RESUMEN
Immune checkpoint inhibitors, which unleash a patient's own T cells to kill tumors, are revolutionizing cancer treatment. Several independent studies suggest that higher non-synonymous mutational burden assessed by whole exome sequencing (WES) in tumors is associated with improved objective response, durable clinical benefit, and progression-free survival in immune checkpoint inhibitors treatment. Next-generation sequencing (NGS) is a promising technology being used in the clinic to direct patient treatment. Cancer genome WES poses a unique challenge due to tumor heterogeneity and sequencing artifacts introduced by formalin-fixed, paraffin-embedded (FFPE) tissue. In order to evaluate the data interoperability of WES data from different sources to survey tumor mutational landscape, we compared WES data of several tumor/normal matched samples from five commercial vendors. A large data discrepancy was observed from vendors' self-reported data. Independent data analysis from vendors' raw NGS data shows that whole exome sequencing data from qualified vendors can be combined and analyzed uniformly to derive comparable quantitative estimates of tumor mutational burden.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias/genética , Línea Celular Tumoral , Supervivencia sin Enfermedad , Humanos , Laboratorios , Mutación , Neoplasias/mortalidad , Neoplasias/patología , Adhesión en Parafina , Análisis de Secuencia de ADNRESUMEN
Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.
Asunto(s)
Cartílago Articular/metabolismo , Dinoprostona/antagonistas & inhibidores , Receptores Nucleares Huérfanos/agonistas , Osteoartritis/complicaciones , Dolor/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAMTS4 , Animales , Benzoatos/farmacología , Bencilaminas/farmacología , Línea Celular Tumoral , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Ligandos , Receptores X del Hígado , Ratones , Ratones Mutantes , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/fisiología , Osteoartritis/metabolismo , Dolor/etiología , Procolágeno N-Endopeptidasa/antagonistas & inhibidores , Prostaglandina-E Sintasas , RatasRESUMEN
Liver X receptors (LXRs) play a critical role in regulating lipid synthesis and transport in numerous tissues. In the skin, activation of LXR induces keratinocyte differentiation and improves epidermal permeability barrier homeostasis. To elucidate the mechanism of LXR action in skin, we mapped its cistrome by identifying LXRß-RXRα binding sites using ChIP-on-chip in normal human epidermal keratinocytes (NHEKs). The cistrome was integrated with transcription data to obtain a global view of LXR action in keratinocyte biology. Here, we identify 2035 LXRß-RXRα binding sites containing 4794 LXR response elements in NHEKs and show the presence of consensus heterodimer active regions in genes involved in keratinocyte lipid transport/synthesis and terminal differentiation. Bioinformatics analysis of the cistrome revealed an enrichment of AP1 cis-regulatory motifs in the vicinity of the LXRß-RXRα binding sites. Importantly, we have demonstrated a direct interaction between LXR and Jun/Fos, indicating that the cooperation between LXR and AP1 may orchestrate keratinocyte differentiation. Finally, we corroborated these results by genome-wide mapping of the c-Fos and c-Jun cistromes in NHEKs, demonstrating that 77% of all the LXRß-RXRα binding regions show the presence of AP1 motifs at adjacent locations. Our findings provide new insight into the mechanism of LXR action in keratinocyte differentiation, lipid production and barrier formation, further strengthening the validation of LXR as a potential therapeutic target for skin disorders including skin aging, psoriasis, and atopic dermatitis.
Asunto(s)
Receptores Nucleares Huérfanos/química , Receptores X Retinoide/química , Factor de Transcripción AP-1/química , Animales , Sitios de Unión , Diferenciación Celular , Dimerización , Regulación de la Expresión Génica , Genoma , Humanos , Queratinocitos/citología , Receptores X del Hígado , Ratones , Ratones Noqueados , Transducción de Señal , Piel/metabolismoRESUMEN
One of the many harmful factors faced by the skin is solar UV radiation, which damages skin by inducing chronic low-grade inflammation through increased expression of proinflammatory cytokines, metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). Estrogen receptors (ERs) alpha and beta are ligand-dependent transcription factors that are expressed in skin, and an ERbeta agonist has previously shown efficacy in vivo in models of pain and inflammation. Because ERbeta does not carry the breast and uterine proliferation liabilities of ERalpha, we decided to explore the possibility of using ERbeta as a target for photoaging. We show that ERbeta-selective compounds suppressed the expression of cytokines and MMPs in activated keratinocytes and fibroblast-based in vitro models of photoaging. Furthermore, in activated dermal fibroblasts, ERbeta-selective compounds also inhibited COX-2. These activities of ERbeta ligands in skin cells correlated with the expression levels of ERbeta and showed reversal by treatment with a potent synthetic ER antagonist. Furthermore, the pharmacology of ERbeta-selective compound was observed in wild-type but not in skin cells obtained from ERbeta knockout mice. Finally, we demonstrate that a synthetic ERbeta agonist inhibited UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the potential of an ERbeta ligand to regulate multiple pathways underlying the cause of photoaging suggests ERbeta to be a novel therapeutic target for the prevention and treatment of photoaging.
Asunto(s)
Receptor beta de Estrógeno/fisiología , Envejecimiento/efectos de la radiación , Animales , Citocinas/genética , Receptor beta de Estrógeno/deficiencia , Receptor beta de Estrógeno/efectos de los fármacos , Receptor beta de Estrógeno/genética , Femenino , Fibroblastos/fisiología , Humanos , Ligandos , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Pelados , Ratones Noqueados , Envejecimiento de la Piel/genética , Envejecimiento de la Piel/fisiología , Luz Solar/efectos adversos , Rayos Ultravioleta/efectos adversosRESUMEN
The use of estrogen-based therapies and the selective estrogen receptor (ER) modulator (SERM), raloxifene, which are approved for postmenopausal osteoporosis, is associated with side effects such as uterine/breast hyperproliferation, thromboembolism, and hot flashes. A combination of a new SERM, bazedoxifene (BZA), and Premarin (conjugated estrogens; CE) is under investigation to mitigate the estrogen/SERM side effects with promising results in Phase III clinical trials. To explore the mechanism of BZA/CE action, we investigated the recruitment of cofactor peptides to ERalpha by components of CE and a mixture containing the 10 major components of CE with or without three different SERMs. Here, we demonstrate differential recruitment of cofactor peptides to ERalpha by the individual CE components using a multiplex nuclear receptor-cofactor peptide interaction assay. We show that estrone and equilin are partial agonists in comparison with 17beta-estradiol in recruiting cofactor peptides to ERalpha. Further, CE was more potent than 17beta-estradiol in mediating ERalpha interaction with cofactor peptides. Interestingly, BZA was less potent than other SERMs in antagonizing the CE-mediated cofactor peptide recruitment to ERalpha. Finally, in accordance with these biochemical findings, 17beta-estradiol and CE, as well as SERM/CE combinations, showed differential gene regulation patterns in MCF-7 cells. In addition, BZA showed antagonism of a unique set of CE-regulated genes and did not down-regulate the expression of a number of CE-regulated genes, the expression of which was effectively antagonized by the other two SERMs. These results indicate that SERMs in combination with CE exhibit differential pharmacology, and therefore, combinations of other SERMs and estrogen preparations may not yield the same beneficial effects that are observed in clinic by pairing BZA with CE.
Asunto(s)
Estrógenos Conjugados (USP)/farmacología , Indoles/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Conservadores de la Densidad Ósea/administración & dosificación , Conservadores de la Densidad Ósea/efectos adversos , Conservadores de la Densidad Ósea/farmacología , Línea Celular , Interacciones Farmacológicas , Quimioterapia Combinada , Estradiol/administración & dosificación , Estradiol/efectos adversos , Estradiol/farmacología , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Estrógenos Conjugados (USP)/administración & dosificación , Estrógenos Conjugados (USP)/efectos adversos , Femenino , Perfilación de la Expresión Génica , Humanos , Indoles/administración & dosificación , Indoles/efectos adversos , Ligandos , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Estructura Terciaria de Proteína , Moduladores Selectivos de los Receptores de Estrógeno/administración & dosificación , Moduladores Selectivos de los Receptores de Estrógeno/efectos adversos , Activación Transcripcional/efectos de los fármacosRESUMEN
Liver X receptors (LXRalpha and -beta) are liposensors that exert their metabolic effects by orchestrating the expression of macrophage genes involved in lipid metabolism and inflammation. LXRs are also expressed in other tissues, including skin, where their natural oxysterol ligands induce keratinocyte differentiation and improve epidermal barrier function. To extend the potential use of LXR ligands to dermatological indications, we explored the possibility of using LXR as a target for skin aging. We demonstrate that LXR signaling is down-regulated in cell-based models of photoaging, i.e. UV-activated keratinocytes and TNFalpha-activated dermal fibroblasts. We show that a synthetic LXR ligand inhibits the expression of cytokines and metalloproteinases in these in vitro models, thus indicating its potential in decreasing cutaneous inflammation associated with the etiology of photoaging. Furthermore, a synthetic LXR ligand induces the expression of differentiation markers, ceramide biosynthesis enzymes, and lipid synthesis and transport genes in keratinocytes. Remarkably, LXRbeta-null mouse skin showed some of the molecular defects that are observed in chronologically aged human skin. Finally, we demonstrate that a synthetic LXR agonist inhibits UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the ability of an LXR ligand to modulate multiple pathways underlying the etiology of skin aging suggests that LXR is a novel target for developing potential therapeutics for photoaging and chronological skin aging indications.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Envejecimiento de la Piel/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Humanos , Técnicas In Vitro , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/efectos de la radiación , Ligandos , Metabolismo de los Lípidos/genética , Receptores X del Hígado , Ratones , Ratones Pelados , Ratones Noqueados , Modelos Biológicos , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Envejecimiento de la Piel/patologíaRESUMEN
Estrogens generally stimulate the proliferation of estrogen receptor (ER)-containing breast cancer cells, but they also suppress proliferation of some ER-positive breast tumors. Using a genome-wide analysis of gene expression in two ER-positive human breast cancer cell lines that differ in their proliferative response to estrogen, we sought to identify genes involved in estrogen-regulated cell proliferation. To this end, we compared the transcriptional profiles of MCF-7 and MDA-MB-231ER+ cells, which have directionally opposite 17beta-estradiol (E2)-dependent proliferation patterns, MCF-7 cells being stimulated and 231ER+ cells suppressed by E2. We identified a set of approximately 70 genes regulated by E2 in both cells, with most being regulated by hormone in an opposite fashion. Using a variety of bioinformatics approaches, we found the E2F binding site to be overrepresented in the potential regulatory regions of many cell cycle-related genes stimulated by estrogen in MCF-7 but inhibited by estrogen in 231ER+ cells. Biochemical analyses confirmed that E2F1 and E2F downstream target genes were increased in MCF-7 and decreased in 231ER+ cells upon estrogen treatment. Furthermore, RNA interference-mediated knockdown of E2F1 blocked estrogen regulation of E2F1 target genes and resulted in loss of estrogen regulation of proliferation. These results demonstrate that regulation by estrogen of E2F1, and subsequently its downstream target genes, is critical for hormone regulation of the proliferative program of these breast cancer cells, and that gene expression profiling combined with bioinformatic analyses of transcription factor binding site enrichment in regulated genes can identify key components associated with nuclear receptor hormonal regulation of important cellular functions.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Factor de Transcripción E2F1/fisiología , Estrógenos/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Factor de Transcripción E2F1/genética , Femenino , Humanos , Receptores de Estrógenos/metabolismoRESUMEN
Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with quantitative PCR verification in many cases, to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. Of the >12,000 genes queried, over 400 showed a robust pattern of regulation, and, notably, the majority (70%) were down-regulated. We observed a general up-regulation of positive proliferation regulators, including survivin, multiple growth factors, genes involved in cell cycle progression, and regulatory factor-receptor loops, and the down-regulation of transcriptional repressors, such as Mad4 and JunB, and of antiproliferative and proapoptotic genes, including B cell translocation gene-1 and -2, cyclin G2, BCL-2 antagonist/killer 1, BCL 2-interacting killer, caspase 9, and TGFbeta family growth inhibitory factors. These together likely contribute to the stimulation of proliferation and the suppression of apoptosis by E2 in these cells. Of interest, E2 appeared to modulate its own activity through the enhanced expression of genes involved in prostaglandin E production and signaling, which could lead to an increase in aromatase expression and E2 production, as well as the decreased expression of several nuclear receptor coactivators that could impact ER activity. Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which this hormone operates to achieve its widespread effects on breast cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Estradiol/farmacología , Perfilación de la Expresión Génica , Expresión Génica/efectos de los fármacos , División Celular/genética , Supervivencia Celular/genética , Citocinas/genética , Regulación hacia Abajo , Femenino , Sustancias de Crecimiento/genética , Hormonas/genética , Humanos , Receptores de Superficie Celular/genética , Transducción de Señal/genética , Factores de Tiempo , Factores de Transcripción/genética , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.
Asunto(s)
Ensayos Clínicos como Asunto , Sistema de Señalización de MAP Quinasas/genética , Línea Celular , Cartilla de ADN , Genes ras , Humanos , Límite de Detección , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas B-raf/genética , Reproducibilidad de los ResultadosRESUMEN
Bazedoxifene (BZA), a new selective estrogen receptor modulator (SERM) was recently approved in Europe for the prevention and treatment of postmenopausal osteoporosis. Combination therapy using BZA and conjugated estrogens (CE) is currently in late stage development representing a new paradigm for the treatment of menopausal symptoms and prevention of osteoporosis. A GeneChip microarray study was designed to compare gene expression profiles of BZA to that of other SERMs, raloxifene (RAL) and lasofoxifene (LAS). In addition, we compared the gene expression profiles of the three SERMs in combination with CE, a mixture of 10 most abundant estrogens present in Premarin. According to the hierarchical clustering heat map analysis, gene clusters that specifically responded to CE treatments or SERM treatments were identified and gene lists sorted based on a set of cutoff filters. A group of genes differentially regulated by CE were also identified to be antagonized by BZA when comparing CE with the BZA+CE treatment. All three SERMs showed significant antagonistic effect against CE-stimulated cell proliferation, based on the MCF-7 cell proliferation assay and GeneChip data, with the order of antagonist activity being BZA>RAL>LAS. These results indicate that SERMs in combination with CE exhibit differential pharmacology, and therefore, combinations of other SERMs and estrogen preparations may not yield the same effects that are observed in clinic by pairing BZA with CE.
Asunto(s)
Neoplasias de la Mama/metabolismo , Estrógenos Conjugados (USP)/antagonistas & inhibidores , Estrógenos Conjugados (USP)/farmacología , Perfilación de la Expresión Génica , Indoles/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Regulación hacia Abajo/genética , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pirrolidinas/farmacología , Clorhidrato de Raloxifeno/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tetrahidronaftalenos/farmacología , Regulación hacia Arriba/genéticaRESUMEN
Nuclear receptor estrogen receptor alpha (ER alpha) controls the expression of hundreds of genes responsible for target cell phenotypic properties, but the relative importance of direct versus tethering mechanisms of DNA binding has not been established. In this first report, we examine the genome-wide chromatin localization of an altered-specificity mutant ER with a DNA binding domain deficient in binding to estrogen response element (ERE)-containing DNA (DBDmut ER) versus wild-type ER alpha. Using high-throughput sequencing of ER chromatin immunoprecipitations (ChIP-Seq) and mRNA transcriptional profiling, we show that direct ERE binding is required for most of (75%) estrogen-dependent gene regulation and 90% of hormone-dependent recruitment of ER to genomic binding sites. De novo motif analysis of the chromatin binding regions in MDA-MB-231 human breast cancer cells defined unique transcription factor profiles responsible for genes regulated through tethering versus direct ERE binding, with Runx motifs enriched in ER-tethered sites. We confirmed a role for Runx1 in mediating ER alpha genomic recruitment and regulation of tethering genes. Our findings delineate the contributions of direct receptor ERE binding versus binding through response elements for other transcription factors in chromatin localization and ER-dependent gene regulation, paradigms likely to underlie the gene regulatory actions of other nuclear receptors as well.