RESUMEN
Carriers of BRCA1 germline pathogenic variants are at substantially higher risk of developing breast and ovarian cancer than the general population. Accurate identification of at-risk individuals is crucial for risk stratification and the implementation of targeted preventive and therapeutic interventions. Despite significant progress in variant classification efforts, a sizable portion of reported BRCA1 variants remain as variants of uncertain clinical significance (VUSs). Variants leading to premature protein termination and loss of essential functional domains are typically classified as pathogenic. However, the impact of frameshift variants that result in an extended incorrect terminus is not clear. Using validated functional assays, we conducted a systematic functional assessment of 17 previously reported BRCA1 extended incorrect terminus variants (EITs) and concluded that 16 constitute loss-of-function variants. This suggests that most EITs are likely to be pathogenic. However, one variant, c.5578dup, displayed a protein expression level, affinity to known binding partners, and activity in transcription and homologous recombination assays comparable to the wild-type BRCA1 protein. Twenty-three additional carriers of c.5578dup were identified at a US clinical diagnostic lab and assessed using a family history likelihood model providing, in combination with the functional data, a likely benign interpretation. These results, consistent with family history data in the current study and available data from ClinVar, indicate that most, but not all, BRCA1 variants leading to an extended incorrect terminus constitute loss-of-function variants and underscore the need for comprehensive assessment of individual variants.
Asunto(s)
Predisposición Genética a la Enfermedad , Neoplasias Ováricas , Femenino , Humanos , Proteína C , Proteína BRCA1/genética , Neoplasias Ováricas/epidemiología , Mutación de Línea Germinal/genéticaRESUMEN
BACKGROUND AND AIMS: BRCA1 (BRCA1 DNA repair associated) and PALB2 (partner and localizer of BRCA2) interact with each other to promote homologous recombination and DNA double-strand breaks repair. The disruption of this interaction has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. APPROACH AND RESULTS: We demonstrated that mice with disrupted BRCA1-PALB2 interaction were more susceptible to HCC than wild-type mice. HCC tumors arising from these mice showed plenty of T-lymphocyte infiltration and a better response to programmed cell death 1 (PD-1) antibody treatment. Mechanistically, disruption of the BRCA1-PALB2 interaction causes persistent high level of DNA damage in HCC cells, leading to activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in both malignant hepatocytes and M1 macrophages in the tumor microenvironment. The activated cGAS-STING pathway induces programmed cell death 1 ligand 1 expression via the STING-interferon regulatory factor 3 (IRF3)-signal transducer and activator of transcription 1 pathway, causing immunosuppression to facilitate tumorigenesis and tumor progression. Meanwhile, M1 macrophages with an activated cGAS-STING pathway could recruit T lymphocytes through the STING-IRF3 pathway, leading to T-lymphocyte infiltration in tumors. After normalizing immune responses by PD-1 antibody treatment, the infiltrating T lymphocytes attack tumor cells rapidly and effectively. CONCLUSIONS: This study reveals that persistent DNA damage caused by a defective BRCA pathway induces tumor immunosuppression and T-lymphocyte infiltration in HCC through the cGAS-STING pathway, providing insight into tumor immune microenvironment remodeling that may help improve HCC response to PD-1 antibody treatment.
Asunto(s)
Proteína BRCA1 , Carcinoma Hepatocelular , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Neoplasias Hepáticas , Animales , Ratones , Carcinogénesis , Carcinoma Hepatocelular/inmunología , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Terapia de Inmunosupresión , Neoplasias Hepáticas/inmunología , Nucleotidiltransferasas/metabolismo , Receptor de Muerte Celular Programada 1 , Linfocitos T , Microambiente Tumoral , Proteína BRCA1/metabolismoRESUMEN
The BRCA1-PALB2-BRCA2 axis plays essential roles in the cellular response to DNA double-strand breaks (DSB), maintenance of genome integrity, and suppression of cancer development. Upon DNA damage, BRCA1 is recruited to DSBs, where it facilitates end resection and recruits PALB2 and its associated BRCA2 to load the central recombination enzyme RAD51 to initiate homologous recombination (HR) repair. In recent years, several BRCA1-independent mechanisms of PALB2 recruitment have also been reported. Collectively, these available data illustrate a series of hierarchical, context-dependent, and cooperating mechanisms of PALB2 recruitment that is critical for HR and therapy response either in the presence or absence of BRCA1. Here, we review these BRCA1-dependent and independent mechanisms and their importance in DSB repair, cancer development, and therapy. As BRCA1-mutant cancer cells regain HR function, for which PALB2 is generally required, and become resistant to targeted therapies, such as PARP inhibitors, targeting BRCA1-independent mechanisms of PALB2 recruitment represents a potential new avenue to improve treatment of BRCA1-mutant tumors.
Asunto(s)
Neoplasias , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , ADN , Daño del ADN , Reparación del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.
Asunto(s)
Proteína BRCA2/genética , ADN Primasa/genética , ADN de Neoplasias/genética , ADN de Cadena Simple/genética , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Enzimas Multifuncionales/genética , Reparación del ADN por Recombinación , Proteína BRCA2/antagonistas & inhibidores , Proteína BRCA2/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Daño del ADN , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Primasa/antagonistas & inhibidores , ADN Primasa/metabolismo , Replicación del ADN , ADN de Neoplasias/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Proteínas de Mantenimiento de Minicromosoma/antagonistas & inhibidores , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Enzimas Multifuncionales/antagonistas & inhibidores , Enzimas Multifuncionales/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purificationmass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Espectrometría de Masas , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Purificación por Afinidad en TándemRESUMEN
Our group described the oncogenic potential of a normal neuronal receptor, metabotropic glutamate receptor 1 (GRM1/mGluR1, gene/protein), when aberrantly expressed in melanocytes led to cell transformation in vitro and spontaneous metastatic tumors in vivo. Earlier, we demonstrated the accumulation of phosphorylated histone H2AX (γH2AX), a marker for DNA damage when mGluR1-expressing melanoma cells were treated with a functional inhibitor, riluzole. The precise mechanisms on how riluzole induces DNA damage in these cells are unknown. In an attempt to begin to identify possible DNA repair pathways that may be involved in riluzole-induced DNA damage, we took advantage of specific inhibitors to two well-known DNA repair pathways, homologous recombination and nonhomologous end joining (NHEJ) repair pathways. Using flow cytometry and a fluorescent antibody to γH2AX, our results demonstrate that NHEJ is likely to be the preferred DNA repair pathway to restore DNA double-stranded breaks induced by riluzole in mGluR1-expressing melanoma cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Melanoma , Riluzol/farmacología , Neoplasias Cutáneas , Línea Celular Tumoral , HumanosRESUMEN
PURPOSE: Inherited pathogenic variants in PALB2 are associated with increased risk of breast and pancreatic cancer. However, the functional and clinical relevance of many missense variants of uncertain significance (VUS) identified through clinical genetic testing is unclear. The ability of patient-derived germline missense VUS to disrupt PALB2 function was assessed to identify variants with potential clinical relevance. METHODS: The influence of 84 VUS on PALB2 function was evaluated using a cellular homology directed DNA repair (HDR) assay and VUS impacting activity were further characterized using secondary functional assays. RESULTS: Four (~5%) variants (p.L24S,c.71T>C; p.L35P,c.104T>C; pI944N,c.2831T>A; and p.L1070P,c.3209T>C) disrupted PALB2-mediated HDR activity. These variants conferred sensitivity to cisplatin and a poly(ADP-ribose) polymerase (PARP) inhibitor and reduced RAD51 foci formation in response to DNA damage. The p.L24S and p.L35P variants disrupted BRCA1-PALB2 protein complexes, p.I944N was associated with protein instability, and both p.I944N and p.L1070P mislocalized PALB2 to the cytoplasm. CONCLUSION: These findings show that the HDR assay is an effective method for screening the influence of inherited variants on PALB2 function, that four missense variants impact PALB2 function and may influence cancer risk and response to therapy, and suggest that few inherited PALB2 missense variants disrupt PALB2 function in DNA repair.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Recombinasa Rad51/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , Femenino , Factor de Transcripción GATA3/genética , Predisposición Genética a la Enfermedad , Humanos , Mutación Missense/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Reparación del ADN por Recombinación/genéticaRESUMEN
Triple negative breast cancer (TNBC) is an aggressive subset for which effective therapeutic approaches are needed. A significant proportion of TNBC patients harbor either germline or somatic mutations in BRCA1, or epigenetic silencing of BRCA1, which renders them deficient in DNA repair. Virtually all BRCA1 deficient breast cancers harbor mutations in TP53 suggesting that inactivation of p53 is a requirement for tumor progression in the setting of BRCA1 deficiency. Due to this dependency, we hypothesized that restoring wild type p53 function in BRCA1 deficient breast cancer would be therapeutic. The majority of TP53 mutations are missense, which generate a defective protein that potentially can be targeted with small molecules. Zinc metallochaperones (ZMCs) are a new class of anti-cancer drugs that specifically reactivate zinc-deficient mutant p53 by restoring zinc binding. Using ZMC1 in human breast cancer cell lines expressing the zinc deficient p53R175H, we demonstrate that loss of BRCA1 sensitizes cells to mutant p53 reactivation. Using murine breast cancer models with Brca1 deficiency, we demonstrate that ZMC1 significantly improves survival of mice bearing tumors harboring the zinc-deficient Trp53 R172H allele but not the Trp53 -/- allele. We synthesized a new formulation of ZMC1 (Zn-1), in which the drug is made in complex with zinc to improve zinc delivery, and demonstrate that Zn-1 has increased efficacy. Furthermore, we show that ZMC1 plus olaparib is a highly effective combination for p53R172H tumor growth inhibition. In conclusion, we have validated preclinically a new therapeutic approach for BRCA1 deficient breast cancer through reactivation of mutant p53.
RESUMEN
The G2/M checkpoint inhibits mitotic entry upon DNA damage, thereby preventing segregation of broken chromosomes and preserving genome stability. The tumor suppressor proteins BRCA1, PALB2 and BRCA2 constitute a BRCA1-PALB2-BRCA2 axis that is essential for homologous recombination (HR)-based DNA doublestrand break repair. Besides HR, BRCA1 has been implicated in both the initial activation and the maintenance of the G2/M checkpoint, while BRCA2 and PALB2 have been shown to be critical for its maintenance. Here we show that all three proteins can play a significant role in both checkpoint activation and checkpoint maintenance, depending on cell type and context, and that PALB2 links BRCA1 and BRCA2 in the checkpoint response. The BRCA1-PALB2 interaction can be important for checkpoint activation, whereas the PALB2-BRCA2 complex formation appears to be more critical for checkpoint maintenance. Interestingly, the function of PALB2 in checkpoint response appears to be independent of CHK1 and CHK2 phosphorylation. Following ionizing radiation, cells with disengaged BRCA1-PALB2 interaction show greatly increased chromosomal abnormalities due apparently to combined defects in HR and checkpoint control. These findings provide new insights into DNA damage checkpoint control and further underscore the critical importance of the proper cooperation of the BRCA and PALB2 proteins in genome maintenance.
Asunto(s)
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Animales , Proteína BRCA1/genética , Proteína BRCA2/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa de Punto de Control 2/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Células HCT116 , Células HEK293 , Humanos , Ratones , Fosforilación , Reparación del ADN por RecombinaciónRESUMEN
BRCA1 plays a critical role in homology-directed repair (HDR) of DNA double strand breaks, and the repair defect of BRCA1-mutant cancer cells is being targeted with platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors. We have employed relatively simple and sensitive assays to determine the function of BRCA1 variants or mutants in two HDR mechanisms, homologous recombination (HR) and single strand annealing (SSA), and in conferring resistance to cisplatin and olaparib in human cancer cells. Our results define the functionality of the top 22 patient-derived BRCA1 missense variants and the contribution of different domains of BRCA1 and its E3 ubiquitin ligase activity to HDR and drug resistance. Importantly, our results also demonstrate that the BRCA1-PALB2 interaction dictates the choice between HR and SSA. These studies establish functional and mutational landscapes of BRCA1 for HDR and therapy resistance, while revealing novel insights into BRCA1 regulatory mechanisms and HDR pathway choice.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Antineoplásicos/metabolismo , Emparejamiento Base , Línea Celular Tumoral , Cisplatino/metabolismo , Resistencia a Antineoplásicos , Recombinación Homóloga , Humanos , Ftalazinas/metabolismo , Piperazinas/metabolismoRESUMEN
NRF2 is a transcription factor serving as a master regulator of the expression of many genes involved in cellular responses to oxidative and other stresses. In the absence of stress, NRF2 is constantly synthesized but maintained at low levels as it is targeted by KEAP1 for ubiquitination and proteasome-mediated degradation. NRF2 binds KEAP1 mainly through a conserved "ETGE" motif that has also been found in several other proteins, such as DPP3, which has been shown to bind KEAP1 and enhance NRF2 function upon overexpression. Here we demonstrate the interaction between endogenous DPP3 and endogenous KEAP1. We further show that the DPP3-KEAP1 interaction is strongly induced by hydrogen peroxide and that DPP3 is required for timely NRF2 induction and nuclear accumulation in the estrogen receptor (ER)-positive MCF7 breast cancer cells. Moreover, we present evidence that the binding of DPP3 to KEAP1 stabilizes the latter. Finally, we show that DPP3 is overexpressed in breast cancer and that elevated levels of DPP3 mRNA correlate with increased NRF2 downstream gene expression and poor prognosis, particularly for ER-positive breast cancer. Our studies reveal novel insights into the regulation of NRF2 and identify DPP3 and an NRF2 transcriptional signature as potential biomarkers for breast cancer prognosis and treatment. Cancer Res; 77(11); 2881-92. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias de la Mama/mortalidad , Supervivencia Celular , Femenino , Células HeLa , Humanos , Células MCF-7 , Estrés Oxidativo , Transducción de Señal , Análisis de Supervivencia , TransfecciónRESUMEN
ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Recombinación , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Transducción de Señal , Factores de Tiempo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.