RESUMO
The mechanisms driving late relapse in uveal melanoma (UM) patients remains a medical mystery and major challenge. Clinically it is inferred that UM disseminated cancer cells (DCCs) persist asymptomatic for years-to-decades mainly in the liver before they manifest as symptomatic metastasis. Here we reveal using Gαq/11 mut /BAP wt human uveal melanoma models and human UM metastatic samples, that the neural crest lineage commitment nuclear receptor NR2F1 is a key regulator of spontaneous UM DCC dormancy in the liver. Using a quiescence reporter, RNA-seq and multiplex imaging we revealed that rare dormant UM DCCs upregulate NR2F1 expression and genes related to neural crest programs while repressing gene related to cell cycle progression. Gain and loss of function assays showed that NR2F1 silences YAP1/TEAD1 transcription downstream of Gαq/11 signaling and that NR2F1 expression can also be repressed by YAP1. YAP1 expression is repressed by NR2F1 binding to its promoter and changing the histone H3 tail activation marks to repress YAP1 transcription. In vivo CRISPR KO of NR2F1 led dormant UM DCCs to awaken and initiate relentless liver metastatic growth. Cut&Run and bulk RNA sequencing further confirmed that NR2F1 epigenetically stimulates neuron axon guidance and neural lineage programs, and it globally represses gene expression linked to G-protein signaling to drive dormancy. Pharmacological inhibition of Gαq/11 mut signaling resulted in NR2F1 upregulation and robust UM growth arrest, which was also achieved using a novel NR2F1 agonist. Our work sheds light on the molecular underpinnings of UM dormancy revealing that transcriptional programs driven by NR2F1 epigenetically short-circuit Gαq/11 signaling to its downstream target YAP1. Highlights: Quiescent solitary uveal melanoma (UM) DCCs in the liver up- and down-regulate neural crest and cell cycle progression programs, respectively.NR2F1 drives solitary UM DCC dormancy by antagonizing the Gαq/11-YAP1 pathway; small molecule Gαq/11 inhibition restores NR2F1 expression and quiescence. NR2F1 short-circuits oncogenic YAP1 and G-protein signaling via a chromatin remodeling program. Loss of function of NR2F1 in dormant UM DCCs leads to aggressive liver metastasis.
RESUMO
Uveal melanoma (UM) is the most common intraocular tumor in adults, and up to 50% of patients develop metastatic disease, which remains uncurable. Because patients with metastatic UM have an average survival of less than 1 year after diagnosis, there is an urgent need to develop new treatment strategies. Although activating mutations in Gαq or Gα11 proteins are major drivers of pathogenesis, the therapeutic intervention of downstream Gαq/11 targets has been unsuccessful in treating UM, possibly due to alternative signaling pathways and/or resistance mechanisms. Activation of the insulin-like growth factor 1 (IGF1) signaling pathway promotes cell growth, metastasis, and drug resistance in many types of cancers, including UM, where expression of the IGF1 receptor (IGF1R) correlates with a poor prognosis. In this article, we show that direct inhibition of Gαq/11 by the cyclic depsipeptide YM-254890 in combination with inhibition of IGF1R by linsitinib cooperatively inhibits downstream signaling and proliferation of UM cells. We further demonstrate that a 2-week combination treatment of 0.3 to 0.4 mg/kg of YM-254890 administered by intraperitoneal injection and 25 to 40 mg/kg linsitinib administered by oral gavage effectively inhibits the growth of metastatic UM tumors in immunodeficient NOD scid gamma (NSG) mice and identifies the IGF1 pathway as a potential resistance mechanism in response to Gαq/11 inhibition in UM. These data suggest that the combination of Gαq/11 and IGF1R inhibition provides a promising therapeutic strategy to treat metastatic UM.
Assuntos
Melanoma , Neoplasias Uveais , Camundongos , Animais , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Transdução de Sinais , Neoplasias Uveais/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Linhagem Celular TumoralRESUMO
SIN3A, a scaffold protein has regulatory functions in tumor biology. Through its Paired amphipathic helix (PAH2) domain, SIN3A interacts with PHF12 (PF1), a protein with SIN3 interaction domain (SID) that forms a complex with MRG15 and KDM5A/B. These components are often overexpressed in cancer. In the present study, we evaluated the role of SIN3A and its interacting partner PF1 in mediating inhibition of tumor growth and invasion in triple negative breast cancer (TNBC). We found profound inhibition of invasion, migration, and induction of cellular senescence by specific disruption of the PF1/SIN3A PAH2 domain interaction in TNBC cells expressing PF1-SID transcript or peptide treatment. Genome-wide transcriptomic analysis by RNA-seq revealed that PF1-SID downregulates several gene sets and pathways linked to invasion and migration. Integrin α6 (ITGA6) and integrin ß1 (ITGB1) and their downstream target proteins were downregulated in PF1-SID cells. We further determined increased presence of SIN3A and transcriptional repressor, KLF9, on promoters of ITGA6 and ITGB1 in PF1-SID cells. Knockdown of KLF9 leads to re-expression of ITGA6 and ITGB1 and restoration of the invasive phenotype, functionally linking KLF9 to this process. Overall, these data demonstrate that specific disruption of PF1/SIN3A, inhibits tumor growth, migration, and invasion. Also, PF1-SID not only inhibits tumor growth by senescence induction and reduced proliferation, but it also targets cancer stem cell gene expression and blocks mammosphere formation. Overall, these data demonstrate a mechanism whereby invasion and metastasis of TNBC can be suppressed by inhibiting SIN3A-PF1 interaction and enhancing KLF9 mediated suppression of ITGA6 and ITGB1.
RESUMO
Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARß is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RARß has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RARß expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RARß induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RARα and RARß from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARß expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RARα agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RARß promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARß activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARß expression. These data suggest that SIN3A/MAD1 acts as a second RARß repressor and may be involved in fine-tuning retinoid sensitivity.
Assuntos
Neoplasias da Mama , Proteínas de Ciclo Celular , Receptores do Ácido Retinoico , Complexo Correpressor Histona Desacetilase e Sin3 , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina , Feminino , Humanos , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/genéticaRESUMO
The multiprotein SWI-independent 3 (Sin3)-HDAC (histone deacetylase) corepressor complex mediates gene repression through its interaction with DNA-binding factors and recruitment of chromatin-modifying proteins on to the promoters of target gene. Previously, an increased expression of Sin3B and tumour suppressor protein, p53 has been established upon adriamycin treatment. We, now provide evidence that Sin3B expression is significantly up-regulated under variety of stress conditions and this response is not stress-type specific. We observed that Sin3B expression is significantly up-regulated both at transcript and at protein level upon DNA damage induced by bleomycin drug, a radiomimetic agent. This increase in Sin3B expression upon stress is found to be p53-dependent and is associated with enhanced interaction of Sin3B with Ser(15) phosphorylated p53. Binding of Sin3-HDAC repressor complex on to the promoters of p53 target genes influences gene regulation by altering histone modifications (H3K9me3 and H3K27me3) at target genes. Furthermore, knockdown of Sin3B by shRNA severely compromises p53-mediated gene repression under stress conditions. Taken together, these results suggest that stress-induced Sin3B activation is p53-dependent and is essential for p53-mediated repression of its selective target genes. The present study has an implication in understanding the transrepression mechanism of p53 under DNA damaging conditions.
Assuntos
Bleomicina/farmacologia , Dano ao DNA , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Linhagem Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Sin3, a large acidic protein, shares structural similarity with the helix-loop-helix dimerization domain of proteins of the Myc family of transcription factors. Sin3/HDAC corepressor complex functions in transcriptional regulation of several genes and is therefore implicated in the regulation of key biological processes. Knockdown studies have confirmed the role of Sin3 in cellular proliferation, differentiation, apoptosis and cell cycle regulation, emphasizing Sin3 as an essential regulator of critical cellular events in normal and pathological processes. The present review covers the diverse functions of this master transcriptional regulator as well as illustrates the redundant and distinct functions of its two mammalian isoforms.
Assuntos
Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3/genética , Animais , Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Redes e Vias Metabólicas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/química , Complexo Correpressor Histona Desacetilase e Sin3/metabolismoRESUMO
Master regulator protein p53, popularly known as the "guardian of genome" is the hub for regulation of diverse cellular pathways. Depending on the cell type and severity of DNA damage, p53 protein mediates cell cycle arrest or apoptosis, besides activating DNA repair, which is apparently achieved by regulation of its target genes, as well as direct interaction with other proteins. p53 is known to repress target genes via multiple mechanisms one of which is via recruitment of chromatin remodelling Sin3/HDAC1/2 complex. Sin3 proteins (Sin3A and Sin3B) regulate gene expression at the chromatin-level by serving as an anchor onto which the core Sin3/HDAC complex is assembled. The Sin3/HDAC co-repressor complex can be recruited by a large number of DNA-binding transcription factors. Sin3A has been closely linked to p53 while Sin3B is considered to be a close associate of E2Fs. The theme of this study was to establish the role of Sin3B in p53-mediated gene repression. We demonstrate a direct protein-protein interaction between human p53 and Sin3B (hSin3B). Amino acids 1-399 of hSin3B protein are involved in its interaction with N-terminal region (amino acids 1-108) of p53. Genotoxic stress induced by Adriamycin treatment increases the levels of hSin3B that is recruited to the promoters of p53-target genes (HSPA8, MAD1 and CRYZ). More importantly recruitment of hSin3B and repression of the three p53-target promoters upon Adriamycin treatment were observed only in p53(+/+) cell lines. Additionally an increased tri-methylation of the H3K9 residue at the promoters of HSPA8 and CRYZ was also observed following Adriamycin treatment. The present study highlights for the first time the essential role of Sin3B as an important associate of p53 in mediating the cellular responses to stress and in the transcriptional repression of genes encoding for heat shock proteins or proteins involved in regulation of cell cycle and apoptosis.