53BP1 Expression Research Articles

High XIST and Low 53BP1 Expression Predict Poor Outcome after High-Dose Alkylating Chemotherapy in Patients with a BRCA1-like Breast Cancer.

In previous studies, high expression of XIST and low expression of 53BP1 were respectively associated with poor systemic therapy outcome in patients and therapy resistance in BRCA1-deficient mouse tumor models, but have not been evaluated in BRCA1-deficient patients. Previously, we demonstrated that classifying breast cancer copy number profiles as BRCA1-like or non-BRCA1-like identified patients enriched for defects in BRCA1 that benefit from high-dose (HD) alkylating chemotherapy compared with a conventional standard regimen. We investigated whether XIST and 53BP1 expression predicted poor outcome of HD chemotherapy within 28 BRCA1-like patients from a trial randomizing between HD [4 cycles 5-fluorouracil, epirubicin, cyclophosphamide (FEC) followed by 1 cycle HD carboplatin, thiotepa, cyclophosphamide] or conventional chemotherapy (5 cycles FEC), for which both XIST and 53BP1 statuses were available. High RNA expression of XIST (n = 5) and low protein expression of 53BP1 (n = 3) expression did not coincide. Patients with either one had poor outcome after treatment with HD chemotherapy, whereas patients with low expression of XIST and high expression of 53BP1 derived substantial benefit of this regimen on recurrence-free survival, disease-free survival, and overall survival, corroborating preclinical findings. XIST and 53BP1 may be predictive biomarkers in BRCA1-like breast cancer.

53BP1 suppresses epithelial-mesenchymal transition by downregulating ZEB1 through microRNA-200b/429 in breast cancer.

Epithelial–mesenchymal transition (EMT) is an important mechanism of cancer invasion and metastasis. Although p53 binding protein 1 (53BP1) has been implicated in several biological processes, its function in EMT of human cancers has not yet been reported. Here, we show that 53BP1 negatively regulated EMT by modulating ZEB1 through targeting microRNA (miR)-200b and miR-429. Furthermore, 53BP1 promoted ZEB1-mediated upregulation of E-cadherin and also inhibited the expressions of mesenchymal markers, leading to increased migration and invasion in MDA-MB-231 breast cancer cells. Consistently, in MCF-7 breast cancer cells, low 53BP1 expression reduced E-cadherin expression, resulting in increased migration and invasion. These effects were reversed by miR-200b and miR-429 inhibition or overexpression. Sections of tumor xenograft model showed increased ZEB1 expression and decreased E-cadherin expression with the downregulation of 53BP1. In 18 clinical tissue samples, expression of 53BP1 was positively correlated with miR-200b and mir-429 and negatively correlated with ZEB1. It was also found that 53BP1 was associated with lymph node metastasis. Taken together, these results suggest that 53BP1 functioned as a tumor suppressor gene by its novel negative control of EMT through regulating the expression of miR-200b/429 and their target gene ZEB1.

Abstract 3955: 53BP1 facilitates the ATM-dependent phosphorylation of APLF in the DNA damage response

Abstract Aprataxin and polynucleotide kinase-like factor (APLF) is a DNA repair factor which facilitates DNA repair in part through interactions with its forkhead-associated (FHA) domain. The APLF FHA domain mediates interactions with specific threonine phosphorylated epitopes via a functionally conserved and essential arginine residue (Arg-27). In the context of DNA double-strand break (DSB) repair, substitution of APLF amino acid residue Arg-27 to alanine abolishes interactions with the DSB repair scaffold protein XRCC4, and impairs DSB repair. APLF is also a component of the ATM-dependent DNA damage response (DDR), which also includes 53BP1, and undergoes ionizing-radiation induced phosphorylation at Ser-116 facilitating its role in DSB repair. Here we demonstrate that the APLF FHA domain is essential for Ser-116 phosphorylation and for the association with 53BP1, which collectively enhance the accumulation of APLF at sites of DNA damage. The level of 53BP1 expression, but not XRCC4, was found to correlate with the phosphorylation of APLF by ATM, and disruption of the APLF-53BP1 interaction was associated with impaired cell survival following DNA damage. These results suggest that the APLF FHA domain directs interactions with multiple partners in the DNA damage response, and that 53BP1 facilitates the ATM-dependent phosphorylation of APLF. Citation Format: Amanda L. Fenton, Diana Tran, Christine Anne Koch. 53BP1 facilitates the ATM-dependent phosphorylation of APLF in the DNA damage response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3955. doi:10.1158/1538-7445.AM2014-3955

BRCA1 promoter hypermethylation, 53BP1 protein expression and PARP-1 activity as biomarkers of DNA repair deficit in breast cancer

BackgroundPoly(adenosine diphosphate–ribose) polymerase 1 (PARP-1) and the balance between BRCA1 and 53BP1 play a key role in the DNA repair and cell stress response. PARP inhibitors show promising clinical activity in metastatic triple negative (TN) or BRCA-mutated breast cancer. However, a comprehensive analysis of PARP-1 activity, BRCA1 promoter methylation and 53BP1 expression in tumours without known BRCA1 mutation has not yet been carried out.MethodsWe investigated cytosolic PARP-1 activity, 53BP1 protein levels and BRCA1 promoter methylation in 155 surgical breast tumour samples from patients without familial breast cancer history or known BRCA1 mutations who were treated between January 2006 and November 2009 and evaluated their statistical association with classical predictive and prognostic factors.ResultsThe mitotic count score was the only parameter clearly associated with PARP-1 activity. BRCA1 promoter hypermethylation (15.4% of all cancers) was significantly associated with uPA and PAI-1 levels, tumour grade, mitotic count score, hormone receptor and HER2 negative status and TN profile (29% of TN tumours showed BRCA1 promoter hypermethylation compared to 5% of grade II-III hormone receptor-positive/HER2-negative and 2% of HER2-positive tumours). No statistical association was found between BRCA1 promoter hypermethylation and PARP-1 activity. High 53BP1 protein levels correlated with lymph node positivity, hormone receptor positivity, molecular grouping, unmethylated BRCA1 promoter and PARP-1 activity. In TN tumours, BRCA1 promoter methylation was only marginally associated with age, PARP-1 activity was not associated with any of the tested clinico-pathological factors and high 53BP1 protein levels were significantly associated with lymph node positivity. Only 3 of the 14 TN tumours with BRCA1 promoter hypermethylation presented high 53BP1 protein levels.ConclusionsBreast cancers that harbour simultaneously high 53BP1 protein level and BRCA1 promoter hypermethylation and are the putative target population of drugs targeting DNA repair appear to be restricted to a small subgroup of TN tumours.

53BP1 Expression Is a Modifier of the Prognostic Value of Lymph Node Ratio and CA 19-9 in Pancreatic Adenocarcinoma

53BP1 binds to the tumor suppressor p53 and has a key role in DNA damage response and repair. Low 53BP1 expression has been associated with decreased survival in breast cancer and has been shown to interact with several prognostic factors in non-small cell lung cancer. The role of 53BP1 in pancreatic ductal adenocarcinoma (PDAC) has yet to be determined. We aimed to investigate whether 53BP1 levels interact with established prognostic factors in PDAC. 106 patients for whom there was tissue available at time of surgical resection for PDAC were included. A tissue microarray was constructed using surgical specimens, stained with antibodies to 53BP1, and scored for expression intensity. Univariate and multivariate statistical analyses were performed to investigate the association between 53BP1 and patient survival with known prognostic factors for survival. The association of 53BP1 with several established prognostic factors was examined, including stage, tumor grade, surgical margin, peripancreatic extension, lymph node ratio (LNR), and CA 19–9. We found that 53BP1 modified the effects of known prognostic variables including LNR and CA 19–9 on survival outcomes. When 53BP1 intensity was low, increased LNR was associated with decreased OS (HR 4.84, 95% CI (2.26, 10.37), p<0.001) and high CA19-9 was associated with decreased OS (HR 1.72, 95% CI (1.18, 2.51), p=0.005). When 53BP1 intensity was high, LNR and CA19-9 were no longer associated with OS (p=0.958 and p=0.606, respectively). In this study, 53BP1, a key player in DNA damage response and repair, was found to modify the prognostic value of two established prognostic factors, LNR and CA 19–9, suggesting 53BP1 may alter tumor behavior and ultimately impact how we interpret the value of other prognostic factors.

Significance of p53‐binding protein 1 (53BP1) expression in thyroid papillary microcarcinoma: association with BRAFV600E mutation status

In a previous report, we proposed that analysis of 53BP1 expression by immunofluorescence could be a useful tool in estimating the level of genomic instability (GIN), as well as the malignant potential, of thyroid tumours. In an attempt to clarify the value of 53BP1 expression as a new molecular marker for the aggressiveness of thyroid papillary microcarcinoma (PMC), we assessed the association between the type of 53BP1 expression and clinicopathological features such as tumour size, extrathyroidal invasion, lymph node metastasis and BRAF(V) (600E) mutation of PMC. A total of 36 surgically resected thyroid tumours, including 13 PMC and 23 conventional papillary thyroid carcinomas (PTC), were available for this study. Analysis using immunofluorescence revealed that the incidence of an abnormal or high DNA damage response (DDR) type of 53BP1 expression was significantly higher in PTC than PMC. BRAF(V) (600E) mutation was not associated significantly with tumour aggressiveness in either PMC or PTC cases. Abnormal/high DDR type of 53BP1 expression was associated closely with both BRAF(V) (600E) mutation and papillary and/or trabecular architecture of PMC. Abnormal/high DDR type of 53BP1 expression might be associated with GIN and papillary/trabecular morphology at an early stage of PTC carcinogenesis through BRAF(V) (600E) mutation.

The predictive value of 53BP1 and BRCA1 mRNA expression in advanced non-small-cell lung cancer patients treated with first-line platinum-based chemotherapy

Platinum-based chemotherapy is the standard first-line treatment for non-oncogene- addicted non-small cell lung cancers (NSCLCs) and the analysis of multiple DNA repair genes could improve current models for predicting chemosensitivity. We investigated the potential predictive role of components of the 53BP1 pathway in conjunction with BRCA1. The mRNA expression of BRCA1, MDC1, CASPASE3, UBC13, RNF8, 53BP1, PIAS4, UBC9 and MMSET was analyzed by real-time PCR in 115 advanced NSCLC patients treated with first-line platinum-based chemotherapy. Patients expressing low levels of both BRCA1 and 53BP1 obtained a median progression-free survival of 10.3 months and overall survival of 19.3 months, while among those with low BRCA1 and high 53BP1 progression-free survival was 5.9 months (P less than 0.0001) and overall survival was 8.2 months (P=0.001). The expression of 53BP1 refines BRCA1-based predictive modeling to identify patients most likely to benefit from platinum-based chemotherapy.

Decreased 53BP1 expression predicts improved survival in sporadic ovarian carcinoma

Objective: 53BP1 is a critical regulator of the balance between homologous recombination (HR) and the more error-prone nonhomologous endjoining (NHEJ) DNA repair. Deletion of 53BP1 in brca1- (but not brca2-) null cells rescues embryonic lethality, partially restores HR, and reverses sensitivity to poly-ADP ribose polymerase inhibitors (PARPi). We characterized 53BP1 and BRCA1 expression in a large number of primary and recurrent ovarian carcinomas to determine if 53BP1 expression is associated with clinical outcomes in sporadic and inherited cases.

Abstract 1770: Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer.

Abstract Purpose: BRCA1 plays a key role in DNA repair and cellular stress response. Inhibitors of PARP show promising clinical activity in metastatic, triple-negative BRCA-mutated breast cancer. P53 binding protein 1 (53BP1) is a protein involved in DNA damage checkpoint activation and DNA repair that is involved in both non homologous end-joining and homology-mediated repair of double-strand DNA breaks. When both BRCA1 and 53BP1 are lost, homology-mediated repair appears to be restored and the sensitivity to DNA damaging agents is reduced. Loss of 53BP1 leads to resistance to cis-platinum and PARP inhibitors in BRCA1-deficient cells, suggesting that 53BP1 is a critical modulator of repair choice in BRCA1 mutant, and possibly epigenetically inactivated cells. Reduced 53BP1 expression has been reported in sporadic basal-like and triple-negative and BRCA-associated breast cancers. It thus appears important to simultaneously evaluate 53BP1 status altogether with BRCA1 mutation/methylation in order to estimate the homology-mediated functionality in breast cancer tumors. Methods: We investigated cytosolic 53BP1 levels and BRCA1 gene promoter hypermethylation profile in 151 surgical tumor samples from patients treated in our institution between January 2006 and November 2009 and evaluated their statistical association with classical breast cancer predictive and prognostic factors. DNA methylation patterns in the CpG islands of BRCA1 gene promoter were determined by a methylation specific PCR distinguishing unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA. Cytosolic 53BP1 level was evaluated using a commercially available assay kit. Results: Median age was 54 years (range 29-75 years). Median 53BP1 protein level (pg/mg of cytosolic protein) was 9.5. Using this cut-off, low 53BP1 levels were significantly associated with negative ER and PR status, triple-negative profile, low cytosolic PARP activity, BRCA1 promoter hypermethylation and absence of nodal metastases. The hypermethylation status was significantly associated with UPA and PAI-1 levels, SBR grade, SBR Mitotic count score, ER, PR and HER-2 negative status, and then conversely triple negative (ER, PR and HER-2 negative) profile. Twenty nine percent of the triple negative tumors presented a hypermethylated status, comparing to 5 and 2% for HR+/HER-2- and HER-2+ tumors respectively. In the triple negative population (N=48), only 2 of the 13 tumors with BRCA1 promoter hypermethylation, presented high (&amp;gt;9.5 pg/mg) 53BP1 protein levels Conclusion: Triple negative breast cancers harboring simultaneously a high level of 53BP1 and BRCA1 promoter hypermethylation are a putative target population for development of drugs targeting DNA repair. However, this population appears to be restricted to a small subgroup of triple negative breast cancers. Citation Format: William Jacot, Simon Thezenas, Anne-Claire Laberenne, Romain Senal, Cathy Viglianti, Frédéric Bibeau, Jean-Pierre Bleuse, Gilles Romieu, Pierre-Jean Lamy. Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer. [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 1770. doi:10.1158/1538-7445.AM2013-1770

Trolox contributes to Nrf2-mediated protection of human and murine primary alveolar type II cells from injury by cigarette smoke

Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)−/− mice. We determined human primary ATII cell injury by CS in vitro and analyzed ATII cells isolated from smoker and non-smoker lung donors ex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2−/− mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential antioxidant prevention strategy against oxidative injury of ATII cells in CS-related lung diseases.

53BP1 expression is a modifier of the prognostic value of lymph node ratio and CA 19-9 in pancreatic adenocarcinoma.

Background53BP1 binds to the tumor suppressor p53 and has a key role in DNA damage response and repair. Low 53BP1 expression has been associated with decreased survival in breast cancer and has been shown to interact with several prognostic factors in non-small cell lung cancer. The role of 53BP1 in pancreatic ductal adenocarcinoma (PDAC) has yet to be determined. We aimed to investigate whether 53BP1 levels interact with established prognostic factors in PDAC.Methods106 patients for whom there was tissue available at time of surgical resection for PDAC were included. A tissue microarray was constructed using surgical specimens, stained with antibodies to 53BP1, and scored for expression intensity. Univariate and multivariate statistical analyses were performed to investigate the association between 53BP1 and patient survival with known prognostic factors for survival.ResultsThe association of 53BP1 with several established prognostic factors was examined, including stage, tumor grade, surgical margin, peripancreatic extension, lymph node ratio (LNR), and CA 19–9. We found that 53BP1 modified the effects of known prognostic variables including LNR and CA 19–9 on survival outcomes. When 53BP1 intensity was low, increased LNR was associated with decreased OS (HR 4.84, 95% CI (2.26, 10.37), p<0.001) and high CA19-9 was associated with decreased OS (HR 1.72, 95% CI (1.18, 2.51), p=0.005). When 53BP1 intensity was high, LNR and CA19-9 were no longer associated with OS (p=0.958 and p=0.606, respectively).ConclusionsIn this study, 53BP1, a key player in DNA damage response and repair, was found to modify the prognostic value of two established prognostic factors, LNR and CA 19–9, suggesting 53BP1 may alter tumor behavior and ultimately impact how we interpret the value of other prognostic factors.

53BP1 expression in sporadic and inherited ovarian carcinoma: Relationship to genetic status and clinical outcomes

Objectives53BP1, a critical mediator of the DNA damage response, functions by regulating the balance between homologous recombination (HR) and the more error-prone non-homologous endjoining (NHEJ). Deletion of 53BP1 in brca1 (but not brca2) null cells partially restores HR and reverses sensitivity to poly-ADP-ribose polymerase inhibitors (PARPi). We characterized 53BP1 and BRCA1 expression and their association with clinical outcomes in sporadic and inherited ovarian carcinomas. MethodsWe evaluated 53BP1 and BRCA1 protein expression using immunohistochemistry in 248 ovarian carcinomas and mRNA expression in 89 cases with quantitative reverse transcriptase PCR. All subjects were comprehensively characterized for germline mutations in BRCA1 and BRCA2. ResultsBRCA1-mutated (but not BRCA2-mutated) ovarian carcinomas had significantly higher 53BP1 protein expression than wildtype carcinomas. 53BP1 message levels were significantly associated with BRCA1 message levels in wildtype and BRCA1-mutated but not BRCA2-mutated carcinomas. In wildtype carcinomas, lower 53BP1 message predicted improved survival (p=0.02, median survival 74 vs. 41months, HR 0.49, 95% CI 0.27–0.88). Survival was not impacted by BRCA1 message level. 53BP1 expression was not associated with primary platinum resistance. In 54 paired primary and recurrent cases, 53BP1 protein expression was equally likely to decrease or increase, and there was no association between decreased 53BP1 at recurrence and the development of platinum resistance. ConclusionsBRCA1-mutated ovarian carcinomas have higher 53BP1 protein expression than wildtype or BRCA2-mutated carcinomas, in opposition to previous findings in breast carcinomas. Higher 53BP1 protein, which promotes NHEJ, could explain the frequent chromosomal aberrations that are characteristic of BRCA1-mutated ovarian carcinomas. In wildtype ovarian carcinomas, decreased 53BP1 message predicts improved survival, but message and protein expression were not associated.

53BP1 Is a Haploinsufficient Tumor Suppressor and Protects Cells from Radiation Response in Glioma

The DNA damage response (DDR) plays a crucial role in tumor development in different tissues. Here, we show that p53-binding protein 1 (53BP1), a key element of the DDR, is heterozygously lost in approximately 20% of human glioblastoma multiforme (GBM) specimens, primarily of the Proneural subtype, and low 53BP1 expression levels are associated with worse prognosis. We present evidence that 53BP1 behaves as haploinsufficient tumor suppressor in a mouse model of platelet-derived growth factor-induced gliomagenesis. We also show that very low level of 53BP1 as found in 53BP1 null gliomas or robust 53BP1 gene silencing in glioma cell lines (but not 53BP1 heterozygous tumors or partial gene knockdown) sensitizes glioma cells to ionizing radiation (IR), both in vitro and in vivo. We further show the 53BP1 gene silencing induces defects in the nonhomologous end-joining (NHEJ) DNA repair pathway. These deficiencies lead to a failure to fully repair the damaged DNA upon exposure of glioma cells to IR with a consequent prolonged cell-cycle arrest and increased apoptosis. Our data suggest that either 53BP1 or other NHEJ components may be critical molecules to be pharmacologically targeted in GBM in combination with standard therapies.

53BP1 functions as a tumor suppressor in breast cancer via the inhibition of NF-κB through miR-146a

p53-binding protein-1 (53BP1) plays a critical role in cell cycle checkpoint and DNA repair activities. Recently, 53BP1 was recognized as a potential tumor suppressor gene. In this study, we investigated its tumor suppressor function in breast cancer. In clinical samples, we observed a lower level of 53BP1 expression in the cancer lesions than in the matched non-tumor tissues. Furthermore, the 53BP1 level showed a gradual decrease during the progression from precancerous to cancer lesion. Ectopic expression of 53BP1 can significantly inhibit cell proliferation and curb the invasiveness in breast cancer cell lines, whereas knockdown of 53BP1 by RNA interference had the opposite effects. Additionally, 53BP1 markedly inhibited xenograft formation and metastasis of breast cancer cells in nude mice. Both in vitro and in vivo studies revealed that the 53BP1 expression level was inversely correlated to the function of nuclear factor-kappaB (NF-κB), which contributes to the invasion and metastasis of breast cancer. Importantly, the inhibitory effect of 53BP1 on NF-κB activity was shown to be mediated by the upregulation of miR-146a. Together, our findings demonstrated that 53BP1 has a potent tumor suppressor activity in breast cancer, and it may serve as a novel target for breast cancer prevention and treatment.

Cre-Mediated Stress Affects Sirtuin Expression Levels, Peroxisome Biogenesis and Metabolism, Antioxidant and Proinflammatory Signaling Pathways

Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts), inducing PPARα/γ, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14) as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase). In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2) and a NF-κB activation were noted. TGFβ1 and proinflammatory cytokines like IL1, IL6 and TNFα were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7) in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt) with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out-mediated effects.

Low p53 Binding Protein 1 (53BP1) Expression Is Associated With Increased Local Recurrence in Breast Cancer Patients Treated With Breast-Conserving Surgery and Radiotherapy

To investigate whether the expression of p53 binding protein 1 (53BP1) has prognostic significance in a cohort of early-stage breast cancer patients treated with breast-conserving surgery and radiotherapy (BCS+RT). A tissue microarray of early-stage breast cancer treated with BCS+RT from a cohort of 514 women was assayed for 53BP1, estrogen receptor, progesterone receptor, and HER2 expression by immunohistochemistry. Through log-rank tests and univariate and multivariate models, the staining profile of each tumor was correlated with clinical endpoints, including ipsilateral breast recurrence-free survival (IBRFS), distant metastasis-free survival (DMFS), cause-specific survival (CSS), recurrence-free survival (RFS), and overall survival (OS). Of the 477 (93%) evaluable tumors, 63 (13%) were scored as low. Low expression of 53BP1 was associated with worse outcomes for all endpoints studied, including 10-year IBRFS (76.8% vs. 90.5%; P=.01), OS (66.4% vs. 81.7%; P=.02), CSS (66.0% vs. 87.4%; P<.01), DMFS (55.9% vs. 87.0%; P<.01), and RFS (45.2% vs. 80.6%; P<.01). Multivariate analysis incorporating various clinico-pathologic markers and 53BP1 expression found that 53BP1 expression was again an independent predictor of all endpoints (IBRFS: P=.0254; OS: P=.0094; CSS: P=.0033; DMFS: P=.0006; RFS: P=.0002). Low 53BP1 expression was also found to correlate with triple-negative (TN) phenotype (P<.01). Furthermore, in subset analysis of all TN breast cancer, negative 53BP1 expression trended for lower IBRFS (72.3% vs. 93.9%; P=.0361) and was significant for worse DMFS (48.2% vs. 86.8%; P=.0035) and RFS (37.8% vs. 83.7%; P=.0014). Our data indicate that low 53BP1 expression is an independent prognostic indicator for local relapse among other endpoints in early-stage breast cancer and TN breast cancer patients treated with BCS+RT. These results should be verified in larger cohorts of patients to validate their clinical significance.

Discordance between phosphorylation and recruitment of 53BP1 in response to DNA double-strand breaks

During the DNA damage response (DDR), chromatin modifications contribute to localization of 53BP1 to sites of DNA double-strand breaks (DSBs). 53BP1 is phosphorylated during the DDR, but it is unclear whether phosphorylation is directly coupled to chromatin binding. In this study, we used human diploid fibroblasts and HCT116 tumor cells to study 53BP1 phosphorylation at Serine-25 and Serine-1778 during endogenous and exogenous DSBs (DNA replication and whole-cell or sub-nuclear microbeam irradiation, respectively). In non-stressed conditions, endogenous DSBs in S-phase cells led to accumulation of 53BP1 and γH2AX into discrete nuclear foci. Only the frank collapse of DNA replication forks following hydroxyurea treatment initiated 53BP1Ser25 and 53BP1Ser1778 phosphorylation. In response to exogenous DSBs, 53BP1Ser25 and 53BP1Ser1778 phosphoforms localized to sites of initial DSBs in a cell cycle-independent manner. 53BP1 phosphoforms also localized to late residual foci and associated with PML-NBs during IR-induced senescence. Using isogenic cell lines and small-molecule inhibitors, we observed that DDR-induced 53BP1 phosphorylation was dependent on ATM and DNA-PKcs kinase activity but independent of MRE11 sensing or RNF168 chromatin remodeling. However, loss of RNF168 blocked recruitment of phosphorylated 53BP1 to sites of DNA damage. Our results uncouple 53BP1 phosphorylation from DSB localization and support parallel pathways for 53BP1 biology during the DDR. As relative 53BP1 expression may be a biomarker of DNA repair capacity in solid tumors, the tracking of 53BP1 phosphoforms in situ may give unique information regarding different cancer phenotypes or response to cancer treatment.

Significance of p53‐binding protein 1 nuclear foci in uterine cervical lesions: endogenous DNA double strand breaks and genomic instability during carcinogenesis

AimsA defective DNA damage response can result in genomic instability (GIN) and lead to transformation to cancer. As p53-binding protein 1 (53BP1) localizes at the sites of DNA double strand breaks (DSBs) and rapidly forms nuclear foci (NF), the presence of 53BP1 NF can be considered to be an indicator of endogenous DSBs reflecting GIN. Our aim was to analyse the presence of DSBs by immunofluorescence for 53BP1 expression in a series of cervical lesions, to evaluate the significance of GIN during carcinogenesis.Methods and resultsA total of 80 archival cervical tissue samples, including 11 normal, 16 cervical intraepithelial neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14 squamous cell carcinoma samples, were analysed for 53BP1 NF, human papillomavirus (HPV) infection, and p16INK4a overexpression. The number of 53BP1 NF in cervical cells appeared to increase with progression during carcinogenesis. The distribution of 53BP1 NF was similar to that of the punctate HPV signals as determined by in-situ hybridization and also to p16INK4a overexpression in CIN, suggesting an association with viral infection and replication stress.ConclusionsImmunofluorescence analysis of 53BP1 expression can be a useful tool with which to estimate the level of GIN. During cervical carcinogenesis, GIN may allow further accumulation of genomic alterations, causing progression to invasive cancer.

Abstract A65: RNF8: The molecular modulator of breast cancer aggressiveness

Abstract Background: Women of Mexican and African-American descent have a higher mortality and a higher proportion of triple negative or basal-like breast cancer with BRCA like features. Basal-like breast cancer subtype is associated with an elevated Ki67 index and extensive genomic instability. This increase in the Ki67 index indicates cell proliferation and perhaps greater DNA damage; double strand breaks are the most consequential DNA lesions. Knockdown of RING-finger 8 (RNF8), an E3 ubiquitin-protein ligase, by siRNA inhibit focus formation at double strand breaks sites suggesting that RNF8 is an important regulator of the DNA repair system. We are investigating the hypothesis that RNF8 is a modulator of genomic integrity by acting as a regulator of BRCA1 and 53BP1 proteins in human breast cancers. Methods: We compared RNF8, p53 binding protein 1 (53BP1), cyclin B1 (CB1) and Ki67 mRNA expression in 57 formalin fixed paraffin embedded human breast cancer taken from a population of Mexican women enriched for basal-like tumors. Further we investigated copy number alterations in the 6p21.2 chromosomal region that contains RNF8 in a subset of 971 early stage breast cancers categorized by tumor subtype considering race/ethnicity. Results: RNF8 and 53BP1 were found to exhibit variable expression at the mRNA level by Ki67 and CB1 expression levels. The combined effect RNF8 high/53BP1 low was associated with high expression of Ki67 (p=0.028). Conversely, RNF8 low expression was associated with low expression of Ki67 (p = 0.017), low CB1 (p = 0.035) and was independent of the 53BP1 expression. In a sample set of 971 stage I/II breast cancers, copy number gain at 6p21.3 (RNF8 locus) was positively associated with triple negative breast cancer (p&amp;lt; 0.0001), high grade tumors (p=0.0017) and large size tumors (p=0.051). Gain in the genomic regions containing the RNF8 locus was significantly associated with greater genomic instability (fdr=0.01). Additionally, though not significant given small sample size, African-Americans were ∼2-fold more likely than Non-Hispanic Whites or Hispanics to be diagnosed with tumors showing gain at the RNF8 locus (p=0.11). Different groups have established that RNF8 is recruited to DNA damaged sites and subsequently mediates DNA repair by accumulation of BRCA1 and 53BP1 at DNA lesions. Recently, two groups have independently determined, in a BRCA deficient environment, that chromosomal instability is promoted in the presence of 53BP1. Additionally, it was shown that 53BP1 depletion can restore the DNA repair functions. In this report, we demonstrate differential expression of RNF8 and 53BP1 in a series of human breast cancers and an association between RNF8/53BP1 levels, proliferation, and tumor subtype. Our findings suggest that some breast tumors present an altered DNA repair pathway resulting in activation of RNF8 and de-regulation of 53BP1, which we hypothesize, may contribute to genomic instability and more proliferative tumors. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A65.

Constitutive expression of γ-H2AX has prognostic relevance in triple negative breast cancer

Background and purposeConstitutive γ-H2AX expression might indicate disruption of the DNA damage repair pathway, genomic instability, or shortened telomeric ends. Here, we quantified expression of endogenous γ-H2AX and its downstream factor 53BP1 in a large number of breast cancer cell lines (n=54) and a node-negative breast cancer cohort that had not received adjuvant systemic treatment (n=122). Materials and methodsFormalin fixed paraffin embedded breast cancer cell lines and tumors were immunohistochemically analyzed for γ-H2AX and 53BP1 expression, and related to cell line, patient and tumor characteristics and to disease progression. ResultsIn breast cancer cell lines, γ-H2AX positivity was associated with the triple negative/basal like subgroup (p=0.005), and with BRCA1 (p=0.011) or p53 (p=0.053) mutations. Specifically in triple negative breast cancer patients a high number of γ-H2AX foci indicated a significantly worse prognosis (p=0.006 for triple negative vs. p=0.417 for estrogen receptor (ER), progesterone receptor (PR) or HER2 positive patients). A similar association with disease progression was found for 53BP1. In a multivariate analysis with tumor size, grade, and triple negativity, only the interaction between triple negativity and γ-H2AX remained significant (p=0.002, Hazard Ratio=6.77, 95% CI=2.07–22.2). ConclusionsConstitutive γ-H2AX and 53BP1 staining reveals a subset of patients with triple negative breast tumors that have a significantly poorer prognosis.