RESUMEN
Genetic loss-of-function studies on development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by histone H3 trimethylated on K9 (H3K9me3). Loss of macroH2A leads to major defects in nuclear organization, including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by DNA repeat sequences are disorganized, expanded and fragmented, and mildly re-expressed when depleted of macroH2A. At the molecular level, we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein lamin B1. Taken together, our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher-order chromatin architecture.
Asunto(s)
Heterocromatina/metabolismo , Histonas/metabolismo , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , Células HEK293 , Células Hep G2 , Heterocromatina/ultraestructura , Humanos , Lamina Tipo B/metabolismo , Lisina/metabolismo , Masculino , Metilación , Unión ProteicaRESUMEN
UNLABELLED: Peptidyl arginine deiminases (PADI) are a family of enzymes that catalyze the poorly understood posttranslational modification converting arginine residues into citrullines. In this study, the role of PADIs in the pathogenesis of colorectal cancer was investigated. Specifically, RNA expression was analyzed and its association with survival in a cohort of 98 colorectal cancer patient specimens with matched adjacent mucosa and 50 controls from donors without cancer. Key results were validated in an independent collection of tumors with matched adjacent mucosa and by mining of a publicly available expression data set. Protein expression was analyzed by immunoblotting for cell lines or IHC for patient specimens that further included 24 cases of adenocarcinoma with adjacent dysplasia and 11 cases of active ulcerative colitis. The data indicate that PADI2 is the dominantly expressed PADI enzyme in colon mucosa and is upregulated during differentiation. PADI2 expression is low or absent in colorectal cancer. Frequently, this occurs already at the stage of low-grade dysplasia. Mucosal PADI2 expression is also low in ulcerative colitis. The expression level of PADI2 in tumor and adjacent mucosa correlates with differential survival: low levels associate with poor prognosis. IMPLICATIONS: Downregulation of PADI2 is an early event in the pathogenesis of colorectal cancer associated with poor prognosis and points toward a possible role of citrullination in modulating tumor cells and their microenvironment. Mol Cancer Res; 14(9); 841-8. ©2016 AACR.
Asunto(s)
Neoplasias Colorrectales/enzimología , Hidrolasas/biosíntesis , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinogénesis , Estudios de Casos y Controles , Diferenciación Celular/fisiología , Línea Celular Tumoral , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación hacia Abajo , Enterocitos/enzimología , Enterocitos/patología , Células HCT116 , Células HT29 , Humanos , Hidrolasas/genética , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Pronóstico , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina ProteicaRESUMEN
Squamous cell carcinomas have a range of histopathological manifestations. The parameters that determine this clinically observed heterogeneity are not fully understood. Here, we report the generation of a cell culture model that reflects part of this heterogeneity. We have used the catalytic subunit of human telomerase hTERT and large T to immortalize primary UV-unexposed keratinocytes. Then, mutant HRAS G12V has been introduced to transform these immortal keratinocytes. When injected into immunosuppressed mice, transformed cells grew as xenografts with distinct histopathological characteristics. We observed three major tissue architectures: solid, sarcomatoid and cystic growth types, which were primarily composed of pleomorphic and basaloid cells but in some cases displayed focal apocrine differentiation. We demonstrate that the cells generated represent different stages of skin cancerogenesis and as such can be used to identify novel tumor-promoting alterations such as the overexpression of the PADI2 oncogene in solid-type SCC. Importantly, the cultured cells maintain the characteristics from the xenograft they were derived from while being amenable to manipulation and analysis. The availability of cell lines representing different clinical manifestations opens a new tool to study the stochastic and deterministic factors that cause case-to-case heterogeneity despite departing from the same set of oncogenes and the same genetic background.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Mutación , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Expresión Génica , Estudios de Asociación Genética , Xenoinjertos , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , RatonesRESUMEN
Epigenetic regulation is one of the most promising and expanding areas of cancer research. One of the emerging, but least understood aspects of epigenetics is the facultative and locus-specific incorporation of histone variants and their function in chromatin. With the characterization of the first loss of function phenotypes of the macroH2A histone variants, previously unrecognized epigenetic mechanisms have now moved into the spotlight of cancer research. Here, we summarize data supporting different molecular mechanisms that could mediate the primarily tumor suppressive function of macroH2A. We further discuss context-dependent and isoform-specific functions. The aim of this review is to provide guidance for those assessing macroH2A's potential as biomarker or therapeutic intervention point.
Asunto(s)
Histonas/genética , Neoplasias/genética , Epigénesis Genética , Epigenómica , HumanosRESUMEN
One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.
Asunto(s)
Células Madre Adultas/citología , Diferenciación Celular/fisiología , Proliferación Celular , Células Madre Embrionarias/citología , Histonas/fisiología , Células Madre Adultas/fisiología , Animales , Cromatina/fisiología , Cuerpos Embrioides/metabolismo , Cuerpos Embrioides/patología , Células Madre Embrionarias/fisiología , Humanos , Queratinocitos/citología , Queratinocitos/fisiología , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Teratoma/metabolismo , Teratoma/patologíaRESUMEN
V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.
Asunto(s)
Furanos/química , Imidazoles/química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Pirazoles/química , Animales , Sitios de Unión , Simulación por Computador , Femenino , Humanos , Ratones , Mutación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirazoles/síntesis química , Pirazoles/farmacocinética , Relación Estructura-ActividadRESUMEN
BRAF is a serine-threonine-specific protein kinase that is mutated in 2% of human cancers. Oncogenic BRAF is a validated therapeutic target that constitutively activates mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling, driving tumor cell proliferation and survival. Drugs designed to target BRAF have been developed, but it is difficult to prove that they mediate their antitumor effects by inhibiting BRAF rather than by working through off-target effects. We generated drug-resistant versions of oncogenic BRAF by mutating the gatekeeper residue. Signaling by the mutant proteins was resistant to the small-molecule inhibitor sorafenib, but sorafenib still inhibited the growth of tumors driven by the mutant protein. In contrast, both BRAF signaling and tumor growth were resistant to another RAF drug, PLX4720. These data provide unequivocal evidence that sorafenib mediates its antitumor effects in a manner that is independent of its ability to target oncogenic BRAF, whereas PLX4720 inhibits tumor growth by targeting oncogenic BRAF directly.