RESUMO
Triple-negative breast cancer (TNBC) is clinically aggressive and relatively unresponsive to current therapies. Therefore, the development of new anticancer agents is needed to satisfy clinical needs. Oxyphenisatin acetate (Acetalax), which had been used as a laxative, has recently been reported to have anticancer activity in murine models. In this study, we demonstrate that Acetalax and its diphenolic laxative structural analogue bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines and cause oncosis, a nonapoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing, leading to mitochondrial dysfunction, ATP depletion, and enhanced immune and inflammatory responses. Mechanistically, we provide evidence that transient receptor potential melastatin member 4 (TRPM4) is poisoned by Acetalax and bisacodyl in MDA-MB468, BT549, and HS578T TNBC cells. MDA-MB231 and MDA-MB436 TNBC cells without endogenous TRPM4 expression as well as TRPM4-knockout TNBC cells were found to be Acetalax- and bisacodyl-resistant. Conversely, ectopic expression of TRPM4 sensitized MDA-MB231 and MDA-MB436 cells to Acetalax. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover, TRPM4 is rapidly degraded by the ubiquitin-proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and bisacodyl efficacy and could be used for the clinical development of these drugs as anticancer agents. SIGNIFICANCE: Acetalax and bisacodyl kill cancer cells by causing oncosis following poisoning of the plasma membrane sodium transporter TRPM4 and represent a new therapeutic approach for TNBC.
Assuntos
Canais de Cátion TRPM , Neoplasias de Mama Triplo Negativas , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/genética , Humanos , Linhagem Celular Tumoral , Feminino , Proliferação de Células/efeitos dos fármacos , Animais , Camundongos , Antineoplásicos/farmacologiaRESUMO
EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.
Assuntos
Doenças Neurodegenerativas , Ácidos Nucleicos , Proteína EWS de Ligação a RNA , Humanos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , RNA Polimerase II/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismoRESUMO
BACKGROUND: The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe elimination (clearance), focusing on the intramural periarterial drainage (IPAD) pathway. METHODS: Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min to 48 h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD-vascular matrix analyzed in 11,668 metastases. RESULTS: In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85-93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways. CONCLUSIONS: Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.
Assuntos
Neoplasias Encefálicas , Neoplasias da Mama , Imunoglobulina G , Receptor ErbB-2 , Trastuzumab , Trastuzumab/farmacocinética , Animais , Camundongos , Humanos , Feminino , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Imunoglobulina G/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos Imunológicos/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.
Assuntos
Replicação do DNA , Neoplasias , Humanos , Replicação do DNA/genética , DNA Helicases/metabolismo , Repetições de Microssatélites , Dano ao DNA , Neoplasias/tratamento farmacológico , Neoplasias/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.
RESUMO
We report systematic analysis of endogenous EWSR1's cellular organization. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Interestingly, EWSR1 and FUS, another FET protein, exhibit distinct spatial organizations. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.
RESUMO
Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.
Assuntos
Corpos de Processamento , Grânulos de Estresse , Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Humanos , Linhagem CelularRESUMO
Tumor-infiltrating lymphocytes (TIL) that can recognize and kill tumor cells have curative potential in subsets of patients treated with adoptive cell transfer (ACT). However, lack of TIL therapeutic efficacy in many patients may be due in large part to a paucity of tumor-reactive T cells in TIL and the exhausted and terminally differentiated status of those tumor-reactive T cells. We sought to reprogram exhausted TIL that possess T-cell receptors (TCR) specific for tumor antigens into induced pluripotent stem cells (iPSC) to rejuvenate them for more potent ACT. We first attempted to reprogram tumor neoantigen-specific TIL by αCD3 Ab prestimulation which resulted in failure of establishing tumor-reactive TIL-iPSCs, instead, T cell-derived iPSCs from bystander T cells were established. To selectively activate and enrich tumor-reactive T cells from the heterogenous TIL population, CD8+ PD-1+ 4-1BB+ TIL population were isolated after coculture with autologous tumor cells, followed by direct reprogramming into iPSCs. TCR sequencing analysis of the resulting iPSC clones revealed that reprogrammed TIL-iPSCs encoded TCRs that were identical to the pre-identified tumor-reactive TCRs found in minimally cultured TIL. Moreover, reprogrammed TIL-iPSCs contained rare tumor antigen-specific TCRs, which were not detectable by TCR sequencing of the starting cell population. Thus, reprogramming of PD-1+ 4-1BB+ TIL after coculture with autologous tumor cells selectively generates tumor antigen-specific TIL-iPSCs, and is a distinctive method to enrich and identify tumor antigen-specific TCRs of low frequency from TIL. Significance: Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias , Humanos , Linfócitos do Interstício Tumoral , Receptor de Morte Celular Programada 1 , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Antígenos de NeoplasiasRESUMO
Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Saco Vitelino , Células-Tronco Hematopoéticas , Organoides , Atividades CotidianasRESUMO
Zinc finger transcription factor CASZ1b is essential for nervous system development and suppresses neuroblastoma growth. Our previous study showed that CASZ1b interacts with DNA repair proteins, however, whether CASZ1b is involved in the DNA damage response remains unclear. In this study, we investigated the kinetic recruitment of CASZ1b to sites of DNA damage upon induction by laser microirradiation. We find that CASZ1b is transiently recruited to sites of DNA damage in multiple cell lines. Mutagenesis of either the poly-(ADP-ribose) (PAR) binding motif or NuRD complex binding region in CASZ1b significantly reduces the recruitment of CASZ1b to these sites of DNA damage (â¼65% and â¼30%, respectively). In addition, treatment of cells with a poly-(ADP-ribose) polymerase (PARP) inhibitor significantly attenuates the recruitment of CASZ1b to these DNA damaged sites. Loss of CASZ1 increases cell sensitivity to DNA damage induced by gamma irradiation as shown by decreased colony formation. Our studies reveal that CASZ1b is transiently recruited to DNA damage sites mainly in a PARP-dependent way and regulates cell sensitivity to DNA damage. Our results suggest that CASZ1b has a role, although perhaps a minor one, in the DNA damage response and ultimately regulating the efficiency of DNA repair during normal development and tumorigenesis.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Dedos de Zinco , Poli(ADP-Ribose) Polimerase-1/metabolismo , Reparo do DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Dano ao DNA , Poli Adenosina Difosfato Ribose/metabolismoRESUMO
Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.
Assuntos
Quebras de DNA de Cadeia Simples , Desmetilação do DNA , Reparo do DNA , Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes Induzidas , Neurônios , Timina DNA Glicosilase , Diferenciação Celular , Neurônios/enzimologia , 5-Metilcitosina/metabolismo , Humanos , Transdiferenciação CelularRESUMO
Epithelial ovarian cancer (EOC) is a global health burden and remains the fifth leading cause of cancer related death in women worldwide with the poorest five-year survival rate of the gynecological malignancies. EOC recurrence is considered to be driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs). We previously showed that disulfiram, an ALDH inhibitor, effectively targeted TICs compared to adherent EOC cells in terms of viability, spheroid formation, oxidative stress and also prevented relapse in an in vivo model of EOC. In this study we sought to determine whether specific targeting of ALDH isoenzyme ALDH1A1 would provide similar benefit to broader pathway inhibition by disulfiram. NCT-505 and NCT-506 are isoenzyme-specific ALDH1A1 inhibitors whose activity was compared to the effects of disulfiram. Following treatment with both the NCTs and disulfiram, the viability of TICs versus adherent cells, sphere formation, and cell death in our in vitro relapse model were measured and compared in EOC cell lines. We found that disulfiram decreased the viability of TICs significantly more effectively versus adherent cells, while no consistent trend was observed when the cells were treated with the NCTs. Disulfiram also affected the expression of proteins associated with NFκB signaling. Comparison of disulfiram to the direct targeting of ALDH1A1 with the NCTs suggests that the broader cellular effects of disulfiram are more suitable as a therapeutic to eradicate TICs from tumors and prevent EOC relapse. In addition to providing insight into a fitting treatment for TICs, the comparison of disulfiram to NCT-505 and -506 has increased our understanding of the mechanism of action of disulfiram. Further elucidation of the mechanism of disulfiram has the potential to reveal additional targets to treat EOC TICs and prevent disease recurrence.
RESUMO
Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.
Assuntos
Neoplasias da Mama/patologia , Heterocromatina/metabolismo , Mecanotransdução Celular/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Citoesqueleto/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Adesões Focais/genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Metiltransferases/metabolismo , Camundongos , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Microambiente TumoralRESUMO
The naïve epiblast transitions to a pluripotent primed state during embryo implantation. Despite the relevance of the FGF pathway during this period, little is known about the downstream effectors regulating this signaling. Here, we examined the molecular mechanisms coordinating the naïve to primed transition by using inducible ESC to genetically eliminate all RAS proteins. We show that differentiated RASKO ESC remain trapped in an intermediate state of pluripotency with naïve-associated features. Elimination of the transcription factor ERF overcomes the developmental blockage of RAS-deficient cells by naïve enhancer decommissioning. Mechanistically, ERF regulates NANOG expression and ensures naïve pluripotency by strengthening naïve transcription factor binding at ESC enhancers. Moreover, ERF negatively regulates the expression of the methyltransferase DNMT3B, which participates in the extinction of the naïve transcriptional program. Collectively, we demonstrated an essential role for ERF controlling the exit from naïve pluripotency in a MAPK-dependent manner during the progression to primed pluripotency.
RESUMO
Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Reprogramação Celular , Células-Tronco Totipotentes/citologia , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Morte Celular , Dano ao DNA , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Células-Tronco Totipotentes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The centrosome is a unique membraneless organelle that plays a pivotal role in the orderly progression of the cell cycle in animal cells. It has been shown that two pericentriolar scaffold proteins, Cep63 and Cep152, generate a heterotetrameric complex to self-assemble into a higher-order cylindrical architecture around a centriole. However, the mechanisms underlying how they reach their threshold concentrations in the vast intracellular space and generate a self-assembled architecture remain mysterious. Here we demonstrate that, like liquid-like assemblies, Cep63 and Cep152 cooperatively generate amorphous aggregates capable of undergoing dynamic turnover and inter-aggregate fusion in vivo and a significant level of internal rearrangemefnt within a condensate in vitro. Consistently, 1,6-hexanediol, a liquid-liquid phase separation disruptor, greatly diminished the ability of endogenous Cep63 and Cep152 to localize to centrosomes. Interestingly, a purified Cep63â¢Cep152 complex generated either a cylindrical structure or a vesicle-like hollow sphere in a spatially controlled manner. It also formed condensate-like solid spheres in the presence of a macromolecular crowder. At the molecular level, two hydrophobic motifs, one each from Cep63 and Cep152, were required for generating phase-separating condensates and a high molecular-weight assembly. Thus, we propose that the self-assembly of the Cep63â¢Cep152 complex is triggered by an intrinsic property of the complex undergoing density transition through the hydrophobic-motif-mediated phase separation. Abbreviations: PCM, pericentriolar material; LLPS, liquid-liquid phase separation; MW, molecular-weight; CLEM, correlative light and electron microscopy; WT, wild-type; CMV, cytomegalovirus; FRAP, fluorescence recovery after photobleaching; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; 3D-SIM, three-dimensional structured illumination microscopy; DMEM, Dulbecco's Modified Eagle Medium; PEI Max, Polyethylenimine Max; PBS, phosphate-buffered saline; RT, room temperature; DAPI, 4', 6-diamidino-2-phenylindole; AOTF, acousto-optic tunable filter; LB, Luria broth; OD, optical density; IPTG, isopropyl ß-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Extração Líquido-Líquido/métodos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Agregados Proteicos/genética , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , TransfecçãoRESUMO
Immunotherapy is reportedly an effective form of therapy for some advanced cancers such as lung adenocarcinoma, malignant melanoma and colorectal adenocarcinoma. In renal cell carcinoma (RCC), the role of immunotherapy is under investigation. Programmed Death-Ligand 1 (PD-L1) is a molecule expressed on the surface of certain tumor cells and binds to the Programmed cell death protein 1 (PD-1) on cytotoxic T-cells, an interaction that inhibits the antitumor immune response. The aim of this study is to evaluate PD-L1 expression in the morphologic spectrum of RCC. A total of 172 cases of RCC comprising all types were studied and the PD-L1 was correlated with immune response for CD4 and CD8. Positive membranous staining for PD-L1 was seen in 59 (34%) of the 172 samples. The positive cases were HLRCC (31/53), Type 1 Papillary RCC (10/31), Chromophobe (7/20), Hybrid (3/9), TFE-3 related cancer (3/8), Undifferentiated (3/5), and TFEB tumors (2/2). Clear cell carcinomas, Oncocytomas and SDHB deficient-RCC didn't show any expression of PD-L1; (0/34;0/7;0/3). Our results demonstrated that aggressive forms of RCC such as HLRCC have high expression of PD-L1, in contrast to clear cell renal carcinomas. Our findings support a possible role of anti-PD-L1/PD-1 immunotherapies in the treatment of PD-L1-positive RCC.
RESUMO
53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.
Assuntos
Recombinação Homóloga/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Animais , Proteína BRCA1/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Recombinação Homóloga/efeitos dos fármacos , Camundongos , Mutação/efeitos dos fármacos , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.
Assuntos
Autoantígenos/genética , RNA Helicases DEAD-box/genética , Herpesvirus Humano 8/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Argonautas/genética , Regulação Viral da Expressão Gênica/genética , Células HeLa , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , RNA Viral/genética , Replicação Viral/genéticaRESUMO
A paradox of tumor immunology is that tumor-infiltrating lymphocytes are dysfunctional in situ, yet are capable of stem cell-like behavior including self-renewal, expansion, and multipotency, resulting in the eradication of large metastatic tumors. We find that the overabundance of potassium in the tumor microenvironment underlies this dichotomy, triggering suppression of T cell effector function while preserving stemness. High levels of extracellular potassium constrain T cell effector programs by limiting nutrient uptake, thereby inducing autophagy and reduction of histone acetylation at effector and exhaustion loci, which in turn produces CD8+ T cells with improved in vivo persistence, multipotency, and tumor clearance. This mechanistic knowledge advances our understanding of T cell dysfunction and may lead to novel approaches that enable the development of enhanced T cell strategies for cancer immunotherapy.