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1.
Mol Cell ; 76(1): 11-26.e7, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31400850

RESUMO

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Autofagia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proliferação de Células , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligases/genética , Ligases/metabolismo , Lisina , Neoplasias/genética , Neoplasias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade Proteica , Proteínas de Ligação a RNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Transdução de Sinais , Sumoilação , Telômero/genética , Telômero/patologia
2.
Mol Cancer ; 23(1): 56, 2024 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-38491381

RESUMO

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.


Assuntos
Anidrases Carbônicas , Carcinoma de Células Renais , Neoplasias Renais , Receptores de Antígenos Quiméricos , Animais , Camundongos , Humanos , Anidrase Carbônica IX/genética , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/patologia , Receptores de Antígenos Quiméricos/genética , Anidrases Carbônicas/metabolismo , Anidrases Carbônicas/uso terapêutico , Antígenos de Neoplasias , Anticorpos , Linfócitos T/metabolismo
5.
J Biol Chem ; 291(52): 26875-26885, 2016 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-27875297

RESUMO

Uracil N-glycosylase 2 (UNG2), the nuclear isoform of UNG, catalyzes the removal of uracil or 5-fluorouracil lesions that accumulate in DNA following treatment with the anticancer agents 5-fluorouracil and 5-fluorodeoxyuridine (floxuridine), a 5-fluorouracil metabolite. By repairing these DNA lesions before they can cause cell death, UNG2 promotes cancer cell survival and is therefore critically involved in tumor resistance to these agents. However, the mechanisms by which UNG2 is regulated remain unclear. Several phosphorylation sites within the N-terminal regulatory domain of UNG2 have been identified, although the effects of these modifications on UNG2 function have not been fully explored, nor have the identities of the kinases involved been determined. Here we show that glycogen synthase kinase 3 (GSK-3) interacts with and phosphorylates UNG2 at Thr60 and that Thr60 phosphorylation requires a Ser64 priming phosphorylation event. We also show that mutating Thr60 or Ser64 to Ala increases the half-life of UNG2, reduces the rate of in vitro uracil excision, and slows UNG2 dissociation from chromatin after DNA replication. Using an UNG2-deficient ovarian cancer cell line that is hypersensitive to floxuridine, we show that GSK-3 phosphorylation facilitates UNG2-dependent repair of floxuridine-induced DNA lesions and promotes tumor cell survival following exposure to this agent. These data suggest that GSK-3 regulates UNG2 and promotes DNA damage repair.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias Ovarianas/patologia , Antimetabólitos Antineoplásicos/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patologia , DNA Glicosilases/genética , Replicação do DNA/efeitos dos fármacos , Feminino , Floxuridina/farmacologia , Fluoruracila/farmacologia , Quinase 3 da Glicogênio Sintase/genética , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fosforilação , Células Tumorais Cultivadas
6.
Trends Cancer ; 6(3): 247-260, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32101727

RESUMO

Alternative lengthening of telomeres (ALT) is a mechanism of telomere maintenance that is observed in many of the most recalcitrant cancer subtypes. Telomeres in ALT cancer cells exhibit a distinctive nucleoprotein architecture shaped by the mismanagement of chromatin that fosters cycles of DNA damage and replicative stress that activate homology-directed repair (HDR). Mutations in specific chromatin-remodeling factors appear to be key determinants of the emergence and survival of ALT cancer cells. However, these may represent vulnerabilities for the targeted elimination of ALT cancer cells that infiltrate tissues and organs to become devastating tumors. In this review we examine recent findings that provide new insights into the factors and mechanisms that mediate telomere length maintenance and survival of ALT cancer cells.


Assuntos
Neoplasias/genética , Homeostase do Telômero , Cromatina/ultraestrutura , Evolução Clonal , Proteínas Correpressoras/antagonistas & inibidores , Proteínas Correpressoras/fisiologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Neoplasias/metabolismo , DNA de Neoplasias/ultraestrutura , Histonas/fisiologia , Recombinação Homóloga , Humanos , Modelos Genéticos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/fisiologia , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neoplasias/ultraestrutura , Conformação de Ácido Nucleico , Telomerase/genética , Telomerase/fisiologia , Proteína Nuclear Ligada ao X/antagonistas & inibidores , Proteína Nuclear Ligada ao X/fisiologia
7.
Nat Struct Mol Biol ; 27(12): 1152-1164, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33046907

RESUMO

The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.


Assuntos
DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerases/genética , Processamento de Proteína Pós-Traducional , Reparo de DNA por Recombinação , Telômero/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , Dano ao DNA , DNA de Neoplasias/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fase G2 , Glicosídeo Hidrolases/metabolismo , Células HeLa , Chaperonas de Histonas/antagonistas & inibidores , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Telômero/ultraestrutura , Homeostase do Telômero , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo
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