RESUMO
We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest fraction of the Drosophila proteome. The network contains 32,668 interactions among 3,644 proteins, organized into 632 clusters representing putative functional modules. Our analysis expands the pool of known protein interactions in Drosophila, provides annotation for poorly studied genes, and postulates previously undescribed protein interaction relationships. The predictive power and functional relevance of this network are probed through the lens of the Notch signaling pathway, and we find that newly identified members of complexes that include known Notch modifiers can also modulate Notch signaling. DPIM2 allows direct comparisons with a recently published human protein interaction network, defining the existence of functional interactions conserved across species. Thus, DPIM2 defines a valuable resource for predicting protein co-complex memberships and functional associations as well as generates functional hypotheses regarding specific protein interactions.
RESUMO
The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.
Assuntos
Arginina , Lipogênese , Proteína de Ligação a Elemento Regulador de Esterol 1 , Lipogênese/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fatores de Processamento de RNA , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Humanos , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
BACKGROUND: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier. METHODS: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties. RESULTS: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing. CONCLUSIONS: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.
Assuntos
Agonismo Inverso de Drogas , Neuralgia , Ratos , Animais , Qualidade de Vida , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/uso terapêutico , Neuralgia/tratamento farmacológico , Fenômenos EletrofisiológicosRESUMO
Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.
Assuntos
Proteostase , Receptor de Insulina , RNA Mensageiro/metabolismo , Receptor de Insulina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sirolimo , Biossíntese de ProteínasRESUMO
BACKGROUND: PD-L1, a transmembrane ligand for immune checkpoint receptor PD1, has been successfully targeted to activate an anti-tumor immune response in a variety of solid tumors, including non-small cell lung cancer (NSCLC). Despite the success of targeting PD-L1, only about 20% of patients achieve a durable response. The reasons for the heterogeneity in response are not understood, although some molecular subtypes (e.g., mutant EGF receptor tumors) are generally poor responders. Although PD-L1 is best characterized as a transmembrane PD1 ligand, the emerging view is that PD-L1 has functions independent of activating PD1 signaling. It is not known whether these cell-intrinsic functions of PD-L1 are shared among non-transformed and transformed cells, if they vary among cancer molecular subtypes, or if they are impacted by anti-PD-L1 therapy. METHODS: Here we use quantitative microscopy techniques and APEX2 proximity mapping to describe the behavior of PD-L1 and to identify PD-L1's proximal proteome in human lung epithelial cells. RESULTS: Our data reveal growth factor control of PD-L1 recycling as a mechanism for acute and reversible regulation of PD-L1 density on the plasma membrane. In addition, we describe novel PD-L1 biology restricted to mutant EGFR cells. Anti-PD-L1 antibody treatment of mutant EGFR cells perturbs cell intrinsic PD-L1 functions, leading to reduced cell migration, increased half-life of EGFR and increased extracellular vesicle biogenesis, whereas anti-PD-L1 antibody does not induce these changes in wild type EGFR cells. CONCLUSIONS: Growth factor acute regulation of PD-L1 trafficking, by contributing to the control of plasma membrane density, might contribute to the regulation of PD-L1's immune checkpoint activity, whereas the specific effects of anti-PD-L1 on mutant EGFR cells might contribute to the poor anti-PD-L1 response of mutant EGFR tumors. Video Abstract.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteoma , Ligantes , Receptores ErbB/genética , Receptores ErbB/metabolismo , Pulmão/metabolismo , Antígeno B7-H1/metabolismo , MutaçãoRESUMO
The systemic metabolic shifts that occur during aging and the local metabolic alterations of a tumor, its stroma and their communication cooperate to establish a unique tumor microenvironment (TME) fostering cancer progression. Here, we show that methylmalonic acid (MMA), an aging-increased oncometabolite also produced by aggressive cancer cells, activates fibroblasts in the TME, which reciprocally secrete IL-6 loaded extracellular vesicles (EVs) that drive cancer progression, drug resistance and metastasis. The cancer-associated fibroblast (CAF)-released EV cargo is modified as a result of reactive oxygen species (ROS) generation and activation of the canonical and noncanonical TGFß signaling pathways. EV-associated IL-6 functions as a stroma-tumor messenger, activating the JAK/STAT3 and TGFß signaling pathways in tumor cells and promoting pro-aggressive behaviors. Our findings define the role of MMA in CAF activation to drive metastatic reprogramming, unveiling potential therapeutic avenues to target MMA at the nexus of aging, the tumor microenvironment and metastasis.
Assuntos
Fibroblastos Associados a Câncer , Vesículas Extracelulares , Neoplasias , Humanos , Fibroblastos Associados a Câncer/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Metilmalônico/metabolismo , Interleucina-6/metabolismo , Microambiente Tumoral , Neoplasias/patologia , Vesículas Extracelulares/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET-rearranged thyroid cancer.
Assuntos
Neoplasias Pulmonares , Neoplasias da Glândula Tireoide , Animais , Proteínas do Citoesqueleto/genética , Humanos , Neoplasias Pulmonares/patologia , Proteômica , Proteínas Proto-Oncogênicas c-ret/genética , Receptores de Fatores de Crescimento de Fibroblastos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genéticaRESUMO
Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials.
Assuntos
Cobre/metabolismo , Mitocôndrias/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Feminino , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Metástase Neoplásica , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosforilação Oxidativa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Metabolic adaptations and the signaling events that control them promote the survival of pancreatic ductal adenocarcinoma (PDAC) at the fibrotic tumor site, overcoming stresses associated with nutrient and oxygen deprivation. Recently, rewiring of NADPH production has been shown to play a key role in this process. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells. However, de novo NADP+ is synthesized only through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). In this study, we show that oncogenic KRAS promotes protein kinase C (PKC)-mediated NADK phosphorylation, leading to its hyperactivation, thus sustaining both NADP+ and NADPH levels in PDAC cells. Together, our data show that increased NADK activity is an important adaptation driven by oncogenic signaling. Our findings indicate that NADK could serve as a much-needed therapeutic target for PDAC.
Assuntos
Adenocarcinoma/enzimologia , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/enzimologia , Neoplasias Pancreáticas/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Adenocarcinoma/patologia , Animais , Vias Biossintéticas , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , NADP/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação , Fosfosserina/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMO
Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4+ T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting â¼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Regulação Neoplásica da Expressão Gênica , Expressão Gênica , Imunoterapia/métodos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Terapia de Alvo Molecular , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/terapia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/terapia , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Prognóstico , Carcinoma Anaplásico da Tireoide/imunologia , Neoplasias da Glândula Tireoide/imunologiaRESUMO
An extra copy of chromosome 21 causes Down syndrome, the most common genetic disease in humans. The mechanisms contributing to aneuploidy-related pathologies in this syndrome, independent of the identity of the triplicated genes, are not well defined. To characterize aneuploidy-driven phenotypes in trisomy 21 cells, we performed global transcriptome, proteome, and phenotypic analyses of primary human fibroblasts from individuals with Patau (trisomy 13), Edwards (trisomy 18), or Down syndromes. On average, mRNA and protein levels were increased by 1.5-fold in all trisomies, with a subset of proteins enriched for subunits of macromolecular complexes showing signs of posttranscriptional regulation. These results support the lack of evidence for widespread dosage compensation or dysregulation of chromosomal domains in human autosomes. Furthermore, we show that several aneuploidy-associated phenotypes are present in trisomy 21 cells, including lower viability and increased dependency on serine-driven lipid synthesis. Our studies establish a critical role of aneuploidy, independent of triplicated gene identity, in driving cellular defects associated with trisomy 21.
Assuntos
Aneuploidia , Fibroblastos/patologia , Trissomia/genética , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Fibroblastos/metabolismo , Dosagem de Genes/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Lipídeos/biossíntese , Substâncias Macromoleculares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Neoantigens generated by somatic nonsynonymous mutations are key targets of tumor-specific T cells, but only a small number of mutations predicted to be immunogenic are presented by MHC molecules on cancer cells. Vaccination studies in mice and patients have shown that the majority of neoepitopes that elicit T cell responses fail to induce significant antitumor activity, for incompletely understood reasons. We report that radiotherapy upregulates the expression of genes containing immunogenic mutations in a poorly immunogenic mouse model of triple-negative breast cancer. Vaccination with neoepitopes encoded by these genes elicited CD8+ and CD4+ T cells that, whereas ineffective in preventing tumor growth, improved the therapeutic efficacy of radiotherapy. Mechanistically, neoantigen-specific CD8+ T cells preferentially killed irradiated tumor cells. Neoantigen-specific CD4+ T cells were required for the therapeutic efficacy of vaccination and acted by producing Th1 cytokines, killing irradiated tumor cells, and promoting epitope spread. Such a cytotoxic activity relied on the ability of radiation to upregulate class II MHC molecules as well as the death receptors FAS/CD95 and DR5 on the surface of tumor cells. These results provide proof-of-principle evidence that radiotherapy works in concert with neoantigen vaccination to improve tumor control.
Assuntos
Antígenos de Neoplasias/farmacologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/terapia , Células Th1/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/efeitos da radiação , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Radioterapia , Células Th1/patologia , VacinaçãoRESUMO
Advanced prostate cancer initially responds to hormonal treatment, but ultimately becomes resistant and requires more potent therapies. One mechanism of resistance observed in around 10-20% of these patients is lineage plasticity, which manifests in a partial or complete small cell or neuroendocrine prostate cancer (NEPC) phenotype. Here, we investigate the role of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex in NEPC. Using large patient datasets, patient-derived organoids and cancer cell lines, we identify mSWI/SNF subunits that are deregulated in NEPC and demonstrate that SMARCA4 (BRG1) overexpression is associated with aggressive disease. We also show that SWI/SNF complexes interact with different lineage-specific factors in NEPC compared to prostate adenocarcinoma. These data point to a role for mSWI/SNF complexes in therapy-related lineage plasticity, which may also be relevant for other solid tumors.
Assuntos
Linhagem da Célula , Plasticidade Celular , Proteínas Cromossômicas não Histona/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Estudos de Coortes , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Modelos Biológicos , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Subunidades Proteicas/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
The risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards1-6. Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy2,3,7,8. For decades, this link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the established role of diet, exercise and small molecules that target the pace of metabolic ageing9-12. Here we show that metabolic alterations that occur with age can produce a systemic environment that favours the progression and aggressiveness of tumours. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is upregulated in the serum of older people and functions as a mediator of tumour progression. We traced this to the ability of MMA to induce SOX4 expression and consequently to elicit transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, the accumulation of MMA represents a link between ageing and cancer progression, suggesting that MMA is a promising therapeutic target for advanced carcinomas.
Assuntos
Envelhecimento/metabolismo , Progressão da Doença , Ácido Metilmalônico/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologia , Adulto , Idoso , Envelhecimento/sangue , Envelhecimento/genética , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Ácido Metilmalônico/sangue , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias/sangue , Neoplasias/genética , Fatores de Transcrição SOXC/metabolismo , Transdução de Sinais , Transcriptoma/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cancer stem cells (CSCs) represent rare tumor cell populations capable of self-renewal, differentiation, and tumor initiation and are highly resistant to chemotherapy and radiotherapy. Thus, therapeutic approaches that can effectively target CSCs and tumor cells could be the key to efficient tumor treatment. In this study, we explored the function of SPHK1 in breast CSCs and non-CSCs. We showed that RNAi-mediated knockdown of SPHK1 inhibited cell proliferation and induced apoptosis in both breast CSCs and non-CSCs, while ectopic expression of SPHK1 enhanced breast CSC survival and mammosphere forming efficiency. We identified STAT1 and IFN signaling as key regulatory targets of SPHK1 and demonstrated that an important mechanism by which SPHK1 promotes cancer cell survival is through the suppression of STAT1. We further demonstrated that SPHK1 inhibitors, FTY720 and PF543, synergized with doxorubicin in targeting both breast CSCs and non-CSCs. In conclusion, we provide important evidence that SPHK1 is a key regulator of cell survival and proliferation in breast CSCs and non-CSCs and is an attractive target for the design of future therapies.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fator de Transcrição STAT1/metabolismo , Apoptose , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Doxorrubicina/farmacologia , Feminino , Células HEK293 , Humanos , Interferons/metabolismo , Lisofosfolipídeos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function1,2. Currently, there are no therapies proven to prevent beta cell loss and some, namely insulin secretagogues, have been linked to accelerated beta cell failure, thereby limiting their use in type 2 diabetes3,4. The adipokine adipsin/complement factor D controls the alternative complement pathway and generation of complement component C3a, which acts to augment beta cell insulin secretion5. In contrast to other insulin secretagogues, we show that chronic replenishment of adipsin in diabetic db/db mice ameliorates hyperglycemia and increases insulin levels while preserving beta cells by blocking dedifferentiation and death. Mechanistically, we find that adipsin/C3a decreases the phosphatase Dusp26; forced expression of Dusp26 in beta cells decreases expression of core beta cell identity genes and sensitizes to cell death. In contrast, pharmacological inhibition of DUSP26 improves hyperglycemia in diabetic mice and protects human islet cells from cell death. Pertaining to human health, we show that higher concentrations of circulating adipsin are associated with a significantly lower risk of developing future diabetes among middle-aged adults after adjusting for body mass index (BMI). Collectively, these data suggest that adipsin/C3a and DUSP26-directed therapies may represent a novel approach to achieve beta cell health to treat and prevent type 2 diabetes.
Assuntos
Complemento C3a/genética , Fator D do Complemento/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fosfatases de Especificidade Dupla/genética , Células Secretoras de Insulina/efeitos dos fármacos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Animais , Índice de Massa Corporal , Desdiferenciação Celular/efeitos dos fármacos , Fator D do Complemento/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Glucose/metabolismo , Humanos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/genética , Hiperglicemia/patologia , Insulina/genética , Resistência à Insulina/genética , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos NODRESUMO
Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.
Assuntos
Carcinoma/patologia , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Metástase Neoplásica/genética , Animais , Carcinogênese/genética , Carcinoma/genética , Linhagem Celular Tumoral , Cromatina/genética , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Progressão da Doença , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , Feminino , Histonas/genética , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas/genética , RNA-Seq , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.
Assuntos
Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Células-Tronco Pluripotentes/metabolismoRESUMO
Despite recent therapeutic advances, prostate cancer remains a leading cause of cancer-related death. A subset of castration resistant prostate cancers become androgen receptor (AR) signaling-independent and develop neuroendocrine prostate cancer (NEPC) features through lineage plasticity. These NEPC tumors, associated with aggressive disease and poor prognosis, are driven, in part, by aberrant expression of N-Myc, through mechanisms that remain unclear. Integrative analysis of the N-Myc transcriptome, cistrome and interactome using in vivo, in vitro and ex vivo models (including patient-derived organoids) identified a lineage switch towards a neural identity associated with epigenetic reprogramming. N-Myc and known AR-co-factors (e.g., FOXA1 and HOXB13) overlapped, independently of AR, at genomic loci implicated in neural lineage specification. Moreover, histone marks specifically associated with lineage-defining genes were reprogrammed by N-Myc. We also demonstrated that the N-Myc-induced molecular program accurately classifies our cohort of patients with advanced prostate cancer. Finally, we revealed the potential for EZH2 inhibition to reverse the N-Myc-induced suppression of epithelial lineage genes. Altogether, our data provide insights on how N-Myc regulates lineage plasticity and epigenetic reprogramming associated with lineage-specification. The N-Myc signature we defined could also help predict the evolution of prostate cancer and thus better guide the choice of future therapeutic strategies.