f 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.T he two major high-penetrance breast cancer susceptibility genes BRCA1 and BRCA2 encode very large proteins with a critical function in homologous recombinational repair (HRR) of DNA double-strand breaks (15). PALB2 was discovered as a major BRCA2 binding partner that controls its intranuclear localization, stability, recombinational repair, and DNA damage checkpoint functions (38). Immediately after its discovery, germ line truncating mutations in PALB2 were identified in familial breast cancer (4,22,31) and the N subtype of Fanconi anemia (FA-N) (23, 37). Later, PALB2 was also found to be mutated in familial pancreatic cancer, being the second most highly mutated pancreatic susceptibility gene after BRCA2 (7,25). Furthermore, hypermethylation of the PALB2 promoter occurs in a significant fraction (ϳ7%) of both sporadic and familial breast/ovarian cancer cases (21). To date, dozens of truncating PALB2 mutations have been identified in cancer families around the world, causing moderate to very high risks of breast cancer (1,26,30). Finally, certain single nucleotide polymorphisms (SNPs) in the gene have been suggested to confer an increased risk of breast cancer (2).The PALB2 protein contains a coiled-coil domain at the N terminus and a series of WD repeats at its C terminus (Fig. 1A). The WD repeats together form a ␤-propeller structure that directly binds BRCA2 (18), and the N-terminal coiled-coil motif was later found to directly bind BRCA1 (28,41,42). Approximately 50% of PALB2 and 50% of BRCA2 are associated with each other in the cell with high affinity (28, 38). The stoichiometry of PALB2-BRCA1 binding appears to be much lower (28, 42). However, the interaction has proved crucial for HRR as several mutations generated in each protein that abrogate the interaction have been shown to greatly compromise repair efficiency (28,41,42). Furthermore, multiple naturally occurring, patient-derived missense variants that disrupt PALB2 binding have been identified in both BRCA1 and BRCA2, and all of them have been found to abrogate HRR (28, 38). Thus, PALB2 links BRCA1 and BRCA2 in HRR and breast cancer s...

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.

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 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.