DNA Repair Signaling Pathways Research Articles

Abstract 2266: Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair

Abstract According to the American Cancer Society, more than 90% of breast cancer patients who succumb to the condition die as a result of the cancer metastasizing to other areas of the body. Our lab has previously shown that the presence of a chemoresistant invasive subpopulation of breast carcinoma cells is correlated with the prevalence of metastasis. Additionally, we have hypothesized that chemoresistance and survival of these cancer cells which eventually leads to relapse and metastasis following initial treatment with chemotherapeutic agents is achieved by enhanced DNA repair capability in this population. We reported that an anti-apoptotic protein ARC (apoptosis repressor with caspase recruitment domain) is over expressed in the invasive mammary cancer cells that are resistant to standard chemotherapeutic agents. Further, we have shown that ARC over expressing cells are resistant to chemotherapy both in vivo and in vitro. In this study, we examined cellular DNA repair signaling pathways in order to delineate the role of ARC in the pathogenesis of breast cancer. Utilizing cell lines where ARC was either knocked down or overexpressed, we investigated potential correlations of ARC with DNA repair capability by means of the single cell gel electrophoresis assay (comet assay). The results of these experiments indicated that over expression of ARC leads to increased cellular DNA repair while knocking it down leads to decreased cellular DNA repair. In order to determine the molecular basis of the role of ARC in the DNA repair pathways, we have performed qRT-PCR based gene expression analysis on ARC modified cell lines using techniques previously established in our lab. The results of these experiments indicated that increased levels of ARC are correlated with upregulation of key genes in single stranded DNA repair pathways such as the base excision and nucleotide excision repair pathways. We are working to identify the specific domain of ARC that serves as its effector in the DNA repair pathways. In particular, we are looking at the caspase recruitment domain (CARD) of ARC which is known to be key for ARC`s function in the apoptotic pathways. Once this domain is identified, it may serve as a potential drug target, the silencing of which could result in the disruption of key pathways required for invasive breast cancer cell survival. Such therapy may facilitate the ablation of chemoresistant breast cancer cell populations, prevent metastasis and tumor relapse, and increase long term patient survival. Citation Format: Eli Grunblatt, Evanka Madan, Sweta Roy, Sumanta Goswami. Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair. [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 2266. doi:10.1158/1538-7445.AM2014-2266

DNA methylation changes in genes frequently mutated in sporadic colorectal cancer and in the DNA repair and Wnt/β-catenin signaling pathway genes

The onset and progression of colorectal cancer (CRC) involves a cascade of genetic and/or epigenetic events. The aim of the present study was to address the DNA methylation status of genes relevant in colorectal carcinogenesis and its progression, such as genes frequently mutated in CRC, genes involved in the DNA repair and Wnt signaling pathway. We analyzed methylation status in totally 160 genes in 12 paired colorectal tumors and adjacent healthy mucosal tissues using the Illumina Infinium Human Methylation 450 BeadChip. We found significantly aberrant methylation in 23 genes (NEIL1, NEIL3, DCLRE1C, NHEJ1, GTF2H5, CCNH, CTNNB1, DKK2, DKK3, FZD5 LRP5, TLE3, WNT2, WNT3A, WNT6, TCF7L1, CASP8, EDNRB1, GPC6, KIAA1804, MYO1B, SMAD2 and TTN). External validation by mRNA expression showed a good agreement between hypermethylation in cancer and down-regulated mRNA expression of the genes EDNRB1, GPC6 and SMAD2, and between hypomethylation and up-regulated mRNA expression of the CASP8 and DCLRE1C genes. Aberrant methylation of the DCLRE1C and GPC6 genes are presented here for the first time and are therefore of special interest for further validation as novel candidate biomarker genes in CRC, and merit further validation with specific assays.

Chemical strategies for development of ATR inhibitors.

ATR protein kinase is one of the key players in maintaining genome integrity and coordinating of the DNA damage response and repair signalling pathways. Inhibition of ATR prevents signalling from stalled replication forks and enhances the formation of DNA damage, particularly under conditions of replication stress present in cancers. For this reason ATR/CHK1 checkpoint inhibitors can potentiate the effect of DNA cross-linking agents, as evidenced by ATR inhibitors recently entering human clinical trials. This review aims to compile the existing literature on small molecule inhibitors of ATR, both from academia and the pharmaceutical industry, and will provide the reader with a comprehensive summary of this promising oncology target.

Abstract P4-06-01: Expression and DNA copy number profiling suggest novel therapeutic approaches for triple negative breast cancer subtypes

Abstract The aggressive triple negative breast cancers (TNBCs), which lack ER, PR and HER2, comprise a high-risk subset of human breast cancers which remain poorly characterized and lack effective treatments. While recent meta-analyses indicate the complexity of these tumors, no robust independently validated phenotypes have been defined. We have identified four distinct molecular subtypes through independent non-negative matrix factorization of expression data from 84 Discovery and 114 Validation Set TNBCs profiled at a single institution, with matching CNV data (SNP array). We then classified 485 publically available TNBCs via a centroid signature of only 80 genes. All three sets supported stratification of tumors by cell cycle, DNA repair, and immunological signaling pathways that have significantly different clinical outcomes. The first subtype, composed of intermediate grade tumors, resembles the “Molecular Apocrine” or “Luminal AR” subtype described previously and was defined by enrichment of prolactin, aryl hydrocarbon receptor, and ERBB4 signaling with activated downstream expression patterns of ESR1 signaling. Large deletions of chromosome 6 were specific to this subtype. While focal deletions at 14q21.2 and 12q13.13 were present in >60% of tumors of the other subtypes, the genes at these loci (FOXA1 and ERBB3) were overexpressed in the first subtype. Inhibitors of AR and MUC1, both overexpressed, may prove effective for these tumors. A second subtype defined as “Claudin-Low” or “Mesenchymal Stem-Like” showed overexpression of markers of mesenchymal lineage (ADIPOQ and OGN). Targets responsive to beta-blockers (ADRB2), and targetable molecules associated with platelet and endothelial function (EDNRB, PLA2G2A, PTGER3/4, PTGFR, PTGFRA) were also upregulated. Two basal-like subtypes were found with significant differences in DFS and OS, even after correction for available clinical covariates. The high-risk (31% 5-year DFS), low immune function subtype was regulated by SOX 10, 8, and 6 and had unique copy-number driven expression of FGFR2. The second, low-risk (78% 5-year DFS) basal-like subtype was enriched for overexpression of many immune pathways, regulated by increased STAT1 and activated STAT downstream signaling, as well as exclusive upregulation of CTLA4. This subtype also had the lowest tumor cell fraction as calculated by allele specific copy number analysis of tumors (ASCAT). Both basal-like subtypes expressed TTK, CHEK1, TOP2A, and AURKA. CDK1 was correlated with copy number variation at 10q21.1. We proposed and validated four molecular subtypes of TNBC before applying the resulting gene signature to 7 external expression sets. The described subtypes vary by clinical behavior and inferred biology. Each subtype appears to have specific gene expression regulated by copy number variation and a set of genes targetable by currently available agents. These findings further define the heterogeneity of TNBCs and suggest potential therapeutic targets for each subtype. This work was supported by a Promise grant from the Susan G. Komen for the Cure Foundation (KG081694). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-06-01.

Aberrant Upregulation Of Alternatively Spliced Isoforms Of Son In Myeloid Leukemia: Implications In Hematopoiesis and Leukemogenesis

▪SON is a large, poorly characterized nuclear speckle protein. The key structural domains comprising the SON protein are the serine/arginine-rich (RS) domain, single stranded RNA-, and double stranded RNA-binding domains, in addition to several long repeating amino acid sequences likely to facilitate its scaffolding function. Previously, we identified SON as a key factor for efficient RNA splicing of diverse genes related to cell cycle, DNA repair, and survival signaling pathways (Molecular Cell, 2011, 42:185). We also demonstrated that SON inhibits transcriptional activation of the promoter associated to the miR-23a-27a-24 cluster, which harbors a regulator for the expression of GATA-2, a known hematopoietic stem cell regulator (J. Biol. Chem. 2013, 288:5381). Moreover, SON is highly expressed in hematopoietic tissues/organs, and particularly upregulated in hematopoietic stem cells and leukemic blasts. These results suggest potential roles of SON in both normal hematopoiesis and hematological malignancies. Based on the nucleotide sequence of the SON gene, several databases predict that the primary transcript of SON contains alternative exons, and inclusion of these alternative exons during RNA splicing may generate C-terminally truncated short isoforms (splice variants) which lack RNA-binding motifs. However, expression of these short SON isoforms has not been confirmed in any types of cells and functions of the isoforms remain unexplored.Using 3' rapid amplification of cDNA end (3' RACE), we confirmed that short SON isoforms are indeed expressed in hematopoietic cells, and the included alternative exon provides the 3' UTR and polyadenylation signal different from those of full-length SON transcript. To further examine SON isoform expression, we designed primers targeting specific alternative exons of SON and screened the level of full-length SON and the short isoforms. Our quantitative PCR data showed that lineage marker negative (Lin-) bone marrow cells highly express full-length SON, but not short isoforms, when compared to total bone marrow cells. Interestingly, we found that the levels of short SON isoforms are noticeably high in acute myeloid leukemia (AML) patient bone marrow cells, while full-length SON is downregulated when compared to normal bone marrow cells. These expression patterns were consistent with data analyses in a cancer microarray database (Oncomine). Furthermore, we found that short isoforms, but not full-length SON, were downregulated during PMA-induced differentiation of HL-60 and K562 cells. These results suggest that while full-length SON is the major form in normal hematopoietic stem cells/progenitors, the production of short SON isoforms through alternative exon inclusion is aberrantly activated in leukemic cells, likely due to a factor associated with impaired differentiation.To address whether the short SON isoforms affect the function of full-length SON in RNA splicing, we examined RNA splicing efficiency using a minigene containing SON-dependent exon-intron junction. As we expected, the short isoform alone, which lacks RNA-binding motifs, was not able to process minigene RNA splicing. However, to our surprise, co-expression of a short isoform together with full-length SON caused an increase in spliced RNA product from a minigene. These results suggest that short isoform expression potentiates the full-length SON function during exon recognition. Taken together, our results reveal that inclusion/skipping of the alternative exons within the primary SON transcript is abnormally regulated in myeloid leukemia, resulting in upregulation of short splice variants of SON. Furthermore, fine-tuning of RNA splicing efficiency by short SON isoforms implicates potential roles of these isoforms in RNA processing and global gene expression during hematopoiesis and leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Molecular Insight into the Association Between Cartilage Regeneration and Ear Wound Healing in Genetic Mouse Models: Targeting New Genes in Regeneration

Tissue regeneration is a complex trait with few genetic models available. Mouse strains LG/J and MRL are exceptional healers. Using recombinant inbred strains from a large (LG/J, healer) and small (SM/J, nonhealer) intercross, we have previously shown a positive genetic correlation between ear wound healing, knee cartilage regeneration, and protection from osteoarthritis. We hypothesize that a common set of genes operates in tissue healing and articular cartilage regeneration. Taking advantage of archived histological sections from recombinant inbred strains, we analyzed expression of candidate genes through branched-chain DNA technology directly from tissue lysates. We determined broad-sense heritability of candidates, Pearson correlation of candidates with healing phenotypes, and Ward minimum variance cluster analysis for strains. A bioinformatic assessment of allelic polymorphisms within and near candidate genes was also performed. The expression of several candidates was significantly heritable among strains. Although several genes correlated with both ear wound healing and cartilage healing at a marginal level, the expression of four genes representing DNA repair (Xrcc2, Pcna) and Wnt signaling (Axin2, Wnt16) pathways was significantly positively correlated with both phenotypes. Cluster analysis accurately classified healers and nonhealers for seven out of eight strains based on gene expression. Specific sequence differences between LG/J and SM/J were identified as potential causal polymorphisms. Our study suggests a common genetic basis between tissue healing and osteoarthritis susceptibility. Mapping genetic variations causing differences in diverse healing responses in multiple tissues may reveal generic healing processes in pursuit of new therapeutic targets designed to induce or enhance regeneration and, potentially, protection from osteoarthritis.

P-16 DNA repair signaling pathway genes are overexpressed in complex aneuploid preimplantation embryos

On the 6th gestational week, a clinical pregnancy was confirmed with a single fetal sac and heart beat. A singleton female ongoing pregnancy beyond 35 weeks of gestation is present to date. Conclusion: To the best of our knowledge, this is the first report presenting an ongoing clinical pregnancy achieved after selection of chromosomally normal/balanced embryos using the aCGH technique in a couple where female was a translocation carrier and male partner was diagnosed with Klinefelter syndrome. This case report validates the use of aCGH in PGD for complex indications requiring extensive screening of whole chromosomes.

Identification of DNA Repair Pathways That Affect the Survival of Ovarian Cancer Cells Treated with a Poly(ADP-Ribose) Polymerase Inhibitor in a Novel Drug Combination

Floxuridine (5-fluorodeoxyuridine, FdUrd), a U.S. Food and Drug Administration-approved drug and metabolite of 5-fluorouracil, causes DNA damage that is repaired by base excision repair (BER). Thus, poly(ADP-ribose) polymerase (PARP) inhibitors, which disrupt BER, markedly sensitize ovarian cancer cells to FdUrd, suggesting that this combination may have activity in this disease. It remains unclear, however, which DNA repair and checkpoint signaling pathways affect killing by these agents individually and in combination. Here we show that depleting ATR, BRCA1, BRCA2, or RAD51 sensitized to ABT-888 (veliparib) alone, FdUrd alone, and FdUrd + ABT-888 (F+A), suggesting that homologous recombination (HR) repair protects cells exposed to these agents. In contrast, disabling the mismatch, nucleotide excision, Fanconi anemia, nonhomologous end joining, or translesion synthesis repair pathways did not sensitize to these agents alone (including ABT-888) or in combination. Further studies demonstrated that in BRCA1-depleted cells, F+A was more effective than other chemotherapy+ABT-888 combinations. Taken together, these studies 1) identify DNA repair and checkpoint pathways that are important in ovarian cancer cells treated with FdUrd, ABT-888, and F+A, 2) show that disabling HR at the level of ATR, BRCA1, BRCA2, or RAD51, but not Chk1, ATM, PTEN, or FANCD2, sensitizes cells to ABT-888, and 3) demonstrate that even though ABT-888 sensitizes ovarian tumor cells with functional HR to FdUrd, the effects of this drug combination are more profound in tumors with HR defects, even compared with other chemotherapy + ABT-888 combinations, including cisplatin + ABT-888.

Abstract 2119: DDB2 and XPC influence ATR and ATM recruitment and regulate DNA damage response upon UV irradiation

Abstract DNA damage response (DDR) pathways control the activation of DNA repair mechanisms, the activation and eventual release of cell cycle checkpoints to prevent replication of damaged DNA, and apoptosis as a last resort. These processes suggest the coordination of numerous proteins required to preserve genomic integrity. ATR and ATM kinases are central to checkpoint activation in response to DNA damage and replication stress. Activated ATR and ATM phosphorylate several proteins involved in DNA repair and cell cycle arrest. The nature of the signal, which activates ATR and ATM in response to UV damage, is not clear. Here, we showed that DDB2 and XPC, two early damage recognition factors, promote ATR and ATM recruitment and phosphorylation. ATR and ATM localize to the damage site and physically interact with XPC and DDB2. ATR and ATM recruitment and their phosphorylation are affected in cells defective in DDB2 and XPC function. In contrast, ATR- and ATM-deficiency do not regulate DDB2 and XPC recruitment to the damage site. Consequently, phosphorylation of ATR and ATM substrates Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutated cells, indicating that defective DDB2 and XPC function impair checkpoint signal transduction cascade in response to UV damage. Furthermore, DDB2 and XPC also regulate BRCA1 and Rad51 recruitment to the damage site, implicating their role in homologous recombination-mediated DNA repair pathway. Collectively, these data showed that ATR and ATM are present in the preincision complex with XPC, and influence DNA repair and checkpoint signaling pathways. In support of this, we revealed that depletion of ATR and ATM influence NER efficiency. These results are consistent with a model in which DDB2 and XPC cross-talk with ATR and ATM during maintenance of genomic integrity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2119. doi:1538-7445.AM2012-2119

Baicalin modulates microRNA expression in UVB irradiated mouse skin

This study aimed to evaluate the effects of baicalin on ultraviolet radiation B (UVB)-mediated microRNA (miRNA) expression in mouse skin. We determined miRNA expression profiles in UVB irradiated mice, baicalin treated irradiated mice, and untreated mice, and conducted TargetScan and Gene Ontology analyses to predict miRNA targets. Three miRNAs (mmu-miR-125a-5p, mmu-miR-146a, and mmu-miR-141) were downregulated and another three (mmu-miR-188-5p, mmu-miR-223 and mmu-miR-22) were upregulated in UVB irradiated mice compared with untreated mice. Additionally, these miRNAs were predicted to be related to photocarcinogenesis, hypomethylation and apoptosis. Three miRNAs (mmu-miR-378, mmu-miR-199a-3p and mmu-miR-181b) were downregulated and one (mmu-miR-23a) was upregulated in baicalin treated mice compared with UVB irradiated mice, and they were predicted to be related to DNA repair signaling pathway. These deregulated miRNAs are potentially involved in the pathogenesis of photodamage, and may aid treatment and prevention of UVB-induced dermatoses.

Radio-Sensitization of Human Leukaemic MOLT-4 Cells by DNA-Dependent Protein Kinase Inhibitor, NU7026

In this paper we describe the influence of NU7026, a specific inhibitor of DNA-dependent protein kinase, phosphoinositide 3-kinase, and ATM-kinase on molecular and cellular mechanisms triggered by ionising irradiation in human T-lymphocyte leukaemic MOLT-4 cells. We studied the effect of this inhibitor (10 1microM) combined with gamma-radiation (1 Gy) leading to DNA damage response and induction of apoptosis. We used methods for apoptosis assessment (cell viability count and flow-cytometric analysis) and cell cycle analysis (DNA content measurement) and we detected expression and post-translational modifications (Western blotting) of proteins involved in DNA repair signalling pathways. Pre-treatment with NU7026 resulted into decreased activation of checkpoint kinase-2 (Thr68), p53 (Ser15 and Ser392), and histone H2A.X (Ser139) 2 hours after irradiation. Subsequently, combination of radiation and inhibitor led to decreased amount of cells in G2-phase arrest and into increased apoptosis after 72 hours. Our results indicate that in leukaemic cells the pre-incubation with inhibitor NU7026 followed by low doses of ionising radiation results in radio-sensitising of MOLT-4 cells via diminished DNA repair and delayed but pronounced apoptosis. This novel approach might offer new strategies in combined treatment of leukaemia diseases.

RAD18–BRCTx interaction is required for efficient repair of UV-induced DNA damage

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage signaling pathways. The BRCT domain-containing protein BRCTx has been shown to interact physically with RAD18, an E3 ligase involved in postreplication repair and homologous recombination repair. However, the physiological relevance of the interaction between RAD18 and BRCTx is largely unknown. In this study, we showed that RAD18 interacts with BRCTx in a phosphorylation-dependent manner and that this interaction, mediated via highly conserved serine residues on the RAD18 C terminus, is required for BRCTx accumulation at DNA damage sites. Furthermore, we uncovered critical roles of the RAD18-BRCTx module in UV-induced DNA damage repair but not PCNA mono-ubiquitination or homologous recombination. Thus, our results suggest that RAD18 has an additional function in the surveillance of the UV-induced DNA damage response signal.

Protein expression pattern in response to ionizing radiation in MCF-7 human breast cancer cells

Breast cancer is one of the most common types of cancer in women and is highly treatable by radiotherapy. However, repeated exposure to radiation results in tumor cell resistance. Understanding the molecular mechanisms involved in the response of tumors to γ-irradiation is important for improving radiotherapy. For this reason, we aimed to identify radiation-responsive genes at the protein level. In the present study, we observed differentially expressed proteins using 2D-PAGE and MALDI-TOF-MS for the global analysis of protein expression patterns in response to ionizing radiation (IR). When the expression patterns of proteins were compared to a control gel, numerous spots were found that differed greatly. Among them, 11 spots were found to be significantly different. One set of proteins (GH2, RGS17, BAK1, CCNH, TSG6, RAD51B, IGFBP1 and CASP14) was upregulated and another set of proteins (C1QRF, PLSCR2 and p34(SE1-1)) was downregulated after exposure to γ-rays. These proteins are known to be related to cell cycle control, apoptosis, DNA repair, cell proliferation and other signaling pathways.

Structural studies of the PARP-1 BRCT domain

BackgroundPoly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins localized to foci of DNA damage. Upon activation by encountering nicked DNA, the PARP-1 mediated trans-poly(ADP-ribosyl)ation of DNA binding proteins occurs, facilitating access and accumulation of DNA repair factors. PARP-1 also auto-(ADP-ribosyl)ates its central BRCT-containing domain forming part of an interaction site for the DNA repair scaffolding protein X-ray cross complementing group 1 protein (XRCC1). The co-localization of XRCC1, as well as bound DNA repair factors, to sites of DNA damage is important for cell survival and genomic integrity.ResultsHere we present the solution structure and biophysical characterization of the BRCT domain of rat PARP-1. The PARP-1 BRCT domain has the globular α/β fold characteristic of BRCT domains and has a thermal melting transition of 43.0°C. In contrast to a previous characterization of this domain, we demonstrate that it is monomeric in solution using both gel-filtration chromatography and small-angle X-ray scattering. Additionally, we report that the first BRCT domain of XRCC1 does not interact significantly with the PARP-1 BRCT domain in the absence of ADP-ribosylation. Moreover, none of the interactions with other longer PARP-1 constructs which previously had been demonstrated in a pull-down assay of mammalian cell extracts were detected.ConclusionsThe PARP-1 BRCT domain has the conserved BRCT fold that is known to be an important protein:protein interaction module in DNA repair and cell signalling pathways. Data indicating no significant protein:protein interactions between PARP-1 and XRCC1 likely results from the absence of poly(ADP-ribose) in one or both binding partners, and further implicates a poly(ADP-ribose)-dependent mechanism for localization of XRCC1 to sites of DNA damage.

Sirtuin 6 and DNA Repair

A genome stability regulator integrates DNA repair and stress signaling pathways.

Common genetic variants related to genomic integrity and risk of papillary thyroid cancer

DNA damage is an important mechanism in carcinogenesis, so genes related to maintaining genomic integrity may influence papillary thyroid cancer (PTC) risk. Candidate gene studies targeting some of these genes have identified only a few polymorphisms associated with risk of PTC. Here, we expanded the scope of previous candidate studies by increasing the number and coverage of genes related to maintenance of genomic integrity. We evaluated 5077 tag single-nucleotide polymorphisms (SNPs) from 340 candidate gene regions hypothesized to be involved in DNA repair, epigenetics, tumor suppression, apoptosis, telomere function and cell cycle control and signaling pathways in a case-control study of 344 PTC cases and 452 matched controls. We estimated odds ratios for associations of single SNPs with PTC risk and combined P values for SNPs in the same gene region or pathway to obtain gene region-specific or pathway-specific P values using adaptive rank-truncated product methods. Nine SNPs had P values <0.0005, three of which were in HDAC4 and were inversely related to PTC risk. After multiple comparisons adjustment, no SNPs remained associated with PTC risk. Seven gene regions were associated with PTC risk at P < 0.01, including HUS1, ALKBH3, HDAC4, BAK1, FAF1_CDKN2C, DACT3 and FZD6. Our results suggest a possible role of genes involved in maintenance of genomic integrity in relation to risk of PTC.

Abstract 591: Expanding the therapeutic window through pharmacological activation of the RB pathway in breast cancer treatment

Abstract Triple negative breast cancer (TNBC) is a highly aggressive disease that is most often treated with cytotoxic chemotherapies, such as anthracyclines and platinum compounds. While effective for certain tumors, these therapies are known to cause severe toxicities in numerous tissues. Thus, targeting pathways to both exploit therapy-sensitive tumors and ameliorate tissue toxicity is of utmost significance. Loss of the retinoblastoma tumor suppressor (RB) protein is a frequent event in TNBC, and is expected to alter therapeutic sensitivity by perturbing cell cycle checkpoints and yielding a predilection towards cell death. Conversely, it is possible that the same principles that drive sensitivity to cytotoxic agents in an RB-deficient setting could be used to prevent toxicities in an RB-proficient setting (i.e. normal tissues). Thus, modulation of the RB pathway could be critical for expanding the therapeutic window in the treatment of breast cancer. To address this hypothesis, combination studies of pharmacological activation of the RB pathway and cytotoxic chemotherapies commonly used to treat TNBC were performed in vitro and in vivo. Results indicate that activation of the RB pathway can modulate the efficacy of cytotoxic compounds in the treatment of TNBC. The mechanisms behind these observations were attributed directly to RB-mediated regulation of DNA damage and repair signaling pathways, as well as apoptosis. RB-deficiency resulted in bypass of these mechanisms and thus enhanced sensitivity to cytotoxic therapies. These results were supported by in silico data demonstrating that RB-deficient TNBC is associated with better clinical outcome in response to chemotherapy. Importantly, functional studies indicated that RB pathway activation can provide protection against the toxicity of frequently used chemotherapeutic compounds in normal tissue. Combined, these studies suggest that RB pathway status could be utilized in the clinic to direct combination treatments that would exploit the sensitivity of RB-deficient tumors to cytotoxic chemotherapies while simultaneously providing protection for normal tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 591. doi:10.1158/1538-7445.AM2011-591

Identification of a gene signature in cell cycle pathway for breast cancer prognosis using gene expression profiling data

BackgroundNumerous studies have used microarrays to identify gene signatures for predicting cancer patient clinical outcome and responses to chemotherapy. However, the potential impact of gene expression profiling in cancer diagnosis, prognosis and development of personalized treatment may not be fully exploited due to the lack of consensus gene signatures and poor understanding of the underlying molecular mechanisms.MethodsWe developed a novel approach to derive gene signatures for breast cancer prognosis in the context of known biological pathways. Using unsupervised methods, cancer patients were separated into distinct groups based on gene expression patterns in one of the following pathways: apoptosis, cell cycle, angiogenesis, metastasis, p53, DNA repair, and several receptor-mediated signaling pathways including chemokines, EGF, FGF, HIF, MAP kinase, JAK and NF-κB. The survival probabilities were then compared between the patient groups to determine if differential gene expression in a specific pathway is correlated with differential survival.ResultsOur results revealed expression of cell cycle genes is strongly predictive of breast cancer outcomes. We further confirmed this observation by building a cell cycle gene signature model using supervised methods. Validated in multiple independent datasets, the cell cycle gene signature is a more accurate predictor for breast cancer clinical outcome than the previously identified Amsterdam 70-gene signature that has been developed into a FDA approved clinical test MammaPrint®.ConclusionTaken together, the gene expression signature model we developed from well defined pathways is not only a consistently powerful prognosticator but also mechanistically linked to cancer biology. Our approach provides an alternative to the current methodology of identifying gene expression markers for cancer prognosis and drug responses using the whole genome gene expression data.

A molecular bar-coded DNA repair resource for pooled toxicogenomic screens

DNA damage from exogenous and endogenous sources can promote mutations and cell death. Fortunately, cells contain DNA repair and damage signaling pathways to reduce the mutagenic and cytotoxic effects of DNA damage. The identification of specific DNA repair proteins and the coordination of DNA repair pathways after damage has been a central theme to the field of genetic toxicology and we have developed a tool for use in this area. We have produced 99 molecular bar-coded Escherichia coli gene-deletion mutants specific to DNA repair and damage signaling pathways, and each bar-coded mutant can be tracked in pooled format using bar-code specific microarrays. Our design adapted bar-codes developed for the Saccharomyces cerevisiae gene-deletion project, which allowed us to utilize an available microarray product for pooled gene-exposure studies. Microarray-based screens were used for en masse identification of individual mutants sensitive to methyl methanesulfonate (MMS). As expected, gene-deletion mutants specific to direct, base excision, and recombinational DNA repair pathways were identified as MMS-sensitive in our pooled assay, thus validating our resource. We have demonstrated that molecular bar-codes designed for S. cerevisiae are transferable to E. coli, and that they can be used with pre-existing microarrays to perform competitive growth experiments. Further, when comparing microarray to traditional plate-based screens both overlapping and distinct results were obtained, which is a novel technical finding, with discrepancies between the two approaches explained by differences in output measurements (DNA content versus cell mass). The microarray-based classification of Δtag and ΔdinG cells as depleted after MMS exposure, contrary to plate-based methods, led to the discovery that Δtag and ΔdinG cells show a filamentation phenotype after MMS exposure, thus accounting for the discrepancy. A novel biological finding is the observation that while ΔdinG cells filament in response to MMS they exhibit wild-type sulA expression after exposure. This decoupling of filamentation from SulA levels suggests that DinG is associated with the SulA-independent filamentation pathway.

Activation Mechanism of Protein Kinase B by DNA-dependent Protein Kinase Involved in the DNA Repair System.

DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be 2nd upstream kinase for protein kinase B (PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells (MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK (M059J) and a wild-type of DNA-PK (M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.