Rad3-related Protein Research Articles

Abstract 1421: Evaluation of DDR-targeting strategy using ATR inhibitor in biliary tract cancer

Abstract Background: The DNA damage response (DDR) is a multicomplex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints and apoptosis. The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key enzyme in the DDR that activates checkpoint kinase 1 (Chk1), resulting in cell cycle arrest. Tumor types with loss of ATM function and/or high replication stress are expected to be more susceptible to DDR targeting. In biliary tract cancer (BTC), DNA repair pathway, which includes BAP1, MSH6, BRCA1, ATM, MLH1, MSH2, is altered in about 20 % of cases. TP53 module is observed in 33.9% of BTC cases (Nat Genet 2015). The purpose of this study is to test DDR targeting strategy using ATR inhibitor in biliary tract cancer. Methods: Using 9 kinds of BTC cells, MTT assay and colony formation assay were done for determining growth inhibitory effect of AZD6738, an ATR inhibitor. Cell cycle analysis was done by FACS Calibur flow cytometer and the methods described by Chou and Talalay were used to determine whether a synergistic effect existed between AZD6738 and cytotoxic chemotherapeutic agents (cisplatin, 5-FU, gemcitabine). The alkaline comet assay was done to measure of DNA damage in individual cells. Tumor xenografts model was used for in vivo test of AZD6738. Results: Among 9 BTC cells, SNU478 and SNU869 were most sensitive to AZD6738, which showed low expression of both ATM and p53. AZD6738 blocked ATR-mediated Chk1 phosphorylation and increased rH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis (cleavage of PARP and caspase-7) and G2/M arrest, increased level of p21, and decreased cdc2. In addition, combination of AZD6738 and cytotoxic chemotherapeutic agents demonstrated synergistic effects in colony formation assay, cell cycle analysis and comet assay. In xenograft model of SNU478, AZD6738 monotherapy decreased tumor growth. The combination of AZD6738 and cisplatin showed more potent growth inhibitory effects, decreased Ki67, increased Tunel than monotherapy of each drug. Conclusion: In BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC. Citation Format: Ah Rong Nam, Ji Eun Park, Ju Hee Bang, Mei Hua Jin, Do Youn Oh, Yung Jue Bang. Evaluation of DDR-targeting strategy using ATR inhibitor in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1421. doi:10.1158/1538-7445.AM2017-1421

Epstein-Barr virus-induced up-regulation of TCAB1 is involved in the DNA damage response in nasopharyngeal carcinoma

Telomerase Cajal body protein 1 (TCAB1), which is involved in Cajal body maintenance, telomere elongation and ribonucleoprotein biogenesis, has been linked to cancer predisposition, including nasopharyngeal carcinoma (NPC), due to its oncogenic properties. However, there are no specific reports to date on the functional relevance of TCAB1 and Epstein–Barr virus (EBV), which is considered to be a risk factor for NPC. In this study, we first examined NPC clinical tissues and found a notable overexpression of TCAB1 in EBV-positive specimens. Secondly, on a cellular level, we also observed that TCAB1 expression rose gradually along with the increased duration of EBV exposure in NPC cell lines. Additionally, EBV infection promoted cell proliferation and telomerase activity, but the activation was significantly inhibited after TCAB1 knockdown. Moreover, depletion of TCAB1 caused both cell cycle arrest and apoptosis, and suppressed the activation of ataxia telangiectasia and Rad3 related protein (ATR) induced by EBV, resulting in accumulation of DNA damage. Taken together, we here demonstrate that up-regulated expression of TCAB1, induced by EBV in the development of NPC, is involved in stimulating telomerase activity and regulating the DNA damage response within the context of EBV infection.

JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell

Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1–20μM) increased the expression of DNA damage-associated protein phosphorylation H2AX (γH2AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC.

Divergence of cAMP signalling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes.

Loss-of-function melanocortin 1 receptor (MC1R) polymorphisms are common in UV-sensitive fair-skinned individuals and are associated with blunted cAMP second messenger signalling and higher lifetime risk of melanoma because of diminished ability of melanocytes to cope with UV damage. cAMP signalling positions melanocytes to resist UV injury by upregulating synthesis of UV-blocking eumelanin pigment and by enhancing the repair of UV-induced DNA damage. cAMP enhances melanocyte nucleotide excision repair (NER), the genome maintenance pathway responsible for the removal of mutagenic UV photolesions, through cAMP-activated protein kinase (protein kinase A)-mediated phosphorylation of the ataxia telangiectasia-mutated and Rad3-related (ATR) protein on the S435 residue. We investigated the interdependence of cAMP-mediated melanin upregulation and cAMP-enhanced DNA repair in primary human melanocytes and a melanoma cell line. We observed that the ATR-dependent molecular pathway linking cAMP signalling to the NER pathway is independent of MITF activation. Similarly, cAMP-mediated upregulation of pigment synthesis is independent of ATR, suggesting that the key molecular events driving MC1R-mediated enhancement of genome maintenance (eg PKA-mediated phosphorylation of ATR) and MC1R-induced pigment induction (eg MITF activation) are distinct.

Abstract B21: The selective ATR inhibitor VX-970 enhances the therapeutic effects of standards of care in glioblastoma

Abstract Glioblastoma (GBM) represents one of the most aggressive cancer types with the vast majority of patients succumbing to disease within the first five years. This dire prognosis reflects the limited efficacy of our frontline therapies which include radiation therapy and temozolomide (TMZ) chemotherapy. The cellular response to these therapies is critically mediated by DNA damage response signaling networks that are regulated by Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia And Rad3-Related Protein (ATR). Preliminary studies from our laboratory suggest the ATR inhibitor VX-970 has single agent efficacy in both established and primary GBM cell lines. Moreover, combined treatment with TMZ demonstrated therapeutic responses that, following regression analysis, were deemed synergistic (p<0.001). Validation studies using siRNA confirmed ATR as a central mediator of TMZ sensitivity as knockdown of ATM elicited no added benefit when tested in combination. VX-970 also enhanced radiation effects in neurosphere formation assays to suggest it may also be used as a radiosensitizer. While the specific mechanism responsible for these observations has yet to be determined, VX-970 treatment was found to exert anti-proliferative effects in a dose-dependent manner. This collection of work suggests that ATR inhibition may represent a worthwhile treatment strategy in GBM. Citation Format: Danielle Burgenske, Ann Mladek, Jann Sarkaria. The selective ATR inhibitor VX-970 enhances the therapeutic effects of standards of care in glioblastoma [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 B21.

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.

Discovery of pyrazolopyrimidine derivatives as novel inhibitors of ataxia telangiectasia and rad3 related protein (ATR)

The ATR pathway is a critical mediator of the replication stress response in cells. In aberrantly proliferating cancer cells, this pathway can help maintain sufficient genomic integrity for cancer cell progression. Herein we describe the discovery of 19, a pyrazolopyrimidine-containing inhibitor of ATR via a strategic repurposing of compounds targeting PI3K.

Inhibition of ATR in Combination with Nucleoside Analogues Eradicates Acute Myeloid Leukaemia in an Orthotopic Murine Xenograft Model

The ataxia telangiectasia and RAD3-related (ATR) protein kinase is a component of the cellular DNA damage response pathway and promotes cell survival by signalling repair of collapsed replication forks generated by replication stress. We hypothesised that inhibition of ATR potentiates the anti-leukaemic activity of chain terminating nucleoside analogues used in the treatment of acute myeloid leukaemia (AML). We used VE-821 and its derivative VX-970 (Vertex Pharmaceuticals, Abingdon, UK) as potent and specific inhibitors of ATR kinase activity to examine the effects of ATR inhibition in AML cell lines, primary AML cells and AML xenografts.Co-treatment with 1mM VE-821 did not consistently potentiate the anti-proliferative effects of cytarabine, clofarabine or fludarabine in a panel of AML cell lines. However, there was consistent potentiation of hydroxyurea and gemcitabine in all 7 AML cell lines tested. Treatment with hydroxyurea, which induces replication stress via depletion of dNTPs, resulted in phosphorylation of CHK1, a downstream target of ATR. CHK1 phosphorylation was attenuated when 1mM VE-821 was co-administered with hydroxyurea. Exposure of cells to gemcitabine or hydroxyurea slowed transit through S phase, which was pronounced in combination with VE-821. HL-60 AML cell clones expressing either a constitutively active or inducible shRNA construct targeting ATR had reduced ATR protein expression compared to control cells and were significantly more sensitive to the anti-proliferative effects of gemcitabine and hydroxyurea, but not to cytarabine, clofarabine or fludarabine.The growth inhibitory effects of hydroxyurea and gemcitabine were also significantly potentiated by VE-821 in primary AML patient samples, which included three adult patients with de novo AML and a paediatric patient with therapy-related AML. In contrast, ATR inhibition did not potentiate the inhibitory effects of hydroxyurea or gemcitabine in primary bone marrow cells from healthy donors ex vivo.We next sought to determine whether ATR inhibition potentiated hydroxyurea and gemcitabine in an orthotopic mouse model of AML. MV4-11 AML cells engineered to express firefly luciferase (MV4-11 pSLIEW) were intrafemorally transplanted into immunodeficient Rag2-/- gc-/- mice. Bioluminescent imaging via IVIS Spectrum (PerkinElmer, Buckinghamshire, UK) demonstrated localised femoral engraftment first detectable 4-5 days post-injection, with luciferase signal developing in other parts of the body (liver, ovaries) between days 15 and 18 in untreated mice. Treatment was initiated 7 days post-injection when disease was localised to the femur and prior to emergence of disseminated luciferase signal. Single agent hydroxyurea (250mg per kg, IP days 0-4 and 7-11) conferred some early disease control compared to controls as determined by luciferase total body flux measured on day 14, but this was not statistically significant (p=0.18) and did not affect overall survival (mean 35 days for controls and 37 days for hydroxyurea, p=0.47). Monotherapy with VX-970 (60 mg per kg, orally on days 0-4 and 7-11) also conferred early disease control compared to vehicle-treated mice (p=0.18), and resulted in significantly longer overall survival (mean 40 days, p=0.017). Combination treatment with hydroxyurea and VX-970 did not result in more effective early disease control or improved overall survival compared to monotherapy with either agent. Treatment with gemcitabine monotherapy (100 mg per kg, intraperitoneal injection on days 0, 3, 7 and 10) conferred significant early disease control (p=0.002) and significantly improved overall survival compared to controls (mean survival 73 days, p<0.001). Nevertheless, leukemic cells persisted and the cause of death in all mice randomised to this arm was relapsed disseminated disease. Treatment with gemcitabine and VX-970 in combination also conferred very significant early disease control (p=0.002), which eradicated luciferase signal by day 21 in all 6 mice randomised to this arm (Figure 1), and also conferred significantly improved overall survival compared to controls (p<0.001) or mice treated with gemcitabine monotherapy (p=0.001), with four mice remaining disease free at day 126. No significant weight loss or toxicity was observed in any of the treatment arms. Taken together, these data suggest ATR inhibition as a promising therapeutic strategy in AML. [Display omitted] DisclosuresPollard:Vertex Pharmaceuticals (Europe) Ltd.: Employment.

Hsa-miR-520d-5p promotes survival in human dermal fibroblasts exposed to a lethal dose of UV irradiation.

We previously reported that hsa-miR-520d-5p is functionally involved in the induction of the epithelial–mesenchymal transition and stemness-mediated processes in normal cells and cancer cells, respectively. On the basis of the synergistic effect of p53 upregulation and demethylation induced by 520d-5p, the current study investigated the effect of this miRNA on apoptotic induction by ultraviolet B (UVB) light in normal human dermal fibroblast (NHDF) cells. 520d-5p was lentivirally transfected into NHDF cells either before or after a lethal dose of UVB irradiation (302 nm) to assess its preventive or therapeutic effects, respectively. The methylation level, gene expression, production of type I collagen and cell cycle distribution were estimated in UV-irradiated cells. NHDF cells transfected with 520d-5p prior to UVB irradiation had apoptotic characteristics, and the transfection exerted no preventive effects. However, transfection with 520d-5p into NHDF cells after UVB exposure resulted in the induction of reprogramming in damaged fibroblasts, the survival of CD105-positive cells, an extended cell lifespan and prevention of cellular damage or malfunction; these outcomes were similar to the effects observed in 520d-5p-transfected NHDF cells (520d/NHDF). The gene expression of c-Abl (Abelson murine leukemia viral oncogene homolog 1), ATR (ataxia telangiectasia and Rad3-related protein), and BRCA1 (breast cancer susceptibility gene I) in transfectants was transcriptionally upregulated in order. These mechanistic findings indicate that ATR-dependent DNA damage repair was activated under this stressor. In conclusion, 520d-5p exerted a therapeutic effect on cells damaged by UVB and restored them to a normal senescent state following functional restoration via survival of CD105-positive cells through c-Abl-ATR-BRCA1 pathway activation, p53 upregulation, and demethylation.

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression.

Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I-related chain (MIC) A/B and UL16-binding proteins (ULBP)1-6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.

Transcriptional and Posttranslational Regulation of Nucleotide Excision Repair: The Guardian of the Genome against Ultraviolet Radiation.

Ultraviolet (UV) radiation from sunlight represents a constant threat to genome stability by generating modified DNA bases such as cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). If unrepaired, these lesions can have deleterious effects, including skin cancer. Mammalian cells are able to neutralize UV-induced photolesions through nucleotide excision repair (NER). The NER pathway has multiple components including seven xeroderma pigmentosum (XP) proteins (XPA to XPG) and numerous auxiliary factors, including ataxia telangiectasia and Rad3-related (ATR) protein kinase and RCC1 like domain (RLD) and homologous to the E6-AP carboxyl terminus (HECT) domain containing E3 ubiquitin protein ligase 2 (HERC2). In this review we highlight recent data on the transcriptional and posttranslational regulation of NER activity.

Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells.

Ataxia telangiectasia and Rad3-related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)-dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti-proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC50 values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK-BR-3 and BT-474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. Sensitive SK-BR-3 but not the less sensitive BT-474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check-point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2-positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2-positive breast cancer treatment.

New approach of delivering cytotoxic drugs towards CAIX expressing cells: A concept of dual-target drugs

Carbonic anhydrase IX (CAIX) is a hypoxia-regulated and tumor-specific protein that maintains the pH balance of cells. Targeting CAIX might be a valuable approach for specific delivery of cytotoxic drugs, thereby reducing normal tissue side-effects. A series of dual-target compounds were designed and synthesized incorporating a sulfonamide, sulfamide, or sulfamate moiety combined with several different anti-cancer drugs, including the chemotherapeutic agents chlorambucil, tirapazamine, and temozolomide, two Ataxia Telangiectasia and Rad3-related protein inhibitors (ATRi), and the anti-diabetic biguanide agent phenformin. An ATRi derivative (12) was the only compound to show a preferred efficacy in CAIX overexpressing cells versus cells without CAIX expression when combined with radiation. Its efficacy might however not solely depend on binding to CAIX, since all described compounds generally display low activity as carbonic anhydrase inhibitors. The hypothesis that dual-target compounds specifically target CAIX expressing tumor cells was therefore not confirmed. Even though dual-target compounds remain an interesting approach, alternative options should also be investigated as novel treatment strategies.

Ewing Tumor-associated Antigen 1 Interacts with Replication Protein A to Promote Restart of Stalled Replication Forks

The replication protein A (RPA) complex binds single-stranded DNA generated at stalled replication forks and recruits other DNA repair proteins to promote recovery of these forks. Here, we identify Ewing tumor-associated antigen 1 (ETAA1), which has been linked to susceptibility to pancreatic cancer, as a new repair protein that is recruited to stalled forks by RPA. We demonstrate that ETAA1 interacts with RPA through two regions, each of which resembles two previously identified RPA-binding domains, RPA70N-binding motif and RPA32C-binding motif, respectively. In response to replication stress, ETAA1 is recruited to stalled forks where it colocalizes with RPA, and this recruitment is diminished when RPA is depleted. Notably, inactivation of the ETAA1 gene increases the collapse level of the stalled replication forks and decreases the recovery efficiency of these forks. Moreover, epistasis analysis shows that ETAA1 stabilizes stalled replication forks in an ataxia telangiectasia and Rad3-related protein (ATR)-independent manner. Thus, our results reveal that ETAA1 is a novel RPA-interacting protein that promotes restart of stalled replication forks.

Chlorogenic acid prevents isoproterenol-induced DNA damage in vascular smooth muscle cells.

Numerous clinical therapeutic agents have been identified as DNA damaging. The present study revealed that isoproterenol (Iso) resulted in DNA damage in vascular smooth muscle cells (VSMCs) and increased the levels of intracellular oxygen free radicals. Administration of chlorogenic acid (CGA) inhibited this effect. Pretreatment with CGA abrogated the increase in protein expression levels of γ-H2A histone family member X, phosphorylated ataxia telangiectasia mutated, phosphorylated Rad3-related protein, breast cancer 1 and C-terminal Src homologous kinase induced by Iso. In addition, the increase in levels of intracellular reactive oxygen species (ROS) induced by Iso was inhibited by CGA pretreatment in a dose-dependent manner. The results of the present study suggest that CGA may inhibit Iso-induced VSMC damage via the suppression of ROS generation. Therefore, CGA may be a novel agent for the treatment of vascular diseases.

Cancer Cells with Alternative Lengthening of Telomeres Do Not Display a General Hypersensitivity to ATR Inhibition.

Telomere maintenance is a hallmark of cancer as it provides cancer cells with cellular immortality. A significant fraction of tumors uses the alternative lengthening of telomeres (ALT) pathway to elongate their telomeres and to gain an unlimited proliferation potential. Since the ALT pathway is unique to cancer cells, it represents a potentially valuable, currently unexploited target for anti-cancer therapies. Recently, it was proposed that ALT renders cells hypersensitive to ataxia telangiectasia- and RAD3-related (ATR) protein inhibitors (Flynn et al., Science 347, 273). Here, we measured the response of various ALT- or telomerase-positive cell lines to the ATR inhibitor VE-821. In addition, we compared the effect of the inhibitor on cell viability in isogenic cell lines, in which ALT was active or suppressed. In these experiments, a general ATR inhibitor sensitivity of cells with ALT could not be confirmed. We rather propose that the observed variations in sensitivity reflect differences between cell lines that are unrelated to ALT.

Synergistic potentiation of (−)-lomaiviticin A cytotoxicity by the ATR inhibitor VE-821

(−)-Lomaiviticin A (1) is a cytotoxic bacterial metabolite that induces double-strand breaks in DNA. Here we show that the cytotoxicity of (−)-lomaiviticin A (1) is synergistically potentiated in the presence of VE-821 (7), an inhibitor of ataxia telangiectasia and Rad3-related protein (ATR). While 0.5nM 1 or 10μM 7 alone are non-lethal to K562 cells, co-incubation of the two leads to high levels of cell kill (81% and 94% after 24 and 48h, respectively). Mechanistic data indicate that cells treated with 1 and 7 suffer extensive DNA double-strand breaks and apoptosis. These data suggest combinations of 1 and 7 may be a valuable chemotherapeutic strategy.

Aspartate Rescues S-phase Arrest Caused by Suppression of Glutamine Utilization in KRas-driven Cancer Cells

During G1-phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to α-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.

MHC class I chain-related molecule A and B expression is upregulated by cisplatin and associated with good prognosis in patients with non-small cell lung cancer.

MHC class I chain-related molecule A and B (MICA/B) are NK group 2 member D (NKG2D) ligands, which are broadly expressed in transformed cells. Both DNA damage-induced ataxia-telangiectasia-mutated (ATM)- and ATM and Rad3-related protein kinases (ATM-ATR) signaling and oncogene-induced PI3K-AKT signaling regulate the expression of NKG2D ligands, which promote NK cell-mediated cytotoxicity via NKG2D-NKG2D ligand interactions. NKG2D ligand overexpression was recently reported to be correlated with good prognosis in several types of cancer. However, the prognostic significance of NKG2D ligands in non-small cell lung cancer (NSCLC) remains unclear. Here, MICA/B expression was evaluated based on immunohistochemistry of 91 NSCLC samples from patients following radical surgery. In addition, expression of MICA/B was assessed in NSCLC cell lines treated with cisplatin in order to evaluate the regulatory mechanisms of MICA/B expression. Overall, 28 out of 91 (30.8%) specimens showed high expression level of MICA/B, which was associated with low (18)F-fluorodeoxyglucose uptake and manifestation of adenocarcinoma. After a median follow-up of 48.2 months, high MICA/B expression was associated with good recurrence-free survival (p = 0.037). In vitro assays using cell lines revealed that MICA/B expression was upregulated by cisplatin via ATM-ATR signaling, resulting in enhanced NK cell-mediated cytotoxicity. Upregulated MICA/B expressions in patients with radically resected NSCLC are predictive of good disease prognosis. Cisplatin-induced MICA/B upregulation is possibly an indirect mechanism by which the innate immune system eliminates tumor cells. NKG2D-NKG2D ligand-targeting therapy is a promising avenue for future immune-chemotherapy development.

Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression.

Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.