Early events in BRCA1-associated tumorigenesis remain poorly understood. To understand the immediate consequences of BRCA1 loss of function, we modeled BRCA1 loss of function in vitro using normal primary human mammary epithelial cells (HMEC). We have found that in HMEC, loss of BRCA1 results in a novel type of senescence. Loss of BRCA1-induced senescence is not associated with DNA damage or p53 upregulation. We find that p53 protein levels are down regulated due to proteasome-mediated degradation. Although p53 levels are down regulated, we find that BRCA1 loss induced expression of a number of p53-dependent anti-oxidant genes. In particular we uncovered that SESN2, a p53 downstream target gene, inhibits loss of BRCA1 induced ROS and activates autophagy. In contrast to human fibroblasts, we found that loss of BRCA1 induced senescence is p53 independent, and can occur in the absence of ROS upregulation and autophagy induction.

Abstract Breast cancer is a common malignancy affecting women around the world. Approximately 10 percent of breast cancer patients have a hereditary form of the disease. Women with an inherited alteration in one of the BRCA1 and BRCA2 genes have an increased risk of developing these cancers at a young age (before menopause), and often have multiple family members with the disease. A total of 6 families with multiple cases of breast cancer were identified from southern Taiwan, and five of these families were found to have missense mutations in the BRCA1 or BRCA2 genes. One novel missense mutation of A5885C (Gln1886Pro), as well as new silent mutation of A4806G (Thr1526), in the exon 11 of the BRCA2 gene was found in one(A) family. The second(F) family was found to have three missense mutations of C2731T (Pro871Leu), A3232G (Glu1038Gly) and A3667G (Lys1183Arg) in the exon 11 of the BRCA1 gene. It is very unusual to have three previously reported BRCA1 mutations in the same family and these three mutations are located on the same chromosome. Two missense mutations of A3232G (Glu1038Gly) in exon 11 and A4956G (Ser1613Gly) in exon 16, as well as silent mutations of T2430C (Leu771) and T4427C (Ser1436), of the BRCA1 gene were found in the third(E) family. The missense mutation of A4956G (Ser1613Gly) in exon 16, as well as silent mutation of T4427C (Ser1436), of the BRCA1 are found in the fourth(C) and fifth(D) family. The sixth(B) families were found to possess only one silent mutation of T4035C (Val1269) in the BRCA2 gene. The amino acid changes might cause the protein structure unstable and these could explain the moderate role of BRCA mutations in the pathogenesis of breast cancer.

Breast cancer is a heterogeneous disease with vastly different tumor progression kinetics and survival outcomes depending upon the differentiation state and gene expression patterns of the tumor. Effective treatments exist for patients with endocrine therapy sensitive or HER2 overexpressing tumors, but targeted treatments are not available for other tumor types. The mechanisms governing mammary tumor phenotype generation could prove critical to finding treatments. The c-Jun N-terminal kinase (JNK) pathway has recently been implicated in the inhibition of breast tumor luminal differentiation (1, 2) and JNK2, in particular, is important in mammary tumorigenesis and tumor progression (3-8). Therefore, the involvement of JNK2 in inhibition of mammary luminal cell differentiation was investigated in normal glands and tumors. Studies found that JNK2 inhibits luminal cell populations in normal mammary ducts. Additionally, JNK2 suppresses Notch activity in stem cell niche of the developing mammary gland. In vitro assays show that control over differentiation by JNK2 is due to suppression of p53-dependent Notch1 expression. Inhibition of luminal cell populations by JNK2 is also apparent in tumor cell models regardless of p53 expression. In the p53-competent Polyoma Middle T-antigen model, Notch1 expression is suppressed by JNK2. In the absence of p53, JNK2 suppresses luminal populations independent of Notch1. In this model, decreased luminal marker expression is accompanied by increased epithelial to mesenchymal transition. It was also found that JNK2-dependent epithelial to mesenchymal transition inhibits luminal populations and is driven by JNK2-dependent suppression of Brca1. JNK2 also confers resistance to estrogen signaling inhibition, and increases the metastatic ability of tumor cells in vivo. These data establish the importance of JNK2 in mammary epithelial cell differentiation in normal glands and tumors. They also suggest that JNK2 may be an effective prognostic marker or treatment target. / text

No description available.

An estimated 5-10% of breast cancers are hereditary in nature and are due to the presence of a mutation in a breast cancer predisposition gene; approximately half of these cases possess a mutation in BRCA1 or BRCA2. Many BRCA1/BRCA2 mutations result in a truncated protein and hence are unequivocally disease-causing. However another class of mutations, the Variants of Unknown Significance (VUS), are more problematic as the effect of these mutations on protein function is unclear. The inability to classify these mutations as disease causing generates significant problems in risk evaluation, counseling and preventive care. Accordingly we sought to determine whether carriers of either a BRCA1 or BRCA2 mutation could be identified from non-carriers based on the gene expression patterns of non-cancerous cells. EBV-transformed lymphoblastoid cell lines established from BRCA1/BRCA2 mutation carriers and normal individuals were obtained through the NIH Breast Cancer Family Registries. Cell lines were mock-irradiated or treated with ionizing radiation (2 Gy). Following a recovery period of 6 hours total RNA was extracted and whole genome gene expression profiling was carried out. Molecular classifiers comparing the baseline expression profiles and the radiation-dependent expression profiles of BRCA1/BRCA2 mutation carriers to control individuals were created using a Support Vector Machine (SVM) coupled with a recursive feature removal (RFR) algorithm. Our results suggest that cell populations derived from BRCA1/BRCA2 mutation carriers display unique expression phenotypes from those of control individuals in both the basal and radiation-induced cases. In the task of classification using baseline expression, the BRCA1-classifier correctly classified 15/18 test samples using feature selection based on the training set only, while feature selection using the entire dataset (AD) improved classification to 16/18 samples. The BRCA2-baseline classifier correctly classified 13/17 and 14/17 (AD) samples, respectively. In the task of radiation-dependent classification, the BRCA1-IR classifier correctly classified 12/18 and 16/18 (AD) test samples respectively while the BRCA2-IR classifier correctly classified 13/17 and 16/17 (AD) test samples respectively. These results suggest the possibility of development of this assay into a novel hereditary breast cancer screening diagnostic able to accurately identify the presence of BRCA1 or BRCA2 mutations via a functional assay thereby improving patient outcomes. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-03-27 15:38:19.269 / Canadian Breast Cancer Foundation-Ontario Chapter, Department of Pathology & Molecular Medicine Clinical Trust Fund

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.