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1.
Article in English | MEDLINE | ID: mdl-38325835

ABSTRACT

Overdose of carbon dioxide gas (CO2) is a common euthanasia method for rodents; however, CO2 exposure activates nociceptors in rats at concentrations equal to or greater than 37% and is reported to be painful in humans at concentrations equal to or greater than 32.5%. Exposure of rats to CO2 could cause pain before loss of consciousness. We used 2 standardized loss of righting reflex (LORR) methods to identify CO2 concentrations associated with unconsciousness in Wistar, Long-Evans, and Sprague-Dawley rats (n = 28 animals per strain). A rotating, motorized cylinder was used to test LORR while the rat was being exposed to increasing concentrations of CO2. LORR was defined based on a 15-second observation period. The 2 methods were 1) a 1-Paw assessment (the righting reflex was considered to be present if one or more paws contacted the cylinder after the rat was positioned in dorsal recumbency), and 2) a 4-Paw assessment (the righting reflex was considered to be present if all 4 paws contacted the cylinder after the rat was positioned in dorsal recumbency). Data were analyzed with Probit regression, and dose-response curves were plotted. 1-Paw EC95 values (CO2 concentration at which LORR occurred for 95% of the population) were Wistar, 27.2%; Long-Evans, 29.2%; and Sprague-Dawley, 35.0%. 4-Paw EC95 values were Wistar, 26.2%; Long-Evans, 25.9%, and Sprague-Dawley, 31.1%. Sprague-Dawley EC95 values were significantly higher in both 1- and 4-Paw tests as compared with Wistar and Long-Evans rats. No differences were detected between sexes for any strain. The 1-Paw EC95 was significantly higher than the 4-Paw EC95 only for Sprague-Dawley rats. These results suggest that a low number of individual rats from the strains studied may experience pain during CO2 euthanasia.

2.
Nat Struct Mol Biol ; 31(3): 498-512, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38182927

ABSTRACT

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.


Subject(s)
Breast Neoplasms , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Decitabine/pharmacology , Decitabine/therapeutic use , Decitabine/metabolism , Epigenome , DNA Methylation/genetics , Chromatin , Epigenesis, Genetic , DNA/metabolism , Gene Expression Regulation, Neoplastic
3.
J Am Assoc Lab Anim Sci ; 62(6): 553-558, 2023 Nov 11.
Article in English | MEDLINE | ID: mdl-37770194

ABSTRACT

Exposure to CO2 gas is a common rodent euthanasia method. CO2 activates nociceptors in rats and is painful to humans at concentrations equal to or greater than 32.5% The concentration of CO2 at which rodents become unconsciousness is inadequately defined. We used loss of righting reflex (LORR) to identify the concentration at which CO2 caused loss of consciousness in C57Bl/6, CD1 and 129P3J mice (16 females and 16 males per strain). We used a custom built, rotating, motorized cylinder to determine LORR as CO2 concentrations were increased. Two LORR assessment methods were used: 1) a 1-Paw assessment in which the righting reflex was considered to be present if one or more paws contacted the cylinder after rotation into dorsal recumbency and 2) a 4-Paw assessment in which the righting reflex was considered to be present only if all 4 paws contacted the cylinder. LORR test data were analyzed with Probit regression and dose response curves were plotted. 1-Paw EC95 values (CO2 concentration at which LORR occurred for 95% of the population) were: C57Bl/6; 30.7%, CD1; 26.2%, 129P3J; 20.1%. The EC95 for C57Bl/6 was significantly higher than that of the 129P3J mice, with no significant differences between other strains. Four-Paw EC95 values were: C57Bl/6; 22.8%, CD1; 25.3%, 129P3J; 20.1%. Values for 129P3J mice were significantly lower than those of CD1 mice), with no significant difference between other strains. The EC95 varied significantly between 1-Paw and 4-Paw methods only for C57Bl/6 mice. These results suggest a potential for nociception and pain to occur in some individuals of some mouse strains during CO2 euthanasia.


Subject(s)
Carbon Dioxide , Animals , Female , Male , Mice , Mice, Inbred C57BL , Pain/veterinary , Reflex , Reflex, Righting/physiology , Unconsciousness
4.
RSC Med Chem ; 14(2): 356-366, 2023 Feb 22.
Article in English | MEDLINE | ID: mdl-36846364

ABSTRACT

Aromatase (CYP19A1) inhibitors are the mainstay therapeutics for the treatment of hormone dependant breast cancer, which accounts for approximately 70% of all breast cancer cases. However, increased resistance to the clinically used aromatase inhibitors, including letrozole and anastrazole, and off target effects, necessitates the development of aromatase inhibitors with improved drug profiles. The development of extended 4th generation pyridine based aromatase inhibitors with dual binding (haem and access channel) is therefore of interest and here we describe the design, synthesis and computational studies. Cytotoxicity and selectivity studies identified the pyridine derivative (4-bromophenyl)(6-(but-2-yn-1-yloxy)benzofuran-2-yl)(pyridin-3-yl)methanol (10c) as optimal with CYP19A1 IC50 0.83 nM (c.f. letrozole IC50 0.70 nM), and an excellent cytotoxicity and selectivity profile. Interestingly, computational studies for the 6-O-butynyloxy (10) and 6-O-pentynyloxy (11) derivatives identified an alternative access channel lined by Phe221, Trp224, Gln225 and Leu477, providing further insight into the potential binding mode and interactions of the non-steroidal aromatase inhibitors.

5.
Gene ; 851: 147022, 2023 Jan 30.
Article in English | MEDLINE | ID: mdl-36347335

ABSTRACT

The response to psychological stress can differ depending on the type and duration of the stressor. Acute stress can facilitate a "fight or flight response" and aid survival, whereas chronic long-term stress with the persistent release of stress hormones such as cortisol has been shown to be detrimental to health. We are now beginning to understand how this stress hormone response impacts important processes such as DNA repair and cell proliferation processes in breast cancer. However, it is not known what epigenetic changes stress hormones induce in breast cancer. Epigenetic mechanisms include modification of DNA and histones within chromatin that may be involved in governing the transcriptional processes in cancer cells in response to changes by endogenous stress hormones. The contribution of endogenous acute or long-term exposure of glucocorticoid stress hormones, and exogenous glucocorticoids to methylation patterns in breast cancer tissues with different aetiologies remains to be evaluated. In vitro and in vivo models were developed to investigate the epigenetic modifications and their contribution to breast cancer progression and aetiology. A panel of triple negative breast cancer cell lines were treated with the glucocorticoid, cortisol which resulted in epigenetic alteration characterised by loss of methylation on promoter regions of tumour suppressor genes including ESR1, and loss of methylation on LINE-1 repetitive element used as a surrogate marker for global methylation. This was verified in vivo in MDA-MB-231 xenografts; the model verified the loss of methylation on ESR1 promoter, and subsequent increase in ESR1 expression in primary tumours in mice subjected to restraint stress. Our study highlights that DNA methylation landscape in breast cancer can be altered in response to stress and glucocorticoid treatment.


Subject(s)
Estrogen Receptor alpha , Triple Negative Breast Neoplasms , Humans , Mice , Animals , Fulvestrant , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Glucocorticoids/pharmacology , Hydrocortisone/pharmacology , DNA Methylation
7.
Eur J Med Chem ; 240: 114569, 2022 Oct 05.
Article in English | MEDLINE | ID: mdl-35834906

ABSTRACT

One in every eight women will be diagnosed with breast cancer during their lifetime and approximately 70% of all patients are oestrogen receptor (ER) positive depending upon oestrogen for their growth accounting for third generation aromatase (CYP19A1) inhibitors being the mainstay in the treatment of ER-positive breast cancer. Despite the success of current aromatase inhibitors, acquired resistance occurs after prolonged therapy. Although the precise mechanisms of resistance are not known, lack of cross resistance among aromatase inhibitors drives the need for a newer generation of inhibitors to overcome this resistance alongside minimising toxicity and adverse effects. Novel triazole-based inhibitors were designed based on previously published parent compound 5a, making use of the now available crystal structure of CYP19A1 (PDB 3S79), to make modifications at specific sites to explore the potential of dual binding at both the active site and the access channel. Modifications included adding long chain substituents e.g. but-2-ynyloxy and pent-2-ynyloxy at different positions including the most active compound 13h with IC50 value in the low picomolar range (0.09 nM). Aromatase inhibition results paired with molecular dynamics studies provided a clear structure activity relationship and favourable dual binding mode was verified. Toxicity assays and CYP selectivity profile studies for some example compounds were performed to assess the safety profile of the prepared inhibitors providing the basis for the 4th generation nonsteroidal aromatase inhibitors.


Subject(s)
Aromatase Inhibitors , Breast Neoplasms , Aromatase/metabolism , Aromatase Inhibitors/chemistry , Aromatase Inhibitors/pharmacology , Breast Neoplasms/metabolism , Female , Humans , Receptors, Estrogen , Triazoles/pharmacology
8.
NPJ Breast Cancer ; 8(1): 57, 2022 May 02.
Article in English | MEDLINE | ID: mdl-35501337

ABSTRACT

Intratumoral heterogeneity is caused by genomic instability and phenotypic plasticity, but how these features co-evolve remains unclear. SOX10 is a neural crest stem cell (NCSC) specifier and candidate mediator of phenotypic plasticity in cancer. We investigated its relevance in breast cancer by immunophenotyping 21 normal breast and 1860 tumour samples. Nuclear SOX10 was detected in normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In tumours, nuclear SOX10 was almost exclusive to TNBC, and predicted poorer outcome amongst cross-sectional (p = 0.0015, hazard ratio 2.02, n = 224) and metaplastic (p = 0.04, n = 66) cases. To understand SOX10's influence over the transcriptome during the transition from normal to malignant states, we performed a systems-level analysis of co-expression data, de-noising the networks with an eigen-decomposition method. This identified a core module in SOX10's normal mammary epithelial network that becomes rewired to NCSC genes in TNBC. Crucially, this reprogramming was proportional to genome-wide promoter methylation loss, particularly at lineage-specifying CpG-island shores. We propose that the progressive, genome-wide methylation loss in TNBC simulates more primitive epigenome architecture, making cells vulnerable to SOX10-driven reprogramming. This study demonstrates potential utility for SOX10 as a prognostic biomarker in TNBC and provides new insights about developmental phenotypic mimicry-a major contributor to intratumoral heterogeneity.

9.
Explor Target Antitumor Ther ; 3(2): 224-239, 2022 Apr 26.
Article in English | MEDLINE | ID: mdl-35591900

ABSTRACT

Aim: Zinc is a key secondary messenger that can regulate multiple signalling pathways within cancer cells, thus its levels need to be strictly controlled. The Zrt, Irt-like protein (ZIP, SLC39A) family of zinc transporters increase cytosolic zinc from either extracellular or intracellular stores. This study examines the relevance of zinc transporters ZIP7 and ZIP6 as therapeutic targets in tamoxifen resistant (TAMR) breast cancer. Methods: A series of in vitro assays, including immunohistochemistry, immunofluorescence, flow cytometry, and western blotting were used to evaluate levels and activity of ZIP7 and ZIP6 in models of TAMR and sensitive (MCF-7) breast cancer. Analyses of these transporters in the clinical setting were performed using publicly available online resources: Gene Expression Profiling Interactive Analysis (GEPIA)2 and Kaplan-Meier Plotter (KmPlot). Results: Both total and activated levels of ZIP7 were significantly elevated in TAMR cells versus responsive MCF-7 cells. This was accompanied by an associated increase in free cytoplasmic zinc leading to amplification of downstream signals. Consistent with our proposed model, activated ZIP6 levels correlated with mitotic cells, which could be efficiently inhibited through use of our anti-ZIP6 monoclonal antibody. Mitotic inhibition translated to impaired proliferation in both models, with TAMR cells displaying increased sensitivity. Analysis of matched tumour and normal breast samples from patients revealed significant increases in both ZIP7 and ZIP6 in tumours, as well as family member ZIP4. Kaplan-Meier analysis revealed that high ZIP7 levels correlated with decreased overall and relapse-free survival (RFS) of patients, including patient groups who had received systemic endocrine therapy or tamoxifen only. In contrast, high ZIP6 levels were significantly linked to improved overall and RFS in all patients, as well as RFS in patients that received systemic endocrine therapy. Conclusions: TAMR cells displayed increased activity of both ZIP7 and ZIP6 transporters compared to anti-hormone responsive cells, suggesting their potential as novel therapeutic targets following development of resistant disease.

10.
Cell Mol Life Sci ; 78(4): 1781-1798, 2021 Feb.
Article in English | MEDLINE | ID: mdl-32797246

ABSTRACT

Zinc has been known to be essential for cell division for over 40 years but the molecular pathways involved remain elusive. Cellular zinc import across biological membranes necessitates the help of zinc transporters such as the SLC39A family of ZIP transporters. We have discovered a molecular process that explains why zinc is required for cell division, involving two highly regulated zinc transporters, as a heteromer of ZIP6 and ZIP10, providing the means of cellular zinc entry at a specific time of the cell cycle that initiates a pathway resulting in the onset of mitosis. Crucially, when the zinc influx across this heteromer is blocked by ZIP6 or ZIP10 specific antibodies, there is no evidence of mitosis, confirming the requirement for zinc influx as a trigger of mitosis. The zinc that influxes into cells to trigger mitosis additionally changes the phosphorylation state of STAT3 converting it from a transcription factor to a protein that complexes with this heteromer and pS38Stathmin, the form allowing microtubule rearrangement as required in mitosis. This discovery now explains the specific cellular role of ZIP6 and ZIP10 and how they have special importance in the mitosis process compared to other ZIP transporter family members. This finding offers new therapeutic opportunities for inhibition of cell division in the many proliferative diseases that exist, such as cancer.


Subject(s)
Carrier Proteins/genetics , Cation Transport Proteins/genetics , Mitosis/genetics , STAT3 Transcription Factor/genetics , Gene Expression Regulation , Humans , MCF-7 Cells , Phosphorylation/genetics , Protein Multimerization/genetics , Signal Transduction/genetics , Zinc/chemistry , Zinc/metabolism
11.
Nat Commun ; 11(1): 320, 2020 01 16.
Article in English | MEDLINE | ID: mdl-31949157

ABSTRACT

Endocrine therapy resistance frequently develops in estrogen receptor positive (ER+) breast cancer, but the underlying molecular mechanisms are largely unknown. Here, we show that 3-dimensional (3D) chromatin interactions both within and between topologically associating domains (TADs) frequently change in ER+ endocrine-resistant breast cancer cells and that the differential interactions are enriched for resistance-associated genetic variants at CTCF-bound anchors. Ectopic chromatin interactions are preferentially enriched at active enhancers and promoters and ER binding sites, and are associated with altered expression of ER-regulated genes, consistent with dynamic remodelling of ER pathways accompanying the development of endocrine resistance. We observe that loss of 3D chromatin interactions often occurs coincidently with hypermethylation and loss of ER binding. Alterations in active A and inactive B chromosomal compartments are also associated with decreased ER binding and atypical interactions and gene expression. Together, our results suggest that 3D epigenome remodelling is a key mechanism underlying endocrine resistance in ER+ breast cancer.


Subject(s)
Binding Sites , Breast Neoplasms/genetics , Chromatin/metabolism , Epigenesis, Genetic , Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/metabolism , CCCTC-Binding Factor/chemistry , CCCTC-Binding Factor/metabolism , Chromatin/chemistry , Chromatin/genetics , DNA Methylation , Epigenesis, Genetic/drug effects , Female , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Neoplasm Proteins/genetics , Promoter Regions, Genetic/drug effects , Protein Interaction Domains and Motifs , Whole Genome Sequencing
12.
Clin Cancer Res ; 26(7): 1574-1585, 2020 04 01.
Article in English | MEDLINE | ID: mdl-31836609

ABSTRACT

PURPOSE: The STAKT study examined short-term exposure (4.5 days) to the oral selective pan-AKT inhibitor capivasertib (AZD5363) to determine if this drug can reach its therapeutic target in sufficient concentration to significantly modulate key biomarkers of the AKT pathway and tumor proliferation. PATIENTS AND METHODS: STAKT was a two-stage, double-blind, randomized, placebo-controlled, "window-of-opportunity" study in patients with newly diagnosed ER+ invasive breast cancer. Stage 1 assessed capivasertib 480 mg b.i.d. (recommended monotherapy dose) and placebo, and stage 2 assessed capivasertib 360 and 240 mg b.i.d. Primary endpoints were changes from baseline in AKT pathway markers pPRAS40, pGSK3ß, and proliferation protein Ki67. Pharmacologic and pharmacodynamic properties were analyzed from blood sampling, and tolerability by adverse-event monitoring. RESULTS: After 4.5 days' exposure, capivasertib 480 mg b.i.d. (n = 17) produced significant decreases from baseline versus placebo (n = 11) in pGSK3ß (H-score absolute change: -55.3, P = 0.006) and pPRAS40 (-83.8, P < 0.0001), and a decrease in Ki67 (absolute change in percentage positive nuclei: -9.6%, P = 0.031). Significant changes also occurred in secondary signaling biomarker pS6 (-42.3, P = 0.004), while pAKT (and nuclear FOXO3a) also increased in accordance with capivasertib's mechanism (pAKT: 81.3, P = 0.005). At doses of 360 mg b.i.d. (n = 5) and 240 mg b.i.d. (n = 6), changes in primary and secondary biomarkers were also observed, albeit of smaller magnitude. Biomarker modulation was dose and concentration dependent, and no new safety signals were evident. CONCLUSIONS: Capivasertib 480 mg b.i.d. rapidly modulates key biomarkers of the AKT pathway and decreases proliferation marker Ki67, suggesting future potential as an effective therapy in AKT-dependent breast cancers.


Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/drug therapy , Estrogen Receptor alpha/metabolism , Ki-67 Antigen/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Pyrroles/pharmacokinetics , Pyrroles/therapeutic use , Breast Neoplasms/pathology , Cell Proliferation , Double-Blind Method , Female , Humans , Middle Aged , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Tissue Distribution , Treatment Outcome
13.
Metallomics ; 11(9): 1579-1592, 2019 09 01.
Article in English | MEDLINE | ID: mdl-31483418

ABSTRACT

ZIP7, a member of the ZIP family of zinc importers, resides on the endoplasmic reticulum membrane and transports zinc from intracellular stores to the cytoplasm after activation by CK2 phosphorylation on two serine residues (S275 and S276). ZIP7 is known to be required for the growth of anti-hormone resistant breast cancer models, especially those with acquired tamoxifen resistance developed from MCF-7. Using our new pS275S276ZIP7 antibody which only recognises activated ZIP7 (pZIP7), we have demonstrated that the hyperactivation of ZIP7 is prevalent in tamoxifen-resistant breast cancer cells. This evidence suggests that pZIP7 might have potential as a biomarker of acquired resistance to such anti-hormones in breast cancer, a current unmet clinical need. In this regard, we have also developed a new immunohistochemical assay for pZIP7 which allowed pZIP7 to be tested on a small clinical series of breast cancer tissues confirming its prevalence in such tumours and relationship to a variety of clinicopathological parameters and biomarkers previously associated with endocrine resistant phenotypes, notably increased activated MAPK signalling, expression of ErbB2, CD71 and the proto-oncogene c-Fos, as well as with increased tumour grade.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Cation Transport Proteins/metabolism , Tamoxifen/pharmacology , Zinc/metabolism , Breast Neoplasms/metabolism , Drug Resistance, Neoplasm , Female , Humans , MCF-7 Cells , Proto-Oncogene Mas
14.
Cancer Lett ; 458: 66-75, 2019 08 28.
Article in English | MEDLINE | ID: mdl-31121213

ABSTRACT

Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due to de novo or acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER + breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER + tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER + metastatic samples. PAK4 activity increases in ER + models of acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER + breast cancers.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Neoplastic Stem Cells/pathology , Receptors, Estrogen/metabolism , Tamoxifen/pharmacology , p21-Activated Kinases/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Disease Progression , Down-Regulation , Drug Resistance, Neoplasm , Estrogen Receptor Antagonists/pharmacology , Female , Fulvestrant/pharmacology , Gene Expression , Humans , MCF-7 Cells , Meta-Analysis as Topic , Neoplasm Metastasis , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Prognosis , Small Molecule Libraries/pharmacology , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/biosynthesis , p21-Activated Kinases/genetics
15.
Br J Cancer ; 120(6): 621-632, 2019 03.
Article in English | MEDLINE | ID: mdl-30783203

ABSTRACT

BACKGROUND: MicroRNAs are potent post-transcriptional regulators involved in all hallmarks of cancer. Mir-196a is transcribed from two loci and has been implicated in a wide range of developmental and pathogenic processes, with targets including Hox, Fox, Cdk inhibitors and annexins. Genetic variants and altered expression of MIR196A are associated with risk and progression of multiple cancers including breast cancer, however little is known about the regulation of the genes encoding this miRNA, nor the impact of variants therein. METHODS: Genomic data and chromatin interaction analysis were used to discover functional promoter and enhancer elements for MIR196A. Expression data were used to associate MIR196A with mechanisms of resistance, breast cancer subtypes and prognosis. RESULTS: Here we demonstrate that MIR196A displays complex and dynamic expression patterns, in part controlled by long-range transcriptional regulation between promoter and enhancer elements bound by ERα. Expression of this miRNA is significantly increased in drug-resistant models of hormone-receptor positive disease. The expression of MIR196A also proves to be a robust prognostic factor for patients with advanced and post-menopausal ER+ disease. CONCLUSION: This work sheds light on the normal and abnormal regulation of MIR196A and provides a novel stratification method for therapeutically resistant breast cancer.


Subject(s)
Breast Neoplasms/genetics , Estrogen Receptor alpha/genetics , MicroRNAs/genetics , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA Methylation , Disease Progression , Drug Resistance, Neoplasm , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/genetics , Humans , MCF-7 Cells , MicroRNAs/biosynthesis , Prognosis , Tamoxifen/pharmacology
16.
Breast Cancer Res ; 20(1): 98, 2018 08 30.
Article in English | MEDLINE | ID: mdl-30165904

ABSTRACT

After the publication of this work [1], an error was noticed in Fig. 2b and Fig. 4b as well as Fig. 4b. and Fig. 5d. Images of the ERK1/2 blots were accidentally duplicated. In Fig. 5a. and Fig. 5c., the last lane for p-ERK1/2 was mistakenly cropped out of the final image. The original blot for Fig. 4b., "total EGFR" (or lane 2) is shown below to avoid any misunderstanding of the data. We apologize for this error, which did not affect any of the interpretations or conclusions of the article.

17.
Clin Cancer Res ; 24(10): 2452-2463, 2018 05 15.
Article in English | MEDLINE | ID: mdl-29363524

ABSTRACT

Purpose: One third of ER-positive breast cancer patients who initially respond to endocrine therapy become resistant to treatment. Such treatment failure is associated with poor prognosis and remains an area of unmet clinical need. Here, we identify a specific posttranslational modification that occurs during endocrine resistance and which results in tumor susceptibility to the apoptosis-inducer TRAIL. This potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer.Experimental Design: Cell line and primary-derived xenograft models of endocrine resistance were investigated for susceptibility to TRAIL. Tumor viability, cancer stem cell (CSC) viability (tumorspheres), tumor growth kinetics, and metastatic burden were assessed. Western blots for the TRAIL-pathway inhibitor, c-FLIP, and upstream regulators were performed. Results were confirmed in primary culture of 26 endocrine-resistant and endocrine-naïve breast tumors.Results: Breast cancer cell lines with acquired resistance to tamoxifen (TAMR) or faslodex were more sensitive to TRAIL than their endocrine-sensitive controls. Moreover, TRAIL eliminated CSC-like activity in TAMR cells, resulting in prolonged remission of xenografts in vivo In primary culture, TRAIL significantly depleted CSCs in 85% endocrine-resistant, compared with 8% endocrine-naïve, tumors, whereas systemic administration of TRAIL in endocrine-resistant patient-derived xenografts reduced tumor growth, CSC-like activity, and metastases. Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of c-FLIP, and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades c-FLIP.Conclusions: These results identify a novel mechanism of acquired vulnerability to an extrinsic cell death stimulus, in endocrine-resistant breast cancers, which has both therapeutic and prognostic potential. Clin Cancer Res; 24(10); 2452-63. ©2018 AACR.


Subject(s)
Breast Neoplasms/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Drug Resistance, Neoplasm , Protein Processing, Post-Translational , Receptors, Estrogen/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Xenograft Model Antitumor Assays
18.
Hum Mol Genet ; 25(15): 3269-3283, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27378691

ABSTRACT

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.


Subject(s)
Biomarkers, Tumor/biosynthesis , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , RNA, Long Noncoding/biosynthesis , RNA, Neoplasm/biosynthesis , Transcription, Genetic , Biomarkers, Tumor/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Forkhead Box Protein M1/biosynthesis , Forkhead Box Protein M1/genetics , Hepatocyte Nuclear Factor 3-alpha/biosynthesis , Hepatocyte Nuclear Factor 3-alpha/genetics , Humans , MCF-7 Cells , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , RNA, Long Noncoding/genetics , RNA, Neoplasm/genetics
19.
Front Oncol ; 6: 145, 2016.
Article in English | MEDLINE | ID: mdl-27379207

ABSTRACT

While endocrine therapy is the mainstay of ER+ breast cancer, the clinical effectiveness of these agents is limited by the phenomenon of acquired resistance that is associated with disease relapse and poor prognosis. Our previous studies revealed that acquired resistance is accompanied by a gain in cellular invasion and migration and also that CD44 family proteins are overexpressed in the resistant phenotype. Given the association of CD44 with tumor progression, we hypothesized that its overexpression may act to promote the aggressive behavior of endocrine-resistant breast cancers. Here, we have investigated further the role of two specific CD44 isoforms, CD44v3 and CD44v6, in the endocrine-resistant phenotype. Our data revealed that overexpression of CD44v6, but not CD44v3, in endocrine-sensitive MCF-7 cells resulted in a gain in EGFR signaling, enhanced their endogenous invasive capacity, and attenuated their response to endocrine treatment. Suppression of CD44v6 in endocrine-resistant cell models was associated with a reduction in their invasive capacity. Our data suggest that upregulation of CD44v6 in acquired resistant breast cancer may contribute to a gain in the aggressive phenotype of these cells and loss of endocrine response through transactivation of the EGFR pathway. Future therapeutic targeting of CD44v6 may prove to be an effective strategy alongside EGFR-targeted agents in delaying/preventing acquired resistance in breast cancer.

20.
Br J Pharmacol ; 173(1): 177-90, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26454020

ABSTRACT

BACKGROUND AND PURPOSE: Small conductance calcium-activated potassium (KCa 2.x) channels have a widely accepted canonical function in regulating cellular excitability. In this study, we address a potential non-canonical function of KCa 2.x channels in breast cancer cell survival, using in vitro models. EXPERIMENTAL APPROACH: The expression of all KCa 2.x channel isoforms was initially probed using RT-PCR, Western blotting and microarray analysis in five widely studied breast cancer cell lines. In order to assess the effect of pharmacological blockade and siRNA-mediated knockdown of KCa 2.x channels on these cell lines, we utilized MTS proliferation assays and also followed the corresponding expression of apoptotic markers. KEY RESULTS: All of the breast cancer cell lines, regardless of their lineage or endocrine responsiveness, were highly sensitive to KCa 2.x channel blockade. UCL1684 caused cytotoxicity, with LD50 values in the low nanomolar range, in all cell lines. The role of KCa 2.x channels was confirmed using pharmacological inhibition and siRNA-mediated knockdown. This reduced cell viability and also reduced expression of Bcl-2 but increased expression of active caspase-7 and caspase-9. Complementary to these results, a variety of cell lines can be protected from apoptosis induced by staurosporine using the KCa 2.x channel activator CyPPA. CONCLUSIONS AND IMPLICATIONS: In addition to a well-established role for KCa 2.x channels in migration, blockade of these channels was potently cytotoxic in breast cancer cell lines, pointing to modulation of KCa 2.x channels as a potential therapeutic approach to breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Gene Knockdown Techniques , Small-Conductance Calcium-Activated Potassium Channels/deficiency , Alkanes/toxicity , Apoptosis/drug effects , Apoptosis Regulatory Proteins/biosynthesis , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Lethal Dose 50 , Protein Isoforms/biosynthesis , Protein Isoforms/deficiency , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Quinolinium Compounds/toxicity , RNA, Small Interfering/pharmacology , Small-Conductance Calcium-Activated Potassium Channels/biosynthesis , Small-Conductance Calcium-Activated Potassium Channels/genetics , Small-Conductance Calcium-Activated Potassium Channels/metabolism , Staurosporine/antagonists & inhibitors , Staurosporine/pharmacology
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