Estrogen Receptor-negative Breast Cancer Cells Research Articles

Strong Protection by 4-Hydroxyestrone against Erastin-Induced Ferroptotic Cell Death in Estrogen Receptor-Negative Human Breast Cancer Cells: Evidence for Protein Disulfide Isomerase as a Mechanistic Target for Protection.

Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E1), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI's catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E1 as well as cystamine (a known PDI inhibitor) against chemically induced ferroptosis in human breast cancer cells. Importantly, inhibition of PDI by 4-OH-E1 and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced ferroptosis. In conclusion, this study demonstrates that 4-OH-E1 is a novel inhibitor of PDI and can strongly inhibit ferroptosis in human breast cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.

Increased matrix stiffness enhances pro-tumorigenic traits in a physiologically relevant breast tissue- monocyte 3D model

High mammographic density, associated with increased tissue stiffness, is a strong risk factor for breast cancer per se. In postmenopausal women there is no differences in the occurrence of ductal carcinoma in situ (DCIS) depending on breast density. Preliminary data suggest that dense breast tissue is associated with a pro-inflammatory microenvironment including infiltrating monocytes. However, the underlying mechanism(s) remains largely unknown. A major roadblock to understanding this risk factor is the lack of relevant in vitro models. A biologically relevant 3D model with tunable stiffness was developed by cross-linking hyaluronic acid. Breast cancer cells were cultured with and without freshly isolated human monocytes. In a unique clinical setting, extracellular proteins were sampled using microdialysis in situ from women with various breast densities. We show that tissue stiffness resembling high mammographic density increases the attachment of monocytes to the cancer cells, increase the expression of adhesion molecules and epithelia-mesenchymal-transition proteins in estrogen receptor (ER) positive breast cancer. Increased tissue stiffness results in increased secretion of similar pro-tumorigenic proteins as those found in human dense breast tissue including inflammatory cytokines, proteases, and growth factors. ER negative breast cancer cells were mostly unaffected suggesting that diverse cancer cell phenotypes may respond differently to tissue stiffness. We introduce a biological relevant model with tunable stiffness that resembles the densities found in normal breast tissue in women. The model will be key for further mechanistic studies. Additionally, our data revealed several pro-tumorigenic pathways that may be exploited for prevention and therapy against breast cancer. Statement of significanceWomen with mammographic high-density breasts have a 4–6-fold higher risk of breast cancer than low-density breasts. Biological mechanisms behind this increase are not fully understood and no preventive therapeutics are available. One major reason being a lack of suitable experimental models. Having such models available would greatly enhance the discovery of relevant targets for breast cancer prevention. We present a biologically relevant 3D-model for studies of human dense breasts, providing a platform for investigating both biophysical and biochemical properties that may affect cancer progression. This model will have a major scientific impact on studies for identification of novel targets for breast cancer prevention.

EGF-Dependent Activation of ELK1 Contributes to the Induction of CLDND1 Expression Involved in Tight Junction Formation.

Claudin proteins are intercellular adhesion molecules. Increased claudin domain-containing 1 (CLDND1) expression is associated with the malignant transformation of estrogen receptor-negative breast cancer cells with low sensitivity to hormone therapy. Abnormal CLDND1 expression is also implicated in vascular diseases. Previously, we investigated the regulatory mechanism underlying CLDND1 expression and identified a strong enhancer region near the promoter. In silico analysis of the sequence showed high homology to the ETS domain-containing protein-1 (ELK1)-binding sequence which is involved in cell growth, differentiation, angiogenesis, and cancer. Transcriptional ELK1 activation is associated with the mitogen-activated protein kinase (MAPK) signaling cascade originating from the epidermal growth factor receptor (EGFR). Here, we evaluated the effect of gefitinib, an EGFR tyrosine kinase inhibitor, on the suppression of CLDND1 expression using ELK1 overexpression in luciferase reporter and chromatin immunoprecipitation assays. ELK1 was found to be an activator of the enhancer region, and its transient expression increased that of CLDND1 at the mRNA and protein levels. CLDND1 expression was increased following EGF-induced ELK1 phosphorylation. Furthermore, this increase in CLDND1 was significantly suppressed by gefitinib. Therefore, EGF-dependent activation of ELK1 contributes to the induction of CLDND1 expression. These findings open avenues for the development of new anticancer agents targeting CLDND1.

Effect of combining CBD with standard breast cancer therapeutics

Breast cancer is the most common malignancy in women worldwide. Sixty-five percent of breast cancers are estrogen and/or progesterone receptor positive. Estrogen receptor expression is a prognostic and predictive biomarker of response to endocrine therapy, which consists of the selective estrogen receptor modulator tamoxifen, aromatase inhibitors, and the selective estrogen receptor degrader fulvestrant. Cannabidiol is a phytocannabinoid that is emerging as a potential therapeutic agent. The aim of this study was to investigate the effect of cannabidiol on estrogen receptor-positive and estrogen receptor-negative representative breast cancer cell lines in combination with standard therapeutic agents used in clinical practice. To compare the effects of cannabidiol on breast cancer cell viability, cancer cell lines were exposed to increasing concentrations of cannabidiol. The effects of cannabidiol in combination with the endocrine therapeutics tamoxifen, fulvestrant, and the cyclin-dependent kinase inhibitor palbociclib on breast cancer cell viability were examined. We demonstrated that cannabidiol dose-dependently decreased the viability of all breast cancer cell lines independent of estrogen receptor expression. The addition of cannabidiol to tamoxifen had an additive negative effect on cell viability in ER+ ​in estrogen receptor positive T-47D line. Cannabidiol did not attenuate the effect of standard treatment of hormone receptor-positive breast cancer with fulvestrant and palbociclib. In addition, cannabidiol did not attenuate the effect of standard treatment of triple-negative breast cancer and human epidermal growth factor receptor 2 positive breast cancer cell lines with trastuzumab and cisplatin.

Re-Expression of ERα and AR in Receptor Negative Endocrine Cancers via GSK3 Inhibition.

DNA methylation, catalyzed by DNA methyltransferase (DNMT), is a well-characterized epigenetic modification in cancer cells. In particular, promoter hypermethylation of AR and ESR1 results in loss of expression on Androgen Receptor (AR) and Estrogen Receptor (ER), respectively, and is associated with a hormone refractory state. We now report that Glycogen Synthase Kinase 3 (GSK3) phosphorylates DNMT1 at S714, which is localized to a 62 amino acid region referred to as auto-inhibitory linker, which functions to occlude the DNA from the active site of DNMT1 to prevent the methylation of unmethylated DNA. Molecular Dynamics simulation indicates that phosphorylation at S714 resulted in conformational rearrangement of the autoinhibitory domain that inactivated its ability to block the methylation of unmethylated DNA and resulted in enhanced DNA binding. Treatment with a novel and more selective inhibitor of GSK3 resulted in decreased methylation of the promoter region of genes encoding the Androgen Receptor (AR) and Estrogen Receptor alpha (ERa) and re-expression of the AR and ERa in AR negative prostate cancer and ER negative breast cancer cells, respectively. As a result, concurrent treatment with the GSK3 inhibitor resulted in responsiveness of AR negative prostate cancer and ER negative breast cancer cells to inhibitors of the AR or ER, respectively, in in vitro and in vivo experimental models.

The analog of cGAMP, c-di-AMP, activates STING mediated cell death pathway in estrogen-receptor negative breast cancer cells.

Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negativebreast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.

Sevoflurane modulates breast cancer cell survival via modulation of intracellular calcium homeostasis

BackgroundSome retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis.MethodsEstrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects.ResultsSevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell.ConclusionSevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.

Abstract 4375: Inhibition of triple negative breast cancer cell progression using estrogen related receptor alpha endogenous ligands

Abstract Triple-negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is considered to be more metastatic, and has poorer prognosis and higher risk of recurrence than other subtypes of breast cancer. The death rate in patients with TNBC is twice that of ERα positive tumors mainly because there are fewer targeted therapies that treat TNBC patients [1]. Therefore, there is a need to discover new drug-targeted therapy for these patients. Several lines of evidence indicate that estrogen related receptors (ERRs), which belong to the orphan nuclear receptor superfamily, play a crucial role in breast cancer, with ERRα overexpression reportedly leading to adverse clinical outcomes in TNBC patients [2]. Despite intensive efforts, no endogenous ligand has been identified for ERRs so far. The discovery of ligands that bind these receptors may lead to novel strategies for the treatment of TNBC. In this study we show two endogenous ligands of ERRs: 1) estradienolone (ED), a novel endogenous steroid during pregnancy which acts as an inverse agonist of ERRs, 2) cholesterol as an agonist of ERRs. Our recent results show that ED acts as an inverse agonist of ERRα and ERRγ by directly interacting with these receptors, and inhibiting their transcriptional activity. We also demonstrate that ED has strong anti-mitogenic properties. ED inhibits the growth of both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells in a dose dependent manner, while of displaying a little effect on normal epithelial breast cells. In addition, we show that cholesterol binds directly and specifically to ERRα, it increases the transcriptional activity of ERRα in a peroxisome proliferator-activated receptor coactivator-1α (PGC-1) dependent manner. Our finding suggests that cholesterol enhances the interaction of ERRα and its coactivator PGC-1, and this leads to induce the expression of ERRα itself (an specific auto-induction) and the metabolic target genes of ERRα to fuel TNBC cells proliferation and migration. Moreover, our results demonstrates that the effect of cholesterol or lovastatin (a drug known to inhibit cholesterol synthesis) in triple negative breast cancer (MDA-MB 231) requires ERRα. These data suggest that both ED-ERR and cholesterol-ERR interactions may represent novel druggable signaling pathway in TNBC. Citation Format: Faegheh Ghanbari, Sylvie Mader, Anie Philip. Inhibition of triple negative breast cancer cell progression using estrogen related receptor alpha endogenous ligands [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4375.

Abstract 4236: Evaluating alterations in mRNA biogenesis with TCGA breast cancer sequencing data

Abstract Alternative splicing leads to the production of multiple mRNA isoforms of a gene transcript with distinct functional characteristics. These events are frequently observed in rapidly dividing cancer cells and are broadly recognized as important signatures for oncogenesis pathways and treatment options. In this study, we utilized data from The Cancer Genome Atlas (TCGA) to identify differences in isoform expression between estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RNA sequencing analysis combined with clinical annotation data revealed alterations in isoform expression that were associated with patient survival. These global changes in isoform expression occurred independently of total gene expression in both estrogen receptor-positive and -negative breast cancer. Furthermore, we identified multiple underrepresented isoforms including CDKAL1 and CSTF3 that were associated with favorable prognostic outcomes despite a lack of association observed with their traditional transcripts. These data suggest an underlying role mediated by alternative spliced isoforms in driving clinical outcome in breast cancer, warranting further investigation in delineating isoform expression profiles in previously established cancer pathways. Citation Format: Thomas J. Yan, Matthew E. Burow, Elizabeth C. Martin. Evaluating alterations in mRNA biogenesis with TCGA breast cancer sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4236.

Abstract 1003: Modulating estrogen related receptors (ERRs) activity in breast cancer using endogenous ligands

Abstract Estrogen-receptor related receptors which consists of ERRα, ERRβ and ERRγ belong to the orphan nuclear receptor subfamily of NR3B (nuclear receptor subfamily 3, group B). The ERRs have been shown to actively modulate estrogenic responses, and to play an essential role in pregnancy, and are implicated in breast cancer progression. Despite intensive efforts, no endogenous ligand has been identified for ERRs so far. The discovery of ligands that bind these orphan receptors will allow the manipulation of this pathway and may lead to novel strategies for the treatment of cancer and other diseases. In this study we show two endogenous ligands of ERRs: 1) estradienolone (ED), a novel endogenous steroid during pregnancy which acts as an inverse agonist of ERRs, 2) cholesterol as an agonist of ERRs. Our recent results show that ED acts as an inverse agonist of ERRα and ERRγ by directly interacting with these receptors, and inhibiting their transcriptional activity. We also demonstrate that ED has strong anti-mitogenic properties. ED inhibits the growth of both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells in a dose dependent manner, while of displaying a little effect on normal epithelial breast cells. In addition, we show that the effect of cholesterol or lovastatin (a drug known to inhibit cholesterol synthesis) in triple negative breast cancer (MDA-MB 231) requires ERRα. These data suggest that both ED-ERR and cholesterol-ERR interactions may represent novel physiologically relevant ERR pathways in the human. Citation Format: Faegheh Ghanbari, Sylvie Mader, Anie Philip. Modulating estrogen related receptors (ERRs) activity in breast cancer using endogenous ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1003.

Isolation and functional characterization of a novel endogenous inverse agonist of estrogen related receptors (ERRs) from human pregnancy urine

Estrogen-receptor related receptors (ERRs) which consists of ERRα, ERRβ and ERRγ belong to the orphan nuclear receptor subfamily 3, group B (NR3B) subfamily, and are constitutively active. ERRs have been shown to actively modulate estrogenic responses, and to play an essential role in pregnancy, and are implicated in breast cancer progression. Despite intensive efforts, no endogenous ligand other than the ubiquitous sterol, cholesterol which binds ERRα, has been identified for ERRs so far. The discovery of ligands that bind these orphan receptors will allow the manipulation of this pathway and may lead to novel strategies for the treatment of cancer and other diseases. We previously reported the identification of a novel endogenous estradienolone-like steroid (ED) that is strongly bound to sex hormone binding globulin, in pregnant women. Our recent results show that ED acts as an inverse agonist of ERRα and ERRγ by directly interacting with these receptors, and inhibiting their transcriptional activity. We also demonstrate that ED inhibits the growth of both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells in a dose dependent manner, while of displaying a little effect on normal epithelial breast cells. Furthermore, the anti-mitogenic effect of ED in breast cancer cells is ERRα-dependent. These data suggest that ED-ERR interaction may represent a novel physiologically relevant hormone response pathway in the human. The finding that ED inhibits both ER negative and ER positive breast cancer cell growth may have important implications in pathophysiology breast cancer.

Retraction: Sensitizing Estrogen Receptor-negative Breast Cancer Cells to Tamoxifen with OSU-03012, a Novel Celecoxib-derived Phosphoinositide-dependent Protein Kinase-1/Akt Signaling Inhibitor.

Tamoxifen is a mainstay in the treatment of estrogen receptor (ER)-positive breast cancer patients. Although the efficacy of tamoxifen has been attributed to induction of tumor cell growth arrest and apoptosis by inhibition of ER signaling, recent evidence indicates that tamoxifen possesses ER-independent antitumor activities. Here, we use OSU-03012, a small-molecule inhibitor of phosphoinositide-dependent protein kinase-1 (PDK-1) to address the hypothesis that PDK-1/Akt signaling represents a therapeutically relevant target to sensitize ER-negative breast cancer to tamoxifen. OSU-03012 sensitized both ER-positive MCF-7 and ER-negative MDA-MB-231 cells to the antiproliferative effects of tamoxifen in an ER-independent manner. Flow cytometric analysis of phosphatidylserine externalization revealed that this augmented suppression of cell viability was attributable to a marked enhancement of tamoxifen-induced apoptosis by OSU-03012. Mechanistically, this OSU-03012-mediated sensitization was associated with suppression of a transient tamoxifen-induced elevation of Akt phosphorylation and enhanced modulation of the functional status of multiple Akt downstream effectors, including FOXO3a, GSK3alpha/beta, and p27. The growth of established MDA-MB-231 tumor xenografts was suppressed by 50% after oral treatment with the combination of tamoxifen (60 mg/kg) and OSU-03012 (100 mg/kg), whereas OSU-03012 and tamoxifen alone suppressed growth by 30% and 0%, respectively. These findings indicate that the inhibition of PDK-1/Akt signaling to sensitize ER-negative breast cancer cells to the ER-independent antitumor activities of tamoxifen represents a feasible approach to extending the use of tamoxifen to a broader population of breast cancer patients. Considering the urgent need for novel therapeutic strategies for ER-negative breast cancer patients, this combinatorial approach is worthy of continued investigation.

HIGH expression of OSM and IL-6 are associated with decreased breast cancer survival: synergistic induction of IL-6 secretion by OSM and IL-1β.

Chronic inflammation has been recognized as a risk factor for the development and maintenance of malignant disease. Cytokines such as interleukin-6 (IL-6), oncostatin M (OSM), and interleukin-1 beta (IL-1β) promote the development of both acute and chronic inflammation while promoting in vitro metrics of breast cancer metastasis. However, anti-IL-6 and anti-IL-1β therapeutics have not yielded significant results against solid tumors in clinical trials. Here we show that these three cytokines are interrelated in expression. Using the Curtis TCGA™ dataset, we have determined that there is a correlation between expression levels of OSM, IL-6, and IL-1β and reduced breast cancer patient survival (r = 0.6, p = 2.2 x 10−23). Importantly, we confirm that OSM induces at least a 4-fold increase in IL-6 production from estrogen receptor-negative (ER−) breast cancer cells in a manner that is dependent on STAT3 signaling. Furthermore, OSM induces STAT3 phosphorylation and IL-1β promotes p65 phosphorylation to synergistically induce IL-6 secretion in ER− MDA-MB-231 and to a lesser extent in ER+ MCF7 human breast cancer cells. Induction may be reduced in the ER+ MCF7 cells due to a previously known suppressive interaction between ER and STAT3. Interestingly, we show in MCF7 cells that ER’s interaction with STAT3 is reduced by 50% through both OSM and IL-1β treatment, suggesting a role for ER in mitigating STAT3-mediated inflammatory cascades. Here, we provide a rationale for a breast cancer treatment regime that simultaneously suppresses multiple targets, as these cytokines possess many overlapping functions that increase metastasis and worsen patient survival.

Abstract P2-02-01: Inhibition of creatine kinase metabolism represses invasion and sensitizes estrogen-receptor negative breast cancer cells to standard chemotherapies

Abstract Dysregulated tumor cell metabolism is a hallmark of cancer progression and therapeutic resistance. In a screen for Hypoxia-Inducible Factor (HIF)-dependent genes regulating metabolism, we identified creatine kinase, brain isoform (CKB) as down-regulated in HIF-1 knockout mammary tumor cells. CKB is a member of a family of cytosolic and mitochondrial creatine kinases (CKs) that reversibly catalyze the transfer of a high-energy phosphoryl group from ATP to creatine, generating local stores of phosphocreatine in the forward reaction, and re-generating ATP in the reverse reaction. Creatine kinases are up-regulated in a variety of solid tumors, including breast cancers. CK activity is inhibited by cyclocreatine (cCr), a creatine kinase substrate that represses the CK-dependent generation of ATP from phosphocreatine >170-fold. Knockdown of CKB in tumor cells derived from the polyoma middle T (PyMT) transgenic model of metastatic breast cancer did not impair cell proliferation or wound healing, but potently suppressed invasion in vitro, suggesting utility for treating stage IV disease. When female FVB/Nj mice were injected with wild type PyMT cells in a tail vein assay and then treated with cCr, lung metastasis was repressed to the same extent as CKB gene knockdown. These data were validated using a variety of human estrogen receptor (ER)-negative breast cancer cell lines. CKB loss- and gain-of-function models were created and effects on cell proliferation, survival, wound healing and ATP production were compared to cCr treatment. Overall, whereas deletion of CKB had no effect on cell proliferation or survival in either adherent or suspension conditions, either deletion of CKB or cCr therapy potently reduced intracellular ATP levels, which was associated with reduced invasive potential. In contrast, over-expression of CKB enhanced chemotaxis to EGF and invasion towards serum. Whereas cCr monotherapy is generally cytostatic, combination with either doxorubicin or paclitaxel is synergistic to kill tumor cells. Together, these data suggest that inhibition of CK activity may be effective in treating stage IV breast cancer patients with established metastatic disease. We are currently exploring the cell signaling and cell survival pathways that are altered by CKB gene deletion or cCr treatment to understand how the creatine kinase arm of metabolism promotes tumor progression, metastasis and response to conventional chemotherapies. Citation Format: Barch HP, Krutilina R, Brooks DL, Parke D, Seagroves TN. Inhibition of creatine kinase metabolism represses invasion and sensitizes estrogen-receptor negative breast cancer cells to standard chemotherapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-01.

The A2B adenosine receptor in MDA-MB-231 breast cancer cells diminishes ERK1/2 phosphorylation by activation of MAPK-phosphatase-1.

It was previously shown that the estrogen-receptor negative breast cancer cell line MBA-MD-231 expresses high levels of A2B adenosine receptors as the sole adenosine receptor subtype. These receptors couple to both, stimulation of adenylyl cyclase and a Ca2+ signal. In order to establish a potential role of A2B adenosine receptors in tumor growth and development MAPK signaling was investigated in these breast cancer cells. Although it is known that A2B adenosine receptors may stimulate MAPK it was found that in MBA-MD-231 cells ERK1/2 phosphorylation is reduced upon agonist-stimulation of A2B adenosine receptors. This reduction is also triggered by forskolin, but abolished by the PKA inhibitor H89, suggesting an important role for the cAMP-PKA pathway. Likewise, a role for intracellular Ca2+ was established as the Ca2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) abolished the reduction of ERK1/2 phosphorylation triggered by A2B stimulation. It was shown that various pathways downstream from A2B adenosine receptors resulted in a stimulation of MAPK phosphatase-1 (MKP-1) which dephosphorylates phospho ERK1/2, and thus plays a critical role in the regulation of the phosphorylation state of ERK1/2. The reduction of ERK1/2 phosphorylation mediated by A2B adenosine receptors might provide an interesting approach for adjuvant treatment leading to reduced growth of certain tumors expressing the A2B subtype.

Effect of serum containing Xihuang pill on proliferation of human breast cancer cell line MDA-MB-435 and MCF-7 Cells

To study the effect of serum containing Xihuang pill on the proliferation of human breast cancer cell lines MDA-MB-435 and MCF-7 and the gene and protein expressions of Bcl-2, Bax, TP53, in order to explore the effect and mechanism of Xihuang pill in resisting breast cancer. The serum of the rats was prepared by the method of MTT assay. The expressions of Bcl-2 and Bax were detected by RT-PCR. The serum levels of Bcl-2 and Bax and the mRNA expression of TP53 were detected by immunofluorescence. The rats with serum containing Xihuang pill could inhibit the proliferation of MDA-MB-435 cells and MCF-7 cells (P<0.05). The serum containing Xihuang pill increased TP53 and Bax in MDA-MB-435 cells (P<0.05), and the ratio of Bcl-2/Bax was decreased (P<0.05). Meanwhile, the serum containing Xihuang pill could up-regulate the mRNA expression of Bax in MCF-7 cells and decrease the expression of Bcl (P<0.05), but there was no significant difference between the expression of TP53mRNA and Bax protein expressions after the treatment of MCF-7 cells with Xihuang pill serum. Serum containing Xihuang pill can induce the apoptosis of human breast cancer cells, and the mechanism of estrogen receptor-negative breast cancer cell apoptosis may be induced by up-regulating the mRNA expression of TP53, which can induce the expression of Bax and promote the metastasis of Bax to mitochondria, and ultimately play the role of inducing apoptosis.

Enhanced ZnR/GPR39 Activity in Breast Cancer, an Alternative Trigger of Signaling Leading to Cell Growth

Acquired resistance to the estrogen receptor (ER) antagonist tamoxifen, is a major obstacle in treatment of breast cancer. Changes in Zn2+ accumulation and distribution are associated with tamoxifen-resistance and breast cancer progression. The Zn2+-sensing G-protein coupled receptor, ZnR/GPR39, triggers signaling leading to cell growth, but a role for this receptor in breast cancer in unknown. Using fluorescence imaging, we found Zn2+-dependent Ca2+ release, mediated by ZnR/GPR39 activity, in TAMR tamoxifen-resistant cells derived from MCF-7 cells, but not in ER-expressing MCF-7 or T47D cells. Furthermore, ZnR/GPR39 signaling was monitored in ER negative BT20, MDA-MB-453 and JIMT-1 cells. Expression of ZnR/GPR39 was increased in grade 3 human breast cancer biopsies compared to grade 2. Consistently, analysis of two breast cancer patient cohorts, GDS4057 and TCGA, indicated that in ER-negative tumors higher ZnR/GPR39 mRNA levels are associated with more aggressive tumors. Activation of ZnR/GPR39 in TAMR cells triggered MAPK, mTOR and PI3K signaling. Importantly, enhanced cell growth and invasiveness was observed in the ER negative breast cancer cells, TAMR, MDA-MB-453 and BT20 cells but not in the ER expressing MCF-7 cells. Thus, we suggest ZnR/GPR39 as a potential therapeutic target for combination treatment in breast cancer, particularly relevant in ER negative tumors.

Local estrogen axis in the human bone microenvironment regulates estrogen\xa0receptor-positive\xa0breast cancer cells

BackgroundApproximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear.MethodsBone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry.ResultsER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation.ConclusionsThese results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

Profiling of gene expression regulated by 17β-estradiol and tamoxifen in estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines.

One area of great importance in breast cancer (BC) research is the study of gene expression regulated by both estrogenic and antiestrogenic agents. Although many studies have been performed in this area, most of them have only addressed the effects of 17β-estradiol (E2) and tamoxifen (TAM) on MCF7 cells. This study aimed to determine the effect of low doses of E2 and TAM on the expression levels of 84 key genes, which are commonly involved in breast carcinogenesis, in four BC cell lines differentially expressing estrogen receptor (ER) α and HER2 (MCF7, T47D, BT474, and SKBR3). The results allowed us to determine the expression patterns modulated by E2 and TAM in ERα+ and ERα− cell lines, as well as to identify differences in expression patterns. Although the MCF7 cell line is the most frequently used model to determine gene expression profiles in response to E2 and TAM, the changes in gene expression patterns identified in ERα+ and ERα− cell lines could reflect distinctive properties of these cells. Our results could provide important markers to be validated in BC patient samples, and subsequently used for predicting the outcome in ERα+ and ERα− tumors after TAM or hormonal therapy. Considering that BC is a molecularly heterogeneous disease, it is important to understand how well, and which cell lines, best model that diversity.

Activation of Akt characterizes estrogen receptor positive human breast cancers which respond to anthracyclines.

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance.The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials.Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30).In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicin-resistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves.