Hormone-responsive Breast Cancer Cells Research Articles

Mitochondrial calcium overload contributes to cannabinoid-induced paraptosis in hormone-responsive breast cancer cells.

Studies have shown that natural products can induce paraptosis in tumour cell lines. Paraptosis is characterized by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and mitochondria. The mechanism of paraptosis is unclear; however, dysregulation of Ca2+ homeostasis is believed to affect paraptosis induction. This study investigated the mechanism of cell death induced by a phytocannabinoid ratio in the MCF7 breast cancer cell line. The crystal violet assay was used to detect changes in viability and morphology changes were investigated using light and transmission electron microscopy. Various inhibitors, fluorescent staining with high-content screening, and Western blot analysis were used to investigate different cell death mechanisms. The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, no apoptosis, necrosis, autophagy, or ferroptosis was detected. Vacuolation induced by phytocannabinoid treatment was inhibited by cycloheximide, suggesting paraptosis induction. The mechanism of paraptosis induction was investigated, and it was found that treatment (1) induced ER dilation and mitochondrial swelling, (2) induced significant ER stress and mitochondrial Ca2+ overload and dysfunction, which appeared to be mediated by the voltage-dependent anion channel, and (3) significantly impaired all mitochondrial metabolic pathways. The data demonstrated that paraptosis induced by the cannabinoid ratio was mediated by Ca2+ flux from the ER to the mitochondria. These findings highlight a novel mechanism of cannabinoid-induced cell death and emphasize the anti-cancer potential of cannabinoid ratios, which exhibited enhanced effects compared to individual cannabinoids.

A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

Abstract IA13: Molecular and genomic mechanisms of estrogen signaling and gene regulation in breast cancer

Abstract Transcriptional enhancers function as nucleation sites for the assembly of transcription-regulating complexes across the genome, which act to drive cell type-specific patterns of gene expression. Although many features of active enhancers [e.g., H3K4me1, H3K27ac, enrichment of p300/CBP and Mediator, and production of enhancer RNAs (eRNAs)] have been defined by genomic assays, the roles of these features in estrogen receptor alpha (ERa) enhancer function are not well understood. We are interested in how the molecular underpinnings of ERa enhancer formation and activity control the gene expression programs that drive breast cancer phenotypes and responses to therapeutic agents. We have used a variety of molecular, biochemical, genomic, genetic, and computational approaches to determine the following aspects of ERa enhancers biology in hormone-responsive breast cancer cells: (1) where ERa enhancers are formed across the genome, (2) the kinetics of ERa enhancer formation and disassembly, (3) the influence of genetic variation on ERa enhancer function, (4) the role of the enhancer features noted above in ERa enhancer function, and (5) how protein complexes and small polypeptides serve as coregulators to drive ERa transcriptional activity. Our current focus has been on eRNAs, which are poorly characterized noncoding RNAs that are thought to regulate enhancer function and gene expression. We have identified a ~40 nucleotide functional eRNA regulatory motif (FERM) present in many estrogen-regulated eRNAs that promotes gene expression by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation. In addition, we have been examining how the dose of estrogen impacts the efficiency of enhancer assembly by ERα. Finally, we have been exploring the role of PARP1, a ubiquitous chromatin-associated regulator, and small polypeptides derived from “non-coding” RNAs, control ERa enhancer function. In all cases, we perform analyses to link enhancer function to gene expression outcomes and breast cancer biology. Collectively, our results demonstrate the biological and mechanistic roles of eRNAs in breast cancer. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Citation Format: W. Lee Kraus. Molecular and genomic mechanisms of estrogen signaling and gene regulation in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA13.

Phytochemicals from Pterocarpus angolensis DC and Their Cytotoxic Activities against Breast Cancer Cells.

Pterocarpus anglonesis DC is an indigenous medicinal plant belonging to the Pterocarpus genus of the Fabaceae family. It is used to treat stomach problems, headaches, mouth ulcers, malaria, blackwater fever, gonorrhea, ringworm, diarrhea, heavy menstruation, and breast milk stimulation. Column chromatography of the stem bark extracts resulted in the isolation of eight compounds, which included friedelan-3-one (1), 3α-hydroxyfriedel-2-one (2), 3-hydroxyfriedel-3-en-2-one (3), lup-20(29)-en-3-ol (4), Stigmasta-5-22-dien-3-ol (5), 4-O-methylangolensis (6), (3β)-3-acetoxyolean-12-en-28-oic acid (7), and tetradecyl (E)-ferulate (8). The structures were established based on NMR, IR, and MS spectroscopic analyses. Triple-negative breast cancer (HCC70), hormone receptor-positive breast cancer (MCF-7), and non-cancerous mammary epithelial cell lines (MCF-12A) were used to test the compounds' cytotoxicity. Overall, the compounds showed either no toxicity or very low toxicity to all three cell lines tested, except for the moderate toxicity displayed by lupeol (4) towards the non-cancerous MCF-12A cells, with an IC50 value of 36.60 μM. Compound (3β)-3-acetoxyolean-12-en-28-oic acid (7) was more toxic towards hormone-responsive (MCF-7) breast cancer cells than either triple-negative breast cancer (HCC70) or non-cancerous breast epithelial (MCF-12A) cells (IC50 values of 83.06 vs. 146.80 and 143.00 μM, respectively).

Genome-wide DNA methylation changes upon DOT1L inhibition in hormone-responsive breast cancer cells

Genome-wide DNA methylation changes upon DOT1L inhibition in hormone-responsive breast cancer cells

Inhibition of Vps34 and p110δ PI3K Impairs Migration, Invasion and Three-Dimensional Spheroid Growth in Breast Cancer Cells.

Breast cancer is a heterogeneous disease that represents the most common cancer around the world; it comprises 12% of new cases according to the World Health Organization. Despite new approaches in early diagnosis and current treatment, breast cancer is still the leading cause of death for cancer mortality. New targeted therapies against key signalling transduction molecules are required. Phosphoinositide 3-kinase (PI3K) regulates multiple biological functions such as proliferation, survival, migration, and growth. It is well established that PI3K isoform-selective inhibitors show fewer toxic side effects compared to broad spectrum inhibition of PI3K (pan-PI3K inhibitors). Therefore, we tested the PI3K p110δ-selective inhibitor, IC87114, and Vps34-selective inhibitor, Vps34-IN1, on the breast cancer cell lines MCF-7 and MDA-MB-231, representing hormone-responsive and triple-negative breast cancer cells, respectively. Our data show that both inhibitors decreased migration of MCF-7 and MDA-MB-231 cells, and Vps34 also significantly impacted MCF-7 cell proliferation. Three-dimensional (3D) in vitro culture models show that IC87114 and Vps34-IN1 treatment reduced the growth of MCF-7 and MDA-MB-231 cells in 3D tumour spheroid cultures. This study identifies IC87114 and Vps34-IN1 as potential therapeutic approaches in breast cancer.

Investigation of Chemopreventive and Antiproliferative Potential of Dicliptera roxburghiana.

Context:Carcinogenesis causes much human misery. It is a process involving multistage alterations. Medicinal plants are candidates for beneficial anticancer agents.Objectives:Investigation of anticancer proficiencies of the plant Dicliptera roxburghiana.Material and methods:Crude extract and derived fractions were inspected for their inhibitory potential against nuclear factor KB (NFκB), nitric oxide synthase inhibition, aromatase inhibition and induction of quinone reductase 1 (QR 1). Antiproliferative activity was determined by using various cancer cell lines for example hormone responsive breast cancer cell line MCF-7, estrogen receptor negative breast cancer cell line MDA-MB-231, murine hepatoma cells Hepa 1c1c7, human neuroblastoma cells SK-N-SH and neuroblastoma cells MYCN-2.Results:Ethyl acetate and n-butanol fractions of D. roxburghiana were strongly active against NFκB with IC50 of 16.6 ± 1.3 and 8.4 ± 0.7 µg/ml respectively with 100% survival. Chloroform fraction of the plant exhibited an induction ratio of 2.4 ± 0.09 with CD value of 17.7 µg/ml. Regarding the nitrite assay, the n-hexane fraction exhibited significant inhibition of NO activity with IC50 of 17.8 ± 1.25 µg/ml. The n-butanol fraction exhibited strong antiproliferative activity against IcIc-7 cell lines with IC50 values of 13.6 ± 1.91 µg/ml; against MYCN-2 a cytotoxic effect developed with dose dependence, with IC50 of 12.6 ± 1.24 µg/ml. In antiproliferative activity against SK-N-SH cell lines, chloroform, ethyl acetate and n-butanol fractions were efficiently active with IC50 values of 11.2 ± 0.84, 14.6 ± 1.71 and 16.3 ± 1.57 respectively.Discussion and Conclusion:It was demonstrated that various fractions of D. roxburghiana displayed appreciable anticancer characteristics and could be a potent source for the development of anticancer leads.

MiR-125a-5p impairs the metastatic potential in breast cancer via IP6K1 targeting.

The deregulation of PI3K/Akt signaling is among the most causes in inducing the acquisition of a metastatic phenotype in breast cancer cells, leading to Epithelial-Mesenchymal Transition (EMT). Inhibition of the PI3K/Akt pathway is known to be beneficial in the clinical setting. However, the activation of secondary pathways and toxicity profiles of available inhibitors, hindering optimal therapeutic results. Preliminary studies showed that myo-Inositol inhibits the PI3K/Akt pathway by exerting a pleiotropic anti-tumor action. Herein, we demonstrate that myo-Inositol triggers a prompt and profound remodeling of delineated expression pattern in triple-negative breast cancer cells (MDA-MB-231). Consequently, it inhibits metastasis and tumor progression through miR-125a-5p transcription and the subsequent inhibition of IP6K1. In contrast, hormone-responsive breast cancer cells (MCF-7) are insensitive to myo-Inositol. This is due to the persistence of MDM2 synthesis promoted by estrogen-dependent pathways. Conversely, the counteraction of estrogen effects recovered the sensitivity to myo-Inositol in the hormone-responsive model. Overall, these results identify a novel axis primed by miR-125a-5p to downregulate IP6K1 gene that inhibits metastasis. Thus, administration of myo-Inositol can activate this axis as a molecular target therapy in breast cancer.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Passive Diffusion vs Active pH-Dependent Encapsulation of Tyrosine Kinase Inhibitors Vandetanib and Lenvatinib into Folate-Targeted Ferritin Delivery System

IntroductionThe present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system.MethodsThe encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities.ResultsThe physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs’ structural and functional properties.ConclusionSince the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.

Amphiregulin retains ERα expression in acquired aromatase inhibitor resistant breast cancer cells.

Acquired resistance to aromatase inhibitors (AIs) is a significant clinical issue in endocrine therapy for estrogen receptor (ER) positive breast cancer which accounts for the majority of breast cancer. Despite estrogen production being suppressed, ERα signaling remains active and plays a key role in most AI-resistant breast tumors. Here, we found that amphiregulin (AREG), an ERα transcriptional target and EGF receptor (EGFR) ligand, is crucial for maintaining ERα expression and signaling in acquired AI-resistant breast cancer cells. AREG was deregulated and critical for cell viability in ER+ AI-resistant breast cancer cells, and ectopic expression of AREG in hormone responsive breast cancer cells promoted endocrine resistance. RNA-sequencing and reverse phase protein array analyses revealed that AREG maintains ERα expression and signaling by activation of PI3K/Akt/mTOR signaling and upregulation of forkhead box M1 (FOXM1) and serum- and glucocorticoid-inducible kinase 3 (SGK3) expression. Our study uncovers a previously unappreciated role of AREG in maintaining ERα expression and signaling, and establishes the AREG-ERα crosstalk as a driver of acquired AI resistance in breast cancer.

Identification of Antiestrogen-Bound Estrogen Receptor α Interactomes in Hormone-Responsive Human Breast Cancer Cell Nuclei.

Estrogen receptor alpha (ERα) is a ligand-inducible transcription factor which mediates estrogen actions in hormone-responsive tumors and is targeted by effective anticancer therapies based on the ERα antagonist ligands, selective estrogen receptor modulators (such as Tamoxifen/TAM) or disruptors (such as Fulvestrant/ICI). Despite its importance for cancer therapy, including acquired resistance to endocrine therapy, the molecular basis of ERα response to different ligands is not fully known to date. Interaction proteomics shows great potential to identify and characterize molecular mechanisms of disease based on physical and functional protein-protein interaction networks. Tandem affinity purification coupled to mass spectrometry is applied here for mapping in hormone-responsive breast cancer cells nuclei, the ERα interactomes, induced by each of the two classes of antiestrogens. The results provide new insights on the molecular bases for antiestrogen-mediated control of ERα function and reveal new potential ways to overcome endocrine therapy resistance in cancer.

Small Non-Coding RNA Profiling Identifies miR-181a-5p as a Mediator of Estrogen Receptor Beta-Induced Inhibition of Cholesterol Biosynthesis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease, representing the most aggressive breast cancer (BC) subtype with limited treatment options due to a lack of estrogen receptor alpha (ERα), progesterone receptor (PR), and Erb-B2 receptor tyrosine kinase 2 (HER2/neu) expression. Estrogen receptor beta (ERβ) is present in a fraction of TNBC patients, where its expression correlates with improved patient outcomes, supported by the fact that it exerts oncosuppressive effects in TNBC cell models in vitro. ERβ is involved in microRNA-mediated regulation of gene expression in hormone-responsive BC cells and could mediate its actions through small noncoding RNAs (sncRNAs) in TNBCs also. To verify this possibility, smallRNA sequencing was performed on three ERβ-expressing cell lines from different TNBC molecular subtypes. Several sncRNAs resulted modulated by ERβ, with a subset being regulated in a tumor subtype-independent manner. Interestingly, sncRNA profiling of 12 ERβ+and 32 ERβ− primary TNBC biopsies identified 7 microRNAs, 1 PIWI-interacting RNA (piRNA), and 1 transfer RNA (tRNA) differentially expressed in ERβ+ compared to ERβ− tumors and cell lines. Among them, miR-181a-5p was found to be overexpressed in ERβ+ tumors and predicted target key components of the cholesterol biosynthesis pathway previously found to be inhibited by ERβ in TNBC cells.

Estrone-Decorated Polyion Complex Micelles for Targeted Melittin Delivery to Hormone-Responsive Breast Cancer Cells.

Tumor targeting has revolutionized cancer research, especially active cellular targeting of nanoparticles, where they are specifically homed to the pathological site to deliver the therapeutics. This strategy, which involves the utilization of affinity ligands on the surface of the nanocarriers, minimizes the nonspecific uptake of nanocarriers and the subsequent harmful side effects in healthy cells. Estrone, one of the mammalian estrogens, has affinity for estrogen receptors (ERα), which are overexpressed in hormone-responsive breast cancers. Despite holding promise, the potential of estrone in active targeting of nanoparticles has barely been explored. Herein, we developed an estrone-appended polyion complex (PIC) micelle to deliver melittin, a cytotoxic peptide, to breast cancer cells. Amino functionalization of estrone was performed to conjugate estrone to the diblock polymer synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization. Estrone-conjugated poly(ethylene glycol) methyl ether methacrylate-b-poly tert-butyl methacrylate (POEGMEMA-PtBuMA) could complex with melittin to form PIC micelles of size around 60 nm ensuing from the electrostatic interaction of the deprotected polymer and melittin in aqueous media. Poly(ethylene glycol) methyl ether acrylate-b-poly acrylic acid (POEGMEA-PAA) was also later incorporated to afford PIC micelles that could exhibit similar cytotoxicity to free melittin in the cytotoxicity studies. The estrone-attached PIC micelles exhibited improved cytotoxicity in two-dimensional (2D) and three-dimensional (3D) cellular models of MCF-7 cells. Cross-linking of the PIC micelles was also performed to improve the stability of the micelles and prevent melittin degradation from enzymatic attack. Flow cytometry demonstrated an enhanced cellular uptake greater than sixfold with the estrone-conjugated PIC micelles, thereby establishing a profound difference in the targeting efficacy of the PIC micelles between MCF-7 and MDA-MB-231 cells. Furthermore, the distribution of the PIC micelles in the spheroids was revealed by light sheet microscopy. The results demonstrate the potential of estrone-anchored PIC micelles for targeted delivery of therapeutics to hormone-responsive breast cancer cells.

Role of mitochondria in rescuing glycolytically inhibited subpopulation of triple negative but not hormone-responsive breast cancer cells

Triple-negative breast cancer (TNBC) subtype is among the most aggressive cancers with the worst prognosis and least therapeutic targetability while being more likely to spread and recur. Cancer transformations profoundly alter cellular metabolism by increasing glucose consumption via glycolysis to support tumorigenesis. Here we confirm that relative to ER-positive cells (MCF7), TNBC cells (MBA-MD-231) rely more on glycolysis thus providing a rationale to target these cells with glycolytic inhibitors. Indeed, iodoacetate (IA), an effective GAPDH inhibitor, caused about 70% drop in MDA-MB-231 cell viability at 20 μM while 40 μM IA was needed to decrease MCF7 cell viability only by 30% within 4 hours of treatment. However, the triple negative cells showed strong ability to recover after 24 h whereas MCF7 cells were completely eliminated at concentrations <10 μM. To understand the mechanism of MDA-MB-231 cell survival, we studied metabolic modulations associated with acute and extended treatment with IA. The resilient TNBC cell population showed a significantly greater count of cells with active mitochondria, lower apoptotic markers, normal cell cycle regulations, moderately lowered ROS, but increased mRNA levels of p27 and PARP1; all compatible with enhanced cell survival. Our results highlight an interplay between PARP and mitochondrial oxidative phosphorylation in TNBC that comes into play in response to glycolytic disruption. In the light of these findings, we suggest that combined treatment with PARP and mitochondrial inhibitors may provide novel therapeutic strategy against TNBC.

MON-218 Inflammatory Role of Sex Steroids in Hidradenitis Suppurativa: An Androgenic Phenotype

Hidradenitis Suppurativa (HS) is an autoinflammatory skin disease more common in women and associated with hyper-androgenic disorders such as polycystic ovary syndrome. HS is characterized by chronic recurrent deep-seated painful nodules and draining tunnels in skin areas that follow the distribution of pubertal terminal hair growth. In women, HS disease activity varies with hormonal fluctuation across the menstrual cycle and pregnancy. Anti-androgens have shown promise in treatment of HS, but the pathophysiologic link between sex hormones and HS is unclear. We sought to further elucidate the role of sex hormones in the pathogenesis of HS. Objective: To determine if there is differential estrogen and androgen regulated gene expression in lesional compared to non-lesional skin transcriptome in HS patients. Methods: (a) We analyzed mRNA microarray data from lesional and non-lesional HS skin (GEO, GSE72702) (Blok JL, Br J Dermatol. 2016;174(6):1392). Inflammatory lesional skin from affected axilla or groin and non-lesional skin from the upper arm or leg was obtained from 17 patients with moderate to severe HS. We then examined differential expression of hormone regulated genes identified in existing androgen and estrogen gene expression signatures from prostate cancer cells [(VCaP treated with dihydrotestosterone (DHT)] and breast cancer cells [MCF7 treated with 17 beta-estradiol, (E2)] in the lesional and non-lesional skin transcriptomes of the HS patients. We identified overlap between the sets of genes that were either both upregulated or both repressed in HS lesions and the hormone-responsive prostate and breast cancer cell line models. Significance of differential gene expression was defined as FDR-adjusted p-value for two-tailed Student t-test less than 0.05. (b) After overlapping genes were identified, we used GeneGo Metacore pathway analysis software to explore the functional significance of the identified genes. Results: (a) Of the 3379 E2-upregulated probe sets in MCF7 breast cancer cells, 647 were significantly upregulated and 584 of 2871 E2-downregulated probe sets were significantly downregulated in HS lesions. Of the 1328 DHT-upregulated probe sets in prostate cancer cells, 341 were significantly upregulated and 196 of the 1251 DHT-downregulated probe sets were significantly downregulated in HS lesions. (b) Initial pathway analysis indicates that DHT upregulated genes in HS lesions are strongly enriched in innate immunity pathways, specifically, type 1 interferon signaling and class I MHC antigen presentation. Conclusion: There is substantial overlap between the transcriptional programme of sex steroids and transcriptional changes observed in HS lesional skin. In particular, androgen target genes are significantly enriched in innate immunity pathways, suggesting a functional link between the androgen signaling and the inflammatory process in HS lesions.

Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells.

Breast cancer (BC) resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor α (ERα) signaling, and ways to block ERα pathway in these tumors are sought after. We identified the H3K79 methyltransferase DOT1L as a novel cofactor of ERα in BC cell chromatin, where the two proteins colocalize to regulate estrogen target gene transcription. DOT1L blockade reduces proliferation of hormone-responsive BC cells in vivo and in vitro, consequent to cell cycle arrest and apoptotic cell death, with widespread effects on ER-dependent gene transcription, including ERα and FOXA1 gene silencing. Antiestrogen-resistant BC cells respond to DOT1L inhibition also in mouse xenografts, with reduction in ERα levels, H3K79 methylation, and tumor growth. These results indicate that DOT1L is an exploitable epigenetic target for treatment of endocrine therapy-resistant ERα-positive BCs.

Estrogen Down-regulator Fulvestrant Potentiates Antitumor Activity of Fluoropyrimidine in Estrogen-responsive MCF-7 Human Breast Cancer Cells.

Endocrine therapy is clinically administered in hormone-responsive breast cancer. Combinations of fluoropyrimidine S-1 and an aromatase inhibitor or anti-estrogen are considered beneficial in Japan. Herein we assessed new combinations of S-1 and fulvestrant. Cytotoxicity of fulvestrant and 5-fluorouracil (5-FU) was assessed in hormone-responsive (MCF-7) and non-responsive (MDA-MB-231) breast cancer cell cultures. Fulvestrant and S-1 were evaluated for antitumor activity in mice and their effects on estrogen receptor (ER)-α and progesterone receptor (PgR) levels in MCF-7 xenografts using immunohistochemical methods. Fulvestrant inhibited growth of MCF-7, but not of MDA-MB-231 xenografts. Combinations of 5-FU and fulvestrant were superior to monotherapy in vitro. In vivo antitumor activity of S-1/fulvestrant combination therapy was significantly (p<0.05) enhanced compared to that of both monotherapies. Fulvestrant partially down-regulated expression of ERα and PgR, but in combination with S-1, it almost completely blocked their expression. Chemo-endocrine combination therapy using S-1 and fulvestrant is beneficial in estrogen-responsive breast cancer.

Stable Isotope-Resolved Metabolomic Differences between Hormone-Responsive and Triple-Negative Breast Cancer Cell Lines.

Purpose To conduct an exploratory study to identify mechanisms that differentiate Luminal A (BT474 and MCF-7) and triple-negative (MDA-MB-231 and MDA-MB-468) breast cancer (BCa) cell lines to potentially provide novel therapeutic targets based on differences in energy utilization. Methods Cells were cultured in media containing either [U-13C]-glucose or [U-13C]-glutamine for 48 hours. Conditioned media and cellular extracts were analyzed by 1H and 13C NMR spectroscopy. Results MCF-7 cells consumed the most glucose, producing the most lactate, demonstrating the greatest Warburg effect-associated energy utilization. BT474 cells had the highest tricarboxylic acid cycle (TCA) activity. The majority of energy utilization patterns in MCF-7 cells were more similar to MDA-MB-468 cells, while the patterns for BT474 cells were more similar to MDA-MB-231 cells. Compared to the Luminal A cell lines, TNBC cell lines consumed more glutamine and less glucose. BT474 and MDA-MB-468 cells produced high amounts of 13C-glycine from media [U-13C]-glucose which was integrated into glutathione, indicating de novo synthesis. Conclusions Stable isotopic resolved metabolomics using 13C substrates provided mechanistic information about energy utilization that was difficult to interpret using 1H data alone. Overall, cell lines that have different hormone receptor status have different energy utilization requirements, even if they are classified by the same clinical BCa subtype; and these differences offer clues about optimizing treatment strategies.

Cation-dependent mannose-6-phosphate receptor expression and distribution are influenced by estradiol in MCF-7 breast cancer cells.

Cancer cells secrete procathepsin D, and its secretion is enhanced by estradiol. Although alterations in the pro-enzyme intracellular transport have been reported, the mechanism by which it is secreted remains poorly understood. In this work, we have studied the influence of estradiol on the expression and distribution of the cation-dependent mannose-6-phosphate receptor (CD-MPR), which would be a key molecule to ensure the proper localization of the enzyme to lysosomes in breast cancer cells. Immunoblotting studies demonstrated that the expression of CD-MPR is higher in MCF-7 cells, as compared to other breast cancer and non-tumorigenic cells. This expression correlated with high levels of cathepsin D (CatD) in these cells. By immunofluorescence, this receptor mostly co-localized with a Golgi marker in all cell types, exhibiting an additional peripheral labelling in MCF-7 cells. In addition, CD-MPR showed great differences regarding to cation-independent mannose-6-phosphate receptor. On the other hand, the treatment with estradiol induced an increase in CD-MPR and CatD expression and a re-distribution of both proteins towards the cell periphery. These effects were blocked by the anti-estrogen tamoxifen. Moreover, a re-distribution of CD-MPR to plasma membrane-enriched fractions, analyzed by gradient centrifugation, was observed after estradiol treatment. We conclude that, in hormone-responsive breast cancer cells, CD-MPR and CatD are distributed together, and that their expression and distribution are influenced by estradiol. These findings strongly support the involvement of the CD-MPR in the pro-enzyme transport in MCF-7 cells, suggesting the participation of this receptor in the procathepsin D secretion previously reported in breast cancer cells.