Search

Immunoprecipitation of BRCA2 . List of proteins identified via mass spectrometry.

Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in BRCA2 confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the BRCA2 gene. In this study, we generated olaparib- and rucaparib-resistant BRCA2-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant BRCA2 allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of BRCA2 or DOT1L was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a BRCA2 reversion, mutation amplification of a mutant-carrying BRCA2 contributes to PARP inhibitor resistance.

Effect of mutating individual cysteine residues of DPP3 on its interaction with KEAP1.

mRNA levels of DPP3, NRF2, NQO1, GCLM, KEAP1 and p62 in MCF7 cell lines stably overexpressing wt and mutant DPP3 species.

Effect of mutating individual cysteine residues of KEAP1 on DPP3 binding.

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.

Effect of N-acetylcystein (NAC) on the DPP3-KEAP1 interaction.

High DPP3 mRNA expression correlates with poor relapse-free survival (RFS) of breast cancer patients in the Kaplan-Meier Plotter database.

Quantification of mRNA amount, qRT-PCR primer sequences, and transfection and immunoprecipitation methods.

Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in BRCA2 confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the BRCA2 gene. In this study, we generated olaparib- and rucaparib-resistant BRCA2-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant BRCA2 allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of BRCA2 or DOT1L was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a BRCA2 reversion, mutation amplification of a mutant-carrying BRCA2 contributes to PARP inhibitor resistance.

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., Tumor suppressor BRCA2 is known to stabilize and restart stalled DNA replication forks. Here the authors show that BRCA2 is recruited to the replication fork through its interaction with MCM10 and inhibits Primase-Polymerase-mediated repriming, lesion bypass and single strand DNA gap formation after DNA damage.

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.

PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.

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 hydrogen peroxide-induced NRF2 nuclear accumulation in the estrogen receptor (ER)-positive MCF7 breast cancer cells. Finally, we present evidence 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.

BackgroundHeterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR), DNA double strand break (DSB) repair and cell cycle regulation following DNA damage.MethodsPALB2 nucleoprotein complexes were isolated using tandem affinity purification from nuclease-solubilized nuclear fraction. Immunofluorescence was used for localization studies of proteins. siRNA-mediated gene silencing and flow cytometry were used for studying DNA repair efficiency and cell cycle distribution/checkpoints. The effect of hnRNP C on mRNA abundance was assayed using quantitative reverse transcriptase PCR.Results and significanceWe identified hnRNP C as a component of a nucleoprotein complex containing breast cancer suppressor proteins PALB2, BRCA2 and BRCA1. Notably, other components of the 40S ribonucleoprotein particle were not present in the complex. hnRNP C was found to undergo significant changes of sub-nuclear localization after ionizing radiation (IR) and to partially localize to DNA damage sites. Depletion of hnRNP C substantially altered the normal balance of repair mechanisms following DSB induction, reducing HR usage in particular, and impaired S phase progression after IR. Moreover, loss of hnRNP C strongly reduced the abundance of key HR proteins BRCA1, BRCA2, RAD51 and BRIP1, which can be attributed, at least in part, to the downregulation of their mRNAs due to aberrant splicing. Our results establish hnRNP C as a key regulator of BRCA gene expression and HR-based DNA repair. They also suggest the existence of an RNA regulatory program at sites of DNA damage, which involves a unique function of hnRNP C that is independent of the 40S ribonucleoprotein particles and most other hnRNP proteins.

The BRCA2 (Breast Cancer 2, early onset) gene is implicated in a variety of familial cancers. Loss of BRCA2 function results in severe defects in DNA double-strand break repair, DNA damage-induced checkpoint response and the stability of stalled DNA replication forks, all of which are important for genomic stability. About 50% of BRCA2 is associated with PALB2 (partner and localizer of BRCA2), which anchors BRCA2 onto chromatin and directs its recruitment to DNA damage sites. By tandem affinity purification of PALB2 we identified MCM10, an essential DNA replication factor, as a component of the PALB2/BRCA2 complex. MCM10 promotes initiation of DNA replication through binding to MCM2-7 helicase complex and recruiting the initial DNA polymerase, pol α, to replication origins. Interestingly, we found that MCM10 binds to BRCA2 instead of PALB2 in the complex. Cells depleted of BRCA2 or MCM10 showed similar instability of stalled replication forks, indicating possible cooperation between the two proteins in fork stabilization following replication stress. Moreover, DNA damage-induced CHK1 activation is markedly reduced in both BRCA2- and MCM10-depleted cells even though RPA (replication protein A) is hyperphosphorylated. Our findings suggest that BRCA2 checkpoint function may be mediated through MCM10 and their interaction may be important for stalled replication fork stability. Domain mapping experiments showed that an evolutionarily conserved coiled-coil motif in the N-terminus of MCM10 which is required for BRCA2 binding and that BRCA2 has at least two MCM10-binding sites, both in its central region. BRCA2 and MCM10 mutants lacking these interaction domains will be used to further test the functional relevance of their interaction. Citation Format: Allen L. Alcivar, Jianglin Ma, Bing Xia. BRCA2 interacts with an essential replication factor, MCM10. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1779. doi:10.1158/1538-7445.AM2013-1779