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1.
Clin Cancer Res ; 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589432

RESUMO

PURPOSE: G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether LC3B, a G9a target gene, can predict treatment response. EXPERIMENTAL DESIGN: Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model. RESULTS: Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of MAP1LC3B or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death. CONCLUSIONS: Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.

2.
Oncoimmunology ; 9(1): 1825178, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-33101776

RESUMO

Tumor-infiltrating lymphocytes (TILs) play indispensable roles in the progression and response to treatment of solid tumors. However, the prognostic significance of CD4+ TILs is not fully disclosed in cancers generally and in CRC in particular, mainly due to the existence of different functional subsets of CD4+ T cells. We performed transcriptomic profiling of CD4+ TILs isolated from CRC patients in order to identify differentially expressed genes and their functional pathways in early versus advanced disease stages. We found that in advanced stages, genes related to immune and inflammatory responses, in particular Th1-mediated immune response and cytotoxicity-mediated genes, were downregulated; while epigenetic-mediated silencing genes were upregulated. Interestingly, we identified genes, which were steadily upregulated or downregulated in CD4+ TILs with CRC progression from stage I to IV. Additionally, of the top 200 deregulated genes, 43 upregulated and 64 downregulated genes showed similar deregulation trends in the cancer genome atlas CRC dataset. From these 97 deregulated genes, we identified a "poor prognosis CD4 gene signature (ppCD4sig)". Patients with high ppCD4sig score showed shorter disease-specific survival (DSS) and progression-free interval (PFI). The ppCD4sig was an independent prognostic indicator for DSS (HR = 1.73, 95% CI 1.32-2.27, P = 0.0001) and PFI (HR = 1.75, 95% CI 1.3-2.35, P = 0.0016). Additionally, patients at advanced stages and at a younger age (<55 years) were more likely to have a high ppCD4sig score. Altogether, our data provide novel insights and a unique prognostic gene signature of CD4+ TILs in the CRC microenvironment.

3.
Ther Adv Med Oncol ; 12: 1758835920946259, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33014146

RESUMO

Background: Human epidermal growth factor receptor-4 (HER4) and yes-associated protein-1 (YAP) are candidate therapeutic targets in oncology. YAP's transcriptional coactivation function is modulated by the HER4 intracellular domain (HER4-ICD) in vitro, but the clinical relevance of this has not been established. This study investigated the potential for targeting the HER4-YAP pathway in brain metastatic breast cancer. Methods: We performed immuno-phenotypic profiling of pathway markers in a consecutive breast cancer series with 25 years of clinical follow up (n = 371), and patient-matched breast and metastatic brain tumours (n = 91; 30 pairs). Results: Membrane localisation of phospho-HER4 [pHER4(Y1162)] was infrequent in primary breast cancer, but very frequent in brain metastases (5.9% versus 75% positive), where it was usually co-expressed with pHER3(Y1289) (p < 0.05). The presence of YAP in tumour cell nuclei was associated directly with nuclear pERK5(T218/Y210) (p = 0.003). However, relationships with disease-specific survival depended on oestrogen receptor (ER) status. Nuclear pYAP(S127) was associated with smaller, good prognostic ER+ breast tumours (log-rank hazard-ratio 0.53; p = 9.6E-03), but larger, poor prognostic triple-negative cancers (log-rank hazard-ratio 2.78; p = 1.7E-02), particularly when co-expressed with nuclear HER4-ICD (p = 0.02). This phenotype was associated with stemness and mitotic instability markers (vimentin, SOX9, ID1, SPAG5, TTK, geminin; p < 0.05). YAP expression in brain metastases was higher than matched primary tumours; specifically, nuclear pYAP(S127) in ER-negative cases (p < 0.05). Nuclear YAP was detected in ~70% of ER-negative, HER4-activated brain metastases. Discussion: Our findings suggest that the canonical-mechanism where Hippo pathway-mediated phosphorylation of YAP ostensibly excludes it from the nucleus is dysfunctional in breast cancer. The data are consistent with pYAP(S127) having independent transcriptional functions, which may include transducing neuregulin signals in brain metastases. Consistent with mechanistic studies implicating it as an ER co-factor, nuclear pYAP(S127) associations with breast cancer clinical outcomes were dependent on ER status. Conclusion: Preclinical studies investigating HER4 and nuclear YAP combination therapy strategies are warranted.

4.
J Immunother Cancer ; 8(2)2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32948653

RESUMO

BACKGROUND: Cytotoxic CD8+ T cell-mediated response is the most important arm of adaptive immunity, which dictates the capacity of the host immune response in eradicating tumor cells. Due to tumor intrinsic and/or extrinsic factors, the density and function of CD8+ tumor-infiltrating lymphocytes (TILs) could be compromised, leading to poor prognosis and survival. METHODS: Using RNA-Seq, transcriptomes of sorted CD3+CD8+ TILs from treatment-naïve colorectal cancer (CRC) patients at advanced stages (III and IV) were compared with those from patients with early stages (I and II). A signature referred to as 'poor prognosis CD8 gene signature (ppCD8sig)' was identified and analyzed in The Cancer Genome Atlas CRC dataset. Scores for the ppCD8sig were calculated and classified as high, intermediate and low, and its prognostic significance was assessed using multivariate analysis and Cox proportional hazard model. Densities of CD3+ and CD8+ T cell infiltration in tumors from patients with high and low ppCD8sig scores were assessed by flow cytometry and immunostaining. RESULTS: Genes related to epigenetic regulation and response to hypoxia were upregulated in CD8+ TILs from patients with advanced stages, while genes related to T cell activation, cell proliferation and cell cycle were downregulated. Patients with high ppCD8sig score had poorer disease-specific survival (DSS) and shorter progression-free interval (PFI). The ppCD8sig was an independent prognostic indicator for DSS (HR 1.83, 95% CI 1.40 to 2.38, p<0.0001) and PFI (HR 1.42, 95% CI 1.04 to 1.93, p=0.026). Additionally, patients with high ppCD8sig score were more likely to have advanced stages (χ2 p<0.0001) and residual disease after primary therapy (χ2 p=0.046). Patients with high ppCD8sig score had reduced levels of CD3+ and CD8+ TILs and low Immunoscores (IS), compared to patients with low ppCD8sig score. CONCLUSIONS: Our data provided insights into the altered regulation of biological mechanisms and signaling pathways in CD8+ TILs during CRC progression, and revealed a gene signature as an independent prognostic indicator. Patients with high ppCD8sig score had lower levels of TILs and low IS. These data further confirm the prognostic value of the identified ppCD8sig and potentially highlight its clinical relevance.

5.
Theranostics ; 10(10): 4515-4529, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32292512

RESUMO

Rationale: Epigenetic mechanisms are fundamental processes that can modulate gene expression, allowing cellular adaptation to environmental conditions. Hypoxia is an important factor known to initiate the metastatic cascade in cancer, activating cell motility and invasion by silencing cell adhesion genes. G9a is a histone methyltransferase previously shown to accumulate in hypoxic conditions. While its oncogenic activity has been previously reported, not much is known about the role G9a plays in the hypoxia-mediated metastatic cascade. Methods: The role of G9a in cell motility in hypoxic condition was determined by inhibiting G9a either by short-hairpin mediated knock down or pharmacologically using a small molecule inhibitor. Through gene expression profiling, we identified CDH10 to be an important G9a target that regulates breast cancer cell motility. Lung metastasis assay in mice was used to determine the physiological significance of G9a. Results: We demonstrate that, while hypoxia enhances breast cancer migratory capacity, blocking G9a severely reduces cellular motility under both normoxic and hypoxic conditions and prevents the hypoxia-mediated induction of cellular movement. Moreover, inhibition of G9a histone methyltransferase activity in mice using a specific small molecule inhibitor significantly reduced growth and colonisation of breast cancer cells in the lung. We identify the type-II cadherin CDH10 as being a novel hypoxia-dependent gene, directly repressed by G9a through histone methylation. CDH10 overexpression significantly reduces cellular movements in breast cancer cell lines and prevents the hypoxia-mediated increase in cell motility. In addition, we show that CDH10 expression is prognostic in breast cancer and that it is inversely correlated to EHMT2 (G9a) transcript levels in many tumor-types, including breast cancer. Conclusion: We propose that G9a promotes cellular motility during hypoxic stress through the silencing of the cell adhesion molecule CDH10 and we describe CDH10 as a novel prognostic biomarker for breast cancer.

6.
Sci Rep ; 9(1): 8541, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189969

RESUMO

During chronic liver injury hepatic stellate cells (HSCs), the principal source of extracellular matrix in the fibrotic liver, transdifferentiate into pro-fibrotic myofibroblast-like cells - a process potentially regulated by microRNAs (miRNAs). Recently, we found serum miRNA-25-3p (miR-25) levels were upregulated in children with Cystic Fibrosis (CF) without liver disease, compared to children with CF-associated liver disease and healthy individuals. Here we examine the role of miR-25 in HSC biology. MiR-25 was detected in the human HSC cell line LX-2 and in primary murine HSCs, and increased with culture-induced activation. Transient overexpression of miR-25 inhibited TGF-ß and its type 1 receptor (TGFBR1) mRNA expression, TGF-ß-induced Smad2 phosphorylation and subsequent collagen1α1 induction in LX-2 cells. Pull-down experiments with biotinylated miR-25 revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as miR-25 targets in HSCs. NanoString analysis confirmed miR-25 regulation of Notch- and Wnt-signaling pathways. Expression of Notch signaling pathway components and endogenous Notch1 signaling was downregulated in miR-25 overexpressing LX-2 cells, as were components of Wnt signaling such as Wnt5a. We propose that miR-25 acts as a negative feedback anti-fibrotic control during HSC activation by reducing the reactivity of HSCs to TGF-ß-induced collagen expression and modulating the cross-talk between Notch, Wnt and TGF-ß signaling.


Assuntos
Colágeno/biossíntese , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Receptor Notch1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Colágeno/genética , Células Estreladas do Fígado , Humanos , Camundongos , MicroRNAs/genética , Receptor Notch1/genética , Fator de Crescimento Transformador beta/genética
7.
Int J Mol Sci ; 20(10)2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31121957

RESUMO

Patients with brain-metastatic breast cancer face a bleak prognosis marked by morbidity and premature death. A deeper understanding of molecular interactions in the metastatic brain tumour microenvironment may inform the development of new therapeutic strategies. In this study, triple-negative MDA-MB-231 breast cancer cells or PBS (modelling traumatic brain injury) were stereotactically injected into the cerebral cortex of NOD/SCID mice to model metastatic colonization. Brain cells were isolated from five tumour-associated samples and five controls (pooled uninvolved and injured tissue) by immunoaffinity chromatography, and proteomic profiles were compared using the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) discovery platform. Ontology and cell type biomarker enrichment analysis of the 125 differentially abundant proteins (p < 0.05) showed the changes largely represent cellular components involved in metabolic reprogramming and cell migration (min q = 4.59 × 10-5), with high-throughput PubMed text mining indicating they have been most frequently studied in the contexts of mitochondrial dysfunction, oxidative stress and autophagy. Analysis of mouse brain cell type-specific biomarkers suggested the changes were paralleled by increased proportions of microglia, mural cells and interneurons. Finally, we orthogonally validated three of the proteins in an independent xenograft cohort, and investigated their expression in craniotomy specimens from triple-negative metastatic breast cancer patients, using a combination of standard and fluorescent multiplex immunohistochemistry. This included 3-Hydroxyisobutyryl-CoA Hydrolase (HIBCH), which is integral for gluconeogenic valine catabolism in the brain, and was strongly induced in both graft-associated brain tissue (13.5-fold by SWATH-MS; p = 7.2 × 10-4), and areas of tumour-associated, reactive gliosis in human clinical samples. HIBCH was also induced in the tumour compartment, with expression frequently localized to margins and haemorrhagic areas. These observations raise the possibility that catabolism of valine is an effective adaptation in metastatic cells able to access it, and that intermediates or products could be transferred from tumour-associated glia. Overall, our findings indicate that metabolic reprogramming dominates the proteomic landscape of graft-associated brain tissue in the intracranial MDA-MB-231 xenograft model. Brain-derived metabolic provisions could represent an exploitable dependency in breast cancer brain metastases.


Assuntos
Neoplasias Encefálicas/secundário , Encéfalo/patologia , Neoplasias da Mama/patologia , Proteínas/análise , Microambiente Tumoral , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteômica
8.
Nat Commun ; 10(1): 1741, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988301

RESUMO

Genome-wide association studies (GWAS) have identified more than 170 breast cancer susceptibility loci. Here we hypothesize that some risk-associated variants might act in non-breast tissues, specifically adipose tissue and immune cells from blood and spleen. Using expression quantitative trait loci (eQTL) reported in these tissues, we identify 26 previously unreported, likely target genes of overall breast cancer risk variants, and 17 for estrogen receptor (ER)-negative breast cancer, several with a known immune function. We determine the directional effect of gene expression on disease risk measured based on single and multiple eQTL. In addition, using a gene-based test of association that considers eQTL from multiple tissues, we identify seven (and four) regions with variants associated with overall (and ER-negative) breast cancer risk, which were not reported in previous GWAS. Further investigation of the function of the implicated genes in breast and immune cells may provide insights into the etiology of breast cancer.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Locos de Características Quantitativas
9.
JCI Insight ; 52019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30830863

RESUMO

Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.


Assuntos
Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Priônicas/metabolismo , Adulto , Antraciclinas/uso terapêutico , Antibióticos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Epirubicina/farmacologia , Epirubicina/uso terapêutico , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Seleção de Pacientes , Proteínas Priônicas/sangue , Proteínas Priônicas/genética , Prognóstico , Ligação Proteica , Isoformas de Proteínas/sangue , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo
10.
J Pathol Clin Res ; 5(1): 25-39, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30246500

RESUMO

Breast cancer metastasis to gynaecological organs is an understudied pattern of tumour spread. We explored clinico-pathological and molecular features of these metastases to better understand whether this pattern of dissemination is organotropic or a consequence of wider metastatic dissemination. Primary and metastatic tumours from 54 breast cancer patients with gynaecological metastases were analysed using immunohistochemistry, DNA copy-number profiling, and targeted sequencing of 386 cancer-related genes. The median age of primary tumour diagnosis amongst patients with gynaecological metastases was significantly younger compared to a general breast cancer population (46.5 versus 60 years; p < 0.0001). Median age at metastatic diagnosis was 54.4, time to progression was 4.8 years (range 0-20 years), and survival following a diagnosis of metastasis was 1.95 years (range 0-18 years). Patients had an average of five involved sites (most frequently ovary, fallopian tube, omentum/peritoneum), with fewer instances of spread to the lungs, liver, or brain. Invasive lobular histology and luminal A-like phenotype were over-represented in this group (42.8 and 87.5%, respectively) and most patients had involved axillary lymph nodes (p < 0.001). Primary tumours frequently co-expressed oestrogen receptor cofactors (GATA3, FOXA1) and harboured amplifications at 8p12, 8q24, and 11q13. In terms of phenotype conversion, oestrogen receptor status was generally maintained in metastases, FOXA1 increased, and expression of progesterone receptor, androgen receptor, and GATA3 decreased. ESR1 and novel AR mutations were identified. Metastasis to gynaecological organs is a complication frequently affecting young women with invasive lobular carcinoma and luminal A-like breast cancer, and hence may be driven by sustained hormonal signalling. Molecular analyses reveal a spectrum of factors that could contribute to de novo or acquired resistance to therapy and disease progression.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/genética , Metástase Linfática/patologia , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Receptores Estrogênicos/genética , Receptores de Progesterona/genética
11.
Cancers (Basel) ; 10(12)2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30562956

RESUMO

The EphA3 receptor has recently emerged as a functional tumour-specific therapeutic target in glioblastoma (GBM). EphA3 is significantly elevated in recurrent disease, is most highly expressed on glioma stem cells (GSCs), and has a functional role in maintaining self-renewal and tumourigenesis. An unlabelled EphA3-targeting therapeutic antibody is currently under clinical assessment in recurrent GBM patients. In this study, we assessed the efficacy of EphA3 antibody drug conjugate (ADC) and radioimmunotherapy (RIT) approaches using orthotopic animal xenograft models. Brain uptake studies, using positron emission tomography/computed tomography (PET/CT) imaging, show EphA3 antibodies are effectively delivered across the blood-tumour barrier and accumulate at the tumour site with no observed normal brain reactivity. A robust anti-tumour response, with no toxicity, was observed using EphA3, ADC, and RIT approaches, leading to a significant increase in overall survival. Our current research provides evidence that GBM patients may benefit from pay-loaded EphA3 antibody therapies.

12.
Nat Genet ; 50(7): 968-978, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29915430

RESUMO

The breast cancer risk variants identified in genome-wide association studies explain only a small fraction of the familial relative risk, and the genes responsible for these associations remain largely unknown. To identify novel risk loci and likely causal genes, we performed a transcriptome-wide association study evaluating associations of genetically predicted gene expression with breast cancer risk in 122,977 cases and 105,974 controls of European ancestry. We used data from the Genotype-Tissue Expression Project to establish genetic models to predict gene expression in breast tissue and evaluated model performance using data from The Cancer Genome Atlas. Of the 8,597 genes evaluated, significant associations were identified for 48 at a Bonferroni-corrected threshold of P < 5.82 × 10-6, including 14 genes at loci not yet reported for breast cancer. We silenced 13 genes and showed an effect for 11 on cell proliferation and/or colony-forming efficiency. Our study provides new insights into breast cancer genetics and biology.


Assuntos
Neoplasias da Mama/genética , Estudos de Casos e Controles , Feminino , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Polimorfismo de Nucleotídeo Único , Risco , Transcriptoma
13.
Breast Cancer Res Treat ; 170(1): 179-188, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29468485

RESUMO

PURPOSE: We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease. METHODS: The cell line, P7731, was derived from a metastatic bone lesion of a breast cancer patient and assessed for marker expression. P7731 was analyzed for DNA copy number variation, somatic mutations, and gene expression and was compared with the primary tumor. RESULTS: P7731 cells are negative for estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 (triple-negative); strongly express vimentin (100% of cells positive) and also express cytokeratins 8/18 and 19 but at lower frequencies. Flow cytometry indicates P7731 cells are predominantly CD44+/CD49f+/EpCAM-, consistent with a primitive, mesenchymal-like phenotype. The cell line is tumorigenic in immunocompromised mice. Exome sequencing identified a total of 45 and 76 somatic mutations in the primary tumor and cell line, respectively, of which 32 were identified in both samples and included mutations in known driver genes PIK3CA, TP53, and ARID1A. P7731 retains the DNA copy number alterations present in the matching primary tumor. Homozygous deletions detected in the cell line and in the primary tumor were found in regions containing three known (CDKN2A, CDKN2B, and CDKN1B) and 23 putative tumor suppressor genes. Cell line-specific gene amplification coupled with mRNA expression analysis revealed genes and pathways with potential pro-metastatic functions. CONCLUSION: This novel human breast cancer-bone metastasis cell line will be a useful model to study aspects of breast cancer biology, particularly metastasis-related changes from breast to bone.


Assuntos
Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proteínas de Neoplasias/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Mama/patologia , Variações do Número de Cópias de DNA/genética , Exoma/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Mutação , Neoplasias de Mama Triplo Negativas/genética
14.
Breast Cancer Res Treat ; 167(1): 289-301, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28889351

RESUMO

PURPOSE: Cell lines are extremely useful tools in breast cancer research. Their key benefits include a high degree of control over experimental variables and reproducibility. However, the advantages must be balanced against the limitations of modelling such a complex disease in vitro. Informed selection of cell line(s) for a given experiment now requires essential knowledge about molecular and phenotypic context in the culture dish. METHODS: We performed multidimensional profiling of 36 widely used breast cancer cell lines that were cultured under standardised conditions. Flow cytometry and digital immunohistochemistry were used to compare the expression of 14 classical breast cancer biomarkers related to intrinsic molecular profiles and differentiation states: EpCAM, CD24, CD49f, CD44, ER, AR, HER2, EGFR, E-cadherin, p53, vimentin, and cytokeratins 5, 8/18 and 19. RESULTS: This cell-by-cell analysis revealed striking heterogeneity within cultures of individual lines that would be otherwise obscured by analysing cell homogenates, particularly amongst the triple-negative lines. High levels of p53 protein, but not RNA, were associated with somatic mutations (p = 0.008). We also identified new subgroups using the nanoString PanCancer Pathways panel (730 transcripts representing 13 canonical cancer pathways). Unsupervised clustering identified five groups: luminal/HER2, immortalised ('normal'), claudin-low and two basal clusters, distinguished mostly by baseline expression of TGF-beta and PI3-kinase pathway genes. CONCLUSION: These features are compared with other published genotype and phenotype information in a user-friendly reference table to help guide selection of the most appropriate models for in vitro and in vivo studies, and as a framework for classifying new patient-derived cancer cell lines and xenografts.


Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Heterogeneidade Genética , Proteínas de Neoplasias/genética , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Humanos , Fenótipo
15.
Nat Genet ; 49(12): 1767-1778, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29058716

RESUMO

Most common breast cancer susceptibility variants have been identified through genome-wide association studies (GWAS) of predominantly estrogen receptor (ER)-positive disease. We conducted a GWAS using 21,468 ER-negative cases and 100,594 controls combined with 18,908 BRCA1 mutation carriers (9,414 with breast cancer), all of European origin. We identified independent associations at P < 5 × 10-8 with ten variants at nine new loci. At P < 0.05, we replicated associations with 10 of 11 variants previously reported in ER-negative disease or BRCA1 mutation carrier GWAS and observed consistent associations with ER-negative disease for 105 susceptibility variants identified by other studies. These 125 variants explain approximately 16% of the familial risk of this breast cancer subtype. There was high genetic correlation (0.72) between risk of ER-negative breast cancer and breast cancer risk for BRCA1 mutation carriers. These findings may lead to improved risk prediction and inform further fine-mapping and functional work to better understand the biological basis of ER-negative breast cancer.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Mutação , Polimorfismo de Nucleotídeo Único , Neoplasias da Mama/etnologia , Neoplasias da Mama/metabolismo , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Predisposição Genética para Doença/etnologia , Estudo de Associação Genômica Ampla/métodos , Heterozigoto , Humanos , Receptores Estrogênicos/metabolismo , Fatores de Risco
16.
Sci Rep ; 7(1): 10909, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28883623

RESUMO

Melanoma tumors are highly heterogeneous, comprising of different cell types that vary in their potential for growth and invasion. Heterogeneous expression of the Microphthalmia-associated Transcription Factor (MITF) and the POU domain transcription factor BRN2 (POU3F2) has been found in malignant melanoma. Changing expression of these transcription factors as the disease progresses has been linked to the metastatic mechanism of phenotype switching. We therefore investigated the effects of MITF and BRN2 expression in melanoma growth and metastasis. Depletion of MITF resulted in a cell population that had a slowed cell cycle progression, was less invasive in vitro and had hindered tumor and metastasis forming ability in mouse xenograft studies. BRN2 depletion left a cell population with intact proliferation and invasion in vitro; however metastatic growth was significantly reduced in the mouse xenograft model. These results suggest that the proliferative population within melanoma tumors express MITF, and both MITF and BRN2 are important for metastatic growth in vivo. This finding highlights the importance of BRN2 and MITF expression in development of melanoma metastasis.


Assuntos
Proliferação de Células , Proteínas de Homeodomínio/metabolismo , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Metástase Neoplásica/patologia , Fatores do Domínio POU/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias
17.
Am J Hum Genet ; 101(2): 255-266, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28777932

RESUMO

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.


Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 11/genética , Ciclina D1/genética , Reparo do DNA/genética , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Elementos Facilitadores Genéticos/genética , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença/genética , Humanos , Células MCF-7 , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Guia/genética , RNA Interferente Pequeno/genética
18.
Proc Natl Acad Sci U S A ; 114(27): 7077-7082, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28630300

RESUMO

G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a protein stability is increased in hypoxia via reduced proline hydroxylation and, hence, inefficient degradation by the proteasome. This inefficiency leads to an increase in H3K9me2 at its target promoters. Blocking the methyltransferase activity of G9a inhibited cellular proliferation and migration in vitro and tumor growth in vivo. Furthermore, an increased level of G9a is a crucial factor in mediating the hypoxic response by down-regulating the expression of specific genes, including ARNTL, CEACAM7, GATA2, HHEX, KLRG1, and OGN This down-regulation can be rescued by a small molecule inhibitor of G9a. Based on the hypothesis that the changes in gene expression would influence patient outcomes, we have developed a prognostic G9a-suppressed gene signature that can stratify breast cancer patients. Together, our findings provide an insight into the role G9a plays as an epigenetic mediator of hypoxic response, which can be used as a diagnostic marker, and proposes G9a as a therapeutic target for solid cancers.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Hipóxia/genética , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células , Intervalo Livre de Doença , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos C57BL , Prognóstico , Prolina/química , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/metabolismo , Recidiva , Microambiente Tumoral
19.
Bioorg Med Chem Lett ; 27(14): 3096-3100, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28545975

RESUMO

RAD51 is a vital component of the homologous recombination DNA repair pathway and is overexpressed in drug-resistant cancers, including aggressive triple negative breast cancer (TNBC). A proposed strategy for improving therapeutic outcomes for patients is through small molecule inhibition of RAD51, thereby sensitizing tumor cells to DNA damaging irradiation and/or chemotherapy. Here we report structure-activity relationships for a library of quinazolinone derivatives. A novel RAD51 inhibitor (17) displays up to 15-fold enhanced inhibition of cell growth in a panel of TNBC cell lines compared to compound B02, and approximately 2-fold increased inhibition of irradiation-induced RAD51 foci formation. Additionally, compound 17 significantly inhibits TNBC cell sensitivity to DNA damage, implying a potentially targeted therapy for cancer treatment.


Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Quinazolinonas/química , Rad51 Recombinase/antagonistas & inibidores , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/síntese química , Cinamatos/química , Cinamatos/toxicidade , Dano ao DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Quinazolinas/síntese química , Quinazolinas/química , Quinazolinas/toxicidade , Quinazolinonas/farmacologia , Rad51 Recombinase/metabolismo , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
20.
Blood ; 129(18): 2479-2492, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28270450

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.


Assuntos
Proliferação de Células/fisiologia , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Sobrevivência Celular/fisiologia , Ilhas de CpG/fisiologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Proteínas Supressoras da Sinalização de Citocina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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