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1.
Mol Oncol ; 18(1): 156-169, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37854018

RESUMO

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision-making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one-third of patients with ERα-positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα-conversion. It remains unclear whether ERα-converted cancers are biologically similar to bona fide ERα-negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα-positive, ERα-converted and ERα-negative cases. Our data revealed that the loss of ERα-dependency in this metastatic site leads to only a partial switch to an ERα-negative molecular phenotype, with preservation of a luminal-like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα-converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα-dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.


Assuntos
Neoplasias da Mama , Derrame Pleural Maligno , Feminino , Humanos , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Glucocorticoides/uso terapêutico , Proteômica
2.
EMBO Mol Med ; 15(12): e17737, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37902007

RESUMO

Glucocorticoid receptor (GR) is a transcription factor that plays a crucial role in cancer biology. In this study, we utilized an in silico-designed GR activity signature to demonstrate that GR relates to the proliferative capacity of numerous primary cancer types. In breast cancer, the GR activity status determines luminal subtype identity and has implications for patient outcomes. We reveal that GR engages with estrogen receptor (ER), leading to redistribution of ER on the chromatin. Notably, GR activation leads to upregulation of the ZBTB16 gene, encoding for a transcriptional repressor, which controls growth in ER-positive breast cancer and associates with prognosis in luminal A patients. In relation to ZBTB16's repressive nature, GR activation leads to epigenetic remodeling and loss of histone acetylation at sites proximal to cancer-driving genes. Based on these findings, epigenetic inhibitors reduce viability of ER-positive breast cancer cells that display absence of GR activity. Our findings provide insights into how GR controls ER-positive breast cancer growth and may have implications for patients' prognostication and provide novel therapeutic candidates for breast cancer treatment.


Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo
3.
bioRxiv ; 2023 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-37333335

RESUMO

The crosstalk between prostate cancer (PCa) cells and the tumor microenvironment plays a pivotal role in disease progression and metastasis and could provide novel opportunities for patient treatment. Macrophages are the most abundant immune cells in the prostate tumor microenvironment (TME) and are capable of killing tumor cells. To identify genes in the tumor cells that are critical for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified AR, PRKCD, and multiple components of the NF-κB pathway as hits, whose expression in the tumor cell are essential for being targeted and killed by macrophages. These data position AR signaling as an immunomodulator, and confirmed by androgen-deprivation experiments, that rendered hormone-deprived tumor cells resistant to macrophage-mediated killing. Proteomic analyses showed a downregulation of oxidative phosphorylation in the PRKCD- and IKBKG-KO cells compared to the control, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Furthermore, phosphoproteomic analyses revealed that all hits impaired ferroptosis signaling, which was validated transcriptionally using samples from a neoadjuvant clinical trial with the AR-inhibitor enzalutamide. Collectively, our data demonstrate that AR functions together with the PRKCD and the NF-κB pathway to evade macrophage-mediated killing. As hormonal intervention represents the mainstay therapy for treatment of prostate cancer patients, our findings may have direct implications and provide a plausible explanation for the clinically observed persistence of tumor cells despite androgen deprivation therapy.

4.
Nucleic Acids Res ; 51(18): 9576-9593, 2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37070193

RESUMO

How steroid hormone receptors (SHRs) regulate transcriptional activity remains partly understood. Upon activation, SHRs bind the genome together with a co-regulator repertoire, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we functionally dissected the Glucocorticoid Receptor (GR) complex. We describe a functional cross-talk between PAXIP1 and the cohesin subunit STAG2, critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on chromatin, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. In lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.

5.
medRxiv ; 2023 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-36865297

RESUMO

Androgen Receptor (AR) signaling inhibitors, including enzalutamide, are treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC), but resistance inevitably develops. Using metastatic samples from a prospective phase II clinical trial, we epigenetically profiled enhancer/promoter activities with H3K27ac chromatin immunoprecipitation followed by sequencing, before and after AR-targeted therapy. We identified a distinct subset of H3K27ac-differentially marked regions that associated with treatment responsiveness. These data were successfully validated in mCRPC patient-derived xenograft models (PDX). In silico analyses revealed HDAC3 as a critical factor that can drive resistance to hormonal interventions, which we validated in vitro . Using cell lines and mCRPC PDX tumors in vitro , we identified drug-drug synergy between enzalutamide and the pan-HDAC inhibitor vorinostat, providing therapeutic proof-of-concept. These findings demonstrate rationale for new therapeutic strategies using a combination of AR and HDAC inhibitors to improve patient outcome in advanced stages of mCRPC.

6.
Cancer Discov ; 12(9): 2074-2097, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-35754340

RESUMO

In prostate cancer, androgen receptor (AR)-targeting agents are very effective in various disease stages. However, therapy resistance inevitably occurs, and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multiomics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosurvival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL. Posttreatment ARNTL levels were associated with patients' clinical outcomes, and ARNTL knockout strongly decreased prostate cancer cell growth. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an acquired dependency on the circadian regulator ARNTL, a novel candidate therapeutic target. SIGNIFICANCE: Understanding how prostate cancers adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenomic plasticity toward prosurvival signaling and uncovered the circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel lead for therapeutic development. See related commentary by Zhang et al., p. 2017. This article is highlighted in the In This Issue feature, p. 2007.


Assuntos
Androgênios , Neoplasias de Próstata Resistentes à Castração , Fatores de Transcrição ARNTL/genética , Androgênios/farmacologia , Androgênios/uso terapêutico , Linhagem Celular Tumoral , Ritmo Circadiano , Resistencia a Medicamentos Antineoplásicos/genética , Epigenômica , Humanos , Masculino , Nitrilas/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética
7.
Nat Commun ; 12(1): 4360, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272384

RESUMO

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Glucocorticoides/farmacologia , Neoplasias Pulmonares/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imidazóis/farmacologia , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Camundongos , Proteômica , Pirazinas/farmacologia , RNA Interferente Pequeno , RNA-Seq , Receptor IGF Tipo 1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Int J Cancer ; 146(8): 2348-2359, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31490549

RESUMO

Preclinical studies indicate that activated IGF-1R can drive endocrine resistance in ER-positive (ER+) breast cancer, but its clinical relevance is unknown. We studied the effect of IGF-1R signaling on tamoxifen benefit in patients and we searched for approaches to overcome IGF-1R-mediated tamoxifen failure in cell lines. Primary tumor blocks from postmenopausal ER+ breast cancer patients randomized between adjuvant tamoxifen versus nil were recollected. Immunohistochemistry for IGF-1R, p-IGF-1R/InsR, p-ERα(Ser118), p-ERα(Ser167) and PI3K/MAPK pathway proteins was performed. Multivariate Cox models were employed to assess tamoxifen efficacy. The association between p-IGF-1R/InsR and PI3K/MAPK pathway activation in MCF-7 and T47D cells was analyzed with Western blots. Cell proliferation experiments were performed under various growth-stimulating and -inhibiting conditions. Patients with ER+, IGF-1R-positive breast cancer without p-IGF-1R/InsR staining (n = 242) had tamoxifen benefit (HR 0.41, p = 0.0038), while the results for p-IGF-1R/InsR-positive patients (n = 125) were not significant (HR 0.95, p = 0.3). High p-ERα(Ser118) or p-ERα(Ser167) expression was associated with less tamoxifen benefit. In MCF-7 cells, IGF-1R stimulation increased phosphorylation of PI3K/MAPK proteins and ERα(Ser167) regardless of IGF-1R overexpression. This could be abrogated by the dual IGF-1R/InsR inhibitor linsitinib, but not by the IGF-IR-selective antibody 1H7. In MCF-7 and T47D cells, stimulation of the IGF-1R/InsR pathway resulted in cell proliferation regardless of tamoxifen. Abrogation of cell growth was regained by addition of linsitinib. In conclusion, p-IGF-1R/InsR positivity in ER+ breast cancer is associated with reduced benefit from adjuvant tamoxifen in postmenopausal patients. In cell lines, stimulation rather than overexpression of IGF-1R is driving tamoxifen resistance to be abrogated by linsitinib.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Imidazóis/farmacologia , Pirazinas/farmacologia , Receptor IGF Tipo 1/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Imidazóis/administração & dosagem , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Pirazinas/administração & dosagem , Receptor IGF Tipo 1/antagonistas & inibidores , Tamoxifeno/administração & dosagem
9.
Nucleic Acids Res ; 47(18): 9557-9572, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31372638

RESUMO

Estrogen receptor α (ERα) is an enhancer activating transcription factor, a key driver of breast cancer and a main target for cancer therapy. ERα-mediated gene regulation requires proper chromatin-conformation to facilitate interactions between ERα-bound enhancers and their target promoters. A major determinant of chromatin structure is the CCCTC-binding factor (CTCF), that dimerizes and together with cohesin stabilizes chromatin loops and forms the boundaries of topologically associated domains. However, whether CTCF-binding elements (CBEs) are essential for ERα-driven cell proliferation is unknown. To address this question in a global manner, we implemented a CRISPR-based functional genetic screen targeting CBEs located in the vicinity of ERα-bound enhancers. We identified four functional CBEs and demonstrated the role of one of them in inducing chromatin conformation changes in favor of activation of PREX1, a key ERα target gene in breast cancer. Indeed, high PREX1 expression is a bona-fide marker of ERα-dependency in cell lines, and is associated with good outcome after anti-hormonal treatment. Altogether, our data show that distinct CTCF-mediated chromatin structures are required for ERα- driven breast cancer cell proliferation.


Assuntos
Neoplasias da Mama/genética , Fator de Ligação a CCCTC/genética , Proliferação de Células/genética , Receptor alfa de Estrogênio/genética , Sítios de Ligação/genética , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas/genética , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Células MCF-7 , Ligação Proteica/genética
10.
Acta Neuropathol Commun ; 7(1): 60, 2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023360

RESUMO

Inter-individual differences in cortisol production by the hypothalamus-pituitary-adrenal (HPA) axis are thought to contribute to clinical and pathological heterogeneity of multiple sclerosis (MS). At the same time, accumulating evidence indicates that MS pathogenesis may originate in the normal-appearing white matter (NAWM). Therefore, we performed a genome-wide transcriptional analysis, by Agilent microarray, of post-mortem NAWM of 9 control subjects and 18 MS patients to investigate to what extent gene expression reflects disease heterogeneity and HPA-axis activity. Activity of the HPA axis was determined by cortisol levels in cerebrospinal fluid and by numbers of corticotropin-releasing neurons in the hypothalamus, while duration of MS and time to EDSS6 served as indicator of disease severity. Applying weighted gene co-expression network analysis led to the identification of a range of gene modules with highly similar co-expression patterns that strongly correlated with various indicators of HPA-axis activity and/or severity of MS. Interestingly, molecular profiles associated with relatively mild MS and high HPA-axis activity were characterized by increased expression of genes that actively regulate inflammation and by molecules involved in myelination, anti-oxidative mechanism, and neuroprotection. Additionally, group-wise comparisons of gene expression in white matter from control subjects and NAWM from (subpopulations of) MS patients uncovered disease-associated gene expression as well as strongly up- or downregulated genes in patients with relatively benign MS and/or high HPA-axis activity, with many differentially expressed genes being previously undescribed in the context of MS. Overall, the data suggest that HPA-axis activity strongly impacts on molecular mechanisms in NAWM of MS patients, but partly also independently of disease severity.


Assuntos
Encéfalo/metabolismo , Hidrocortisona/genética , Esclerose Múltipla Crônica Progressiva/genética , Transcriptoma , Substância Branca/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Humanos , Hidrocortisona/metabolismo , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/metabolismo , Índice de Gravidade de Doença
11.
Nat Commun ; 10(1): 1139, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867424

RESUMO

Here we report the transcriptional profile of human microglia, isolated from normal-appearing grey matter (GM) and white matter (WM) of multiple sclerosis (MS) and non-neurological control donors, to find possible early changes related to MS pathology. Microglia show a clear region-specific profile, indicated by higher expression of type-I interferon genes in GM and higher expression of NF-κB pathway genes in WM. Transcriptional changes in MS microglia also differ between GM and WM. MS WM microglia show increased lipid metabolism gene expression, which relates to MS pathology since active MS lesion-derived microglial nuclei show similar altered gene expression. Microglia from MS GM show increased expression of genes associated with glycolysis and iron homeostasis, possibly reflecting microglia reacting to iron depositions. Except for ADGRG1/GPR56, expression of homeostatic genes, such as P2RY12 and TMEM119, is unaltered in normal-appearing MS tissue, demonstrating overall preservation of microglia homeostatic functions in the initiation phase of MS.


Assuntos
Regulação da Expressão Gênica , Redes e Vias Metabólicas/genética , Microglia/patologia , Esclerose Múltipla/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica/métodos , Glicólise/genética , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Humanos , Ferro/metabolismo , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Análise de Sequência de RNA/métodos , Substância Branca/metabolismo , Substância Branca/patologia
12.
Life Sci Alliance ; 2(1): e201800115, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30620009

RESUMO

Chromatin immunoprecipitation (ChIP)-seq analyses of transcription factors in clinical specimens are challenging due to the technical limitations and low quantities of starting material, often resulting in low enrichments and poor signal-to-noise ratio. Here, we present an optimized protocol for transcription factor ChIP-seq analyses in human tissue, yielding an ∼100% success rate for all transcription factors analyzed. As proof of concept and to illustrate general applicability of the approach, human tissue from the breast, prostate, and endometrial cancers were analyzed. In addition to standard formaldehyde fixation, disuccinimidyl glutarate was included in the procedure, greatly increasing data quality. To illustrate the sensitivity of the optimized protocol, we provide high-quality ChIP-seq data for three independent factors (AR, FOXA1, and H3K27ac) from a single core needle prostate cancer biopsy specimen. In summary, double-cross-linking strongly improved transcription factor ChIP-seq quality on human tumor samples, further facilitating and enhancing translational research on limited amounts of tissue.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Fatores de Transcrição/genética , Sequência de Bases/genética , Sítios de Ligação/genética , Biópsia com Agulha de Grande Calibre , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Confiabilidade dos Dados , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Fator 3-alfa Nuclear de Hepatócito/genética , Histonas/genética , Humanos , Células MCF-7 , Masculino , Receptores Androgênicos/genética , Sensibilidade e Especificidade
13.
Nat Commun ; 9(1): 4900, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30464211

RESUMO

The Androgen Receptor (AR) is the key-driving transcription factor in prostate cancer, tightly controlled by epigenetic regulation. To date, most epigenetic profiling has been performed in cell lines or limited tissue samples. Here, to comprehensively study the epigenetic landscape, we perform RNA-seq with ChIP-seq for AR and histone modification marks (H3K27ac, H3K4me3, H3K27me3) in 100 primary prostate carcinomas. Integrative molecular subtyping of the five data streams revealed three major subtypes of which two were clearly TMPRSS2-ERG dictated. Importantly, we identify a third subtype with low chromatin binding and activity of AR, but with high activity of FGF and WNT signaling. While positive for neuroendocrine-hallmark genes, these tumors were copy number-neutral with low mutational burden, significantly depleted for genes characteristic of poor-outcome associated luminal B-subtype. We present a unique resource on transcriptional and epigenetic control in prostate cancer, revealing tight control of gene regulation differentially dictated by AR over three subtypes.


Assuntos
Carcinoma/metabolismo , Epigenômica , Histonas/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Idoso , Epigênese Genética , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Análise de Sequência de RNA
14.
Mol Oncol ; 12(11): 1884-1894, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29972720

RESUMO

Estrogen receptor-alpha (ERα)-positive breast cancer is often treated with antihormonal regimens. However, resistance to treatment is common, leading to metastatic disease. ERα activity requires the functional involvement of pioneer factors FOXA1 and GATA3, which enable ERα-chromatin binding and are crucial for ERα-driven cell proliferation. FOXA1 was found increased in metastatic breast cancers in relation to the primary tumor, but a comprehensive clinical assessment thereof, in relation to different metastatic sites and endocrine therapy usage, is currently lacking. Prior cell line-based reports, however, have revealed that FOXA1 is required for tamoxifen-resistant tumor cell proliferation. We studied expression levels of ERα, GATA3, and FOXA1 by immunohistochemistry in samples from both primary tumors and various metastatic sites. For all factors, expression levels varied between the metastatic sites. For pleural metastases, strong variation was found in FOXA1 and GATA3 levels. Although GATA3 levels remained unaltered between primary breast cancer and pleural metastases, FOXA1 levels were reduced exclusively in metastases of patients who received endocrine therapies in the adjuvant setting, even though ERα was still expressed. Importantly, decreased FOXA1 levels in pleural metastases correlated with hormone irresponsiveness in the palliative setting, while no such correlation was found for GATA3. With this, we show divergent clinical correlations of the two ERα pioneer factors FOXA1 and GATA3 in metastatic breast cancer, where endocrine therapy resistance was associated with decreased FOXA1 levels in pleural metastases.


Assuntos
Neoplasias da Mama , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/mortalidade , Neoplasias Pleurais/patologia , Neoplasias Pleurais/secundário , Taxa de Sobrevida
15.
Nat Commun ; 9(1): 482, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29396493

RESUMO

Male breast cancer (MBC) is rare and poorly characterized. Like the female counterpart, most MBCs are hormonally driven, but relapse after hormonal treatment is also noted. The pan-hormonal action of steroid hormonal receptors, including estrogen receptor alpha (ERα), androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) in this understudied tumor type remains wholly unexamined. This study reveals genomic cross-talk of steroid hormone receptor action and interplay in human tumors, here in the context of MBC, in relation to the female disease and patient outcome. Here we report the characterization of human breast tumors of both genders for cistromic make-up of hormonal regulation in human tumors, revealing genome-wide chromatin binding landscapes of ERα, AR, PR, GR, FOXA1, and GATA3 and enhancer-enriched histone mark H3K4me1. We integrate these data with transcriptomics to reveal gender-selective and genomic location-specific hormone receptor actions, which associate with survival in MBC patients.


Assuntos
Neoplasias da Mama Masculina/metabolismo , Neoplasias da Mama/metabolismo , Cromatina/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Fator de Transcrição GATA3/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Sobrevida
16.
Oncotarget ; 8(33): 55550-55561, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28903441

RESUMO

Discordance in estrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2) status between primary breast cancers and solid distant metastases ("conversion") has been reported previously. Even though metastatic spread to the peritoneal and pleural cavities occurs frequently and is associated with high mortality, the rate of receptor conversion and the prognostic implications thereof remain elusive. We therefore determined receptor conversion in 91 effusion metastases (78 pleural, 13 peritoneal effusions) of 69 patients by immunohistochemistry (IHC) and in situ hybridization. Data were coupled to clinical variables and treatment history. ERα, PR and AR receptor status converted from positive in the primary tumor to negative in the effusion metastases or vice versa in 25-30%, 30-35% and 46-51% of cases for the 1% and 10% thresholds for positivity, respectively. 19-25% of patients converted clinically relevant from "ERα+ or PR+" to ERα-/PR- and 3-4% from ERα-/PR- to "ERα+ or PR+". For HER2, conversion was observed in 6% of cases. Importantly, receptor conversion for ERα (p = 0.058) and AR (p < 0.001) was more often seen in patients adjuvantly treated with endocrine therapy. Analogous to this observation, HER2-loss was more frequent in patients adjuvantly treated with trastuzumab (p < 0.001). Alike solid distant metastases, receptor conversion for ERα, PR, AR and HER2 is a frequent phenomenon in peritoneal and pleural effusion metastases. Adjuvant endocrine and trastuzumab therapy imposes an evolutionary selection pressure on the tumor, leading to receptor loss in effusion metastases. Determination of receptor status in malignant effusion specimens will facilitate endocrine treatment decision-making at this lethal state of the disease, and is hence recommended whenever possible.

17.
J Neuroimmunol ; 309: 12-22, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28601280

RESUMO

HLA-DR, Iba1 and CD68 are widely used microglia markers in human tissue. However, due to differences in gene regulation, they may identify different activation stages of microglia. Here, we directly compared the expression of HLA-DR, Iba1 and CD68 in microglia with different phenotypes, ranging from ramified to amoeboid, to foamy phagocytizing macrophages, in adjacent sections immunocytochemically double stained for two of the markers. Material was used from patients diagnosed with multiple sclerosis (MS) and Alzheimer's disease (AD) patients and control subjects because together they contain all the microglia activation stages in an acute and a chronic inflammatory setting. We found a similar, yet not identical, overall expression pattern. All three markers were expressed by ramified/amoeboid microglia around chronic active MS lesions, but overlap between HLA-DR and Iba1 was limited. Foamy macrophages in the demyelinating rims of active MS lesions of MS expressed more HLA-DR and CD68 than Iba1. All markers were expressed by small microglia accumulations (nodules) in MS NAWM. Dense core AD plaques in the hippocampus were mostly associated with microglia expressing HLA-DR. Diffuse AD plaques were not specifically associated with microglia at all. These results indicate that microglia markers have different potential for neuropathological analysis, with HLA-DR and CD68 reflecting immune activation and response to tissue damage, and Iba1 providing a marker more suited for structural studies in the absence of pathology.


Assuntos
Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Proteínas de Ligação a DNA/biossíntese , Antígenos HLA-DR/biossíntese , Microglia/metabolismo , Microglia/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA/análise , Feminino , Expressão Gênica , Antígenos HLA-DR/análise , Humanos , Masculino , Proteínas dos Microfilamentos , Microglia/química , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Coloração e Rotulagem/métodos
18.
Cell Chem Biol ; 24(6): 725-736.e7, 2017 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-28552582

RESUMO

Drugs that increase 26S proteasome activity have potential therapeutic applications in the treatment of neurodegenerative diseases. A chemical genetics screen of over 2,750 compounds using a proteasome activity probe as a readout in a high-throughput live-cell fluorescence-activated cell sorting-based assay revealed more than ten compounds that increase proteasome activity, with the p38 MAPK inhibitor PD169316 being one of the most potent ones. Genetic and chemical inhibition of either p38 MAPK, its upstream regulators, ASK1 and MKK6, and downstream target, MK2, enhance proteasome activity. Chemical activation of the 26S proteasome increases PROTAC-mediated and ubiquitin-dependent protein degradation and decreases the levels of both overexpressed and endogenous α-synuclein, without affecting the overall protein turnover. In addition, survival of cells overexpressing toxic α-synuclein assemblies is increased in the presence of p38 MAPK inhibitors. These findings highlight the potential of activation of 26S proteasome activity and that this can be achieved through multiple mechanisms by distinct molecules.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
19.
Acta Neuropathol Commun ; 5(1): 16, 2017 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-28212663

RESUMO

Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor.


Assuntos
Encéfalo , Separação Celular , Microglia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Encéfalo/patologia , Antígeno CD11b/metabolismo , Separação Celular/métodos , Células Cultivadas , Líquido Cefalorraquidiano/química , Criopreservação , Feminino , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Antígenos Comuns de Leucócito/metabolismo , Masculino , Microglia/metabolismo , Microglia/patologia , Fatores de Tempo , Bancos de Tecidos , Doadores de Tecidos
20.
Front Immunol ; 8: 1810, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29312322

RESUMO

In multiple sclerosis (MS), activated microglia and infiltrating macrophages phagocytose myelin focally in (chronic) active lesions. These demyelinating sites expand in time, but at some point turn inactive into a sclerotic scar. To identify molecular mechanisms underlying lesion activity and halt, we analyzed genome-wide gene expression in rim and peri-lesional regions of chronic active and inactive MS lesions, as well as in control tissue. Gene clustering revealed patterns of gene expression specifically associated with MS and with the presumed, subsequent stages of lesion development. Next to genes involved in immune functions, we found regulation of novel genes in and around the rim of chronic active lesions, such as NPY, KANK4, NCAN, TKTL1, and ANO4. Of note, the presence of many foamy macrophages in active rims was accompanied by a congruent upregulation of genes related to lipid binding, such as MSR1, CD68, CXCL16, and OLR1, and lipid uptake, such as CHIT1, GPNMB, and CCL18. Except CCL18, these genes were already upregulated in regions around active MS lesions, showing that such lesions are indeed expanding. In vitro downregulation of the scavenger receptors MSR1 and CXCL16 reduced myelin uptake. In conclusion, this study provides the gene expression profile of different aspects of MS pathology and indicates that early demyelination, mediated by scavenger receptors, is already present in regions around active MS lesions. Genes involved in early demyelination events in regions surrounding chronic active MS lesions might be promising therapeutic targets to stop lesion expansion.

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