RESUMO
Cholesterol biosynthesis is a highly regulated, oxygen-dependent pathway, vital for cell membrane integrity and growth. In fungi, the dependency on oxygen for sterol production has resulted in a shared transcriptional response, resembling prolyl hydroxylation of Hypoxia Inducible Factors (HIFs) in metazoans. Whether an analogous metazoan pathway exists is unknown. Here, we identify Sterol Regulatory Element Binding Protein 2 (SREBP2), the key transcription factor driving sterol production in mammals, as an oxygen-sensitive regulator of cholesterol synthesis. SREBP2 degradation in hypoxia overrides the normal sterol-sensing response, and is HIF independent. We identify MARCHF6, through its NADPH-mediated activation in hypoxia, as the main ubiquitin ligase controlling SREBP2 stability. Hypoxia-mediated degradation of SREBP2 protects cells from statin-induced cell death by forcing cells to rely on exogenous cholesterol uptake, explaining why many solid organ tumours become auxotrophic for cholesterol. Our findings therefore uncover an oxygen-sensitive pathway for governing cholesterol synthesis through regulated SREBP2-dependent protein degradation.
Assuntos
Oxigênio , Fatores de Transcrição , Animais , Humanos , Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Hipóxia , Colesterol/metabolismo , Esteróis , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mamíferos/metabolismoRESUMO
In solid tumors, as the tumor grows and the disease progresses, hypoxic regions are often generated, but in contrast to most normal cells which cannot survive under these conditions, tumour cells adapt to hypoxia by HIF-driven mechanisms. Hypoxia can further promote cancer development by generating an immunosuppressive environment within the tumour mass, which allows tumour cells to escape the immune system recognition. This is achieved by recruiting immunosuppressive cells and by upregulating molecules which block immune cell activation. Hypoxia can also confer resistance to antitumor therapies by inducing the expression of membrane proteins that increase drug efflux or by inhibiting the apoptosis of treated cells. In addition, tumor cells require an active interferon (IFN) signalling pathway for the success of many anticancer therapies, such as radiotherapy or chemotherapy. Therefore, hypoxic effects on this pathway needs to be addressed for a successful treatment.
Assuntos
Interferons , Neoplasias , Apoptose , Humanos , Hipóxia/metabolismo , Interferons/farmacologia , Transdução de SinaisRESUMO
Hypoxia is a common phenomenon in solid tumours strongly linked to the hallmarks of cancer. Hypoxia promotes local immunosuppression and downregulates type I interferon (IFN) expression and signalling, which contribute to the success of many cancer therapies. Double-stranded RNA (dsRNA), transiently generated during mitochondrial transcription, endogenously activates the type I IFN pathway. We report the effects of hypoxia on the generation of mitochondrial dsRNA (mtdsRNA) in breast cancer. We found a significant decrease in dsRNA production in different cell lines under hypoxia. This effect was HIF1α/2α-independent. mtdsRNA was responsible for induction of type I IFN and significantly decreased after hypoxia. Mitochondrially encoded gene expression was downregulated and mtdsRNA bound by the dsRNA-specific J2 antibody was decreased during hypoxia. These findings reveal a new mechanism of hypoxia-induced immunosuppression that could be targeted by hypoxia-activated therapies.
RESUMO
BACKGROUND: In clear cell renal cell carcinoma, 80% of cases have biallelic inactivation of the VHL gene, leading to constitutive activation of both HIF1α and HIF2α. As HIF2α is the driver of the disease promoting tumour growth and metastasis, drugs targeting HIF2α have been developed. However, resistance is common, therefore new therapies are needed. METHODS: We assessed the effect of the HIF2α antagonist PT2385 in several steps of tumour development and performed RNAseq to identify genes differentially expressed upon treatment. A drug screening was used to identify drugs with antiproliferative effects on VHL-mutated HIF2α-expressing cells and could increase effectiveness of PT2385. RESULTS: PT2385 did not reduce cell proliferation or clonogenicity but, in contrast to the genetic silencing of HIF2α, it reduced in vitro cell invasion. Many HIF-inducible genes were down-regulated upon PT2385 treatment, whereas some genes involved in cell migration or extracellular matrix were up-regulated. HIF2α was associated with resistance to statins, addition to PT2385 did not increase the sensitivity. CONCLUSIONS: this study shows key differences between inhibiting a target versus knockdown, which are potentially targetable.
Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Inativação Gênica , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reposicionamento de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Humanos , Indanos/farmacologia , Indanos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Ativação Transcricional , Transcriptoma , Resultado do TratamentoRESUMO
Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Hipóxia/genética , Acetilação , Animais , Humanos , Hipóxia/metabolismo , Metilação , Camundongos , Camundongos Knockout , Modelos Animais , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Hypoxia is a common phenomenon in solid tumors and is strongly linked to hallmarks of cancer. Recent evidence has shown that hypoxia promotes local immune suppression. Type I IFN supports cytotoxic T lymphocytes by stimulating the maturation of dendritic cells and enhancing their capacity to process and present antigens. However, little is known about the relationship between hypoxia and the type I IFN pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA) followed by IFNα/ß secretion and transcriptional activation of IFN-stimulated genes (ISG). In this study, we determined the effects of hypoxia on the type I IFN pathway in breast cancer and the mechanisms involved. In cancer cell lines and xenograft models, mRNA and protein expressions of the type I IFN pathway were downregulated under hypoxic conditions. This pathway was suppressed at each level of signaling, from the dsRNA sensors RIG-I and MDA5, the adaptor MAVS, transcription factors IRF3, IRF7, and STAT1, and several ISG including RIG-I, IRF7, STAT1, and ADAR-p150. Importantly, IFN secretion was reduced under hypoxic conditions. HIF1α- and HIF2α-mediated regulation of gene expression did not explain most of the effects. However, ATAC-seq data revealed in hypoxia that peaks with STAT1 and IRF3 motifs had decreased accessibility. Collectively, these results indicate that hypoxia leads to an overall downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in an HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumors. SIGNIFICANCE: These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine/paracrine effects on tumor growth.
Assuntos
Interferon Tipo I/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Hipóxia Tumoral , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Camundongos , RNA Mensageiro , Transdução de Sinais/imunologia , Análise de Célula Única , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Non-invasive biomarkers or liquid biopsies have the potential to revolutionise cancer patient management as repeated sampling allows real-time monitoring of disease progression and response to treatment. This allows for earlier intervention and dynamic treatment management; both cornerstones of personalised medicine. The circulating transcriptome represents a rich source of potential cancer biomarkers that includes many classes of RNA, both coding and non-coding, that are only now beginning to be explored. In particular the increasing power and availability of RNAseq techniques have pushed studies beyond circulating miRNAs, to other classes of RNA including mRNA, snRNA, snoRNA, piRNA, YRNA, lncRNA and circRNA. In this review we focus on the emerging potential for these different classes of RNA as cancer biomarkers, and in particular the barriers and limitations that remain to be overcome if these molecules are to become part of routine clinical practice.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , Transcriptoma/genética , Progressão da Doença , Humanos , Biópsia Líquida/métodos , Neoplasias/patologia , RNA/genéticaRESUMO
Circular RNAs (circRNAs) are a novel class of regulatory RNAs that despite being relatively abundant have only recently begun to be explored. There are many thousands of genes that appear capable of producing circRNAs, however the function of all but a handful remain to be determined. What is emerging about these highly conserved molecules is that they play important roles in biology and cancer biology in particular. The most explored function of circRNAs is as master regulators of gene expression that act to sequester or ´sponge´ other gene expression regulators, in particular miRNAs. They have also been demonstrated to function via direct modulation of transcription, and by interfering with splicing mechanisms. Although generally expressed in low abundance when compared to their linear counterparts, they are often expressed in a tissue- and developmental stage- specific manner. Coupled with their remarkable resistance to RNAse activity due to a covalent closed cyclic structure, circRNAs show great promise as novel biomarkers of cancer and other diseases. In this review we consider the current state of knowledge regarding these molecules, their synthesis, function, and association with cancer. We will also review some of the challenges that remain to be resolved if this emerging class of RNAs are really to become useful in the clinic.
Assuntos
Biomarcadores Tumorais/genética , Regulação da Expressão Gênica/genética , Neoplasias/genética , RNA Circular/genética , Animais , Expressão Gênica/genética , Humanos , MicroRNAs/genética , RNA/genéticaRESUMO
B-cell lymphomas represent a diverse group of neoplasms classified primarily by histopatholgy and are often challenging to accurately diagnose. Despite having been recognized less than 20 years ago, microRNAs (miRNAs) have emerged as one of the most promising class of cancer molecular biomarkers and are particularly attractive as they can be readily detected in formalin-fixed paraffin-embedded biopsy material and biological fluids such as blood. Many of the identified B-cell lymphoma miRNA biomarkers also play crucial regulatory roles in normal B-cell development. Below we consider the identity, function, and biomarker potential of miRNAs in B-cell lymphoma and most importantly the barriers that remain to be overcome if they are really to become part of routine clinical practice.
RESUMO
A method to separate FAME and the linoleic and linolenic acids isomers by GCxGC using an apparatus equipped with a capillary flow technology (CFT) based modulator and a FID detector has been developed. Four different column combinations (one conventional and three inverted phase sets) were used in these experiments. The conventional set first involved a DB5-MS non-polar column followed by a highly polar HP-INNOWax column in the second dimension. The inverted phase set comprised of a highly polar BPX-70 column in the first dimension and a non-polar ZB5-MS column for the second dimension. Furthermore, the influence of the length of the second dimension column on FAME isomer separation was studied in the inverted phase sets, along with other parameters like the modulation time and column flow. The best results in terms of the time required for the analysis and number of FAME identified with the inverted set were achieved with the shorter second dimension column. After supercritical fluid extraction, the method was applied to identify FAMEs in broccoli leaves from three different cultivars (Naxos, Nubia and Viola).