RESUMO
PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Oncogenic PELP1 is frequently overexpressed in TNBC, and it has been demonstrated that PELP1 signaling is essential for TNBC progression. The therapeutic utility of targeting PELP1 in TNBC, however, remains unknown. In this study, we investigated the effectiveness of SMIP34, a recently developed PELP1 inhibitor for the treatment of TNBC. METHODS: To ascertain the impact of SMIP34 treatment, we used seven different TNBC models for testing cell viability, colony formation, invasion, apoptosis, and cell cycle analysis. Western blotting and RT-qPCR were used to determine the mechanistic insights of SMIP34 action. Using xenograft and PDX tumors, the ability of SMIP34 in suppressing proliferation was examined both ex vivo and in vivo. RESULTS: TNBC cells' viability, colony formation, and invasiveness were all decreased by SMIP34 in in vitro cell-based assays, while apoptosis was increased. SMIP34 treatment promoted the degradation of PELP1 through the proteasome pathway. RT-qPCR analyses confirmed that SMIP34 treatment downregulated PELP1 target genes. Further, SMIP34 treatment substantially downregulated PELP1 mediated extranuclear signaling including ERK, mTOR, S6 and 4EBP1. Mechanistic studies confirmed downregulation of PELP1 mediated ribosomal biogenesis functions including downregulation of cMyc and Rix complex proteins LAS1L, TEX-10, and SENP3. The proliferation of TNBC tumor tissues was decreased in explant experiments by SMIP34. Additionally, SMIP34 treatment markedly decreased tumor progression in both TNBC xenograft and PDX models. CONCLUSIONS: Together, these findings from in vitro, ex vivo, and in vivo models show that SMIP34 may be a useful therapeutic agent for inhibiting PELP1 signaling in TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Correpressoras , Cisteína Endopeptidases/metabolismo , Transdução de Sinais , Fatores de Transcrição , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Endometrial cancer (ECa) is the most common female gynecologic cancer. When comparing the two histological subtypes of endometrial cancer, Type II tumors are biologically more aggressive and have a worse prognosis than Type I tumors. Current treatments for Type II tumors are ineffective, and new targeted therapies are urgently needed. LIFR and its ligand, LIF, have been shown to play a critical role in the progression of multiple solid cancers and therapy resistance. The role of LIF/LIFR in the progression of Type II ECa, on the other hand, is unknown. We investigated the role of LIF/LIFR signaling in Type II ECa and tested the efficacy of EC359, a novel small-molecule LIFR inhibitor, against Type II ECa. The analysis of tumor databases has uncovered a correlation between diminished survival rates and increased expression of leukemia inhibitory factor (LIF), suggesting a potential connection between altered LIF expression and unfavorable overall survival in Type II ECa. The results obtained from cell viability and colony formation assays demonstrated a significant decrease in the growth of Type II ECa LIFR knockdown cells in comparison to vector control cells. Furthermore, in both primary and established Type II ECa cells, pharmacological inhibition of the LIF/LIFR axis with EC359 markedly decreased cell viability, long-term cell survival, and invasion, and promoted apoptosis. Additionally, EC359 treatment reduced the activation of pathways driven by LIF/LIFR, such as AKT, mTOR, and STAT3. Tumor progression was markedly inhibited by EC359 treatment in two different patient-derived xenograft models in vivo and patient-derived organoids ex vivo. Collectively, these results suggest LIFR inhibitor EC359 as a possible new small-molecule therapeutics for the management of Type II ECa.
Assuntos
Neoplasias do Endométrio , Transdução de Sinais , Humanos , Feminino , Receptores de OSM-LIF/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Neoplasias do Endométrio/tratamento farmacológicoRESUMO
BACKGROUND: Methyltransferase SETDB1 is highly expressed in breast cancer (BC), however, the mechanisms by which SETDB1 promotes BC progression to endocrine therapy resistance remains elusive. In this study, we examined the mechanisms by which SETDB1 contribute to BC endocrine therapy resistance. METHODS: We utilized therapy sensitive (MCF7 and ZR75), therapy resistant (MCF7-TamR, MCF7-FR, MCF7-PELP1cyto, MCF7-SETDB1) estrogen receptor alpha positive (ER+)BC models and conducted in vitro cell viability, colony formation, 3-dimensional cell growth assays to investigate the role of SETDB1 in endocrine resistance. RNA-seq of parental and SETDB1 knock down ER+ BC cells was used to identify unique pathways. SETDB1 interaction with PELP1 was identified by yeast-two hybrid screen and confirmed by immunoprecipitation and GST-pull down assays. Mechanistic studies were conducted using Western blotting, reporter gene assays, RT-qPCR, and in vitro methylation assays. Xenograft assays were used to establish the role of PELP1 in SETDB1 mediated BC progression. RESULTS: RNA-seq analyses showed that SETDB1 regulates expression of a subset of estrogen receptor (ER) and Akt target genes that contribute to endocrine therapy resistance. Importantly, using yeast-two hybrid screen, we identified ER coregulator PELP1 as a novel interacting protein of SETDB1. Biochemical analyses confirmed SETDB1 and PELP1 interactions in multiple BC cells. Mechanistic studies confirmed that PELP1 is necessary for SETDB1 mediated Akt methylation and phosphorylation. Further, SETDB1 overexpression promotes tamoxifen resistance in BC cells, and PELP1 knockdown abolished these effects. Using xenograft model, we provided genetic evidence that PELP1 is essential for SETDB1 mediated BC progression in vivo. Analyses of TCGA datasets revealed SETDB1 expression is positively correlated with PELP1 expression in ER+ BC patients. CONCLUSIONS: This study suggests that the PELP1/SETDB1 axis play an important role in aberrant Akt activation and serves as a novel target for treating endocrine therapy resistance in breast cancer.
Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas Correpressoras/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Tamoxifeno/farmacologia , Fatores de Transcrição/genéticaRESUMO
Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-κB (NF-κB), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-κB target genes, NF-κB reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.
Assuntos
Neoplasias Cerebelares/metabolismo , Proteínas Correpressoras/metabolismo , Meduloblastoma/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Correpressoras/análise , Proteínas Correpressoras/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
Methyltranscriptome is an exciting new area that studies the mechanisms and functions of methylation in transcripts. The MethylTranscriptome DataBase (MeT-DB, http://compgenomics.utsa.edu/methylation/) is the first comprehensive resource for N6-methyladenosine (m(6)A) in mammalian transcriptome. It includes a database that records publicaly available data sets from methylated RNA immunoprecipitation sequencing (MeRIP-Seq), a recently developed technology for interrogating m(6)A methyltranscriptome. MeT-DB includes â¼ 300 k m(6)A methylation sites in 74 MeRIP-Seq samples from 22 different experimental conditions predicted by exomePeak and MACS2 algorithms. To explore this rich information, MeT-DB also provides a genome browser to query and visualize context-specific m(6)A methylation under different conditions. MeT-DB also includes the binding site data of microRNA, splicing factor and RNA binding proteins in the browser window for comparison with m(6)A sites and for exploring the potential functions of m(6)A. Analysis of differential m(6)A methylation and the related differential gene expression under two conditions is also available in the browser. A global perspective of the genome-wide distribution of m(6)A methylation in all the data is provided in circular ideograms, which also act as a navigation portal. The query results and the entire data set can be exported to assist publication and additional analysis.
Assuntos
Adenosina/análogos & derivados , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Sítios de Ligação , Humanos , Imunoprecipitação , Metilação , Camundongos , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: The recent advent of the state-of-art high throughput sequencing technology, known as Methylated RNA Immunoprecipitation combined with RNA sequencing (MeRIP-seq) revolutionizes the area of mRNA epigenetics and enables the biologists and biomedical researchers to have a global view of N (6)-Methyladenosine (m(6)A) on transcriptome. Yet there is a significant need for new computation tools for processing and analysing MeRIP-Seq data to gain a further insight into the function and m(6)A mRNA methylation. RESULTS: We developed a novel algorithm and an open source R package ( http://compgenomics.utsa.edu/metcluster ) for uncovering the potential types of m(6)A methylation by clustering the degree of m(6)A methylation peaks in MeRIP-Seq data. This algorithm utilizes a hierarchical graphical model to model the reads account variance and the underlying clusters of the methylation peaks. Rigorous statistical inference is performed to estimate the model parameter and detect the number of clusters. MeTCluster is evaluated on both simulated and real MeRIP-seq datasets and the results demonstrate its high accuracy in characterizing the clusters of methylation peaks. Our algorithm was applied to two different sets of real MeRIP-seq datasets and reveals a novel pattern that methylation peaks with less peak enrichment tend to clustered in the 5' end of both in both mRNAs and lncRNAs, whereas those with higher peak enrichment are more likely to be distributed in CDS and towards the 3'end of mRNAs and lncRNAs. This result might suggest that m(6)A's functions could be location specific. CONCLUSIONS: In this paper, a novel hierarchical graphical model based algorithm was developed for clustering the enrichment of methylation peaks in MeRIP-seq data. MeTCluster is written in R and is publicly available.
Assuntos
Algoritmos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transcriptoma/genética , Análise por Conglomerados , Epigênese Genética/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Imunoprecipitação , Metilação , RNA Mensageiro/genética , SoftwareRESUMO
Genome-wide studies have revealed that genes commonly have a high density of RNA polymerase II just downstream of the transcription start site. This has raised the possibility that genes are commonly regulated by transcriptional elongation, but this remains largely untested in vivo, particularly in vertebrates. Here, we show that the proximal promoter from the Rhox5 homeobox gene recruits polymerase II and begins elongating in all tissues and cell lines that we tested, but it only completes elongation in a tissue-specific and developmentally regulated manner. Relief of the elongation block is associated with recruitment of the elongation factor P-TEFb, the co-activator GRIP1, the chromatin remodeling factor BRG1, and specific histone modifications. We provide evidence that two mechanisms relieve the elongation block at the proximal promoter: demethylation and recruitment of androgen receptor. Together, our findings support a model in which promoter proximal pausing helps confer tissue-specific and developmental gene expression through a mechanism regulated by DNA demethylation-dependent nuclear hormone receptor recruitment.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Especificidade de Órgãos , Testosterona/farmacologia , Elongação da Transcrição Genética/efeitos dos fármacos , Androgênios/farmacologia , Animais , Linhagem Celular , Células HeLa , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Camundongos , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Seminais/crescimento & desenvolvimento , Glândulas Seminais/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Methylated RNA Immunoprecipatation combined with RNA sequencing (MeRIP-seq) is revolutionizing the de novo study of RNA epigenomics at a higher resolution. However, this new technology poses unique bioinformatics problems that call for novel and sophisticated statistical computational solutions, aiming at identifying and characterizing transcriptome-wide methyltranscriptome. RESULTS: We developed HEP, a Hidden Markov Model (HMM)-based Exome Peak-finding algorithm for predicting transcriptome methylation sites using MeRIP-seq data. In contrast to exomePeak, our previously developed MeRIP-seq peak calling algorithm, HEPeak models the correlation between continuous bins in an m6A peak region and it is a model-based approach, which admits rigorous statistical inference. HEPeak was evaluated on a simulated MeRIP-seq dataset and achieved higher sensitivity and specificity than exomePeak. HEPeak was also applied to real MeRIP-seq datasets from human HEK293T cell line and mouse midbrain cells and was shown to be able to recapitulate known m6A distribution in transcripts and identify novel m6A sites in long non-coding RNAs. CONCLUSIONS: In this paper, a novel HMM-based peak calling algorithm, HEPeak, was developed for peak calling for MeRIP-seq data. HEPeak is written in R and is publicly available.
Assuntos
Algoritmos , Epigenômica/métodos , Metilação , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Animais , Células Cultivadas , Exoma , Células HEK293 , Humanos , Cadeias de Markov , Mesencéfalo/citologia , Camundongos , RNA Mensageiro/químicaRESUMO
The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility.
Assuntos
Fertilidade/genética , Proteínas de Homeodomínio/metabolismo , Infertilidade Masculina/metabolismo , Células de Sertoli/metabolismo , Animais , Proteínas de Homeodomínio/genética , Infertilidade Masculina/genética , Masculino , Camundongos , Regiões Promotoras Genéticas , Interferência de RNA , Espermatogênese/genéticaRESUMO
Despite the prevalent studies of DNA/Chromatin related epigenetics, such as, histone modifications and DNA methylation, RNA epigenetics has not drawn deserved attention until a new affinity-based sequencing approach MeRIP-Seq was developed and applied to survey the global mRNA N6-methyladenosine (m(6)A) in mammalian cells. As a marriage of ChIP-Seq and RNA-Seq, MeRIP-Seq has the potential to study the transcriptome-wide distribution of various post-transcriptional RNA modifications. We have previously developed an R/Bioconductor package 'exomePeak' for detecting RNA methylation sites under a specific experimental condition or the identifying the differential RNA methylation sites in a case control study from MeRIP-Seq data. Compared with other relatively well studied data types such as ChIP-Seq and RNA-Seq, the study of MeRIP-Seq data is still at very early stage, and existing protocols are not optimized for dealing with the intrinsic characteristic of MeRIP-Seq data. We therein provide here a detailed and easy-to-use protocol of using exomePeak R/Bioconductor package along with other software programs for analysis of MeRIP-Seq data, which covers raw reads alignment, RNA methylation site detection, motif discovery, differential RNA methylation analysis, and functional analysis. Particularly, the rationales behind each processing step as well as the specific method used, the best practice, and possible alternative strategies are briefly discussed. The exomePeak R/Bioconductor package is freely available from Bioconductor: http://www.bioconductor.org/packages/release/bioc/html/exomePeak.html.
Assuntos
Epigenômica/métodos , RNA Mensageiro/genética , RNA/genética , Análise de Sequência de RNA/métodos , Animais , Sequência de Bases , Metilação de DNA/genética , Humanos , Processamento Pós-Transcricional do RNA/genética , SoftwareRESUMO
Decoupling of transcription and translation during postmeiotic germ cell differentiation is critical for successful spermatogenesis. Here we establish that the interaction between microRNAs and actin-associated protein Arpc5 sets the stage for an elaborate translational control mechanism by facilitating the sequestration of germ cell mRNAs into translationally inert ribonucleoprotein particles until they are later translated. Our studies reveal that loss of microRNA-dependent regulation of Arpc5, which controls the distribution of germ cell mRNAs between translationally active and inactive pools, results in abnormal round spermatid differentiation and impaired fertility. Interestingly, Arpc5 functions as a broadly acting translational suppressor, as it inhibits translation initiation by blocking 80S formation and facilitates the transport of mRNAs to chromatoid/P bodies. These findings identify a unique role for actin-associated proteins in translational regulation, and suggest that mRNA-specific and general translational control mechanisms work in tandem to regulate critical germ cell differentiation events and diverse somatic cell functions.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Diferenciação Celular/genética , MicroRNAs/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patologia , Animais , Sequência de Bases , Cromatina/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Haploidia , Células HeLa , Humanos , Masculino , Meiose/genética , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Protaminas/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução , Ribonuclease III/metabolismo , Ribossomos/metabolismo , Cabeça do Espermatozoide/metabolismo , Cabeça do Espermatozoide/patologia , Cabeça do Espermatozoide/ultraestrutura , Testículo/anormalidades , Testículo/patologia , Testículo/ultraestruturaRESUMO
Defects in cellular metabolism have been widely implicated in causing male infertility, but there has been little progress in understanding the underlying mechanism. Here we report that several key metabolism genes are regulated in the testis by Rhox5, the founding member of a large X-linked homeobox gene cluster. Among these Rhox5-regulated genes are insulin 2 (Ins2), resistin (Retn), and adiponectin (Adipoq), all of which encode secreted proteins that have profound and wide-ranging effects on cellular metabolism. The ability of Rhox5 to regulate their levels in the testis has the potential to dictate metabolism locally in this organ, given the existence of the blood-testes barrier. We demonstrate that Ins2 is a direct target of Rhox5 in Sertoli cells, and we show that this regulation is physiologically significant, because Rhox5-null mice fail to up-regulate Ins2 expression during the first wave of spermatogenesis and have insulin-signaling defects. We identify other Rhox family members that induce Ins2 transcription, define protein domains and homeodomain amino acid residues crucial for this property, and demonstrate that this regulation is conserved. Rhox5-null mice also exhibit altered expression of other metabolism genes, including those encoding the master transcriptional regulators of metabolism, PPARG and PPARGC1A, as well as SCD1, the rate-limiting enzyme for fatty acid metabolism. These results, coupled with the known roles of RHOX5 and its target metabolism genes in spermatogenesis in vivo, lead us to propose a model in which RHOX5 is a central transcription factor that promotes the survival of male germ cells via its effects on cellular metabolism.
Assuntos
Adiponectina/metabolismo , Proteínas de Homeodomínio/genética , Insulina/metabolismo , Resistina/metabolismo , Testículo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Células de Sertoli/metabolismo , Espermatogênese/genética , Estearoil-CoA Dessaturase/metabolismo , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
MOTIVATION: Fragmented RNA immunoprecipitation combined with RNA sequencing enabled the unbiased study of RNA epigenome at a near single-base resolution; however, unique features of this new type of data call for novel computational techniques. RESULT: Through examining the connections of RNA epigenome sequencing data with two well-studied data types, ChIP-Seq and RNA-Seq, we unveiled the salient characteristics of this new data type. The computational strategies were discussed accordingly, and a novel data processing pipeline was proposed that combines several existing tools with a newly developed exome-based approach 'exomePeak' for detecting, representing and visualizing the post-transcriptional RNA modification sites on the transcriptome. AVAILABILITY: The MATLAB package 'exomePeak' and additional details are available at http://compgenomics.utsa.edu/exomePeak/.
Assuntos
Epigênese Genética , Exoma , Processamento Pós-Transcricional do RNA , Análise de Sequência de RNA/métodos , Células HEK293 , Humanos , Imunoprecipitação/métodos , Software , TranscriptomaRESUMO
Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody's inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma.
Assuntos
Trifosfato de Adenosina , Neoplasias Ósseas , Neoplasias da Mama , Conexina 43 , Osteócitos , Osteossarcoma , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Animais , Osteócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Conexina 43/metabolismo , Camundongos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Microambiente Tumoral , Anticorpos/farmacologiaRESUMO
Eukaryotic cells maintain cellular fitness by employing well-coordinated and evolutionarily conserved processes that negotiate stress induced by internal or external environments. These processes include the unfolded protein response, autophagy, endoplasmic reticulum-associated degradation (ERAD) of unfolded proteins and altered mitochondrial functions that together constitute the ER stress response. Here, we show that the RNA demethylase ALKBH5 regulates the crosstalk among these processes to maintain normal ER function. We demonstrate that ALKBH5 regulates ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain the calcium flux between the ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently, mitochondrial biogenesis. Thus, these findings reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness.
Assuntos
Estresse do Retículo Endoplasmático , Degradação Associada com o Retículo Endoplasmático , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Transdução de Sinais , Mitocôndrias/metabolismo , HomeostaseRESUMO
Concordant transcriptional regulation can generate multiple gene products that collaborate to achieve a common goal. Here we report a case of concordant transcriptional regulation that instead drives a single protein to be produced in the same cell type from divergent promoters. This gene product-the RHOX5 homeobox transcription factor-is translated from 2 different mRNAs with different 5' untranslated regions (UTRs) transcribed from alternative promoters. Despite the fact that these 2 promoters-the proximal promoter (Pp) and the distal promoter (Pd)-exhibit different patterns of tissue-specific activity, share no obvious sequence identity, and depend on distinct transcription factors for expression, they exhibit a remarkably similar expression pattern in the testes. In particular, both depend on androgen signaling for expression in the testes, where they are specifically expressed in Sertoli cells and have a similar stage-specific expression pattern during the seminiferous epithelial cycle. We report evidence for 3 mechanisms that collaborate to drive concordant Pp/Pd expression. First, both promoters have an intrinsic ability to respond to androgen receptor and androgen. Second, the Pp acts as an enhancer to promote androgen-dependent transcription from the Pd. Third, Pd transcription is positively autoregulated by the RHOX5 protein, which is first produced developmentally from the Pp. Together, our data support a model in which the Rhox5 homeobox gene evolved multiple mechanisms to activate both of its promoters in Sertoli cells to produce Rhox5 in an androgen-dependent manner during different phases of spermatogenesis.
Assuntos
Androgênios/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Células de Sertoli/metabolismo , Fatores de Transcrição/genética , Regiões 5' não Traduzidas , Animais , Metilação de DNA , Genes Homeobox , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/metabolismo , Isoformas de Proteínas , Receptores Androgênicos/metabolismo , Túbulos Seminíferos/metabolismo , Espermatogênese , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.
Assuntos
Metilação de DNA/fisiologia , Metiltransferases/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxiadenosinas/metabolismo , Humanos , RNA/química , RNA/metabolismoRESUMO
Osteosarcoma is the most common malignancy of the bone, yet the survival for patients with osteosarcoma is virtually unchanged over the past 30 years. This is principally because development of new therapies is hampered by a lack of recurrent mutations that can be targeted in osteosarcoma. Here, we report that epigenetic changes via mRNA methylation holds great promise to better understand the mechanisms of osteosarcoma growth and to develop targeted therapeutics. In patients with osteosarcoma, the RNA demethylase ALKBH5 was amplified and higher expression correlated with copy-number changes. ALKBH5 was critical for promoting osteosarcoma growth and metastasis, yet it was dispensable for normal cell survival. Methyl RNA immunoprecipitation sequencing analysis and functional studies showed that ALKBH5 mediates its protumorigenic function by regulating m6A levels of histone deubiquitinase USP22 and the ubiquitin ligase RNF40. ALKBH5-mediated m6A deficiency in osteosarcoma led to increased expression of USP22 and RNF40 that resulted in inhibition of histone H2A monoubiquitination and induction of key protumorigenic genes, consequently driving unchecked cell-cycle progression, incessant replication, and DNA repair. RNF40, which is historically known to ubiquitinate H2B, inhibited H2A ubiquitination in cancer by interacting with and affecting the stability of DDB1-CUL4-based ubiquitin E3 ligase complex. Taken together, this study directly links increased activity of ALKBH5 with dysregulation of USP22/RNF40 and histone ubiquitination in cancers. More broadly, these results suggest that m6A RNA methylation works in concert with other epigenetic mechanisms to control cancer growth. SIGNIFICANCE: RNA demethylase ALKBH5 upregulates USP22 and RNF40 to inhibit histone H2A ubiquitination and induces expression of key replication and DNA repair-associated genes, driving osteosarcoma progression.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Osteossarcoma , Homólogo AlkB 5 da RNA Desmetilase/genética , Histonas/metabolismo , Humanos , Metilação , Osteossarcoma/genética , RNA/genética , RNA/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Ubiquitinas/genéticaRESUMO
Aberrant activities of various cell cycle and DNA repair proteins promote cancer growth and progression and render them resistant to therapies. Here, we demonstrate that the anti-depressant imipramine blocks growth of triple-negative (TNBC) and estrogen receptor-positive (ER+) breast cancers by inducing cell cycle arrest and by blocking heightened homologous recombination (HR) and non-homologous end joining-mediated (NHEJ) DNA repair activities. Our results reveal that imipramine inhibits the expression of several cell cycle- and DNA repair-associated proteins including E2F1, CDK1, Cyclin D1, and RAD51. In addition, we show that imipramine inhibits the growth of ER + breast cancers by inhibiting the estrogen receptor- α (ER-α) signaling. Our studies in preclinical mouse models and ex vivo explants from breast cancer patients show that imipramine sensitizes TNBC to the PARP inhibitor olaparib and endocrine resistant ER + breast cancer to anti-estrogens. Our studies suggest that repurposing imipramine could enhance routine care for breast cancer patients. Based on these results, we designed an ongoing clinical trial, where we are testing the efficacy of imipramine for treating patients with triple-negative and estrogen receptor-positive breast cancer. Since aberrant DNA repair activity is used by many cancers to survive and become resistant to therapy, imipramine could be used alone and/or with currently used drugs for treating many aggressive cancers.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Animais , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Reparo do DNA , Feminino , Humanos , Imipramina/farmacologia , Imipramina/uso terapêutico , Camundongos , Receptores de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Most patients with estrogen receptor alpha-positive (ER+) breast cancers initially respond to treatment but eventually develop therapy resistance with disease progression. Overexpression of oncogenic ER coregulators, including proline, glutamic acid, and leucine-rich protein 1 (PELP1), are implicated in breast cancer progression. The lack of small molecules that inhibits PELP1 represents a major knowledge gap. Here, using a yeast-two-hybrid screen, we identified novel peptide inhibitors of PELP1 (PIP). Biochemical assays demonstrated that one of these peptides, PIP1, directly interacted with PELP1 to block PELP1 oncogenic functions. Computational modeling of PIP1 revealed key residues contributing to its activity and facilitated the development of a small-molecule inhibitor of PELP1, SMIP34, and further analyses confirmed that SMIP34 directly bound to PELP1. In breast cancer cells, SMIP34 reduced cell growth in a dose-dependent manner. SMIP34 inhibited proliferation of not only wild-type (WT) but also mutant (MT) ER+ and therapy-resistant breast cancer cells, in part by inducing PELP1 degradation via the proteasome pathway. RNA sequencing analyses showed that SMIP34 treatment altered the expression of genes associated with estrogen response, cell cycle, and apoptosis pathways. In cell line-derived and patient-derived xenografts of both WT and MT ER+ breast cancer models, SMIP34 reduced proliferation and significantly suppressed tumor progression. Collectively, these results demonstrate SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling in advanced breast cancer. SIGNIFICANCE: Development of a novel inhibitor of oncogenic PELP1 provides potential therapeutic avenues for treating therapy-resistant, advanced ER+ breast cancer.