DNA Repair Signaling Pathways Research Articles

Personalised medicine in urothelial bladder cancer

Available treatment options and outcomes for patients suffering from urothelial bladder cancer, especially in the metastatic stage, have hardly improved over decades. However, the increasing use of high-throughput analyses and the concept of immune surveillance against tumours have recently changed our understanding of tumour biology in terms of tumour development and progression. Our knowledge of genetic mutations and molecular subtypes provides the possibility of tailor-made therapeutic approaches for patients suffering from bladder cancer. For example, changes in DNA repair signalling pathways are possible predictors of chemotherapy response, and targeted therapies using FGFR or PARP inhibitors are currently being tested in clinical trials. The extent to which molecular subtypes will find their way into clinical practice depends on the prospective evaluation of their prognostic and predictive value. The introduction of immune checkpoint inhibitors is probably the most significant expansion of available treatment options in bladder cancer. Despite their promising results, however, a lot of questions remain to be answered, as only 25 % of patients respond. Again, this highlights the need for predictive biomarkers. The large inter- and intratumoural heterogeneity represents a particular challenge for the clinical implementation of personalised treatment options in bladder cancer. All in all, some important steps towards personalised medicine in urothelial bladder cancer have been taken in the past few years, but, for the most part, their prospective evaluation is still pending.

Targeting PARP1 As an Effective Treatment for Diffuse Large B-Cell Lymphoma with High Level of γH2AX

IntroductionDNA damage repair process has been reported to be the cause of malignant cells resistance to genomic DNA-targeted chemicals. Whether DNA damage repair mediates chemo-resistance in diffuse large B-cell lymphoma (DLBCL) remains elusive. H2AX phosphorylation (γH2AX) is an early event of double-strand DNA repair signaling, which can trigger homologous recombination (HR), classical non-homologous end joining (cNHEJ), or alternative non-homologous end joining (aNHEJ) in a context-dependent manner. The priority choice of PARP1 following H2AX phosphorylation promotes aNHEJ signaling, a well-known oncogenic DNA repair process. PARP inhibitor has increased the efficacy of some neoplasms with HR deficiency. The study addressed the association of γH2AX level with DNA repair signaling and its prognostic value in DLBCL. The role of PARP1 as a therapeutic target was also explored in the study.MethodsγH2AX protein was examined in new-diagnosed patients with DLBCL (n=90) using immunohistochemistry method. Its prognostic value was evaluated with multivariate analysis. Gene expression profiling was performed on 50 patients and DNA repair signaling was compared based on γH2AX expression. H2AX shRNA was used to downregulate the expression of H2AX and γH2AX through lentivirus in vitro on cell line to observe the effect on doxorubicin-induced apoptosis using flow cytometry. γH2AX and PARP1 were detected using Western blotting and immunoflurescent assay, while DNA repair signaling was detected using PCR array in vitro on cell lines. PARP inhibitor-induced apoptosis was also compared among cell lines using flow cytometry.ResultγH2AX level suggested a poor prognosis in new-diagnosed patients with diffuse large B-cell lymphomaγH2AX was detected in 43.34% (39/90) of new-diagnosed DLBCL patients (Figure 1A). Patients clinical features were listed in Table 1. The complete remission (CR) rate was lower in positive patients (31.37% vs 68.62%, p=0.009) after anthrocycline-based chemotherapy. Multivariate analysis showed γH2AX to have an independent inverse association with overall survival after adjusting age, ECOG score, IPI score and Ann Arbor stage in these patients [HR=2.202 (1.161-4.184), p=0.016] (Figure 1B).DNA repair signaling pathway was enriched in patients with γH2AX expressionGene expression profiling was performed in 50 new-diagnosed patients with DLBCL. Gene clusters were compared between patients with and without γH2AX expression. The result displayed that DNA repair signaling pathway was enriched in patients with γH2AX expression (NSE=1.901, FDR q-value=0.010) (Figure 1C).The linkage of γH2AX with DNA repair signaling was confirmed in vitro on cell linesThe expression of H2AX and γH2AX was tested in OCI-ly7, OCI-ly3 and OCI-ly8 cell lines. The highest level of H2AX and γH2AX was observed in OCI-ly7 cells, followed by OCI-ly8 and OCI-ly3 cells (Figure 2A). DNA repair genes were proven to be highly expressed in OCI-ly7 cell line using DNA repair signaling PCR array compared with the other two cell lines (Figure 2B).Downregulating γH2AX expression increased doxorubicin-induced apoptosis in vitroThe reduction of γH2AX level was demonstrated in H2AX shRNA-transduced OCI-ly7 cell line (Figure 2C). The proportion of doxorubicin-induced (50ng/ml) apoptosis was increased in these cells compared with control (5.87±2.37% vs 39.18±3.78%, p<0.05). (Figure 2D)PARP1 level presented a positive correlation with γH2AX expression and PARP inhibitor-induced apoptosis in vitroThe level of PARP1 expression was proportional to γH2AX expression in OCI-ly7, OCI-ly3 and OCI-ly8 cell lines (Figure 3A). The result indicates an abnormal aNHEJ oncogenic DNA repair to be superior in DLBCL cells with high γH2AX expression. FEN, another key component of aNHEJ signaling was also overexpressed in cell line and patients with high expression of γH2AX. PARP inhibitor (25μM) induced the highest proportion of apoptosis in OCI-ly7 cell line, compared with OCI-ly8 and OCI-ly3 cell lines (42.90±5.29% vs 29.52±3.95% vs 9.41±2.35%, p<0.05) (Figure 3B).ConclusionγH2AX as a biomarker for abnormally activated DNA repair signaling is associated with poor outcome in the setting of conventional chemotherapy in DLBCL. PARP1-mediated aNHEJ signaling performs critical functions in DLBCL with γH2AX expression. Here, we demonstrated that PARP inhibitor may be efficacious in these patients. DisclosuresNo relevant conflicts of interest to declare.

Pja2 Inhibits Wnt/β-catenin Signaling by Reducing the Level of TCF/LEF1.

Ubiquitination of proteins plays an essential role in various cellular processes, including protein degradation, DNA repair, and cell signaling pathways. Previous studies have shown that protein ubiquitination is implicated in regulating pluripotency as well as fate determination of stem cells. To identify how protein ubiquitination affects differentiation of embryonic stem cells, we analyzed microarray data, which are available in the public domain, of E3 ligases and deubiquitinases whose levels changed during stem cell differentiation. Expression of pja2, a member of the RING-type E3 ligase family, was up-regulated during differentiation of stem cells. Wnt/β-catenin signaling is one of the most important signaling pathways for regulation of the self-renewal and differentiation of embryonic stem cells. Pja2 was shown to bind to TCF/LEF1, which are transcriptional factors for Wnt/β-catenin signaling, and regulate protein levels by ubiquitination, leading to down-regulation of Wnt signaling activity. Based on these results, we suggest that E3 ligase Pja2 regulates stem cell differentiation by controlling the level of TCF/LEF1 by ubiquitination.

Circulating tumor DNA analysis depicts subclonal architecture and genomic evolution of small cell lung cancer

Subclonal architecture and genomic evolution of small-cell lung cancer (SCLC) under treatment has not been well studied primarily due to lack of tumor specimens, particularly longitudinal samples acquired during treatment. SCLC is characterized by early hematogenous spread, which makes circulating cell-free tumor DNA (ctDNA) sequencing a promising modality for genomic profiling. Here, we perform targeted deep sequencing of 430 cancer genes on pre-treatment tumor biopsies, as well as on plasma samples collected prior to and during treatment from 22 SCLC patients. Similar subclonal architecture is observed between pre-treatment ctDNA and paired tumor DNA. Mean variant allele frequency of clonal mutations from pre-treatment ctDNA is associated with progression-free survival and overall survival. Pre- and post-treatment ctDNA mutational analysis demonstrate that mutations of DNA repair and NOTCH signaling pathways are enriched in post-treatment samples. These data suggest that ctDNA sequencing is promising to delineate genomic landscape, subclonal architecture, and genomic evolution of SCLC.

Trax: A versatile signaling protein plays key roles in synaptic plasticity and DNA repair.

Translin-associated protein X (TSNAX), also called trax, was first identified as a protein that interacts with translin. Subsequent studies demonstrated that these proteins form a heteromeric RNase complex that mediates degradation of microRNAs, a pivotal finding that has stimulated interest in understanding the role of translin and trax in cell signaling. Recent studies addressing this question have revealed that trax plays key roles in both synaptic plasticity and DNA repair signaling pathways. In the context of synaptic plasticity, trax works together with its partner protein, translin, to degrade a subset of microRNAs. Activation of the translin/trax RNase complex reverses microRNA-mediated translational silencing to trigger dendritic protein synthesis critical for synaptic plasticity. In the context of DNA repair, trax binds to and activates ATM, a central component of the double-stranded DNA repair process. Thus, these studies focus attention on trax as a critical signaling protein that interacts with multiple partners to impact diverse signaling pathways. To stimulate interest in deciphering the multifaceted role of trax in cell signaling, we summarize the current understanding of trax biology and highlight gaps in our knowledge about this protean protein.

Abstract 3556: Sam68 is required for the growth and survival of non-melanoma skin cancer

Abstract Skin cancer is the most commonly diagnosed malignancy in the United States, and the incidence of skin cancer has increased dramatically over the last few decades. Non-melanoma skin cancer (NMSC), which includes squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), is the most common type of skin cancer with substantial associated morbidity and mortality. Whereas targeting DNA repair signaling pathways has emerged as a promising therapeutic for skin cancer, the relevance of DNA damage responses (DDR) in the development and survival of non-melanoma skin cancer (NMSC) remains obscure. Here we report that Src-associated substrate during mitosis of 68 kDa (Sam68), an early signaling molecule in DDR, is elevated in skin tumor tissues derived from NMSC patients and skin lesions from Gli2-transgenic mice. Downregulation of Sam68 impacts the growth and survival of human tumor keratinocytes and genetic ablation of Sam68 delays the onset of basal cell carcinomas (BCC) in Gli2-transgenic mice. Moreover, Sam68 plays a critical role in DNA damage-induced DNA repair and nuclear factor kappa B (NF-κB) signaling pathways in keratinocytes, hence conferring keratinocyte sensitivity to DNA damaging agents. Together our data reveal a novel function of Sam68 in regulating DDR in keratinocytes that is crucial for the growth and survival of NMSC. Citation Format: Kai Fu, Fengyi Wan. Sam68 is required for the growth and survival of non-melanoma skin cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3556.

Central and Peripheral Expression of DNA Double-Strand Breaks in Human and Mouse Tissues.

Mammalian cells accumulate DNA lesions when they undergo phases of the cell cycle or during normal cellular activity. In this regard, several DNA repair signaling pathways have evolved to maintain genome stability and avoid the potential acquisition of mutations. To define and further characterize the expression of DNA double-strand breaks in humans and mice, we used immunocytochemistry to localize a DNA damage signal within the spatial confines of the cell nucleus. We show that DNA double-strand breaks are abundantly expressed in postmitotic neurons of the human and mouse brain. Notably, DNA double-strand breaks are present in human hypothalamic and mouse striatal and hippocampal cells, with stable expression of the nuclear signal detected throughout the mammalian brain. Analysis of the mouse tongue, heart, and testis shows that expression of DNA double-strand breaks is only demonstrated in circumscribed populations of peripheral cells. These data suggest that levels of DNA double-strand breaks are tissue-specific with the tongue, heart and testicular tissue having different thresholds of DNA repair and DNA damage from those outlined at the brain level. Anat Rec, 301:1251-1257, 2018. © 2018 Wiley Periodicals, Inc.

Oncoprotein Tudor-SN is a key determinant providing survival advantage under DNA damaging stress.

Herein, Tudor-SN was identified as a DNA damage response (DDR)-related protein that plays important roles in the early stage of DDR. X-ray or laser irradiation could evoke the accumulation of Tudor-SN to DNA damage sites in a poly(ADP-ribosyl)ation-dependent manner via interaction with PARP-1. Additionally, we illustrated that the SN domain of Tudor-SN mediated the association of these two proteins. The accumulated Tudor-SN further recruited SMARCA5 (ATP-dependent chromatin remodeller) and GCN5 (histone acetyltransferase) to DNA damage sites, resulting in chromatin relaxation, and consequently activating the ATM kinase and downstream DNA repair signalling pathways to promote cell survival. Consistently, the loss-of-function of Tudor-SN attenuated the enrichment of SMARCA5, GCN5 and acetylation of histone H3 (acH3) at DNA break sites and abolished chromatin relaxation; as a result, the cells exhibited DNA repair and cell survival deficiency. As Tudor-SN protein is highly expressed in different tumours, it is likely to be involved in the radioresistance of cancer treatment.

Advances in molecular genetics of myelodysplastic syndromes

In recent years, with the application of whole-genome sequencing technology, more and more gene mutations in myelodysplastic syndrome (MDS) have been studied and analyzed. Common gene mutations involve RNA splicing, DNA methylation, histone modification, transcription factor, adhesion protein complex, signal transduction pathway kinase, DNA repair and RAS signaling pathway and so on. More and more studies have confirmed that the gene mutations are associated with the unique clinical features of MDS and prognosis. For example, TP53 and arginine/serine rich splicing factor (SRSF)2 gene mutations are associated with the progression of MDS, MDS patients with TET2 mutations respond well to methylation drug treatment; and enhancer of zeste homolog (EZH)2, ETS variant gene (ETV)6, RUNX1 mutations associated with poor prognosis of MDS. This article reviews literatures on advances in molecular genetics of MDS. Key words: Myelodysplastic syndromes; Mutation; Diagnosis; Prognosis

Targeting DNA Repair through Podophyllotoxin and Rutin Formulation in Hematopoietic Radioprotection: An in Silico, in Vitro, and in Vivo Study.

Drug discovery field has tremendously progressed during last few decades, however, an effective radiation countermeasure agent for the safe administration to the victims of radiation exposure is still unavailable. This multi-model study is aimed at elucidating the mechanistic aspects of a novel podophyllotoxin and rutin combination (henceforth referred as G-003M) in the hematopoietic radioprotection and its involvement in the DNA damage and repair signaling pathways. Using in silico study, we identified the binding sites and structural components of G-003M and validated in vitro. We further studied various in vivo endpoints related to the DNA repair and cell death pathways in mice pre-administered with G-003M, irradiated and subsequently euthanized to collect blood and bone marrow cells. In silico study showed the binding of podophyllotoxin to β-tubulin and presence of a functional hydroxyl group in the rutin, suggested their involvement in G2/M arrest and the free radical scavenging respectively. This experimentation was further validated through in vitro studies. In vivo mice studies confirmed that G-003M pre-administration attenuated DNA damage and enhanced repair after whole body exposure. We further noticed a decrease in the levels of γH2AX, p53BP1, and ATM kinase and an increase in the levels of DNA pk, Ku 80, Ligase IV, Mre 11, Rad 50 and NBS 1 in the blood and bone marrow cells of the G-003M pre-administered and irradiated mice. We noticed an overall increase in the pro-survival factors in the G-003M pre-treated and irradiated groups establishing the radioprotective efficacy of this formulation. The lead obtained from this study will certainly help in developing this formulation as a safe and effective radioprotector which could be used for humans against any planned or emergency exposure of radiation.

APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells.

Monocrotophos (MCP) and chlorpyrifos (CP) are widely used organophosphate pesticides (OPPs), speculated to be linked with human pathologies including cancer. Owing to the fact that lung cells are most vulnerable to the environmental toxins, the development and progression of lung cancer can be caused by the exposure of OPPs. The present study investigates the oxidative DNA damage response evoked by MCP and CP in human non-small cell lung carcinoma A549 cells. A549 cells were exposed to MCP and CP; cytotoxicity and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether MCP and CP can initiate the DNA repair and cell survival signalling pathways in A549 cells, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes and transcription factors (TFs) involved in cell survival mechanisms. A significant increase in cell viability and ROS generation was observed when exposed to low and moderate doses of MCP and CP at different time points (24, 48 and 72h) studied. A549 cells displayed a dose-dependent accumulation of apurinic/apyrimidinic (AP) sites after 24h exposure to MCP advocating for the activation of AP endonuclease-mediated DNA BER-pathway. Cellular responses to MCP- and CP-induced oxidative stress resulted in an imbalance in the mRNA and protein expression of BER-pathway enzymes, viz. PARP1, OGG1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points. The treatment of OPPs resulted in the upregulation of TFs, viz. Nrf2, c-jun, phospho-c-jun and inducible nitric oxide synthase. Immunofluorescent confocal imaging of A549 cells indicated that MCP and CP induces the translocation of APE1 within the cytoplasm at an early 6h time point, whereas it promotes nuclear localization after 24h of treatment, which suggests that APE1 subcellular distribution is dynamically regulated in response to OPP-induced oxidative stress. Furthermore, nuclear colocalization of APE1 and the TF c-jun was observed in response to the treatment of CP and MCP for different time points in A549 cells. Therefore, in this study we demonstrate that MCP- and CP-induced oxidative stress alters APE1-dependent BER-pathway and also mediates cell survival signalling mechanisms via APE1 regulation, thereby promoting lung cancer cell survival and proliferation.

Abstract B15: Dissecting the molecular mechanism of dianhydrogalactitol (VAL-083) activity in cancer treatment

Abstract Introduction: Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent causing N7-guanine-methylation and inter-strand DNA crosslinks. In China, VAL-083 is approved to use in the chemotherapeutic treatment of lung cancer and chronic myelogenous leukemia. In the United States, VAL-083 is currently undergoing investigation as a new therapy in the treatment of temozolomide refractory glioblastoma (GBM). VAL-083 is a small water-soluble molecule that readily crosses blood-brain-barrier and accumulates in the tumor tissues in brain, making it a good candidate for targeting brain malignancies, such as GBM and medulloblastoma. Historical data from preclinical studies and clinical trials sponsored by the US National Cancer Institute (NCI) support anti-neoplastic effects of VAL-083 in a variety of cancer types in addition to GBM, including lung cancer, leukemia, cervical cancer, and ovarian cancer. However, the detailed molecular mechanisms mediating VAL-083 sensitivity or resistance in cancer cells is still unclear. Therefore, we investigated the distinct mechanism of action of VAL-083 in different cancer cell lines. Methods: VAL-083 cytotoxicity was evaluated in a panel of human non-small cell lung cancer (NSCLC) cell lines (A549, H2122, H1792, and H23) and prostate cancer cell lines (PC3 and LNCaP) by crystal violet assays. Cell cycle analysis and DNA damage response were investigated by propidium iodide (PI) and immunofluorescent (IF) staining. Western blot and IF staining analyses were employed to elucidate the DNA repair mechanism involved in VAL-083-treated cancer cells. Results: In this study, we report new insights into VAL-083's mechanisms of action by showing that VAL-083 induces irreversible cell-cycle arrest and cell death caused by replication-dependent DNA double-strand breaks (DSBs). In all the cancer cells tested, VAL-083 showed broad cytotoxicity with an IC50 range of 3.1 - 25.7 μM. In lung cancer (H2122, H1792, and A549) and prostate cancer (PC3 and LNCaP) cell lines, VAL-083 treatment caused irreversible cell cycle arrest at S/G2 phase as measured by PI and IF staining in synchronized cells, indicating that VAL-083-induced inter-strand crosslinks result in more difficult to repair DNA lesions during replication, including DSBs. Western blot and IF analyses of DNA repair markers were employed to investigate the DNA damage response induced by VAL-083 in cancer cells. The S/G2 phase cell cycle arrest and the increased γH2A.X (an indication of DSB lesions) expression persisted for 48-72 h after treatment with VAL-083, indicating prolonged unrepaired DNA lesions caused by VAL-083. VAL-083 pulse-treatment led to persistent phosphorylation of DSB sensors ataxia telangiectasia mutated (ATM), single-strand DNA-binding replication protein A (RPA32), and H2A.X. Furthermore, Western blot analyses also demonstrated activation of the downstream effectors of ATM and ataxia telangiectasia and Rad3-related protein (ATR) kinases, Chk2 (T68) and Chk1 (S317 and S345). These results suggest that VAL-083-induced persistent and irreversible DNA damage activated the homologous recombination DNA repair signaling pathway in the panel of cancer cells studied. Conclusions: VAL-083 displayed broad anti-neoplastic activity in different lung and prostate cancer cells through the replication-dependent DSBs. Elucidation of the molecular mechanisms underlying VAL-083 cytotoxicity provides guidance for improved treatment strategies for cancer patients with VAL-083 in either single or combination regimens. Citation Format: Beibei Zhai, Anne Steino, Jeffrey Bacha, Dennis Brown, Mads Daugaard. Dissecting the molecular mechanism of dianhydrogalactitol (VAL-083) activity in cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B15.

Hepatic gene expression profiles of a non-model cyprinid (Barbus plebejus) chronically exposed to river sediments

In this study, we characterized the gene expression responses of the Padanian barbel (Barbus plebejus), a native benthivorous cyprinid with a very compromised presence within the fish community of the River Po. Barbel juveniles were exposed in the laboratory to two river sediments reflecting an upstream/downstream gradient of increasing contamination and collected from one of the most anthropized tributaries of the River Po. After 7months of exposure, hepatic transcriptional changes that were diagnostic of sediment exposure were assessed. We investigated a set of 24 genes involved in xenobiotic biotransformation (cyp1a, gstα, ugt), antioxidant defense (gpx, sod, cat, hsp70), trace metal exposure (mt-I, mt-II), DNA repair (xpa, xpc), apoptosis (bax, casp3), growth (igf2), and steroid (erα, erβ1, erβ2, ar, vtg) and thyroid (dio1, dio2, trα, trβ, nis) hormone signaling pathways. In a consistent overall picture, the results showed that long-term sediment exposure mainly increased the levels of mRNAs encoding proteins involved in xenobiotic metabolism, oxidative stress defense, repair of DNA damage and activation of the apoptotic process. Transcript up-regulation of three receptor genes (erβ2, ar, trβ), likely representing compensatory responses to antagonistic/toxic effects, was also observed, confirming the exposure to disruptors of the reproductive and thyroidal axes. In contrast to expectations, a few genes showed no response (e.g., casp3) or even downregulation (vtg), further suggesting that the timing of exposure/assessment, potential compensatory effects or post-transcriptional modifications interact to modify the gene expression profiles, particularly during exposure to mixtures of contaminants.

A novel alkylating agent Melflufen induces irreversible DNA damage and cytotoxicity in multiple myeloma cells.

Our prior study utilized both invitro and invivo multiple myeloma (MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti-MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2AX/ATR/CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2AX. Melflufen induces γ-H2AX, ATR, and CHK1 as early as after 2h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2AX in melphalan-sensitive cells at 6h and 24h; no γ-H2AX induction was observed in melphalan-resistant cells even after 24h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. Our data therefore suggest that melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in MM cells.

Circadian-disruption-induced gene expression changes in rodent mammary tissues.

Evidence is mounting that circadian disruption (CD) is a potential carcinogen in breast cancer development. However, despite the growing concern, to our knowledge, no studies have attempted a genome-wide analysis of CD-induced gene expression changes in mammary tissues. Using a rodent model system, a proven photoperiod-shifting paradigm, varying degrees of CD, and Illumina sequencing, we performed an exploratory genome-wide mRNA analysis in mammary tissues. Even though our analysis did not identify any significant patterns in mRNA levels based on the degree of CD, and the majority of groups did not show changes in gene expression on a large-scale, one group (two-week chronic ZT19) displayed 196 differentially expressed genes, 51 of which have been linked to breast cancer. Through gene-specific pathway analysis, the data illustrate that CD may promote breast cancer development through downregulation of DNA repair and p53 signaling pathways, thus promoting genomic instability and cancer development. Although these results have to be interpreted with caution because only a single group illustrated drastic changes in transcript levels, they indicate that chronic CD may directly induce changes in gene expression on a large-scale with potentially malignant consequences.

A Cross-Cancer Genetic Association Analysis of the DNA Repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast, and Colorectal Cancer.

DNA damage is an established mediator of carcinogenesis, although genome-wide association studies (GWAS) have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. We conducted a cross-cancer analysis of 60,297 single nucleotide polymorphisms, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. We identified three susceptibility DNA repair genes, RAD51B (P < 5.09 × 10(-6)), MSH5 (P < 5.09 × 10(-6)), and BRCA2 (P = 5.70 × 10(-6)). Hierarchical modeling identified several pleiotropic associations with cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Only three susceptibility loci were identified, which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria.

The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation

The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling.

DNA repair in species with extreme lifespan differences.

Differences in DNA repair capacity have been hypothesized to underlie the great range of maximum lifespans among mammals. However, measurements of individual DNA repair activities in cells and animals have not substantiated such a relationship because utilization of repair pathways among animals--depending on habitats, anatomical characteristics, and life styles--varies greatly between mammalian species. Recent advances in high-throughput genomics, in combination with increased knowledge of the genetic pathways involved in genome maintenance, now enable a comprehensive comparison of DNA repair transcriptomes in animal species with extreme lifespan differences. Here we compare transcriptomes of liver, an organ with high oxidative metabolism and abundant spontaneous DNA damage, from humans, naked mole rats, and mice, with maximum lifespans of ~120, 30, and 3 years, respectively, with a focus on genes involved in DNA repair. The results show that the longer-lived species, human and naked mole rat, share higher expression of DNA repair genes, including core genes in several DNA repair pathways. A more systematic approach of signaling pathway analysis indicates statistically significant upregulation of several DNA repair signaling pathways in human and naked mole rat compared with mouse. The results of this present work indicate, for the first time, that DNA repair is upregulated in a major metabolic organ in long-lived humans and naked mole rats compared with short-lived mice. These results strongly suggest that DNA repair can be considered a genuine longevity assurance system.

Abstract A1-63: Characterizing the DNA damage repair defect in HPV-positive oropharyngeal squamous cell carcinoma

Abstract Although tobacco-associated head and neck cancers (HNCs) are declining in incidence, overall HNC rates are escalating due to increasing prevalence of human papillomavirus (HPV)-associated tumors, especially oropharyngeal squamous cell carcinomas (OPSCCs). Clinically, patients with HPV-associated OPSCCs have improved overall survival and increased response to therapy, especially agents which act by damaging DNA. Based on these observations, we hypothesized that HPV-positive OPSCCs harbor a defect in DNA repair activity and are sensitive to DNA repair-targeted therapy. We evaluated DNA repair activity by immunofluorescent staining for DNA damage-induced formation of DNA repair protein foci. Baseline expression of DNA repair proteins was determined by NanoString nCounter analysis of transcript levels and immunoblotting for protein levels. Therapeutic sensitivity was assessed in vitro using the colony formation assay and in vivo using both cell line-derived xenografts and a patient-derived xenograft. Consistent with our hypothesis, in vitro disease models demonstrated delayed resolution of radiation-induced DNA double strand breaks (DSBs) as assessed by γH2AX foci staining and neutral comet assay. Investigation of the two main DSB repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination (HR), indicated intact activation of both pathways but strikingly diminished recruitment of downstream repair factors DNA-Pk (NHEJ) and BRCA2 (HR) to sites of damage. In addition, protein expression of both DNA-PK and BRCA2 was decreased in HPV-positive compared to HPV-negative HNC cell lines. We next studied susceptibility of HPV-positive OPSCCs to PARP inhibition, a class of anticancer agents which block DNA repair signaling pathways and have proven effective clinically in DNA repair-deficient cancers. In vitro colony formation assays revealed a negative effect on cell survival in HPV-positive but not HPV-negative HNCs treated with the PARP inhibitor veliparib. Importantly, these results were confirmed in vivo in both cell line-derived xenografts and a patient-derived xenograft. In summary, our findings demonstrate for the first time the presence of a significant DNA DSB repair defect in HPV-positive OPSCCs encompassing both NHEJ and HR repair, and suggest therapies targeting DNA repair pathways may help improve therapeutic ratio in this disease. Citation Format: Alice N. Weaver, Tiffiny S. Cooper, Hoa Q. Trummell, James A. Bonner, Eben L. Rosenthal, Eddy S. Yang. Characterizing the DNA damage repair defect in HPV-positive oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-63.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.