RESUMO
Strong connections exist between R-loops (three-stranded structures harbouring an RNA:DNA hybrid and a displaced single-strand DNA), genome instability and human disease1-5. Indeed, R-loops are favoured in relevant genomic regions as regulators of certain physiological processes through which homeostasis is typically maintained. For example, transcription termination pause sites regulated by R-loops can induce the synthesis of antisense transcripts that enable the formation of local, RNA interference (RNAi)-driven heterochromation6. Pause sites are also protected against endogenous single-stranded DNA breaks by BRCA17. Hypotheses about how DNA repair is enacted at pause sites include a role for RNA, which is emerging as a normal, albeit unexplained, regulator of genome integrity8. Here we report that a species of single-stranded, DNA-damage-associated small RNA (sdRNA) is generated by a BRCA1-RNAi protein complex. sdRNAs promote DNA repair driven by the PALB2-RAD52 complex at transcriptional termination pause sites that form R-loops and are rich in single-stranded DNA breaks. sdRNA repair operates in both quiescent (G0) and proliferating cells. Thus, sdRNA repair can occur in intact tissue and/or stem cells, and may contribute to tumour suppression mediated by BRCA1.
Assuntos
Proteína BRCA1/metabolismo , Reparo do DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Interferência de RNA , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas Argonautas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Fatores de Iniciação em Eucariotos/metabolismo , Células HeLa , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fase de Repouso do Ciclo Celular , Ribonuclease III/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/fisiologia , Proteína FUS de Ligação a RNA/fisiologia , Linhagem Celular , Humanos , Neurônios Motores/metabolismo , Transporte Proteico , Transcrição GênicaRESUMO
UNLABELLED: An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy-number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted by activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRAS(G12C)-addicted cell line after KRAS downregulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action. SIGNIFICANCE: MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45-58. ©2015 AACR.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Citosina/análogos & derivados , Amplificação de Genes , Proteína 2 de Ligação a Metil-CpG/genética , Neoplasias/genética , Proteínas ras/genética , 5-Metilcitosina/análogos & derivados , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Citosina/metabolismo , Epigênese Genética , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Transplante de Neoplasias , Isoformas de Proteínas/metabolismo , Transdução de SinaisRESUMO
Natural isoflavones have demonstrated numerous pharmacological activities in breast cancer cells, including antiproliferative activities and binding affinities for estrogen receptors (ERs). Chemical modifications on the isoflavone ring system have been prepared and explored for the development of new therapeutics for hormone-dependent breast cancer. The antiproliferative actions of the synthesized isoflavones on MCF-7 and MDA-MB-231 breast cancer cells were examined, as well as cytotoxicity, interaction with estrogen receptors, and proapoptotic activity. The compounds were screened in the absence and in the presence of estradiol to evaluate whether or not estradiol could rescue cell proliferation on MCF-7 cells. Several compounds were able to inhibit cell proliferation in a dose-dependent manner, and compounds containing the bulky 7-phenylmethoxy substituent resulted in cell toxicity not only in MCF-7 cells but also in MDA-MB-231 cells. Selected synthetic isoflavones were able to bind to estrogen receptor with low affinity. Apoptotic pathways were also activated by these compounds in breast cancer cells. The majority of the compounds can bind to both ERs with low affinity, and their effects on hormone-independent breast cancer cells suggest that their ability to inhibit cell growth in breast cancer cells is not exclusively mediated by ERs. Thus, the synthetic trisubstituted isoflavones act on multiple signaling pathways leading to activation of mechanisms of cell-death and ultimately affecting breast cancer cell survival.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Isoflavonas/farmacologia , Receptores de Estrogênio/metabolismo , Antineoplásicos/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Congêneres do Estradiol/farmacologia , Humanos , Isoflavonas/síntese química , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Previous studies have demonstrated that cyclooxygenase-2 (COX-2) inhibitor NS-398 decrease aromatase activity at the transcript level in breast cancer cells. However, N-Methyl NS-398, which does not have COX-2 inhibitory activity but has very similar structure to NS-398, decreases aromatase activity and transcription in MCF-7 and MDA-MB-231 breast cells to the same extent as NS-398. This suggests that NS-398 decrease aromatase expression in breast cancer cells via other mechanism(s). Further investigations find that both compounds only decrease aromatase activity stimulated by forskolin/phorbol ester at the transcript level in both breast cancer cell lines and in breast stromal cells from patients. They do not affect aromatase expression and activity stimulated by dexamethasone. Both compounds also suppress MCF-7 cell proliferation stimulated by testosterone. Aromatase inhibition studies using placental microsomes demonstrate that the compounds show only weak direct aromatase inhibition. These results suggest that NS-398 and its N-methyl analog suppress aromatase expression and activity with multiple mechanisms.
Assuntos
Aromatase/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Nitrobenzenos/farmacologia , Sulfonamidas/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colforsina/farmacologia , Inibidores de Ciclo-Oxigenase 2/química , Dexametasona/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Estrutura Molecular , Nitrobenzenos/química , Forbóis/farmacologia , Sulfonamidas/química , Testosterona/farmacologiaRESUMO
Aromatase converts androgens to estrogens and is a particularly attractive target in the treatment of estrogen receptor positive breast cancer. The enzyme is encoded by the CYP19 gene, which is expressed in a tissue-specific manner. Prostaglandin E2 (PGE2), the major product of cyclooxygenase-2 (COX-2), stimulates aromatase gene expression via protein kinase A and C signaling pathways. Our previous study demonstrated that COX-2 selective inhibitor nimesulide decreased aromatase activity from the transcriptional level in breast cancer cells. In this manuscript, the synthesis and biological evaluation of a series of nimesulide analogues as potential selective aromatase expression regulators are described. Several novel sulfonanilide compounds demonstrate IC50 values from 0.33 to 2.68 microM in suppressing aromatase enzyme activity in SK-BR-3 breast cancer cells and are 10- to 80-fold more active than nimesulide. Also, the sulfonanilide compounds selectively decrease aromatase gene expression in breast cancer cells, without exhibiting cytotoxic or apoptotic effects at low micromole concentrations.
Assuntos
Antineoplásicos/síntese química , Inibidores da Aromatase/síntese química , Aromatase/biossíntese , Sulfonamidas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Aromatase/genética , Inibidores da Aromatase/química , Inibidores da Aromatase/farmacologia , Neoplasias da Mama , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Natural product drug discovery efforts frequently utilize noncellular screening assays. Fatty acids are commonly found in natural product extracts, and some have been shown to interfere with noncellular assays. Several pure fatty acids were tested using a noncellular aromatase assay, with the unsaturated analogues showing strong inhibitory activity, while the saturated analogues were inactive. Unsaturated fatty acids were further tested against SK-BR-3 hormone-independent human breast cancer cells that overexpress aromatase and were found to be inactive. In natural product screening efforts, especially using plant seeds, it is recommended that extracts active in noncellular bioassays should be dereplicated for the presence of fatty acids prior to bioassay-guided fractionation.
Assuntos
Aromatase/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Neoplasias da Mama , Feminino , Humanos , Microssomos/enzimologia , Placenta/enzimologia , Células Tumorais CultivadasRESUMO
Aromatase is a particularly attractive target in the treatment of estrogen receptor positive breast cancer. Aromatase levels in breast cancer cells are enhanced by prostaglandins and reduced by COX inhibitors. The synthesis and biological evaluation of a novel series of sulfonanilide analogues derived from the COX-2 selective inhibitor NS-398 are described. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose- and time-dependent manner. The effect of these compounds on COX-2 inhibition is investigated in breast cancer cells as well. Structure-activity analysis does not find a correlation between aromatase suppression and COX-2 inhibition. Microsomal aromatase inhibition studies rule out the possibility of direct enzyme inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogues decrease aromatase gene transcription in SK-BR-3 cells. These studies suggest that the novel sulfonanilide compounds suppress aromatase activity and transcription in SK-BR-3 breast cancer cells independent of COX-2 inhibition.