RESUMO
BACKGROUND: Monoallelic germline pathogenic variants (GPVs) in five Fanconi anemia (FA) genes (BRCA1/FANCS, BRCA2/FANCD1, PALB2/FANCN, BRIP1/FANCJ, and RAD51C/FANCO) confer an increased risk of breast (BC) and/or ovarian (OC) cancer, but the role of GPVs in 17 other FA genes remains unclear. METHODS: Here, we investigated the association of germline variants in FANCG/XRCC9 with BC and OC risk. RESULTS: The frequency of truncating GPVs in FANCG did not differ between BC (20/10,204; 0.20%) and OC (8/2966; 0.27%) patients compared to controls (6/3250; 0.18%). In addition, only one out of five tumor samples showed loss-of-heterozygosity of the wild-type FANCG allele. Finally, none of the nine functionally tested rare recurrent missense FANCG variants impaired DNA repair activities (FANCD2 monoubiquitination and FANCD2 foci formation) upon DNA damage, in contrast to all tested FANCG truncations. CONCLUSION: Our study suggests that heterozygous germline FANCG variants are unlikely to contribute to the development of BC or OC.
Assuntos
Neoplasias da Mama , Proteína do Grupo de Complementação G da Anemia de Fanconi , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Pessoa de Meia-Idade , Adulto , Reparo do DNA/genética , Estudos de Casos e Controles , IdosoRESUMO
RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.
Assuntos
Cromatina , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Histonas , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Cromatina/metabolismo , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Histonas/metabolismo , Recombinação Homóloga/genética , Reparo de DNA por Recombinação , Replicação do DNA , Reparo do DNA , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Instabilidade Genômica , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismoRESUMO
Hematopoietic stem cells (HSCs) ensure blood cell production during the life-time of an organism, and to do so they need to balance self-renewal, proliferation, differentiation, and migration in a steady state as well as in response to stress or injury. Importantly, aberrant proliferation of HSCs leads to hematological malignancies, and thus, tight regulation by various tumor suppressor pathways, including p53, is essential. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and promotes cell survival upon induction of genotoxic stress. Truncating mutations in the last exon of PPM1D lead to the production of a stable, enzymatically active protein and are commonly associated with clonal hematopoiesis. Using a transgenic mouse model, we demonstrate that truncated PPM1D reduces self-renewal of HSCs in basal conditions but promotes the development of aggressive AML after exposure to ionizing radiation. Inhibition of PPM1D suppressed the colony growth of leukemic stem and progenitor cells carrying the truncated PPM1D, and remarkably, it provided protection against irradiation-induced cell growth. Altogether, we demonstrate that truncated PPM1D affects HSC maintenance, disrupts normal hematopoiesis, and that its inhibition could be beneficial in the context of therapy-induced AML.
Assuntos
Leucemia Mieloide Aguda , Proteína Supressora de Tumor p53 , Animais , Camundongos , Proliferação de Células , Dano ao DNA , Leucemia Mieloide Aguda/genética , Mutação , Proteína Supressora de Tumor p53/genéticaRESUMO
The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.
Assuntos
Proteínas de Ciclo Celular , Proteínas Supressoras de Tumor , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Supressoras de Tumor/genética , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.
Assuntos
Complexo Shelterina , Proteínas de Ligação a Telômeros , Proteína 2 de Ligação a Repetições Teloméricas , Dano ao DNA , Fosforilação , Proteômica , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , HumanosRESUMO
Cyclin-dependent kinases (CDKs) are master regulators of proliferation, and therefore they represent attractive targets for cancer therapy. Deve-lopment of selective CDK4/6 inhibitors including palbociclib revolutionized the treatment of advanced HR+/HER2- breast cancer. Inhibition of CDK4/6 leads to cell cycle arrest in G0/G1 phase and eventually to a permanent cell cycle exit called senescence. One of the main features of the senescence is an increased cell size. For many years, it was believed that the non-dividing cells simply continue to grow and as a result, they become excessively large. There is now emerging evidence that the increased cell size is a cause rather than consequence of the cell cycle arrest. This review aims to summarize recent advances in our understanding of senescence induction, in particular that resulting from treatment with CDK4/6 inhibitors.
Assuntos
Neoplasias da Mama , Inibidores de Proteínas Quinases , Humanos , Feminino , Inibidores de Proteínas Quinases/farmacologia , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Ciclo Celular , Neoplasias da Mama/tratamento farmacológicoRESUMO
ER distribution depends on microtubules, and ER homeostasis disturbance activates the unfolded protein response resulting in ER remodeling. CDK5RAP3 (C53) implicated in various signaling pathways interacts with UFM1-protein ligase 1 (UFL1), which mediates the ufmylation of proteins in response to ER stress. Here we find that UFL1 and C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or C53 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation accompanied by γ-tubulin accumulation, microtubule formation, and ER expansion. C53, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in cells lacking UFL1. Pharmacological induction of ER stress by tunicamycin also leads to increased microtubule nucleation and ER expansion. Furthermore, tunicamycin suppresses the association of C53 with the centrosome. These findings point to a novel mechanism for the relief of ER stress by stimulation of centrosomal microtubule nucleation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Microtúbulos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , HumanosRESUMO
Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bioensaio/métodos , Neoplasias Ósseas/patologia , Núcleo Celular/metabolismo , Osteossarcoma/patologia , Proteína Fosfatase 2C/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Núcleo Celular/genética , Dano ao DNA , Reparo do DNA , Humanos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 2C/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina A2/metabolismo , Citoplasma/metabolismo , Fase G2/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S/genética , Transdução de Sinais/genética , Proteína Quinase CDC2/deficiência , Proteína Quinase CDC2/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Ciclina A2/genética , Quinase 2 Dependente de Ciclina/deficiência , Quinase 2 Dependente de Ciclina/genética , Dano ao DNA/genética , Ativação Enzimática/genética , Células HeLa , Humanos , Mitose/genética , Fosforilação/genética , Ligação Proteica , Transfecção , Quinase 1 Polo-LikeRESUMO
Germline alterations in many genes coding for proteins regulating DNA repair and DNA damage response (DDR) to DNA double-strand breaks (DDSB) have been recognized as pathogenic factors in hereditary cancer predisposition. The ATM-CHEK2-p53 axis has been documented as a backbone for DDR and hypothesized as a barrier against cancer initiation. However, although CHK2 kinase coded by the CHEK2 gene expedites the DDR signal, its function in activation of p53-dependent cell cycle arrest is dispensable. CHEK2 mutations rank among the most frequent germline alterations revealed by germline genetic testing for various hereditary cancer predispositions, but their interpretation is not trivial. From the perspective of interpretation of germline CHEK2 variants, we review the current knowledge related to the structure of the CHEK2 gene, the function of CHK2 kinase, and the clinical significance of CHEK2 germline mutations in patients with hereditary breast, prostate, kidney, thyroid, and colon cancers.
Assuntos
Quinase do Ponto de Checagem 2/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Neoplasias/enzimologia , Neoplasias/genética , Animais , Quinase do Ponto de Checagem 2/química , Quinase do Ponto de Checagem 2/metabolismo , Humanos , Taxa de Mutação , Especificidade por SubstratoRESUMO
Cutaneous melanoma is the deadliest skin malignity with a rising prevalence worldwide. Patients carrying germline mutations in melanoma-susceptibility genes face an increased risk of melanoma and other cancers. To assess the spectrum of germline variants, we analyzed 264 Czech melanoma patients indicated for testing due to early melanoma (at <25 years) or the presence of multiple primary melanoma/melanoma and other cancer in their personal and/or family history. All patients were analyzed by panel next-generation sequencing targeting 217 genes in four groups: high-to-moderate melanoma risk genes, low melanoma risk genes, cancer syndrome genes, and other genes with an uncertain melanoma risk. Population frequencies were assessed in 1479 population-matched controls. Selected POT1 and CHEK2 variants were characterized by functional assays. Mutations in clinically relevant genes were significantly more frequent in melanoma patients than in controls (31/264; 11.7% vs. 58/1479; 3.9%; p = 2.0 × 10-6). A total of 9 patients (3.4%) carried mutations in high-to-moderate melanoma risk genes (CDKN2A, POT1, ACD) and 22 (8.3%) patients in other cancer syndrome genes (NBN, BRCA1/2, CHEK2, ATM, WRN, RB1). Mutations in high-to-moderate melanoma risk genes (OR = 52.2; 95%CI 6.6-413.1; p = 3.2 × 10-7) and in other cancer syndrome genes (OR = 2.3; 95%CI 1.4-3.8; p = 0.003) were significantly associated with melanoma risk. We found an increased potential to carry these mutations (OR = 2.9; 95%CI 1.2-6.8) in patients with double primary melanoma, melanoma and other primary cancer, but not in patients with early age at onset. The analysis revealed affected genes in Czech melanoma patients and identified individuals who may benefit from genetic testing and future surveillance management of mutation carriers.
RESUMO
Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in Ppm1dT/+ mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of Ppm1dT/+ mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in Ppm1dT/+Trp53+/- mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.
Assuntos
Apoptose , Linfoma/metabolismo , Proteína Fosfatase 2C/fisiologia , Timócitos/citologia , Timo , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Dano ao DNA , Reparo do DNA , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Induzidas por Radiação/metabolismo , Radiação Ionizante , Timócitos/metabolismo , Timo/citologia , Timo/metabolismoRESUMO
Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.
Assuntos
Dano ao DNA/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Linhagem Celular , Humanos , Fosforilação , Proteínas Supressoras de TumorRESUMO
Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates treatment and preventive strategies reducing OC mortality in mutation carriers. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We aimed to analyze 219 genes in 1333 Czech OC patients and 2278 population-matched controls using next-generation sequencing. We revealed germline mutations in 18 OC/BC predisposition genes in 32.0% of patients and in 2.5% of controls. Mutations in BRCA1/BRCA2, RAD51C/RAD51D, BARD1, and mismatch repair genes conferred high OC risk (OR > 5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR = 3.5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Our findings demonstrate the high proportion of patients with hereditary OC in Slavic population justifying genetic testing in all patients with OC, including BTO.
RESUMO
The most common histological subtypes of cutaneous melanoma include superficial spreading and nodular melanoma. However, the spectrum of somatic mutations developed in those lesions and all potential druggable targets have not yet been fully elucidated. We present the results of a sequence capture NGS analysis of 114 primary nodular and superficial spreading melanomas identifying driver mutations using biostatistical, immunohistochemical and/or functional approach. The spectrum and frequency of pathogenic or likely pathogenic variants were identified across 54 evaluated genes, including 59 novel mutations, and the newly identified TP53 loss-of-function mutations p.(L194P) and p.(R280K). Frequently mutated genes most commonly affected the MAPK pathway, followed by chromatin remodeling, and cell cycle regulation. Frequent aberrations were also detected in the genes coding for proteins involved in DNA repair and the regulation and modification of cellular tight junctions. Furthermore, relatively frequent mutations were described in KDR and MET, which represent potential clinically important targets. Those results suggest that with the development of new therapeutic possibilities, not only BRAF testing, but complex molecular testing of cutaneous melanoma may become an integral part of the decision process concerning the treatment of patients with melanoma.
Assuntos
Predisposição Genética para Doença/genética , Mutação com Perda de Função/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Ciclo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Reparo do DNA/genética , Feminino , Frequência do Gene/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/patologia , Junções Íntimas/genética , Proteína Supressora de Tumor p53/genética , Adulto Jovem , Melanoma Maligno CutâneoRESUMO
Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates cell response to genotoxic stress by negatively regulating the tumor suppressor p53 and other targets at chromatin. Mutations in the exon 6 of the PPM1D result in production of a highly stable, C-terminally truncated PPM1D. These gain-of-function PPM1D mutations are present in various human cancers but their role in tumorigenesis remains unresolved. Here we show that truncated PPM1D impairs activation of the cell cycle checkpoints in human non-transformed RPE cells and allows proliferation in the presence of DNA damage. Next, we developed a mouse model by introducing a truncating mutation in the PPM1D locus and tested contribution of the oncogenic PPM1DT allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1DT resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apcmin mice and diminished survival. Moreover, tumor organoids derived from colon of the ApcminPpm1dT/+ mice were less sensitive to 5-fluorouracil when compared to ApcminPpm1d+/+and the sensitivity to 5-fluorouracil was restored by inhibition of PPM1D. Finally, we screened colorectal cancer patients and identified recurrent somatic PPM1D mutations in a fraction of colon adenocarcinomas that are p53 proficient and show defects in mismatch DNA repair. In summary, we provide the first in vivo evidence that truncated PPM1D can promote tumor growth and modulate sensitivity to chemotherapy.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/tratamento farmacológico , Proteína Fosfatase 2C/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Éxons/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mutação/genéticaRESUMO
Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib.
Assuntos
Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína Fosfatase 2C/antagonistas & inibidores , Proteína Fosfatase 2C/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HEK293 , Recombinação Homóloga/genética , Humanos , Proteína Fosfatase 2C/genética , Pontos de Checagem da Fase S do Ciclo Celular , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Hereditary mutations in the CHEK2 gene (which encodes CHK2 kinase) contribute to a moderately increased risk of breast cancer (BC) and other cancers. Large variations in the frequency of CHEK2 mutations and the occurrence of variants of unknown clinical significance (VUS) complicate estimation of cancer risk in carriers of germline CHEK2 mutations. PATIENTS AND METHODS: We performed mutation analysis of 1,526 high-risk Czech BC patients and 3,360 Czech controls. Functional analysis was performed for identified VUS using a model system based on a human RPE1-CHEK2-KO cell line harboring biallelic inactivation of endogenous CHEK2. RESULTS: The frequency of ten truncating CHEK2 variants differed markedly between BC patients (2.26%) and controls (0.11%; p = 4.1 × 1012). We also found 23 different missense variants in 4.5% patients and in 4.0% of controls. The most common was p.I157T, which was found in patients and controls with the same frequency. Functional analysis identified nine functionally deleterious VUS, another nine functionally neutral VUS, and four intermediate VUS (including p.I157T). We found that carriers of truncating CHEK2 mutations had a high BC risk (OR 8.19; 95% CI 4.11-17.75), and that carriers of functionally deleterious missense variants had a moderate risk (OR 4.06; 95% CI, 1.37-13.39). Carriers of these mutations developed BC at 44.4 and 50.7 years, respectively. Functionally neutral and functionally intermediate missense variants did not increase the BC risk. BC in CHEK2 mutation carriers was frequently ER-positive and of higher grade. Notably, carriers of CHEK2 mutations developed second cancers more frequently than BRCA1/BRCA2/PALB2/p53 or mutation non-carriers. CONCLUSION: Hereditary CHEK2 mutations contribute to the development of hereditary BC. The associated cancer risk in mutation carriers increases with the number of affected individuals in a family. Annual follow-up with breast ultrasound, mammography, or magnetic resonance imaging is recommended for asymptomatic mutation carriers from the age of 40. Surgical prevention and specific follow-up of other tumors should be considered based on family cancer history. The work was supported by grants from the Czech Health Research Council of the Ministry of Health of the Czech Republic NR 15-28830A, 16-29959A, NV19-03-00279, projects of the PROGRES Q28/LF1, GAUK 762216, SVV2019 / 260367, PRIMUS/17/MED/9, UNCE/MED/016, Progress Q26, LQ1604 NPU II and project AVČR Qualitas. The analysis of a set of unselected controls was made possible by the existence and support of the scientific infrastructure of the National Center for Medical Genomics (LM2015091) and its project aimed at creating a reference database of genetic variants of the Czech Republic (CZ.02.1.01/0.0/0.0/16_013/0001634). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 4. 2019 Accepted: 14. 5. 2019.