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1.
iScience ; 24(10): 103212, 2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34755086

RESUMO

Activity of liver x receptor (LXR), the homeostatic regulator of cholesterol metabolism, is elevated in triple-negative breast cancer (BCa) relative to other BCa subtypes, driving drug resistance and metastatic gene signatures. The loci encoding LXRα and LXRß produce multiple alternatively spliced proteins, but the true range of variants and their relevance to cancer remain poorly defined. Here, we report seven LXR splice variants, three of which have not previously been reported and five that were prognostic for disease-free survival. Expression of full-length LXRα splice variants was associated with poor prognosis, consistent with a role as an oncogenic driver of triple-negative tumor pathophysiology. Contrary to this was the observation that high expression of truncated LXRα splice variants or any LXRß splice variant was associated with longer survival. These findings indicate that LXR isoform abundance is an important aspect of understanding the link between dysregulated cholesterol metabolism and cancer pathophysiology.

2.
Pilot Feasibility Stud ; 7(1): 193, 2021 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-34715934

RESUMO

BACKGROUND: Ergothioneine is a naturally occurring metabolite of histidine found in many foods and in high amounts in mushrooms. In vivo, ergothioneine acts as an antioxidant and is widely distributed in most mammalian tissues. While ergothioneine is sold as a dietary supplement for its antioxidant and anti-inflammatory properties, to date there are no published intervention trials examining its health benefits in humans. The aim of this work was to develop a study protocol for a pilot interventional trial that will establish the primary and secondary outcomes, and the power required, for a definitive randomised controlled trial to test the hypothesis that ergothioneine supplementation is beneficial for people with metabolic syndrome. METHODS: We have designed the ErgMS study as a single-centre, randomised, double-blind, placebo-controlled, 3-arm parallel, pilot intervention trial, which aims to supplement participants with either placebo, 5 or 30 mg/day ergothioneine for 12 weeks. Measurements of metabolic syndrome risk factors, serum markers of oxidative stress (lipid peroxidation), inflammation, blood platelet function and liver function will take place at baseline, and after 6 weeks and 12 weeks of supplementation. In addition, we will examine if there are any changes in the serum metabolome in response to ergothioneine supplementation. Linear regression and two-way ANOVA will be utilised to analyse the association between ergothioneine and measured variables. DISCUSSION: The ErgMS study will be the first study to address the question does ergothioneine supplementation have health benefits for people with metabolic syndrome. Study results will provide preliminary data as to which dose may improve inflammatory markers in adults with metabolic syndrome and will inform dose and primary outcome selection for a definitive randomised controlled trial. TRIAL REGISTRATION: ISRCTN, ISRCTN25890011 Registered February 10th, 2021.

3.
Biochem Pharmacol ; : 114731, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34407453

RESUMO

Cholesterol esterification proteins Sterol-O acyltransferases (SOAT) 1 and 2 are emerging prognostic markers in many cancers. These enzymes utilise fatty acids conjugated to coenzyme A to esterify cholesterol. Cholesterol esterification is tightly regulated and enables formation of lipid droplets that act as storage organelles for lipid soluble vitamins and minerals, and as cholesterol reservoirs. In cancer, this provides rapid access to cholesterol to maintain continual synthesis of the plasma membrane. In this systematic review and meta-analysis, we summarise the current depth of understanding of the role of this metabolic pathway in pan-cancer development. A systematic search of PubMed, Scopus, Web of Science, and Cochrane Library for preclinical studies identified eight studies where cholesteryl ester concentrations were compared between tumour and adjacent-normal tissue, and 24 studies where cholesterol esterification was blocked by pharmacological or genetic approaches. Tumour tissue had a significantly greater concentration of cholesteryl esters than non-tumour tissue (p < 0.0001). Pharmacological or genetic inhibition of SOAT was associated with significantly smaller tumours of all types (p ≤ 0.002). SOAT inhibition increased tumour apoptosis (p = 0.007), CD8 + lymphocyte infiltration and cytotoxicity (p ≤ 0.05), and reduced proliferation (p = 0.0003) and metastasis (p < 0.0001). Significant risk of publication bias was found and may have contributed to a 32% overestimation of the meta-analysed effect size. Avasimibe, the most frequently used SOAT inhibitor, was effective at doses equivalent to those previously reported to be safe and tolerable in humans. This work indicates that SOAT inhibition should be explored in clinical trials as an adjunct to existing anti-neoplastic agents.

4.
Br J Cancer ; 125(7): 983-993, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34253873

RESUMO

BACKGROUND: Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. METHODS: Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. RESULTS: Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. CONCLUSIONS: This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

5.
Oncogene ; 40(16): 2872-2883, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33742124

RESUMO

Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.


Assuntos
Hidroxicolesteróis/metabolismo , Receptores X do Fígado/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Epirubicina/farmacologia , Feminino , Expressão Gênica , Humanos , Receptores X do Fígado/agonistas , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
6.
J Immunother Cancer ; 9(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33589522

RESUMO

BACKGROUND: Immunotherapy in prostate cancer (PCa) lags behind the progresses obtained in other cancer types partially because of its limited immune infiltration. Tumor-resident immune cells have been detected in the prostate, but the regulatory mechanisms that govern tumor infiltration are still poorly understood. To address this gap, we investigated the role of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase enzyme that targets dimethyl and trimethyl H3K36. WHSC1 is known to promote malignant growth and progression in multiple tumors, but its role in the interface between PCa and immune system is unknown. METHODS: RNA Sequencing (RNASeq) data from patients with PCa from The Cancer Genome Atlas (TCGA) were collected and divided into top/bottom 30% based on the expression of WHSC1 and disease-free survival was calculated. Publicly available chromatin immunoprecipitation (ChIPSeq) data were obtained from Cistrome and integrated with the available RNASeq data. RNASeq, ATACSeq and methylomic were analyzed using R Bioconductor packages comparing C42 cells with or without stable knockdown on WHSC1. Flow cytometry was used to measure Major Histocompatibility complex (MHC) levels, MHC-bound ovalbumin and tumor infiltration. C57B6 and NOD scid gamma (NSG) mice were subcutaneously grafted with TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) C2 cells and treated with MCTP39 (10 mg/kg); tumor size was monitored over time and curves were compared using permutation analyses. All analyses used a significance threshold of 0.05. RESULTS: Leveraging TCGA data, we demonstrated that elevated WHSC1 levels positively correlate with the presence of an immunosuppressive microenvironment. We validated those results in vitro, demonstrating that genetic and pharmacological inhibition of WHSC1 restores antigen presentation. This occurs via an elegant epigenetic regulation of gene expression at the chromatin and DNA methylation levels. In vivo studies in immunocompetent mice also show an increased frequency of CD8+ T cells in tumors from mice treated with WHSC1 inhibitor, supporting the hypothesis that the antitumor effect following WHSC1 inhibition requires a fully functional immune system. CONCLUSIONS: This study demonstrates a novel role for WHSC1 in defining immune infiltration in PCa, with significant future implications for the use of immunotherapies in prostate malignancies.

7.
Br J Cancer ; 124(6): 1110-1120, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33398063

RESUMO

BACKGROUND: Triple negative breast cancers (TNBC) have poor prognoses despite aggressive treatment with cytotoxic chemotherapy. Cancer-associated fibroblasts (CAFs) are prominent in tumour stroma. Our hypothesis was that CAFs modulate chemotherapy sensitivity. METHODS: TNBC cells and breast fibroblasts were cultured; survival after chemotherapeutics was assessed using luciferase or clonogenic assays. Signalling was investigated using transcriptomics, reporters, recombinant proteins and blocking antibodies. Clinical relevance was investigated using immunohistochemistry. RESULTS: Breast CAFs dose-dependently protected TNBC cell lines MDA-MB-231 and MDA-MB-157, but not MDA-MB-468s, from chemotherapy. CAF-induced protection was associated with interferon (IFN) activation. CAFs were induced to express IFNß1 by chemotherapy and TNBC co-culture, leading to paracrine activation in cancer cells. Recombinant IFNs were sufficient to protect MDA-MB-231 and MDA-MB-157 but not MDA-MB-468 cells. In TNBC patients, IFNß1 expression in CAFs correlated with cancer cell expression of MX1, a marker of activated IFN signalling. High expression of IFNß1 (CAFs) or MX1 (tumour cells) correlated with reduced survival after chemotherapy, especially in claudin-low tumours (which MDA-MB-231 and MDA-MB-157 cells represent). Antibodies that block IFN receptors reduced CAF-dependent chemoprotection. CONCLUSIONS: CAF-induced activation of IFN signalling in claudin-low TNBCs results in chemoresistance. Inhibition of this pathway represents a novel method to improve breast cancer outcomes.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Fibroblastos Associados a Câncer/patologia , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon beta/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Proliferação de Células , Técnicas de Cocultura , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Interferon beta/genética , Proteínas de Resistência a Myxovirus/genética , Comunicação Parácrina , Prognóstico , Transcriptoma , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas
8.
Crit Rev Food Sci Nutr ; : 1-21, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33238719

RESUMO

Phytosterols and phytostanols are natural products present in vegetable oils, nuts, and seeds, or added to consumer food products whose intake is inversely associated with incidence and prognosis of several cancers. Randomized cancer prevention trials in humans are unfeasible due to time and cost yet the cellular processes and signaling cascades that underpin anti-cancer effects of these phytochemicals have been explored extensively in vitro and in preclinical in vivo models. Here we have performed an original systematic review, meta-analysis, and qualitative interpretation of literature published up to June 2020. MEDLINE, Scopus, and hand-searching identified 408 unique records that were screened leading to 32 original articles that had investigated the effects of phytosterols or phytostanols on cancer biology in preclinical models. Data was extracted from 22 publications for meta-analysis. Phytosterols were most commonly studied and found to reduce primary and metastatic tumor burden in all cancer sites evaluated. Expression of pAKT, and markers of metastasis (alkaline phosphatase, matrix metalloproteases, epithelial to mesenchymal transcription factors, lung and brain colonization), angiogenesis (vascular endothelial growth factor, CD31), and proliferation (Ki67, proliferating cell nuclear antigen) were consistently reduced by phytosterol treatment in breast and colorectal cancer. Very high dose treatment (equivalent to 0.2-1 g/kg body weight not easily achievable through diet or supplementation in humans) was associated with adverse events including poor gut health and intestinal adenoma development. Phytosterols and phytostanols are already clinically recommended for cardiovascular disease risk reduction, and represent promising anti-cancer agents that could be delivered in clinic and to the general population at low cost, with a well understood safety profile, and now with a robust understanding of mechanism-of-action.

9.
Breast Cancer Res Treat ; 183(3): 607-616, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32734521

RESUMO

PURPOSE: More than a third of primary breast cancer patients are treated with cytotoxic chemotherapy, typically without guidance from predictive markers. Increased use of neoadjuvant chemotherapy provides opportunities for identification of molecules associated with treatment response, by comparing matched tumour samples before and after therapy. Our hypothesis was that somatic variants of increased prevalence after therapy promote resistance, while variants with reduced prevalence cause sensitivity. METHODS: We performed systematic analyses of matched pairs of cancer exomes from primary oestrogen receptor-positive/HER2-negative breast cancers (n = 6) treated with neoadjuvant epirubicin/cyclophosphamide. We identified candidate genes as mediators of chemotherapy response by consistent subclonal changes in somatic variant prevalence through therapy, predicted variant impact on gene function, and enrichment of specific functional pathways. Influence of candidate genes on breast cancer outcome was tested using publicly available breast cancer expression data (n = 1903). RESULTS: We identified 14 genes as the strongest candidate mediators of chemoresponse: TCHH, MUC17, ARAP2, FLG2, ABL1, CENPF, COL6A3, DMBT1, ITGA7, PLXNA1, S100PBP, SYNE1, ZFHX4, and CACNA1C. Genes contained somatic variants showing prevalence changes in up to 4 patients, with up to 3 being predicted as damaging. Genes coding for extra-cellular matrix components or related signalling pathways were significantly over-represented among variants showing prevalence changes. Expression of 5 genes (TCHH, ABL1, CENPF, S100PBP, and ZFHX4) was significantly associated with patient survival. CONCLUSIONS: Genomic analysis of paired pre- and post-therapy samples resulting from neoadjuvant therapy provides a powerful method for identification of mediators of response. Genes we identified should be assessed as predictive markers or targets in chemo-sensitization.


Assuntos
Neoplasias da Mama , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Epirubicina/uso terapêutico , Exoma , Feminino , Genômica , Humanos , Terapia Neoadjuvante , Receptor ErbB-2/genética , Resultado do Tratamento , Proteínas Supressoras de Tumor
10.
Proc Nutr Soc ; 79(3): 367-372, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32183926

RESUMO

The Nutrition Society's 1st Annual Nutrition and Cancer Networking Conference brought together scientists from the fields of Nutrition, Epidemiology, Public Health, Medical Oncology and Surgery with representatives of the public, cancer survivors and cancer charities. Speakers representing these different groups presented the challenges to collaboration, how the needs of patients and the public can be met, and the most promising routes for future research. The conference programme promoted debate on these issues to highlight current gaps in understanding and barriers to generating and implementing evidence-based nutrition advice. The main conclusions were that the fundamental biology of how nutrition influences the complex cancer risk profiles of diverse populations needs to be better understood. Individual and population level genetics interact with the environment over a lifespan to dictate cancer risk. Large charities and government have a role to play in diminishing our current potently obesogenic environment and exploiting nutrition to reduce cancer deaths. Understanding how best to communicate, advise and support individuals wishing to make dietary and lifestyle changes, can reduce cancer risk, enhance recovery and improve the lives of those living with and beyond cancer.


Assuntos
Dieta , Neoplasias , Estado Nutricional , Feminino , Comunicação em Saúde , Disparidades nos Níveis de Saúde , Humanos , Estilo de Vida , Masculino , Neoplasias/mortalidade , Neoplasias/prevenção & controle , Neoplasias/terapia , Fatores de Risco
11.
Nutrients ; 11(11)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31683867

RESUMO

Interventions that alter cholesterol have differential impacts on hormone receptor positive- and negative-breast cancer risk and prognosis. This implies differential regulation or response to cholesterol within different breast cancer subtypes. We evaluated differences in side-chain hydroxycholesterol and liver X nuclear receptor signalling between Oestrogen Receptor (ER)-positive and ER-negative breast cancers and cell lines. Cell line models of ER-positive and ER-negative disease were treated with Liver X Receptor (LXR) ligands and transcriptional activity assessed using luciferase reporters, qPCR and MTT. Publicly available datasets were mined to identify differences between ER-negative and ER-positive tumours and siRNA was used to suppress candidate regulators. Compared to ER-positive breast cancer, ER-negative breast cancer cells were highly responsive to LXR agonists. In primary disease and cell lines LXRA expression was strongly correlated with its target genes in ER-negative but not ER-positive disease. Expression of LXR's corepressors (NCOR1, NCOR2 and LCOR) was significantly higher in ER-positive disease relative to ER-negative, and their knock-down equalized sensitivity to ligand between subtypes in reporter, gene expression and viability assays. Our data support further evaluation of dietary and pharmacological targeting of cholesterol metabolism as an adjunct to existing therapies for ER-negative and ER-positive breast cancer patients.


Assuntos
Neoplasias da Mama/metabolismo , Colesterol/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores X do Fígado/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Prognóstico , RNA Interferente Pequeno , Transdução de Sinais , Transcrição Genética
12.
Curr Opin Clin Nutr Metab Care ; 22(6): 449-458, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31589177

RESUMO

PURPOSE OF REVIEW: Vitamin D deficiency may impact disease progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to review recent studies examining either vitamin D status or the effects of supplementation in patients with NAFLD, along with investigating the roles of genetic polymorphisms and the gut microbiome. RECENT FINDINGS: Six heterogeneous observational studies of vitamin D status, and four randomized controlled intervention trials of vitamin D supplementation in NAFLD were conflicting. All studies were hampered by the challenges of diagnosing NAFLD, were underpowered, and lacked data on clinically important outcomes. The results of three cross-sectional studies, including a Mendelian randomization study, provide limited evidence for a role for genetic modifiers of vitamin D status in NAFLD. Genetic and experimental evidence suggests that vitamin D and the vitamin D receptor (VDR) may influence the gut microbiome in health and disease. SUMMARY: The evidence relating either lower vitamin D status to the prevalence and severity of NAFLD, or examining vitamin D supplementation in patients with NAFLD is inconclusive. Larger, higher quality trials with relevant endpoints are needed. Further mechanistic studies on the roles of vitamin D and VDR in influencing the gut-liver axis in NAFLD are warranted.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Vitamina D , Suplementos Nutricionais , Microbioma Gastrointestinal , Humanos , Polimorfismo de Nucleotídeo Único/genética , Receptores de Calcitriol/genética , Deficiência de Vitamina D
13.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269628

RESUMO

Low fruit and vegetable consumption and high saturated fat consumption causes elevated circulating cholesterol and are breast cancer risk factors. During cholesterol metabolism, oxysterols form that bind and activate the liver X receptors (LXRs). Oxysterols halt breast cancer cell proliferation but enhance metastatic colonization, indicating tumour suppressing and promoting roles. Phytosterols and phytostanols in plants, like cholesterol in mammals, are essential components of the plasma membrane and biochemical precursors, and in human cells can alter LXR transcriptional activity. Here, a panel of breast cancer cell lines were treated with four dietary plant sterols and a stanol, alone or in combination with oxysterols. LXR activation and repression were measured by gene expression and LXR-luciferase reporter assays. Oxysterols activated LXR in all cell lines, but surprisingly phytosterols failed to modulate LXR activity. However, phytosterols significantly inhibited the ability of oxysterols to drive LXR transcription. These data support a role for phytosterols in modulating cancer cell behaviour via LXR, and therefore suggest merit in accurate dietary recordings of these molecules in cancer patients during treatment and perhaps supplementation to benefit recovery.


Assuntos
Neoplasias da Mama/genética , Receptores X do Fígado/genética , Oxisteróis/metabolismo , Fitosteróis/farmacologia , Ativação Transcricional/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Receptores X do Fígado/metabolismo
15.
Methods Mol Biol ; 1951: 15-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825141

RESUMO

Nuclear receptors (NRs) are ligand-activated transcription factors. Class 2 NRs, such as the liver X receptor (LXR)α and (LXR)ß, are typically retained in the nucleus bound to the DNA in both the presence and absence of ligand. Upon binding ligands including hydroxylated cholesterol, LXR releases corepressor proteins in exchange for coactivators resulting in target gene transcription. Activity of the LXRs therefore depends on a combination of the local ligand concentration(s) and cofactor expression, which itself is a function of cell and tissue type, mutation load, and epigenetic regulation. Cross talk with other transcription factors or signaling pathways can also alter LXR activity. The role that LXR plays in both normal physiology and disease progression is becoming increasingly apparent, and a better understanding of how and when LXR is activated or repressed is pressing biological and clinical questions.The complexity of LXR regulation makes identifying novel ligands and determining LXR activity in new cell types challenging. Generating cell lines that contain a stably integrated luciferase reporter gene with an upstream LXR-dependent promoter provides a quick, cheap, robust, efficient, and high-throughput solution to identify novel ligands and assess ligand activity in new cell types. Transplant of these stable cell culture cell lines as xenografts allows reporter activation to be assessed in vivo. Here we describe the generation of stable LXR reporter cell lines, how to confirm transgene insertion and select single cell clones, as well a method to assess transgene activity in vitro.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Receptores X do Fígado/genética , Oxisteróis/farmacologia , Análise de Dados , Dosagem de Genes , Vetores Genéticos/genética , Humanos , Ligantes , Receptores X do Fígado/metabolismo , Transdução Genética
16.
J Steroid Biochem Mol Biol ; 192: 105309, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30779932

RESUMO

Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system ("AFFL"). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.


Assuntos
Neoplasias da Mama/metabolismo , Cromatografia Líquida/métodos , Oxisteróis/análise , Oxisteróis/química , Espectrometria de Massas em Tandem/métodos , Neoplasias da Mama/patologia , Feminino , Humanos , Hidroxicolesteróis/metabolismo
17.
Biochim Biophys Acta Gene Regul Mech ; 1861(11): 996-1006, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30343695

RESUMO

MicroRNAs and RNA-binding proteins exert regulation on >60% of coding genes, yet interplay between them is little studied. Canonical microRNA binding occurs by base-pairing of microRNA 3'-ends to complementary "seed regions" in mRNA 3'UTRs, resulting in translational repression. Similarly, regulatory RNA-binding proteins bind to mRNAs, modifying stability or translation. We investigated post-transcriptional regulation acting on the xenobiotic pump ABCB1/P-glycoprotein, which is implicated in cancer therapy resistance. We characterised the ABCB1 UTRs in primary breast cancer cells and identified UTR sequences that responded to miR-19b despite lacking a canonical binding site. Sequences did, however, contain consensus sites for the RNA-binding protein HuR. We demonstrated that a tripartite complex of HuR, miR-19b and UTR directs repression of ABCB1/P-glycoprotein expression, with HuR essential for non-canonical miR-19b binding thereby controlling chemosensitivity of breast cancer cells. This exemplifies a new cooperative model between RNA-binding proteins and microRNAs to expand the repertoire of mRNAs that can be regulated. This study suggests a novel therapeutic target to impair P-glycoprotein mediated drug efflux, and also indicates that current microRNA binding predictions that rely on seed regions alone may be too conservative.


Assuntos
Neoplasias da Mama/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , MicroRNAs/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Mama/metabolismo , Linhagem Celular , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , RNA Mensageiro/metabolismo
18.
Biochimie ; 153: 150-161, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29730298

RESUMO

Cancer is a global problem with no sign that incidences are reducing. The great costs associated with curing cancer, through developing novel treatments and applying patented therapies, is an increasing burden to developed and developing nations alike. These financial and societal problems will be alleviated by research efforts into prevention, or treatments that utilise off-patent or repurposed agents. Phytosterols are natural components of the diet found in an array of seeds, nuts and vegetables and have been added to several consumer food products for the management of cardio-vascular disease through their ability to lower LDL-cholesterol levels. In this review, we provide a connected view between the fields of structural biophysics and cellular and molecular biology to evaluate the growing evidence that phytosterols impair oncogenic pathways in a range of cancer types. The current state of understanding of how phytosterols alter the biophysical properties of plasma membrane is described, and the potential for phytosterols to be repurposed from cardio-vascular to oncology therapeutics. Through an overview of the types of biophysical and molecular biology experiments that have been performed to date, this review informs the reader of the molecular and biophysical mechanisms through which phytosterols could have anti-cancer properties via their interactions with the plasma cell membrane. We also outline emerging and under-explored areas such as computational modelling, improved biomimetic membranes and ex vivo tissue evaluation. Focus of future research in these areas should improve understanding, not just of phytosterols in cancer cell biology but also to give insights into the interaction between the plasma membrane and the genome. These fields are increasingly providing meaningful biological and clinical data but iterative experiments between molecular biology assays, biosynthetic membrane studies and computational membrane modelling improve and refine our understanding of the role of different sterol components of the plasma membrane.


Assuntos
Neoplasias/prevenção & controle , Fitosteróis/metabolismo , Fenômenos Biofísicos , Membrana Celular/metabolismo , Dieta , Humanos , Fitosteróis/administração & dosagem
19.
BMC Cancer ; 15: 634, 2015 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-26362310

RESUMO

BACKGROUND: Multi-drug Resistance associated Protein-1 (MRP1) can export chemotherapeutics from cancer cells and is implicated in chemoresistance, particularly as is it known to be up-regulated by chemotherapeutics. Our aims in this study were to determine whether activation of Notch signalling is responsible for chemotherapy-induced MRP1 expression Notch in breast cancers, and whether this pathway can be manipulated with an inhibitor of Notch activity. METHODS: MRP1 and Notch1 were investigated in 29 patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, using immunohistochemistry on matched biopsy (pre-NAC) and surgical samples (post-NAC). Breast epithelial cell cultures (T47D, HB2) were treated with doxorubicin in the presence and absence of functional Notch1, and qPCR, siRNA, Western blots, ELISAs and flow-cytometry were used to establish interactions. RESULTS: In clinical samples, Notch1 was activated by neoadjuvant chemotherapy (Wilcoxon signed-rank p < 0.0001) and this correlated with induction of MRP1 expression (rho = 0.6 p = 0.0008). In breast cell lines, doxorubicin induced MRP1 expression and function (non-linear regression p < 0.004). In the breast cancer line T47D, doxorubicin activated Notch1 and, critically, inhibition of Notch1 activation with the γ-secretase inhibitor DAPT abolished the doxorubicin-induced increase in MRP1 expression and function (t-test p < 0.05), resulting in enhanced cellular retention of doxorubicin and increased doxorubicin-induced apoptosis (t-test p = 0.0002). In HB2 cells, an immortal but non-cancer derived breast cell line, Notch1-independent MRP1 induction was noted and DAPT did not enhance doxorubicin-induced apoptosis. CONCLUSIONS: Notch inhibitors may have potential in sensitizing breast cancer cells to chemotherapeutics and therefore in tackling chemoresistance.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Receptor Notch1/metabolismo , Regulação para Cima/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Adulto Jovem
20.
Int J Cancer ; 137(7): 1519-27, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24895240

RESUMO

In 1927 Otto Warburg established that tumours derive energy primarily from the conversion of glucose to lactic acid and only partially through cellular respiration involving oxygen. In the 1950s he proposed that all causes of cancer reflected different mechanisms of disabling cellular respiration in favour of fermentation (now termed aerobic glycolysis). The role of aberrant glucose metabolism in cancer is now firmly established. The shift away from oxidative phosphorylation towards the metabolically expensive aerobic glycolysis is somewhat counter-intuitive given its wasteful nature. Multiple control processes are in place to maintain cellular efficiency and it is likely that these mechanisms are disrupted to facilitate the shift to the reliance on aerobic glycolysis. One such process of cell control is mediated by the nuclear receptor superfamily. This large family of transcription factors plays a significant role in sensing environmental cues and controlling decisions on proliferation, differentiation and cell death for example, to regulate glucose uptake and metabolism and to modulate the actions of oncogenes and tumour suppressors. In this review we highlight mechanisms by which nuclear receptors actions are altered during tumorigenic transformation and can serve to enhance the shift to aerobic glycolysis. At the simplest level, a basic alteration in NR behaviour can serve to enhance glycolytic flux thus providing a basis for enhanced survival within the tumour micro-environment. Ameliorating the enhanced NR activity in this context may help to sensitize cancer cells to Warburg targeted therapies and may provide future drug targets.


Assuntos
Neoplasias/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Metabolismo Energético , Humanos
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