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1.  PALB2: THE HUB OF A NETWORK OF TUMOR SUPPRESSORS INVOLVED IN DNA DAMAGE RESPONSES 
Biochimica et biophysica acta  2014;1846(1):263-275.
PALB2 was first identified as a partner of BRCA2 that mediates its recruitment to sites of DNA damage. PALB2 was subsequently found as a tumor suppressor gene. Inherited heterozygosity for this gene is associated with an increased risk of cancer of the breast and other sites. Additionally, biallelic mutation of PALB2 is linked to Fanconi anemia, which also has an increased risk of developing malignant disease. Recent work has identified numerous interactions of PALB2, suggesting that it functions in a network of proteins encoded by tumor suppressors. Notably, many of these tumor suppressors are related to the cellular response to DNA damage. The recruitment of PALB2 to DNA double-strand breaks at the head of this network is via a ubiquitin-dependent signaling pathway that involves the RAP80, Abraxas and BRCA1 tumor suppressors. Next, PALB2 interacts with BRCA2, which is a tumor suppressor, and with the RAD51 recombinase. These interactions promote DNA repair by homologous recombination (HR). More recently, PALB2 has been found to bind the RAD51 paralog, RAD51C, as well as the translesion polymerase pol η, both of which are tumor suppressors with functions in HR. Further, an interaction with MRG15, which is related to chromatin regulation, may facilitate DNA repair in damaged chromatin. Finally, PALB2 interacts with KEAP1, a regulator of the response to oxidative stress. The PALB2 network appears to mediate the maintenance of genome stability, may explain the association of many of the corresponding genes with similar spectra of tumors, and could present novel therapeutic opportunities.
doi:10.1016/j.bbcan.2014.06.003
PMCID: PMC4183126  PMID: 24998779
PALB2; tumor suppressor; DNA repair; homologous recombination; genome stability; breast cancer
2.  BREAST CANCER-ASSOCIATED MISSENSE MUTANTS OF THE PALB2 WD40 DOMAIN, WHICH DIRECTLY BINDS RAD51C, RAD51 AND BRCA2, DISRUPT DNA REPAIR 
Oncogene  2013;33(40):4803-4812.
Heterozygous carriers of germ-line mutations in the BRCA2/FANCD1, PALB2/FANCN, and RAD51C/FANCO DNA repair genes have an increased life-time risk to develop breast, ovarian and other cancers; bi-allelic mutations in these genes clinically manifest as Fanconi anemia (FA). Here, we demonstrate that RAD51C is part of a novel protein complex that contains PALB2 and BRCA2. Further, the PALB2 WD40 domain can directly and independently bind RAD51C and BRCA2. To understand the role of these homologous recombination (HR) proteins in DNA repair, we functionally characterize effects of missense mutations of the PALB2 WD40 domain that have been reported in breast cancer patients. In contrast to large truncations of PALB2, which display a complete loss of interaction, the L939W, T1030I, and L1143P missense mutants/variants of PALB2 WD40 domain are associated with altered direct binding patterns to the RAD51C, RAD51 and BRCA2 HR proteins in biochemical assays. Further, the T1030I missense mutant is unstable, while the L939W and L1143P proteins are stable but partially disrupt the PALB2-RAD51C-BRCA2 complex in cells. Functionally, the L939W and L1143P mutants display a decreased capacity for DNA double-strand break-induced HR and an increased cellular sensitivity to ionizing radiation. As further evidence for the functional importance of the HR complex, RAD51C mutants that are associated with cancer susceptibility and FA also display decreased complex formation with PALB2. Together, our results suggest that three different cancer susceptibility and FA proteins function in a DNA repair pathway based upon the PALB2 WD40 domain binding to RAD51C and BRCA2.
doi:10.1038/onc.2013.421
PMCID: PMC3994186  PMID: 24141787
PALB2; RAD51C; BRCA2; homologous recombination; BRCA genes; DNA repair
3.  PALB2 Links BRCA1 and BRCA2 in the DNA-Damage Response 
Current biology : CB  2009;19(6):524-529.
Summary
BRCA1 and BRCA2 are often mutated in familial breast and ovarian cancer. Both tumor suppressors play key roles in the DNA-damage response [1, 2]. However, it remains unclear whether these two tumor suppressor function together in the same DNA-damage response pathway. Here, we show that BRCA1 associates with BRCA2 through PALB2/FANCN, a major binding partner of BRCA2 [3]. The interaction between BRCA1 and BRCA2 is abrogated in PALB2-deficient Fanconi anemia cells and in the cells depleted of PALB2 by small interfering RNA. Moreover, we show that BRCA1 promotes the concentration of PALB2 and BRCA2 at DNA-damage sites and the interaction between BRCA1 and PALB2 is important for the homologous recombination repair. Taken together, our results indicate that BRCA1 is an upstream regulator of BRCA2 in the DNA-damage response, and PALB2 is the linker between BRCA1 and BRCA2.
doi:10.1016/j.cub.2009.02.018
PMCID: PMC2750839  PMID: 19268590
4.  Dysfunctional KEAP1–NRF2 Interaction in Non-Small-Cell Lung Cancer 
PLoS Medicine  2006;3(10):e420.
Background
Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.
Methods and Findings
Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.
Conclusions
This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.
Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.
Editors' Summary
Background.
Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.
Why Was This Study Done?
To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.
What Did the Researchers Do and Find?
The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.
What Do These Findings Mean?
These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030420.
US National Cancer Institute information on lung cancer and on cancer treatment
MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer
Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030420
PMCID: PMC1584412  PMID: 17020408
5.  A Hypomorphic PALB2 Allele Gives Rise to an Unusual Form of FA-N Associated with Lymphoid Tumour Development 
PLoS Genetics  2016;12(3):e1005945.
Patients with biallelic truncating mutations in PALB2 have a severe form of Fanconi anaemia (FA-N), with a predisposition for developing embryonal-type tumours in infancy. Here we describe two unusual patients from a single family, carrying biallelic PALB2 mutations, one truncating, c.1676_1677delAAinsG;(p.Gln559ArgfsTer2), and the second, c.2586+1G>A; p.Thr839_Lys862del resulting in an in frame skip of exon 6 (24 amino acids). Strikingly, the affected individuals did not exhibit the severe developmental defects typical of FA-N patients and initially presented with B cell non-Hodgkin lymphoma. The expressed p.Thr839_Lys862del mutant PALB2 protein retained the ability to interact with BRCA2, previously unreported in FA-N patients. There was also a large increased chromosomal radiosensitivity following irradiation in G2 and increased sensitivity to mitomycin C. Although patient cells were unable to form Rad51 foci following exposure to either DNA damaging agent, U2OS cells, in which the mutant PALB2 with in frame skip of exon 6 was induced, did show recruitment of Rad51 to foci following damage. We conclude that a very mild form of FA-N exists arising from a hypomorphic PALB2 allele.
Author Summary
PALB2 is a protein that creates a molecular bridge that promotes the recruitment of Homologous Recombination Repair (HRR) proteins BRCA1, BRCA2 and Rad51 to sites of DNA damage. Cells with functional loss of PALB2 show a defect in HRR and are characterized by an increased number of spontaneous chromosome breaks and hypersensitivity to cross-linking agents. Typically, inherited mutations in PALB2 are associated with a severe Fanconi Anaemia phenotype. Here we describe for the first time the effect of biallelic mutations of PALB2 in which one hypomorphic allele resulted in a low level of expression of the mutant PALB2 protein with some retained function, as shown by its interaction with BRCA2, as well as its ability to facilitate Rad51 focus formation following damage. This resulted in a considerably milder clinical phenotype with increased longevity compared with biallelic PALB2 null patients and an altered tumour spectrum towards development of lymphoid malignancies.
doi:10.1371/journal.pgen.1005945
PMCID: PMC4798644  PMID: 26990772
6.  Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination 
Cancer research  2013;73(7):2199-2210.
Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2 and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an ‘ETGE’ motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA over-expression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that ‘ETGE’-containing proteins like DPP3 contribute to NRF2 activity in cancer.
doi:10.1158/0008-5472.CAN-12-4400
PMCID: PMC3618590  PMID: 23382044
7.  Expression of xCT and activity of system xc− are regulated by NRF2 in human breast cancer cells in response to oxidative stress 
Redox Biology  2015;5:33-42.
Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc−, an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc−, is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc−. We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.
Graphical abstract
Highlights
•Acute H2O2 treatment of MCF-7 human breast cancer cells increases NRF2 nuclear translocation.•H2O2 also increases levels of xCT mRNA and protein, and extracellular glutamate release.•NRF2 overexpression in MCF-7 cells up-regulates the activity of the human xCT promoter, which contains a proximal ARE.•KEAP1 overexpression in MCF-7 cells represses promoter activity, correlating with decreased xCT protein levels.•siRNA-mediated knockdown of KEAP1 up-regulates xCT protein levels and glutamate release.
doi:10.1016/j.redox.2015.03.003
PMCID: PMC4392061  PMID: 25827424
System xc−; xCT; NRF2; KEAP1; Oxidative stress; Hydrogen peroxide
8.  KPNA6 (Importin α7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿  
Molecular and Cellular Biology  2011;31(9):1800-1811.
The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.
doi:10.1128/MCB.05036-11
PMCID: PMC3133232  PMID: 21383067
9.  Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination 
Nature structural & molecular biology  2010;17(10):1247-1254.
Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. The tumor-suppressing capability of PALB2 is thought to be based on its ability to enable BRCA2 function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to strongly stimulates strand invasion, a vital step of homologous recombination. Such stimulation occur by reinforcing biochemical mechanisms as PALB2 alleviates the inhibitory role of RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Collectively, our studies provide the first biochemical insights into the homologous recombination mediator functions of PALB2 with piBRCA2 in DNA double-strand break repair.
doi:10.1038/nsmb.1915
PMCID: PMC4094107  PMID: 20871615
RAD51; PALB2; BRCA2; homologous recombination
10.  Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains 
PLoS Genetics  2011;7(12):e1002409.
The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.
Author Summary
The breast tumor suppressor BRCA2 has a major role in DNA repair by homologous recombination (HR). BRCA2 is a large protein with multiple domains that interact with several proteins as well as with DNA, complicating our understanding of how the protein functions in cells. To investigate the mechanism by which BRCA2 functions in HR in cells, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that DNA binding by BRCA2 is critical when a BRCA2 peptide is deficient in binding another breast cancer suppressor, PALB2, but not when the peptide can bind PALB2, suggesting alternative mechanisms of activity. Binding the small peptide DSS1 is also essential for HR only in some contexts, as are activities in the BRCA2 C terminus. Our results reveal redundancy of BRCA2 domains and emphasize plasticity within this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.
doi:10.1371/journal.pgen.1002409
PMCID: PMC3240595  PMID: 22194698
11.  Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2▿  
Molecular and Cellular Biology  2007;27(18):6334-6349.
The transcription factor Nrf2 regulates cellular redox homeostasis. Under basal conditions, Keap1 recruits Nrf2 into the Cul3-containing E3 ubiquitin ligase complex for ubiquitin conjugation and subsequent proteasomal degradation. Oxidative stress triggers activation of Nrf2 through inhibition of E3 ubiquitin ligase activity, resulting in increased levels of Nrf2 and transcriptional activation of Nrf2-dependent genes. In this study, we identify Keap1 as a key postinduction repressor of Nrf2 and demonstrate that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2-antioxidant response element (ARE) signaling by escorting nuclear export of Nrf2. We provide evidence that ubiquitination of Nrf2 is carried out in the cytosol. Furthermore, we show that Keap1 nuclear translocation is independent of Nrf2 and the Nrf2-Keap1 complex does not bind the ARE. Collectively, our results suggest the following mechanism of postinduction repression: upon recovery of cellular redox homeostasis, Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. Once in the cytoplasm, the Keap1-Nrf2 complex associates with the E3 ubiquitin ligase, resulting in degradation of Nrf2 and termination of the Nrf2 signaling pathway. Hence, postinduction repression of the Nrf2-mediated antioxidant response is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination and degradation machinery.
doi:10.1128/MCB.00630-07
PMCID: PMC2099624  PMID: 17636022
12.  PALB2/FANCN - recombining cancer and Fanconi anemia 
Cancer research  2010;70(19):7353-7359.
PALB2 was originally identified as a BRCA2-interacting protein which is crucial for key BRCA2 genome caretaker functions. It subsequently became clear that PALB2 was another Fanconi anemia (FA) gene (FANCN), and that monoallelic PALB2 mutations are associated with increased risk of breast and pancreatic cancer. Mutations in PALB2 have been identified in breast cancer families worldwide and recent studies have shown that PALB2 also interacts with BRCA1. Here we summarize the molecular functions and clinical phenotypes of this key DNA repair pathway component and discuss how its discovery has advanced our knowledge of both FA and adult cancer predisposition.
doi:10.1158/0008-5472.CAN-10-1012
PMCID: PMC2948578  PMID: 20858716
13.  Transcription Factor Nrf2-Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy 
Purpose.
Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2–related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy.
Methods.
Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy.
Results.
Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA.
Conclusions.
Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
doi:10.1167/iovs.13-11598
PMCID: PMC3676188  PMID: 23633659
antioxidant defense; diabetic retinopathy; Nrf2
14.  Prevalence of PALB2 mutations in Australasian multiple-case breast cancer families 
Introduction
Population-based studies of breast cancer have estimated that some PALB2 mutations confer a breast cancer risk (penetrance) comparable to the average pathogenic mutation in BRCA2. As this risk is of clinical relevance, we sought to identify mono-allelic PALB2 mutations and determine their frequencies in multiple-case breast cancer families attending Familial Cancer Clinics in Australia and New Zealand.
Methods
The youngest affected woman, not known to carry a mutation in BRCA1 or BRCA2, from 747 multiple-case breast cancer families participating in kConFab were selected for PALB2 mutation screening. The coding and flanking intronic regions of PALB2 in DNA extracted from blood were screened using high-resolution melt curve analysis with Sanger sequencing confirmation. Where possible, relatives of women found to carry PALB2 mutations were genotyped for the family-specific mutation, mutant transcripts were characterised and breast tumours arising in mutation carriers were recalled and reviewed. Missense mutations were assessed for potential to disrupt protein function via SIFT, Align GVGD and Polyphen-2.
Results
The mutation screen identified two nonsense mutations (PALB2 c.3113G>A in eight women and PALB2 c.196C>T in one woman), two frameshift mutations (PALB2 c.1947_1948insA and PALB2 c.2982_2983insT each in one woman), 10 missense variants, eight synonymous variants and four variants in intronic regions. Of the four PALB2 mutations identified that were predicted to produce truncated protein products, only PALB2 c.1947_1948insA had not previously been reported. PALB2 c.3113G>A and PALB2 c.196C>T were previously identified in the Australian population whereas PALB2 c.2982_2983insT was previously reported in the UK population. Transcripts derived from three of these mutant PALB2 alleles were vulnerable to nonsense-mediated decay. One missense mutation (PALB2 c.2993G>A) was predicted to disrupt protein function via the three in silico assessment methods applied. The majority of breast cancers arising in carriers that were available for review were high-grade invasive ductal carcinomas. Conclusions: About 1.5% (95% CI 0.6to 2.4) of Australasian multiple-case breast cancer families attending clinics are segregating protein-truncating mutations in PALB2, most being PALB2 c.3113G>A, p.Trp1038*. Given the prevalence, breast cancer risk, and tumour grade associated with this mutation, consideration of clinical PALB2 testing is warranted.
doi:10.1186/bcr3392
PMCID: PMC3672826  PMID: 23448497
15.  Regulation of Nrf2 – An update 
Free radical biology & medicine  2013;66:10.1016/j.freeradbiomed.2013.02.008.
Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes which encode detoxifying enzymes, drug transporters (MRPs), anti-apoptotic proteins and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase mediated degradation of Nrf2. Chemicals including antioxidants, tocopherols including α-tocopherol (vitamin E), phytochemicals and radiations antagonize the Nrf2:INrf2 interaction and leads to the stabilization and activation of Nrf2. The signaling events involve pre-induction, induction and post-induction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinase(s) in a pre-induction response which phosphorylates specific residues on Nrf2 negative-regulators, INrf2, Fyn and Bach1, leading to their nuclear export, ubiquitination and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2cysteine151 followed by PKC phosphorylation of Nrf2serine40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus resulting in a coordinated activation of gene expression. This is followed by a post-induction response that controls the ‘switching off’ of Nrf2-activated gene expression. GSK3β under the control of AKT and PI3K, phosphorylates Fyn leading to Fyn nuclear localization. Fyn phosphorylates Nrf2Y568 resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provides protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.
doi:10.1016/j.freeradbiomed.2013.02.008
PMCID: PMC3773280  PMID: 23434765
Nrf2; INrf2(Keap1); Antioxidants; Vitamins; Phytochemicals; ROS; Signaling; Regulation; Chemoprotection; Oncogenesis
16.  PALB2 self-interaction controls homologous recombination 
Nucleic Acids Research  2012;40(20):10312-10323.
PALB2 is essential for BRCA2 anchorage to nuclear structures and for homologous recombinational repair of DNA double-strand breaks. Here, we report that the N-terminal coiled-coil motif of PALB2 regulates its self-association and homologous recombination. Monomeric PALB2 shows higher efficiency to bind DNA and promotes RAD51 filament formation with or without the inhibitory effect of Replication Protein A. Moreover, overexpression of the PALB2 coiled-coil domain severely affects RAD51 loading to DNA damage sites suggesting a competition between PALB2 self-interaction and PALB2–BRCA1 interaction. In the presence of DNA damage, the switch between PALB2–PALB2 and PALB2–BRCA1 interactions allows the activation of HR. Controlling HR via PALB2 self-interactions could be important to prevent aberrant recombination in normal conditions and activate DNA repair when required.
doi:10.1093/nar/gks807
PMCID: PMC3488246  PMID: 22941656
17.  EMSY overexpression disrupts the BRCA2/RAD51 pathway in the DNA-damage response: implications for chromosomal instability/recombination syndromes as checkpoint diseases 
Molecular Genetics and Genomics  2011;285(4):325-340.
EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention.
doi:10.1007/s00438-011-0612-5
PMCID: PMC3064890  PMID: 21409565
Tumor-suppressor genes; Oncogenes; Development/abortion; Aging; Estrogens
18.  Enhancement of the RAD51 Recombinase Activity by the Tumor Suppressor PALB2 
Nature structural & molecular biology  2010;17(10):1255-1259.
Homologous recombination mediated by the RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-stranded breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2 in the enhancement of RAD51's ability to form the D-loop. We show that PALB2 binds DNA and physically interacts with RAD51. Importantly, while PALB2 alone stimulates D-loop formation, a co-operative effect is seen with RAD51AP1, an enhancer of RAD51. This stimulation stems from PALB2's ability to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results help unveil a multi-faceted role of PALB2 in chromosome damage repair. Since PALB2 mutations can cause breast and other tumors or lead to Fanconi anemia, our findings are important for understanding the mechanism of tumor suppression in humans.
doi:10.1038/nsmb.1916
PMCID: PMC2950913  PMID: 20871616
19.  Kinetic, Thermodynamic, and Structural Characterizations of the Association between Nrf2-DLGex Degron and Keap1 
Molecular and Cellular Biology  2014;34(5):832-846.
Transcription factor Nrf2 (NF-E2-related factor 2) coordinately regulates cytoprotective gene expression, but under unstressed conditions, Nrf2 is degraded rapidly through Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination. Nrf2 harbors two Keap1-binding motifs, DLG and ETGE. Interactions between these two motifs and Keap1 constitute a key regulatory nexus for cellular Nrf2 activity through the formation of a two-site binding hinge-and-latch mechanism. In this study, we determined the minimum Keap1-binding sequence of the DLG motif, the low-affinity latch site, and defined a new DLGex motif that covers a sequence much longer than that previously defined. We have successfully clarified the crystal structure of the Keap1-DC-DLGex complex at 1.6 Å. DLGex possesses a complicated helix structure, which interprets well the human-cancer-derived loss-of-function mutations in DLGex. In thermodynamic analyses, Keap1-DLGex binding is characterized as enthalpy and entropy driven, while Keap1-ETGE binding is characterized as purely enthalpy driven. In kinetic analyses, Keap1-DLGex binding follows a fast-association and fast-dissociation model, while Keap1-ETGE binding contains a slow-reaction step that leads to a stable conformation. These results demonstrate that the mode of DLGex binding to Keap1 is distinct from that of ETGE structurally, thermodynamically, and kinetically and support our contention that the DLGex motif serves as a converter transmitting environmental stress to Nrf2 induction as the latch site.
doi:10.1128/MCB.01191-13
PMCID: PMC4023822  PMID: 24366543
20.  Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently Infected Endothelial Cells through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1 
Journal of Virology  2014;89(4):2268-2286.
ABSTRACT
Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase Cζ (PKCζ) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (γGCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63–ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2, which is involved in the regulation of genes participating in KSHV oncogenesis.
IMPORTANCE The transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated during de novo KSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell line latently infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently infected endothelial cells independently of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQSTM1 to induce Nrf2 activation, thereby manipulating the infected host gene regulation to promote KS pathogenesis.
doi:10.1128/JVI.02742-14
PMCID: PMC4338888  PMID: 25505069
21.  Prevalence of PALB2 Mutations in Breast Cancer Patients in Multi-Ethnic Asian Population in Malaysia and Singapore 
PLoS ONE  2013;8(8):e73638.
Background
The partner and localizer of breast cancer 2 (PALB2) is responsible for facilitating BRCA2-mediated DNA repair by serving as a bridging molecule, acting as the physical and functional link between the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) proteins. Truncating mutations in the PALB2 gene are rare but are thought to be associated with increased risks of developing breast cancer in various populations.
Methods
We evaluated the contribution of PALB2 germline mutations in 122 Asian women with breast cancer, all of whom had significant family history of breast and other cancers. Further screening for nine PALB2 mutations was conducted in 874 Malaysian and 532 Singaporean breast cancer patients, and in 1342 unaffected Malaysian and 541 unaffected Singaporean women.
Results
By analyzing the entire coding region of PALB2, we found two novel truncating mutations and ten missense mutations in families tested negative for BRCA1/2-mutations. One additional novel truncating PALB2 mutation was identified in one patient through genotyping analysis. Our results indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the Malaysian and Singaporean populations.
doi:10.1371/journal.pone.0073638
PMCID: PMC3748013  PMID: 23977390
22.  Autophagy opposes p53-mediated tumor barrier to facilitate tumorigenesis in a model of PALB2-associated hereditary breast cancer 
Cancer discovery  2013;3(8):894-907.
Hereditary breast cancers stem from germline mutations in susceptibility genes such as BRCA1, BRCA2 and PALB2, whose products function in the DNA damage response and redox regulation. Autophagy is an intracellular waste disposal and stress mitigation mechanism important for alleviating oxidative stress and DNA damage response activation; it can either suppress or promote cancer, but its role in breast cancer is unknown. Here we show that, similar to Brca1 and Brca2, ablation of Palb2 in mouse mammary gland resulted in tumor development with long latency and the tumors harbored mutations in Trp53. Interestingly, impaired autophagy, due to monoallelic loss of the essential autophagy gene Becn1, reduced Palb2-associated mammary tumorigenesis in Trp53-wild type but not conditionally null background. These results indicate that, in the face of DNA damage and oxidative stress elicited by PALB2 loss, p53 is a barrier to cancer development, whereas autophagy facilitates cell survival and tumorigenesis.
doi:10.1158/2159-8290.CD-13-0011
PMCID: PMC3740014  PMID: 23650262
23.  No evidence for PALB2 methylation in high-grade serous ovarian cancer 
Background
High-grade serous ovarian cancers are a distinct histological subtype of ovarian cancer often characterised by a dysfunctional BRCA/Fanconi anaemia (BRCA/FA) pathway, which is critical to the homologous recombination DNA repair machinery. An impaired BRCA/FA pathway sensitises tumours to the treatment with DNA cross-linking agents and to PARP inhibitors. The vast majority of inactivating mutations in the BRCA/FA pathway are in the BRCA1 and BRCA2 genes and occur predominantly in high-grade serous cancer. Another member of the BRCA/FA pathway, PALB2 (FANCN), was reported to have been inactivated by DNA methylation in some sporadic ovarian cancers. We therefore sought to investigate the role of PALB2 methylation in high-grade serous ovarian cancers.
Finding
PALB2 methylation was investigated in 92 high-grade serous ovarian cancer samples using methylation-sensitive high-resolution melting analysis. DNA methylation of PALB2 was not detected in any of the ovarian cancer samples investigated.
Conclusion
Epigenetic silencing by DNA methylation of PALB2 is not a common event in high-grade serous ovarian cancers.
doi:10.1186/1757-2215-6-26
PMCID: PMC3636006  PMID: 23587053
DNA methylation; Ovarian cancer; Fanconi anaemia; PALB2; MS-HRM
24.  PALB2 mutations in familial breast and pancreatic cancer 
Familial cancer  2011;10(2):10.1007/s10689-011-9426-1.
PALB2 (Partner And Localizer of BRCA2) binds to and colocalizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1–2% of familial breast cancer and 3–4% of familial pancreatic cancer cases. The goal of this study was to evaluate the prevalence of PALB2 mutations in women with breast cancer without BRCA1/2 mutations who also had a personal or family history of pancreatic cancer. PALB2 mutation analysis was performed in 94 non-BRCA1/2 breast cancer patients with a personal or family history of pancreatic cancer. Two truncating PALB2 mutations, c.3549C>CA and c.2962C>CT, were identified resulting in a mutation prevalence of 2.1%. The proband found to carry the c.3549C>CA PALB2 mutation had a mother diagnosed with both breast and pancreatic cancer; this relative was subsequently confirmed to carry the identical mutation. The proband with the c.2962C>CT mutation had a father and paternal aunt diagnosed with pancreatic cancer; neither relative was available for testing. Two novel PALB2 missense variants were also found, one of which was deemed potentially deleterious. The prevalence rate of PALB2 mutations in a non-BRCA1/2 breast cancer population specifically selected for a family history of pancreatic cancer does not appear to be significantly increased compared to that observed in other breast cancer populations studied thus far. Further evaluation is needed to determine the prevalence of PALB2 mutations and the clinical utility of such testing in those individuals affected with both breast and pancreatic cancers.
doi:10.1007/s10689-011-9426-1
PMCID: PMC3836668  PMID: 21365267
BRCA2; Breast cancer; PALB2; Pancreatic cancer
25.  Assessment of PALB2 as a Candidate Melanoma Susceptibility Gene 
PLoS ONE  2014;9(6):e100683.
Partner and localizer of BRCA2 (PALB2) interacts with BRCA2 to enable double strand break repair through homologous recombination. Similar to BRCA2, germline mutations in PALB2 have been shown to predispose to Fanconi anaemia as well as pancreatic and breast cancer. The PALB2/BRCA2 protein interaction, as well as the increased melanoma risk observed in families harbouring BRCA2 mutations, makes PALB2 a candidate for melanoma susceptibility. In order to assess PALB2 as a melanoma predisposition gene, we sequenced the entire protein-coding sequence of PALB2 in probands from 182 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, and BAP1. In addition, we interrogated whole-genome and exome data from another 19 kindreds with a strong family history of melanoma for deleterious mutations in PALB2. Here we report a rare known deleterious PALB2 mutation (rs118203998) causing a premature truncation of the protein (p.Y1183X) in an individual who had developed four different cancer types, including melanoma. Three other family members affected with melanoma did not carry the variant. Overall our data do not support a case for PALB2 being associated with melanoma predisposition.
doi:10.1371/journal.pone.0100683
PMCID: PMC4065098  PMID: 24949998

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