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1.
Arch Toxicol ; 95(10): 3285-3302, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34480604

RESUMO

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

2.
Cancer Res ; 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548335

RESUMO

The BRCA1 tumor suppressor gene encodes a multi-domain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks (DSB), which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathological features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR-incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer.

3.
Mutagenesis ; 2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34448005

RESUMO

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein (GFP) reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolization and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g., 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.

4.
Biochem Pharmacol ; 190: 114591, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33957093

RESUMO

Drug-induced liver injury (DILI) is the most prevalent adversity encountered in drug development and clinical settings leading to urgent needs to understand the underlying mechanisms. In this study, we have systematically investigated the dynamics of the activation of cellular stress response pathways and cell death outcomes upon exposure of a panel of liver toxicants using live cell imaging of fluorescent reporter cell lines. We established a comprehensive temporal dynamic response profile of a large set of BAC-GFP HepG2 cell lines representing the following components of stress signaling: i) unfolded protein response (UPR) [ATF4, XBP1, BIP and CHOP]; ii) oxidative stress [NRF2, SRXN1, HMOX1]; iii) DNA damage [P53, P21, BTG2, MDM2]; and iv) NF-κB pathway [A20, ICAM1]. We quantified the single cell GFP expression as a surrogate for endogenous protein expression using live cell imaging over > 60 h upon exposure to 14 DILI compounds at multiple concentrations. Using logic-based ordinary differential equation (Logic-ODE), we modelled the dynamic profiles of the different stress responses and extracted specific descriptors potentially predicting the progressive outcomes. We identified the activation of ATF4-CHOP axis of the UPR as the key pathway showing the highest correlation with cell death upon DILI compound perturbation. Knocking down main components of the UPR provided partial protection from compound-induced cytotoxicity, indicating a complex interplay among UPR components as well as other stress pathways. Our results suggest that a systematic analysis of the temporal dynamics of ATF4-CHOP axis activation can support the identification of DILI risk for new candidate drugs.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Modelos Biológicos , Estresse Oxidativo/fisiologia , Análise de Célula Única/métodos , Fator de Transcrição CHOP/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Previsões , Células Hep G2 , Humanos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/fisiologia
5.
Toxicol Sci ; 181(2): 187-198, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33769548

RESUMO

Estrogen receptor alpha (ERα) belongs to the nuclear hormone receptor family of ligand-inducible transcription factors and regulates gene networks in biological processes such as cell growth and proliferation. Disruption of these networks by chemical compounds with estrogenic activity can result in adverse outcomes such as unscheduled cell proliferation, ultimately culminating in tumor formation. To distinguish disruptive activation from normal physiological responses, it is essential to quantify relationships between different key events leading to a particular adverse outcome. For this purpose, we established fluorescent protein MCF7 reporter cell lines for ERα-induced proliferation by bacterial artificial chromosome-based tagging of 3 ERα target genes: GREB1, PGR, and TFF1. These target genes are inducible by the non-genotoxic carcinogen and ERα agonist 17ß-estradiol in an ERα-dependent manner and are essential for ERα-dependent cell-cycle progression and proliferation. The 3 GFP reporter cell lines were characterized in detail and showed different activation dynamics upon exposure to 17ß-estradiol. In addition, they demonstrated specific activation in response to other established reference estrogenic compounds of different potencies, with similar sensitivities as validated OECD test methods. This study shows that these fluorescent reporter cell lines can be used to monitor the spatial and temporal dynamics of ERα pathway activation at the single-cell level for more mechanistic insight, thereby allowing a detailed assessment of the potential carcinogenic activity of estrogenic compounds in humans.


Assuntos
Receptor alfa de Estrogênio , Estrogênios , Carcinógenos , Linhagem Celular Tumoral , Estradiol/toxicidade , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio , Estrogênios/toxicidade , Humanos
6.
Cell Rep ; 32(8): 108068, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32846126

RESUMO

Using genome-wide radiogenetic profiling, we functionally dissect vulnerabilities of cancer cells to ionizing radiation (IR). We identify ERCC6L2 as a major determinant of IR response, together with classical DNA damage response genes and members of the recently identified shieldin and CTC1-STN1-TEN1 (CST) complexes. We show that ERCC6L2 contributes to non-homologous end joining (NHEJ), and it may exert this function through interactions with SFPQ. In addition to causing radiosensitivity, ERCC6L2 loss restores DNA end resection and partially rescues homologous recombination (HR) in BRCA1-deficient cells. As a consequence, ERCC6L2 deficiency confers resistance to poly (ADP-ribose) polymerase (PARP) inhibition in tumors deficient for both BRCA1 and p53. Moreover, we show that ERCC6L2 mutations are found in human tumors and correlate with a better overall survival in patients treated with radiotherapy (RT); this finding suggests that ERCC6L2 is a predictive biomarker of RT response.


Assuntos
Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Helicases/metabolismo , Animais , Humanos , Camundongos
7.
Clin Cancer Res ; 26(17): 4559-4568, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32546644

RESUMO

PURPOSE: Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway. EXPERIMENTAL DESIGN: Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families. RESULTS: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants. CONCLUSIONS: We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.

9.
Clin Cancer Res ; 25(14): 4351-4362, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31036541

RESUMO

PURPOSE: BRCA1-deficient breast cancers carry a specific DNA copy-number signature ("BRCA1-like") and are hypersensitive to DNA double-strand break (DSB) inducing compounds. Here, we explored whether (i) EZH2 is overexpressed in human BRCA1-deficient breast tumors and might predict sensitivity to DSB-inducing drugs; (ii) EZH2 inhibition potentiates cisplatin efficacy in Brca1-deficient murine mammary tumors. EXPERIMENTAL DESIGN: EZH2 expression was analyzed in 497 breast cancers using IHC or RNA sequencing. We classified 370 tumors by copy-number profiles as BRCA1-like or non-BRCA1-like and examined its association with EZH2 expression. Additionally, we assessed BRCA1 loss through mutation or promoter methylation status and investigated the predictive value of EZH2 expression in a study population of breast cancer patients treated with adjuvant high-dose platinum-based chemotherapy compared with standard anthracycline-based chemotherapy. To explore whether EZH2 inhibition by GSK126 enhances sensitivity to platinum drugs in EZH2-overexpressing breast cancers we used a Brca1-deficient mouse model. RESULTS: The highest EZH2 expression was found in BRCA1-associated tumors harboring a BRCA1 mutation, BRCA1-promoter methylation or were classified as BRCA1 like. We observed a greater benefit from high-dose platinum-based chemotherapy in BRCA1-like and non-BRCA1-like patients with high EZH2 expression. Combined treatment with the EZH2 inhibitor GSK126 and cisplatin decreased cell proliferation and improved survival in Brca1-deficient mice in comparison with single agents. CONCLUSIONS: Our findings demonstrate that EZH2 is expressed at significantly higher levels in BRCA1-deficient breast cancers. EZH2 overexpression can identify patients with breast cancer who benefit significantly from intensified DSB-inducing platinum-based chemotherapy independent of BRCA1-like status. EZH2 inhibition improves the antitumor effect of platinum drugs in Brca1-deficient breast tumors in vivo.


Assuntos
Proteína BRCA1/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias Mamárias Animais/tratamento farmacológico , Platina/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Taxa de Sobrevida , Resultado do Tratamento
10.
Cancer Discov ; 9(6): 722-737, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31015319

RESUMO

Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T-cell infiltration and activation in vivo, and that CD8+ T-cell depletion severely compromises antitumor efficacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared with HR-proficient TNBC cells and in vivo models. CRISPR-mediated knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T-cell infiltration in vivo. These findings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. SIGNIFICANCE: This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8+ T-cell recruitment and antitumor efficacy. The data provide insight into the mechanism of action of PARP inhibitors in BRCA-associated breast cancer.This article is highlighted in the In This Issue feature, p. 681.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/etiologia , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Biomarcadores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
11.
Cancer Res ; 79(3): 452-460, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30530501

RESUMO

The defect in homologous recombination (HR) found in BRCA1-associated cancers can be therapeutically exploited by treatment with DNA-damaging agents and PARP inhibitors. We and others previously reported that BRCA1-deficient tumors are initially hypersensitive to the inhibition of topoisomerase I/II and PARP, but acquire drug resistance through restoration of HR activity by the loss of end-resection antagonists of the 53BP1/RIF1/REV7/Shieldin/CST pathway. Here, we identify radiotherapy as an acquired vulnerability of 53BP1;BRCA1-deficient cells in vitro and in vivo. In contrast to the radioresistance caused by HR restoration through BRCA1 reconstitution, HR restoration by 53BP1 pathway inactivation further increases radiosensitivity. This highlights the relevance of this pathway for the repair of radiotherapy-induced damage. Moreover, our data show that BRCA1-mutated tumors that acquire drug resistance due to BRCA1-independent HR restoration can be targeted by radiotherapy. SIGNIFICANCE: These findings uncover radiosensitivity as a novel, therapeutically viable vulnerability of BRCA1-deficient mouse mammary cells that have acquired drug resistance due to the loss of the 53BP1 pathway.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Proteínas Supressoras de Tumor/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Proteína BRCA1 , Proteínas de Ciclo Celular/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Recombinação Homóloga/genética , Humanos , Proteínas Mad2/genética , Camundongos , Neoplasias/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Tolerância a Radiação/genética , Proteínas de Ligação a Telômeros/genética
12.
J Pathol ; 246(1): 41-53, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29877575

RESUMO

Hereditary breast cancers in BRCA1 mutation carriers are mostly estrogen receptor α (ERα)-negative and progesterone receptor (PR)-negative; however, hormone depletion via bilateral oophorectomy does result in a marked reduction in breast cancer risk, suggesting that BRCA1-associated breast tumorigenesis is dependent on hormone signaling. We used geneticaly engineered mouse models to determine the individual influences of ERα and PR signaling on the development of BRCA1-deficient breast cancer. In line with the human data, BRCA1-deficient mouse mammary tumors are ERα-negative, and bilateral ovariectomy leads to abrogation of mammary tumor development. Hormonal replacement experiments in ovariectomized mice showed that BRCA1-deficient mammary tumor formation is promoted by estrogen but not by progesterone. In line with these data, mammary tumorigenesis was significantly delayed by the selective ERα downregulator fulvestrant, but not by the selective PR antagonist Org33628. Together, our results illustrate that BRCA1-associated tumorigenesis is dependent on estrogen signaling rather than on progesterone signaling, and call into question the utility of PR antagonists as a tumor prevention strategy for BRCA1 mutation carriers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Carcinoma in Situ/induzido quimicamente , Transformação Celular Neoplásica/induzido quimicamente , Estradiol/toxicidade , Terapia de Reposição de Estrogênios/efeitos adversos , Neoplasias Mamárias Experimentais/induzido quimicamente , Progesterona/toxicidade , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Animais , Proteína BRCA1 , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Implantes de Medicamento , Estradiol/administração & dosagem , Estrenos/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Feminino , Fulvestranto/farmacologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos da Linhagem 129 , Camundongos Transgênicos , Ovariectomia , Progesterona/administração & dosagem , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Proteínas Supressoras de Tumor/deficiência
13.
EMBO Mol Med ; 9(10): 1398-1414, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28729482

RESUMO

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.


Assuntos
Acetaldeído/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Rad51 Recombinase/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos , Recombinação Homóloga , Humanos , Camundongos , Camundongos Nus , Rad51 Recombinase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Nat Commun ; 8: 15981, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28714471

RESUMO

Mutations in homologous recombination (HR) genes BRCA1 and BRCA2 predispose to tumorigenesis. HR-deficient cancers are hypersensitive to Poly (ADP ribose)-polymerase (PARP) inhibitors, but can acquire resistance and relapse. Mechanistic understanding how PARP inhibition induces cytotoxicity in HR-deficient cancer cells is incomplete. Here we find PARP inhibition to compromise replication fork stability in HR-deficient cancer cells, leading to mitotic DNA damage and consequent chromatin bridges and lagging chromosomes in anaphase, frequently leading to cytokinesis failure, multinucleation and cell death. PARP-inhibitor-induced multinucleated cells fail clonogenic outgrowth, and high percentages of multinucleated cells are found in vivo in remnants of PARP inhibitor-treated Brca2-/-;p53-/- and Brca1-/-;p53-/- mammary mouse tumours, suggesting that mitotic progression promotes PARP-inhibitor-induced cell death. Indeed, enforced mitotic bypass through EMI1 depletion abrogates PARP-inhibitor-induced cytotoxicity. These findings provide insight into the cytotoxic effects of PARP inhibition, and point at combination therapies to potentiate PARP inhibitor treatment of HR-deficient tumours.


Assuntos
Anáfase/efeitos dos fármacos , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Citocinese/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Experimentais/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mitose/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo de DNA por Recombinação/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
15.
J Pathol ; 241(4): 511-521, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27943283

RESUMO

Women with heterozygous germline mutations in the BRCA1 tumour suppressor gene are strongly predisposed to developing early-onset breast cancer through loss of the remaining wild-type BRCA1 allele and inactivation of TP53. Although tumour prevention strategies in BRCA1-mutation carriers are still limited to prophylactic surgery, several therapeutic strategies have been developed to target the DNA repair defects (also known as 'BRCAness') of BRCA1-deficient tumours. In particular, DNA-damaging agents such as platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors show strong activity against BRCA1-mutated tumours. However, it is unclear whether drugs that target BRCAness can also be used to prevent tumour formation in BRCA1-mutation carriers, especially as loss of wild-type BRCA1 may not be the first event in BRCA1-associated tumourigenesis. We performed prophylactic treatments in a genetically engineered mouse model in which de novo development of BRCA1-deficient mammary tumours is induced by stochastic loss of BRCA1 and p53. We found that prophylactic window therapy with nimustine, cisplatin or olaparib reduced the amount and size of mammary gland lesions, and significantly increased the median tumour latency. Similar results were obtained with intermittent prophylactic treatment with olaparib. Importantly, prophylactic window therapy with nimustine and cisplatin resulted in an increased fraction of BRCA1-proficient mammary tumours, suggesting selective survival and malignant transformation of BRCA1-proficient lesions upon prophylactic treatment with DNA-damaging agents. Prophylactic therapy with olaparib significantly prolonged mammary tumour-free survival without any significant increase in the fraction of BRCA1-proficient tumours, warranting the evaluation of this PARP inhibitor in prophylactic trials in BRCA1-mutation carriers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Mamárias Animais/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Supressoras de Tumor/genética , Animais , Antineoplásicos/uso terapêutico , Proteína BRCA1 , Cisplatino/farmacologia , Reparo do DNA , Modelos Animais de Doenças , Feminino , Mutação em Linhagem Germinativa , Humanos , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/prevenção & controle , Camundongos , Nimustina/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Supressoras de Tumor/metabolismo
16.
J Clin Invest ; 126(8): 2903-18, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27454287

RESUMO

Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , Deleção de Genes , Neoplasias Mamárias Animais/genética , Alelos , Animais , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Cruzamentos Genéticos , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Efeito Fundador , Mutação da Fase de Leitura , Engenharia Genética , Humanos , Masculino , Neoplasias Mamárias Animais/tratamento farmacológico , Camundongos , Mutação , Transplante de Neoplasias , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Recombinação Genética
17.
Cancer Res ; 76(9): 2778-90, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27197267

RESUMO

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778-90. ©2016 AACR.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/genética , Processamento Alternativo/genética , Animais , Proteína BRCA1/metabolismo , Western Blotting , Cisplatino/farmacologia , Feminino , Imunofluorescência , Mutação em Linhagem Germinativa , Humanos , Imuno-Histoquímica , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Nature ; 521(7553): 541-544, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25799992

RESUMO

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas Mad2/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Reparo de DNA por Recombinação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/metabolismo , Humanos , Switching de Imunoglobulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mad2/deficiência , Proteínas Mad2/genética , Camundongos , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/metabolismo
20.
EMBO J ; 34(3): 410-24, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25582120

RESUMO

Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.


Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , Telômero/metabolismo , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Autoantígeno Ku , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Telômero/genética , Proteínas de Xenopus
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