide therapeutic decision-making. The prognostic power of PETsign is stable across independent BC cohorts and disease stages including the earliest BC stage, arguing that PETsign is an ab initio metabolic signature. Transcriptomic and metabolomic analysis of BC cells reveals that PETsign predicts enhanced glycolytic dependence and reduced reliance on fatty acid oxidation. Moreover, coamplification of PETsign genes occurs frequently in BC arguing for their causal role in pathogenesis. CXCL8 and EGFR signaling pathways feature strongly in PETsign, and their activation in BC cells causes a shift toward a glycolytic phenotype. Thus, PETsign serves as a molecular surrogate for FDG-PET that could inform clinical management strategies for BC patients.

NREP, transcriptionally upregulated by HIF-1α, aggravates breast cancer cell growth and metastasis by promoting glycolysis

Breast cancer (BC) poses a great threat to women’s health. Neuronal regeneration related protein (NREP) is a multifunctional protein that is involved in embryonic development, regeneration, and human disease. However, the biological function of NREP in tumors is rarely reported and its role in BC remains unknown. Bioinformatics analysis showed that NREP is highly expressed and closely correlated with poor survival in BC patients. Under hypoxic conditions, NREP was upregulated in BC cells, and this promotion was reversed by hypoxia-inducible factor HIF-1α suppression. Luciferase reporter system and chromatin immunoprecipitation assays confirmed that HIF-1α directly binds to the promoter of NREP to increase the transcriptional activity of NREP. NREP suppression inhibited cell proliferation, arrested the cell cycle at the G1/S phase, and promoted apoptosis and caspase-3 activity in BC cells. Suppression of NREP decreased the tube formation ability of HUVECs. In addition, NREP downregulation showed an inhibition effect on cell migration, invasion, and EMT of BC cells. In NREP overexpressed cells, all these changes were reversed. In vivo, animal experiments also confirmed that NREP promotes BC tumor growth and metastasis. In addition, NREP promoted cellular glycolysis and enhanced the levels of glucose consumption, ATP, lactate production, and glucose transporters expression in NREP-overexpressed BC cells. In summary, our results demonstrated that NREP could be transcriptional activated by HIF-1α, which may aggravate BC tumor growth and metastasis by promoting cellular glycolysis. This result suggested that NREP may play an essential part in BC progression.

High-contrast NIR fluorescent probes for selective detection of NQO1 in breast cancer

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a potential biomarker for breast cancer (BC) diagnosis and prognosis. However, existing fluorescent probes for NQO1 detection have limitations such as short emission wavelength, weak fluorescence response, or large background interference. Here, we developed two novel near-infrared (NIR) fluorescent probes, DCl-Q and DCl2-Q, that selectively detect NQO1 activity in BC cells and tissues. They consist of a trimethyl-locked quinone as the recognition group and a donor-π-acceptor structure with halogen atoms as the reporter group. They exhibit strong fluorescence emission at around 660 nm upon binding to NQO1. We demonstrated that they can distinguish BC cells with different NQO1 expression levels and image endogenous NQO1 in tumor-bearing mice. Our probes provide a convenient and highly sensitive tool for BC diagnosis and prognosis based on NQO1 detection.

Elucidation of interleukin-19 as a therapeutic target for breast cancer by computational analysis and experimental validation

Interleukin 19 (IL-19) is a cytokine produced by monocytes and belongs to the family of IL-10. The IL-19 protein stimulates fibronectin (FN) expression and assembly, metastasis, and cell division in breast cancer (BC) cells. IL-19, which is connected to breast pathogenesis and has an autocrine action in BC cells, is a key predictor of prognosis for many tumour forms, including breast cancer. Augmented IL-19 expression has been related to poorer clinical outcomes for patients with BC and directly enhances proliferation and migration while also serving as a microenvironment for tumour formation. The main aim of our study was to examine the expression profile, functional role, and prognostic significance of interleukin-19 in BC pathogenesis and also to find out the molecular mechanism of IL-19 in BC. In this work, we used the various computational approach and tools, to evaluate the expression profile and prognostic implication of IL-19 in BC and discover the role of IL-19 in BC pathogenesis. IL-19 was shown to be highly upregulated in BC as compared to other interleukins. Also, its levels were highly overexpressed in liminal BC patients, mostly in 3rd stage groups under the age group of 21–40 years. IL-19 levels were increased in BC and elevated expression of IL-19 was examined to have worse overall survival (OS). The KEGG analysis andgene ontology of IL-19 depict that IL-19 is significantly augmented in cytokine activity and receptor-ligand activity and also in the JAK-STAT signaling pathway. Moreover, IL-19 showed a high correlation with IL20RA, as later is involved with the JAK-STAT signaling pathway. The in-vivo and in-vitro studies have also reflected that upregulation of IL-19 enhances tumor development and affects clinical outcomes in BC patients through several pathways including the JAK TAT signalling pathway. Overall, our study indicates that IL-19 increases tumour growth and that inhibiting it in addition to standard treatments will greatly improve BC patient’s therapeutic responses.

Overexpression of ferritin light chain as a poor prognostic factor for breast cancer.

Ferritin light chain (FTL) is involved in tumor progression, but the specific molecular processes by which FTL affects the development of breast cancer (BRCA) have remained unknown. In this research, the clinicopathological significance of FTL overexpression in BRCA was investigated. To investigate the role of FTL in BRCA, we utilized multiple online databases to analyse FTL expression levels in BRCA. Next, we reviewed the expression and localization of the FTL protein in BRCA by immunohistochemistry (IHC), Western blot (WB) and immunofluorescence (IF) staining. To assess the impact of FTL on patient prognosis, we conducted Kaplan‒Meier, univariate and multivariate survival analyses. The relationship between FTL and immune infiltration in BRCA was also analysed in the TISCH and SangerBox databases. MTT, malondialdehyde (MDA) and reactive oxygen species (ROS) assays were carried out to investigate the molecular mechanisms of FTL action in BRCA cells. FTL was significantly upregulated in BRCA compared to normal tissues. Its expression significantly linked to histological grade (P = 0.038), PR expression (P = 0.021), Her2 expression (P = 0.012) and Ki-67 expression (P = 0.040) in patients with BRCA. Furthermore, the expression of the FTL protein was higher in the BRCA cell lines than in the normal breast cells and mainly localized in the cytoplasm. Compared to patients with a low level of FTL expression, patients with a high level of FTL expression showed lower overall survival (OS). More convincingly, univariate and multivariate statistical analyses revealed that FTL expression (P = 0.000), ER expression (P = 0.036) and Her2 expression (P = 0.028) were meaningful independent prognostic factors in patients with BRCA. FTL was associated with immune infiltration in BRCA. Functional experiments further revealed that FTL knockdown inhibited the capacity of proliferation and increased the level of oxidative stress in BRCA cells. Overexpression of FTL was associated with the progression of BRCA. FTL overexpression may become a biomarker for the evaluation of poor prognosis in patients with BRCA.

Understanding the Phytoestrogen Genistein Actions on Breast Cancer: Insights on Estrogen Receptor Equivalence, Pleiotropic Essence and Emerging Paradigms in Bioavailability Modulation.

Prevalent as a major phenolic ingredient of soy and soy products, genistein is recognized as an eminent phytoestrogen owing to its interacting ability with estrogen receptors (ERs). The metabolic conversion of plant-derived genistin to genistein by gut microbes and intestinal enzymes enhances its absorption at intestinal pH of ~7.5-7.8. Genistein interferes in breast cancer (BC) development via pleiotropic actions on cell proliferation, survival, angiogenesis, and apoptosis. Though multiple investigations have demonstrated genistein intake-driven reduced BC risk, similar efficacy has not been replicated in clinical trials. Furthermore, multiple studies have structurally and functionally equated genistein extents with 17-β-estradiol (E2), the most available physiological estrogen in females, culminating in aggravated BC growth. Of note, both genistein and E2 function via interacting with ERs (ERα and ERβ). However, although E2 shows almost equal affinity towards both ERα and ERβ, genistein shows more affinity towards ERβ than ERα. Our cautious literature survey revealed typical intake mode, ER expression pattern and the ratio of ERα and ERβ, transactivators/ regulators of ERα and ERβ expression and activities, patient age, and menopausal status as decisive factors affecting genistein BC activities. Of further interest are the mechanisms by which genistein inhibits triple-negative breast cancers (TNBCs), which lack ERs, progesterone receptors (PRs), and human epidermal growth factor receptors (HER2). Herein, we attempt to understand the dosage-specific genistein actions in BC cells and patients with an insight into its better response via derivative development, nanocarrier-assisted, and combinatorial delivery with chemotherapeutic drugs.

The Effect of Silica Nanoparticles (SiNPs) on Cytotoxicity, Induction of Oxidative Stress and Apoptosis in Breast Cancer Cell Lines

Breast cancer is one of the most common cancers in women. Silica nanoparticles (SiNPs) belong to the group of often-used nanoparticles in biomedical applications. The mechanisms of the cytotoxicity, apoptosis, and oxidative stress induced by the 5-15 nm SiNPs still remain unclear. The aim of the study was to evaluate the anti-cancer effect and mechanism of action of SiNPs in breast cancer cell lines. The breast cancer MDA-MB-231 and ZR-75-1 cell lines were analyzed using MTT assay, flow cytometry, and spectrophotometric methods. In this paper, we presented findings about the cytotoxicity, apoptosis, and oxidative stress in both breast cancer cell lines. We indicated that 5-15 nm SiNPs induced dose-dependent cytotoxicity in MDA-MB-231 and ZR-75-1 cells. Moreover, we demonstrated that the process of apoptosis in the studied cell lines was associated with a decrease in the mitochondrial membrane potential (ΔΨm) and an increase in the activity of caspase-9 and caspase-3. Based on the obtained results, 5-15 nm SiNPs are able to induce the mitochondrial apoptosis pathway. Analyzed nanoparticles have also been found to cause an increase in selected oxidative stress parameters in both breast cancer cell lines. The presented study provides an explanation of the possible mechanisms of 5-15 nm SiNPs action in breast cancer cells.

Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation.

Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Here, we report an unexpected intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients, and CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. Depletion of endogenous CCDC3 by CRISPR-Cas9 increased proliferation and drug resistance of BrC cells by alleviating 5-Fluorouracil (5-FU)-induced p53 level and activity. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. p53 induced CCDC3 expression by binding to its promoter in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth.

The cytotoxicity of karanjin toward breast cancer cells is involved in the PI3K/Akt signaling pathway.

Karanjin is a bioactive furanoflavonoid with various pharmacological activities including anticancer activities. However, the effect and the related mechanism of karanjin in breast cancer (BC) have not been revealed. The potential targets of karanjin and BC were predicted using SwissTargetPrediction and GeneCards databases, respectively. The overlapping targets between karanjin and BC were identified using the Venn diagram. DAVID database was used for the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis. Cell viability, proliferation, and apoptosis were examined by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide), EdU (5-ethynyl-2'-deoxyuridine) incorporation, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labeling) assays, respectively. The protein levels were measured by western blot analysis. We screened out 28 overlapping targets between karanjin and BC. KEGG analysis showed that the targets of karanjin in BC were associated with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Karanjin inhibited cell viability and impeded the proliferative ability of BC cells. Moreover, karanjin treatment induced apoptosis in BC cells. Additionally, karanjin treatment blocked the PI3K/Akt signaling pathway and activation of the PI3K/Akt pathway reversed the antitumor effect of karanjin on BC cells. In conclusion, karanjin exerted antitumor activity in BC cells by regulating the PI3K/Akt signaling pathway.

Abstract 856: Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation

Abstract Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Our recent studies revealed an intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients. CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. In contrast, knockdown of endogenous CCDC3 using CRISPR led to reduction of p53 level and activity, consequently promoting the proliferation of BrC cells. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. Also, knockdown of CCDC3 caused the resistance of BrC cells to 5-FU to some degrees by alleviating p53 response to this drug. p53 induced CCDC3 expression by binding to its promoter specifically in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth and suggest that CCDC3 deficiency could be a factor for drug resistance of BrCs. Citation Format: Hyemin Lee, Caiyue Li, Ji Hoon Jung, Yiwei Zhang, Jieqiong Wang, Chang Liu, Allyson M. Segall, Roger L. Sheffmaker, Shelya X. Zeng, Hua Lu. Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 856.

Ribosome assembly factor PNO1 is associated with progression and promotes tumorigenesis in triple‑negative breast cancer.

The aim of the present study was to investigate the expression of ribosome assembly factor partner of NOB1 homolog (PNO1) and its association with the progression of breast cancer (BC) in patients, as well as its biological function and underlying mechanism of action in BC cells. Bioinformatics and immunohistochemical analyses revealed that PNO1 expression was significantly increased in BC tissues and its high mRNA expression was associated with shorter overall survival (OS) and relapse-free survival (RFS) of patients with BC, as well as multiple clinical characteristics (including advanced stage of NPI and SBR, etc.) of patients with BC. Biological functional studies revealed that transduction of lentivirus encoding sh-PNO1 significantly downregulated PNO1 expression, reduced cell confluency and the number of BC cells in vitro and inhibited tumor growth in vivo. Moreover, PNO1 knockdown decreased the cell viability and arrested cell cycle progression at the G2/M phase, as well as downregulated cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDK1) protein expression in BC cells. Correlation analysis demonstrated that PNO1 expression was positively correlated with both CDK1 and CCNB1 expression in BC samples. Collectively, PNO1 was upregulated in BC and associated with BC patient survival, and PNO1 knockdown suppressed tumor growth in vitro and in vivo. In addition, positive regulation of CCNB1 and CDK1 may be one of the underlying mechanisms.

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer.

5'-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.

Heat shock factor 1 (HSF1) cooperates with estrogen receptor α (ERα) in the regulation of estrogen action in breast cancer cells.

Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was linked to estrogen (E2) signaling through estrogen receptor α (ERα). We found that an HSF1 deficiency may decrease ERα level, attenuate the mitogenic action of E2, counteract E2-stimulated cell scattering, and reduce adhesion to collagens and cell motility in ER-positive breast cancer cells. The stimulatory effect of E2 on the transcriptome is largely weaker in HSF1-deficient cells, in part due to the higher basal expression of E2-dependent genes, which correlates with the enhanced binding of unliganded ERα to chromatin in such cells. HSF1 and ERα can cooperate directly in E2-stimulated regulation of transcription, and HSF1 potentiates the action of ERα through a mechanism involving chromatin reorganization. Furthermore, HSF1 deficiency may increase the sensitivity to hormonal therapy (4-hydroxytamoxifen) or CDK4/6 inhibitors (palbociclib). Analyses of data from The Cancer Genome Atlas database indicate that HSF1 increases the transcriptome disparity in ER-positive breast cancer and can enhance the genomic action of ERα. Moreover, only in ER-positive cancers an elevated HSF1 level is associated with metastatic disease.

Antidiabetic DPP-4 Inhibitors Reprogram Tumor Microenvironment That Facilitates Murine Breast Cancer Metastasis Through Interaction With Cancer Cells via a ROS–NF-кB–NLRP3 Axis

Improvement of understanding of the safety profile and biological significance of antidiabetic agents in breast cancer (BC) progression may shed new light on minimizing the unexpected side effect of antidiabetic reagents in diabetic patients with BC. Our recent finding showed that Saxagliptin (Sax) and Sitagliptin (Sit), two common antidiabetic dipeptidyl peptidase-4 inhibitors (DPP-4i) compounds, promoted murine BC 4T1 metastasis via a ROS–NRF2–HO-1 axis in nonobese diabetic–severe combined immunodeficiency (NOD-SCID) mice. However, the potential role of DPP-4i in BC progression under immune-competent status remains largely unknown. Herein, we extended our investigation and revealed that Sax and Sit also accelerated murine BC 4T1 metastasis in orthotopic, syngeneic, and immune-competent BALB/c mice. Mechanically, we found that DPP-4i not only activated ROS–NRF2–HO-1 axis but also triggered reactive oxygen species (ROS)-dependent nuclear factor kappa B (NF-κB) activation and its downstream metastasis-associated gene levels in vitro and in vivo, while NF-кB inhibition significantly abrogated DPP-4i-driven BC metastasis in vitro. Meanwhile, inhibition of NRF2–HO-1 activation attenuated DPP-4i-driven NF-кB activation, while NRF2 activator ALA enhanced NF-кB activation, indicating an essential role of ROS–NRF2–HO-1 axis in DPP-4i-driven NF-кB activation. Furthermore, we also found that DPP-4i increased tumor-infiltrating CD45, MPO, F4/80, CD4, and Foxp3-positive cells and myeloid-derived suppressor cells (MDSCs), and decreased CD8-positive lymphocytes in metastatic sites, but did not significantly alter cell viability, apoptosis, differentiation, and suppressive activation of 4T1-induced splenic MDSCs. Moreover, we revealed that DPP-4i triggered ROS-NF-κB-dependent NLRP3 inflammasome activation in BC cells, leading to increase in inflammation cytokines such as interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), IL-1β and IL-33, and MDSCs inductors granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and M-CSF, which play a crucial role in the remodeling of tumor immune-suppressive microenvironment. Thus, our findings suggest that antidiabetic DPP-4i reprograms tumor microenvironment that facilitates murine BC metastasis by interaction with BC cells via a ROS–NRF2–HO-1–NF-κB–NLRP3 axis. This finding not only provides a mechanistic insight into the oncogenic ROS–NRF2–HO-1 in DPP-4i-driven BC progression but also offers novel insights relevant for the improvement of tumor microenvironment to alleviate DPP-4i-induced BC metastasis.

Anticancer agents based on Plastoquinone analogs with N-phenylpiperazine: Structure-activity relationship and mechanism of action in breast cancer cells

2,3-Dimethyl-1,4-benzoquinones named as Plastoquinone (PQ) analogs have antiproliferative activity and are promising new members of molecules that can be used to cope with cancer. In an attempt to develop effective and potentially safe antiproliferative agents, previously reported twelve Plastoquinone analogs (PQ1-12) have been obtained to understand their antiproliferative profile. All PQ analogs have been selected by the National Cancer Institute (NCI) of Bethesda based on the NCI Developmental Therapeutics Program and tested against the panel of 60 cancer cell lines. Based on those studies, the cytotoxicity of the selected PQ analogs (PQ8, PQ9, PQ11, and PQ12) was determined using four breast cancer cell lines (MCF7, UACC-2087, MDA-MB-231, and MDA-MB-435) and a normal cell line (HaCaT). For better understanding, apoptosis induction, changes in cell proliferation, cell migration, and reactive oxygen species (ROS) generation were investigated for the selected PQ analog (PQ11) on MCF7 and UACC-2087 cell lines. According to the study results, PQ11 showed the most promising anticancer activity against MCF7 cell line through increased oxidative stress and apoptosis and suppression of cell proliferation. Based on the biological activity profile, we hypothesize that PQ11 could be a modulator of the cannabinoid 2 (CB2) receptor. Accordingly, we analyzed molecular level interaction of PQ11 with CB2 receptor through molecular docking simulation and it was also predicted to have a favorable ADMET profile. Overall, our findings suggest that integration of the N-phenylpiperazine moiety can be a good strategy for the structural optimization of PQ analogs as anticancer agents, especially in breast cancer.

ERα36-GPER1 Collaboration Inhibits TLR4/NFκB-Induced Pro-Inflammatory Activity in Breast Cancer Cells.

Inflammation is important for the initiation and progression of breast cancer. We have previously reported that in monocytes, estrogen regulates TLR4/NFκB-mediated inflammation via the interaction of the Erα isoform ERα36 with GPER1. We therefore investigated whether a similar mechanism is present in breast cancer epithelial cells, and the effect of ERα36 expression on the classic 66 kD ERα isoform (ERα66) functions. We report that estrogen inhibits LPS-induced NFκB activity and the expression of downstream molecules TNFα and IL-6. In the absence of ERα66, ERα36 and GPER1 are both indispensable for this effect. In the presence of ERα66, ERα36 or GPER1 knock-down partially inhibits NFκB-mediated inflammation. In both cases, ERα36 overexpression enhances the inhibitory effect of estrogen on inflammation. We also verify that ERα36 and GPER1 physically interact, especially after LPS treatment, and that GPER1 interacts directly with NFκB. When both ERα66 and ERα36 are expressed, the latter acts as an inhibitor of ERα66 via its binding to estrogen response elements. We also report that the activation of ERα36 leads to the inhibition of breast cancer cell proliferation. Our data support that ERα36 is an inhibitory estrogen receptor that, in collaboration with GPER1, inhibits NFκB-mediated inflammation and ERα66 actions in breast cancer cells.

Insight into the mechanism of tetrachlorobisphenol A (TCBPA)-induced proliferation of breast cancer cells by GPER-mediated signaling pathways.

Tetrachlorobisphenol A (TCBPA), a chlorinated derivative of bisphenol A, is an endocrine disruptor based on interaction with nuclear estrogen receptor alpha (ERα). However, there is only limited data on the mechanisms through which TCBPA-associated estrogenic activity is related to the membrane G protein-coupled estrogen receptor (GPER) pathway. In this study, three human breast cancer cell lines-MCF-7, SKBR3, and MDA-MB-231cells were used to evaluate whether, as well as how, TCBPA at concentration range of 0.001-50μM affect cell proliferation. The role of GPER signaling in TCBPA-induced cell proliferation was studied by analyzing the protein expression and mRNA levels of relevant signal targets. The results showed that low concentrations of TCBPA significantly induced the proliferation of MCF-7, SKBR3, and MDA-MB-231cells, with MCF-7cells being the most sensitive to TCBPA exposure. Low-concentration TCBPA also upregulated the expression of GPER, CyclinD1, c-Myc, and c-Fos proteins, as well as increased the phosphorylation of extracellular signal-regulated-kinase 1/2 (Erk1/2) and protein kinase B (Akt). Additionally, the mRNA levels of genes associated with estrogen signaling pathways also increased upon exposure to TCBPA. However, the phosphorylation of Erk1/2 and Akt decreased when the cells were treated with GPER inhibitor G15 and phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin (WM) prior to TCBPA exposure. Besides, the increased proliferation of breast cancer cells induced by TCBPA were also inhibited. In ERα-positive MCF-7cells, TCBPA also upregulated ERα expression, and ERα was found to interact with GPER-mediated signaling. The results indicate that GPER activates the PI3K/Akt and Erk1/2 signal cascades to drive the cell proliferation observed for low concentrations of TCBPA. The presented results suggest a new mechanism by which TCBPA exerts estrogenic action in breast cancer cells, namely, GPER signaling in an ERα-independent manner, and also highlights the potential risks to human health of the usage of TCBPA.

SIRT6 enhances oxidative phosphorylation in breast cancer and promotes mammary tumorigenesis in mice

BackgroundSirtuin 6 (SIRT6) is a NAD+-dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors.MethodsWe evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration.ResultsThe heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice.ConclusionsVia its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.

Glucose Concentration in Cell Culture Medium Influences the BRCA1-Mediated Regulation of the Lipogenic Action of IGF-I in Breast Cancer Cells.

Hyperglycaemia is a common metabolic alteration associated with breast cancer risk and progression. We have previously reported that BRCA1 restrains metabolic activity and proliferative response to IGF-I anabolic actions in breast cancer cells cultured in high glucose. Here, we evaluated the impact of normal physiological glucose on these tumour suppressive roles of BRCA1. Human breast cancer cells cultured in normal physiological and high glucose were treated with IGF-I (0–500 ng/mL). Cellular responses were evaluated using immunoblotting, co-immunoprecipitation, and cell viability assay. As we previously reported, IGF-I induced ACCA dephosphorylation by reducing the association between BRCA1 and phosphorylated ACCA in high glucose, and upregulated FASN abundance downstream of ACCA. However, these effects were not observed in normal glucose. Normal physiological glucose conditions completely blocked IGF-I-induced ACCA dephosphorylation and FASN upregulation. Co-immunoprecipitation studies showed that normal physiological glucose blocked ACCA dephosphorylation by increasing the association between BRCA1 and phosphorylated ACCA. Compared to high glucose, the proliferative response of breast cancer cells to IGF-I was reduced in normal glucose, whereas no difference was observed in normal mammary epithelial cells. Considering these results collectively, we conclude that normal physiological glucose promotes the novel function of BRCA1 as a metabolic restraint of IGF-I actions. These data suggest that maintaining normal glucose levels may improve BRCA1 function in breast cancer and slow down cancer progression.

The quantitation of thioredoxin 1 from serum as a novel means to detect breast cancer (BC).

557 Background: Breast cancer (BC) is the most common cancer and the second leading cause of cancer death in women. Enormous effort has been conducted without success to develop a means to detect BC using the blood. We have reported that the level of thioredoxin 1 (Trx1) in serum could be a novel standard to evaluate the risk of BC. Therefore, we have investigated the clinical utility of Trx1 as a biomarker to detect BC by testing sera from normal women, women with BC, women with five other types of cancer. Methods: We have developed an ELISA kit that quantitates Trx1 in sera. The level of Trx1 was determined in each serum from normal healthy women (n = 114), as well as patients with BC (n = 106), cervical cancer (n = 17), lung cancer (n = 14), stomach cancer (n = 9), and thyroid cancer (n = 4). BC patients were recruited according to their age and cancer stage. Each test was duplicated more than three times, and test results were analyzed by ROC analysis, one-way ANOVA tests, and unpaired t-tests. Results: The mean value of Trx1 from normal women was 5.60±4.39(±SD) and that from BC was 22.25±7.07. The Trx1 level was effective to distinguish BC serum from healthy serum with a sensitivity of 94.3% and specificity of 93.9% (AUC 0.985, p< 0.001). The levels of Trx1 from BC patients were higher than the cut-off value of 14.13 U/ml regardless of age, stage, histological grade, type, ER/PR/HER2 expression profile, and proliferation activity of BC cells. The levels of Trx1 from the other five types of cancers (2.34±1.82 - 3.64±2.99) were low enough to be distinguishable from BC. Especially, Trx1 levels could rescue patients whose mammography resulted in a false judgement. Conclusions: These results indicated that the blood level of Trx1 is an effective and accurate method to detect breast cancer, and particularly as a complement to mammography.