Multi-gene fluorescence in situ hybridization to detect cell cycle gene copy number aberrations in young breast cancer patients

Breast cancer is a disease of cell cycle, and the dysfunction of cell cycle checkpoints plays a vital role in the occurrence and development of breast cancer. We employed multi-gene fluorescence in situ hybridization (M-FISH) to investigate gene copy number aberrations (CNAs) of 4 genes (Rb1, CHEK2, c-Myc, CCND1) that are involved in the regulation of cell cycle, in order to analyze the impact of gene aberrations on prognosis in the young breast cancer patients. Gene copy number aberrations of these 4 genes were more frequently observed in young breast cancer patients when compared with the older group. Further, these CNAs were more frequently seen in Luminal B type, Her2 overexpression, and tiple-negative breast cancer (TNBC) type in young breast cancer patients. The variations of CCND1, Rb1, and CHEK2 were significantly correlated with poor survival in the young breast cancer patient group, while the amplification of c-Myc was not obviously correlated with poor survival in young breast cancer patients. Thus, gene copy number aberrations (CNAs) of cell cycle-regulated genes can serve as an important tool for prognosis in young breast cancer patients.     

Structural basis for recruitment of BRCA2 by PALB2

The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy‐terminal β‐propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein–protein interaction and explains the effects of both cancer‐associated truncating mutants in PALB2 and missense mutations in the amino‐terminal region of BRCA2.     

Deciphering the BRCA1 Tumor Suppressor Network

The BRCA1 tumor suppressor protein is a central constituent of several distinct macromolecular protein complexes that execute homology-directed DNA damage repair and cell cycle checkpoints. Recent years have borne witness to an exciting phase of discovery at the basic molecular level for how this network of DNA repair proteins acts to maintain genome stability and suppress cancer. The clinical dividends of this investment are now being realized with the approval of first-in-class BRCA-targeted therapies for ovarian cancer and identification of molecular events that determine responsiveness to these agents. Further delineation of the basic science underlying BRCA network function holds promise to maximally exploit genome instability for hereditary and sporadic cancer therapy.     

APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer

Mutations in BRCA2 confer an increased risk of cancer development, at least in part because the BRCA2 protein is required for the maintenance of genomic integrity. Here, we use proteomic profiling to identify APRIN (PDS5B), a cohesion-associated protein, as a BRCA2-associated protein. After exposure of cells to hydroxyurea or aphidicolin, APRIN and other cohesin components associate with BRCA2 in early S-phase. We demonstrate that APRIN expression is required for the normal response to DNA-damaging agents, the nuclear localisation of RAD51 and BRCA2 and efficient homologous recombination. The clinical significance of these findings is indicated by the observation that the BRCA2/APRIN interaction is compromised by BRCA2 missense variants of previously unknown significance and that APRIN expression levels are associated with histological grade in breast cancer and the outcome of breast cancer patients treated with DNA-damaging chemotherapy.     

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.     

In vivo analysis of mammary and non-mammary tumorigenesis in MMTV-cyclin D1 transgenic mice deficient in p53

Overexpression of the cyclin D1 oncogene and inactivation of the p53 tumor suppressor have both been implicated in substantial proportions of sporadic human breast cancers. Transgenic mice with cyclin D1 overexpression targeted to mammary tissue by the MMTV enhancer-promoter have been shown to develop mammary cancers. To investigate the relationship between pathways driven by cyclin D1 overexpression and p53 loss during the development of breast cancers, we crossed MMTV-cyclin D1 mice with p53 heterozygous null (p53^+/–) mice. In such crossed mice, cyclin D1-driven mammary neoplasia would need to be substantially accelerated by p53 loss in order for mammary tumors to develop prior to the expected onset of non-mammary tumors characteristic of the p53-deficient background alone. Instead, in mice heterozygous or homozygous for p53 deficiency and simultaneously carrying the MMTV-cyclin D1 transgene, only tumors typically found in p53-deficient mice developed and mammary tumors were not observed. Interestingly, MMTV-cyclin D1/p53^+/– mice appeared to develop these non-mammary tumors more rapidly than p53^+/– mice, and a majority of the sampled non-mammary tumors from MMTV-cyclin D1/p53^+/– mice showed ectopic expression of the MMTV-driven cyclin D1 transgene. Within the constraints of possible genetic background effects and limited sensitivity due to the early emergence of non-mammary tumors, these observations provide no evidence that inactivation of p53 confers a major additional selective advantage to mammary cells overexpressing cyclin D1 in this animal model of human breast cancer. Interestingly, the results do raise the possibility that p53 inactivation might complement or cooperate with cyclin D1 deregulation during the development of some types of non-mammary tumors.     

乳腺癌易感蛋白BRCA1的BRCT2结构域染色质伸展活性的定位

40 %～ 5 0 %的遗传性乳腺癌和至少 80 %的既有乳腺癌又有卵巢癌家族史的患者是由BRCA1突变引起的 .BRCA1C末端含有 2个BRCT结构域 (BRCT1和BRCT2 ) ,它们与BRCA1的重要功能密切相关 .许多乳腺癌易感突变发生在BRCA1的BRCT结构域中 .利用染色质结构检测技术表明 ,BRCT结构域具有染色质伸展活性 .利用缺失突变技术构建了 6种BRCT2结构域 (175 6～ 185 2位氨基酸残基 )缺失突变体并将BRCT2结构域中与染色质伸展相关的重要区域定位到 175 6～ 180 8之间的氨基酸残基 ;用丙氨酸扫描技术构建了 6种BRCT2结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到 1784～ 1788之间的VQLCG .BRCT2结构域的定位有助于预测BRCT2结构域突变后发生乳腺癌的风险 ,也为进一步研究BRCT2结构域的功能机制提供了有用的材料 .