Abstract BackgroundAim The expression patterns of thekey DNA damage - PDF document

Abstract. Background/Aim: The expression patterns of thekey DNA damage response-related proteins, ataxia-telangiectasia and tfiih/ner complex atp-dependent 5-3 dnahelicase subunit rad3 (RAD3)-related (ATR) and ataxia-telangiectasia-mutated (ATM) proteins in ovarian cancer arenot well-known. This study aimed to evaluate the expressionsof ATR and ATM proteins, and to investigate their clinicalsignificance in epithelial ovarian carcinoma (EOC).Materials and Methods: The expressions of nuclear/cytoplasmic Ser428-phosphorylated ATR (p-ATR) and 3909 These Authors contributed equally to this work and are co-first authors.Correspondence to: Yong-Beom Kim, MD, Ph.D., Department ofObstetrics and Gynecology, Seoul National University BundangGyeonggi-do, Republic of Korea. Tel: +82 317877253, Fax: +82317874054, e-mail: ybkimlh@snubh.orgKey Words: DNA damage response-related proteins, ovarian cancer,immunohistochemistry, prognosis. Ataxia-Telangiectasia and RAD3-Related and Ataxia-Telangiectasia-Mutated Proteins in Epithelial OvarianCarcinoma: Their Expression and Clinical Significance BANGHYUN LEE, HYE-YON CHOJAE HONG NO, HAERYOUNG KIMandYONG-BEOM KIM 0250-7005/2015 $2.00+.40 DNA damage may partially explain the mechanism bymutations induces breast and ovarian cancer(5). Genistein, an isoflavonoid in soybean, has beenreported to induce apoptosis of HO-8910 human ovariancancer cells through activation of ATR-CHK1 and ATM-CHK2 checkpoint pathways (6). Furthermore, in A2780ovarian carcinoma cells, the ATM-CHK2 pathway wasactivated by DSBs induced by camptothecin or ionizingradiation (7). The mRNA levels of BRCT-repeat inhibitorof hTERT expression (), a proximal factor in theATR and ATM pathway, were also found to be markedlydecreased in serous ovarian cancer compared to benignovarian tissues (8). suppression and therapy. However, the expression patterns ofDDR-related proteins in ovarian cancer are still unclear.Therefore, the present study aimed to evaluate the expressionsof the key DDR-related proteins, Ser428-phosphorylated ATR(p-ATR) and Ser1981-phosphorylated ATM (p-ATM), and toinvestigate their clinical significance in EOC. Materials and MethodsPatients. This study was approved by the Institutional Review Boardof Seoul National University Bundang Hospital (B-1401/234-301).Our retrospective cohort included patients who underwent stagingoperations for ovarian cancer at the Seoul National UniversityBundang Hospital from May 1, 2003 to December 31, 2009. Theinclusion criteria were as follows: histologically-confirmed EOC,for which formalin-fixed paraffin-embedded (FFPE) tissue blocksand slides were available, and administration of platinum-basedchemotherapy (cisplatin and carboplatin). Patients who underwentneoadjuvant chemotherapy were excluded from the study. 100 patients who were eligible for the current study were reviewed.Representative FFPE blocks from each case were selected for tissuemicroarray (TMA) construction. Clinical response to chemotherapywas evaluated using the revised RECIST guidelines (version 1.1)(9). Recurrence of tumor within 6

months of the end of first-linechemotherapy was considered to indicate platinum-resistance.Disease-free survival (DFS) was defined as the time from surgeryto the first observation of recurrence. Overall survival (OS) wasdefined as the time from surgery to death or to the date the studywas completed. A tumor with size larger than 1 cm was consideredto be a large residual mass.. Tissue cores measuring 2 mm in diameter wererecipient TMA blocks using a trephine apparatus (SuperbiochipsLaboratories, Seoul, Korea). One or two cores were taken from eachspecimen, and normal ovarian tissue was also included in the TMAblock to serve as a control. Immunohistochemistry. Four-micrometer-thick sections were cutfrom TMA blocks for immunohistochemical analysis. Immunohisto -chemistry was performed using a Benchmark XT autostainer(Ventana, Tucson, AZ, USA). The following primary antibodieswere used: rabbit polyclonal anti-phospho-ATR (Ser428) (CST2853;1:30 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA)and mouse monoclonal anti-phospho-ATM (Ser1981) (clone10H11.E12, CST4526; 1:200 dilution; Cell Signaling). Antigenretrieval for both antibodies was performed using EDTA at pH 8.4.UltraView Universal DAB Detection Kit (cat #760-500; Ventana)was used for signal detection. Interpretation of staining and classification of subgroups for eachvariable.independently by two pathologists who were blinded to the clinicalcharacteristics of the patients. The expressions of p-ATR and p-ATM protein were evaluated in both nuclear and cytoplasmiccompartments (Figure 1). The percentage of cells stained (0-100%)and staining intensity (category 0-3) were used for H-scoring. Ascore ranging from 0 to 300 was then calculated by multiplyingthese two variables. Based on the receiver operating characteristic(ROC) curve for resistance to platinum-based chemotherapy,cytoplasmic p-ATR (cutoff score=17.5) and nuclear p-ATM (cutoffscore=225.0) expressions were divided into high and low H-scoregroups. Cytoplasmic p-ATM protein was not detected byimmunohistochemistry. Based on the ROC curve for resistance toplatinum-based chemotherapy, a value of 52 years was used toFurthermore, a value of 292 U/ml was used to classify the patientsinto groups with high and low preoperative serum CA125 levels.The cutoff values were chosen to maximize the sum of sensitivityand specificity, as indicated by the ROC curves.SPSS Statistics 21 (IBM Corporation, Inc., Chicago, IL, USA). Datafor continuous variables are expressed as the mean±the standarddeviation (SD). Data for categorical variables are expressed asnumbers and percentages. Continuous variables were analyzed using-tests. Categorical variables were analyzed using thePearson chi-squared test or Fishers exact test (two-sided). Toevaluate whether specific variables were independent risk factors forDFS and OS, Cox regression analyses were performed with andwithout adjustment for confounding factors, such as age, stage andtype of ovarian cancer, size of the residual mass, preoperative serumCA125 level, and cytoplasmic ATR expression. A value

of was considered statistically significant. ResultsExpression of ATR and ATM.For cytoplasmic p-ATRprotein (mean H-score=41.1), 62.0% of the patients hadlow expression, including 45.0% with negative expression(no staining), and 38.0% had high expression. Nuclear p-ATR protein (mean H-score=5.1) was expressed in 13.0%of the patients and was not expressed in 87.0% of thepatients. Nuclear p-ATM protein (mean H-score=177.8)was highly expressed in 45.0% of the patients. In contrast,low expression of nuclear p-ATM protein was seen in55.0% of the patients, including 5.0% who had negativeexpression with no staining in the cytoplasm (Figure 1,Tables I and II). Associations of ATR and ATM expression with clinicopatho -logical characteristics. Cytoplasmic expression of p-ATRprotein was significantly decreased in patients withadvanced-stage disease, serous-type disease, large residualmass, high preoperative serum CA125 levels, developmentof recurrence, and death (Table I). However, cytoplasmicexpression of p-ATR protein was not significantlyThe following characteristics were not observed to differsignificantly according to the degree of nuclear p-ATRprotein expression: age (0.114) of ovarian cancer, size of residual mass (preoperative serum CA125 levels (0.126), development of0.936) of ovariancancer, size of residual mass (0.246), preoperative serumCA125 levels (0.439), development of recurrencenuclear p-ATM protein expression. Association of platinum resistance with clinicopathologicalcharacteristics. significantly higher incidences of advanced-stage disease,mucinous and clear cell-type disease, and large residualmass. However, age, preoperative serum CA125 levels, andthe degree of cytoplasmic/nuclear p-ATR and nuclear : ATR and ATM in Epithelial Ovarian Cancer 3911 Table I. Association of cytoplasmic expression of p-ATR protein withclinicopathological characteristics. Cytoplasmic p-ATR -Value protein expressionLow (n=62), High (n=38), n (%)n (%)Age (years) 0.83052 34 (54.8)20 (52.6)�5228 (45.2)18 (47.4)FIGO stageEarly (I-II)17 (27.4)24 (63.2)Advanced (III-IV)45 (72.6)14 (36.8)Histological type0.001Serous44 (71.0)12 (31.6)Non-serous18 (29.0)26 (68.4)Mucinous3 ( 4.8)9 (23.7)Endometrioid5 ( 8.1)3 (7.9)Clear cell4 ( 6.5)9 (23.7)Other6 ( 9.7)5 (13.2)Residual mass0.0071 cm34 (54.8)31 (81.6)�.00;က1 cm28 (45.2)7 (18.4)Preoperative serum CA125 0.001292 U/ml28 (45.2)30 (78.9)�.00;က292 U/ml34 (54.8)8 (21.1)Recurrence0.002Yes39 (62.9)12 (31.6)No23 (37.1)26 (68.4)Death0.005Yes28 (45.2)7 (18.4) No34 (54.8)31 (81.6)p-ATR, Phosphorylated ataxia-telangiectasia and tfiih/ner complex atp-dependent 5-3 dna helicase subunit rad3 (RAD3)-related protein;by Fishers exact test. Table II. Association between platinum resistance and clinicopathologicalcharacteristics. Total Platinum-Platinum- -Value(n=100), sensitive resistant n (%)(n=86), n (%)(n=14), n (%)Age (years), 51.5 ± 12.353.1 ± 11.30.6465254 (54.0)48 (55.8)6 (42.9)0.400�5246 (46.0)38 (44.2)8 (57.1)FIGO stage0.028Early (I-II)41 (41.0)39 (45.3)2 (14.3)Advanced (III-IV)59 (59.0)47 (54

.7)12 (85.7)Histological type0.034Serous56 (56.0)52 (60.5)4 (28.6)Non-serous44 (44.0)34 (39.5)10 (71.4)Mucinous12 (12.0)8 ( 9.3)4 (28.6)Endometrioid8 (8.0)8 ( 9.3)0 (0.0)Clear cell13 (13.0)9 (10.5)4 (28.6)Other11 (11.0)9 (10.5)2 (14.3)Residual mass0.031*1 cm65 (65.0)60 (69.8)5 (35.7)�1 cm35 (35.0)26 (30.2)9 (64.3)Preoperative 0.068292 U/ml58 (58.0)53 (61.6)5 (35.7)�292 U/ml42 (42.0)33 (38.4)9 (64.3)Cytoplasmic p-ATR 0.849protein expressionHigh38 (38.0)33 (38.4)5 (35.7)Low 62 (62.0)53 (61.6)9 (64.3)Nuclear p-ATR 0.386protein expressionYes13 (13.0)10 (11.6)3 (21.4)No 87 (87.0)76 (88.4)11 (78.6)Nuclear p-ATM 0.325protein expressionHigh45 (45.0)37 (43.0)8 (57.1) Low 55 (55.0)49 (57.0)6 (42.9)p-ATR, Phosphorylated ataxia-telangiectasia and tfiih/ner complex atp-dependent 5-3 dna helicase subunit rad3 (RAD3)-related protein; p-ATM,months after the end of first-line chemotherapy was considered to indicateAnalysis by Fishers exact test. p- ATM protein expression were not significantly associatedwith resistance to platinum-based chemotherapy (Table II). Survival rates.In the total study population, the median DFS was28 months (range=1-117 months). Univariate analyses indicatedthat older age, advanced-stage cancer, serous-type disease, largeresidual mass, high preoperative serum CA125 level, and lowexpression of cytoplasmic p-ATR protein were significantlyicantlyp0.05; low cytoplasmic p-ATR protein expression: hazard ratio(HR)=2.2, 95% confidence interval (CI)=1.2 to 4.2]. In amultivariate analysis that was adjusted for confounding factors,advanced-stage cancer remained significantly associated with an0.001; Table III and Figure 2). In the total study population, the median OS was 62.5months (range=1-117 months). Univariate analyses indicatedthat advanced-stage cancer, large residual mass, highpreoperative serum CA125 level, and low cytoplasmicexpression of p-ATR protein were significantly associated0.05; lowcytoplasmic p-ATR protein expression: HR=2.3, 95% CI=1.2to 6.1). In a multivariate analysis that was adjusted forconfounding factors, advanced-stage cancer remainedsignificantly associated with an increased risk of death and0.001; Table IVDiscussionunacceptable, there exists an outstanding need for targeted-therapies, in addition to conventional chemotherapy. The ATRand ATM pathways are activated in response to the inhibition . Immunohistochemical staining results. A: Diffuse strong nucleocytoplasmic expression of phosphorylated ataxia-telangiectasia and tfiih/nercomplex atp-dependent 5-3 dna helicase subunit rad3 (RAD3)-related (p-ATR) is seen in the tumor cells, while the adjacent stroma is negative. B:Weak nuclear p-ATR is seen in this case, without cytoplasmic p-ATR expression. C: Diffuse strong nuclear expression of phosphortelangiectasia-mutated (p-ATM) is seen in the tumor cells. D: p-ATM is expressed in occasional nuclei in this case. Original magnification, ×400. of replication, such as is prompted by chemotherapy. Thesenormal ATR and ATM pathways can serve to increase cancercell survival, suggesting a mechanism of

resistance tochemotherapy. Therefore, targeting the ATR and ATMpathways is considered to constitute a promising therapeuticmethod. Many studies have demonstrated that tumor cells canbe sensitized to drugs by inhibiting the ATR and ATMpathways in combination with the administration of variouschemotherapeutic agents (10-12). ATR and ATM inhibitorshave also been shown to radiosensitize tumor cells (13).Therefore, targeting ATR and ATM while administeringchemotherapy or radiotherapy is considered to be an attractivesiRNA to inhibit ATR sensitizes HeLa cells to platinum(14). However, another study found that targeted functionalinhibition of ATM in p53-deficient cells did not increasesensitivity to platinum compounds (15). Therefore, weexpected that the expression of ATR in EOC would at leastplatinum-sensitive group. However, our results showed thatp-ATR and p-ATM expressions were not dependent onplatinum agent use, suggesting that the regulation of platinumresistance occurs through processes other than the ATR andATM pathways in EOC (Table II). Several types of cancers induce a typical stress responseinvolving an acute DDR that is followed by a chronicresponse in which selected DNA-repair pathways aresuppressed in coordination (such as the ATM, CHK1,CHK2, and p53 pathways). However, the exactmight also be possible for these types of cancer to showan altered response to conventional genotoxic therapiesbecause the signaling pathways that are responsive to DNAdamage might be suppressed (17). Therefore, at the startof the current study, we presumed that ATR and ATMproteins would exhibit low expression in advanced EOCand, furthermore, that low expression of ATR and ATMwould be associated with chemotherapy resistance; thepatterns of DDR would differ in specific cell types,depending on specific conditions. A study of gastric cancerrecently showed that ATM, CHK1, and p53 losses weresignificantly associated with advanced stage and poorDFS, suggesting that they have roles in the progression ofgastric cancer (18). Interestingly, in our study, lowexpression of cytoplasmic p-ATR was significantlyassociated with advanced-stage disease, as well asOS; Table I, III and IV, and Figure 2). However, lowexpression of p-ATM was not significantly associated withthese outcomes. The partial or complete loss of ATM-CHK2 signaling is associated with poorer survival for cellsand organisms, although cancer may frequently develop: ATR and ATM in Epithelial Ovarian Cancer 3913 . Survival analysis according to cytoplasmic expression of phosphorylated ataxia-telangiectasia and tfiih/ner complex atp-dependent 5-3 dnahelicase subunit rad3 (RAD3)-related protein. A: Disease-free survival (DFS). B: Overall survival (OS). Univariate Cox regression analyses were performed. mutations (17). However, ATR-CHK1 signaling has beenshown to be essential for the proliferation and survival ofmany cell types, presumably because partial or completeinactivation of ATR may lead to cell death (such asapoptosis) rather than to oncogenic mutations. Thisobservation suggests that ATR is a promisin

g target foranticancer chemotherapy (17, 19, 20). However, eventhough they develop rarely, somatic mutations of ATR-CHK1 signaling have been reported in sporadic stomach,inhibition of ATR-CHK1 expression in carcinomas thathave a mismatch repair deficiency induces chromosomeinstability, leading to genomic instability andtumorigenesis (22). In the current study, low expression ofcytoplasmic p-ATR was significantly associated withadvanced stage and poor outcome of EOC, suggesting thatinhibition of ATR may play a role as a risk factor in cancerwithout mismatch repair deficiency (Tables I, III, and IV;Figure 2). Further studies are needed to clarify cytoplasmicDNA damage (e.g.mitochondrial DNA) related tocytoplasmic ATR in EOC. ATR protein that is expressed in the nucleus haspathways in DDR (19, 20, 23). Recently, the function ofcytoplasmic ATR as an antiapoptotic protein has beenreported. Cytoplasmic ATR had a checkpoint kinase-leukemia/lymphoma 2 (BCL2) associated X (BAX)-mediated apoptotic pathways in mitochondria followingUV irradiation (20). In addition, ATM protein ispredominant in the nucleus. Moreover, the presence ofATM protein has also been documented in the cytoplasmof some cell types. Cytoplasmic functions of ATM in theactivation of PI3K- v-akt murine thymoma viral oncogenehomolog 1 (AKT) have also been discovered, suggestingthe potential for cytoplasmic ATM protein kinase as atherapeutic target of cancer (24). In addition, differentiallocalization of ATM in the cytoplasm and nucleus isreportedly correlated with activation of distinctdownstream signaling pathways (25). Therefore, weinvestigated the expressions of ATR and ATM proteins incytoplasmic and nuclear compartments of EOC. In ourstudy, low cytoplasmic p-ATR protein expression wasdominant (compared to high expression) and cytoplasmicp-ATM protein expression was not detected. Lowcytoplasmic p-ATR protein expression was alsosignificantly associated with aggressive clinicopathologicalcharacteristics, including advanced stage of EOC and Table IV. Univariate and multivariate analyses of clinicopathologicalrisk factors of death as a function of overall survival. No.Univariate Multivariate analysisanalysis HR (95% CI) HR (95% CI)-ValueFIGO stageEarly (I-II)41ReferenceReferenceAdvanced (III-IV)5911.6 (3.5 to 38.0)8.8 (2.6 to 30.0)Residual mass0.3471 cm65ReferenceReference�.00;က1 cm353.9 (2.0 to 7.8)1.4 (0.7 to 3.1)Preoperative serum 0.058292 U/ml58ReferenceReference�.00;က292 U/ml423.7 (1.9 to 7.6)2.0 (1.0 to 4.2)Cytoplasmic p-ATR 0.731protein expressionHigh 38ReferenceReference Low622.3 (1.2 to 6.1)1.2 (0.5 to 3.1)p-ATR, Phosphorylated ataxia-telangiectasia and tfiih/ner complex atp-dependent 5-3 dna helicase subunit rad3 (RAD3)-related protein; FIGO, Table III. Univariate and multivariate analyses of clinicopathologicalrisk factors of recurrence as a function of disease-free survival. No.Univariate Multivariate analysisanalysis HR (95% CI) HR (95% CI)-ValueAge (years)0.15352 54ReferenceReference�52462.1 (1.2 to 3.6)1.6 (0.9 to2.8)FIGO stageEarly (I-II)41Refer

enceReferenceAdvanced (III-IV)598.9 (3.8 to 21.0)6.5 (2.5 to 16.8)Histological type0.725Non-serous44ReferenceReferenceSerous561.8 (1.0 to 3.3)1.1 (0.6 to 2.2)Residual mass0.4431 cm65ReferenceReference�.00;က1 cm353.5 (2.0 to 6.0)1.3 (0.7 to 2.3)Preoperative 0.097292 U/ml58ReferenceReference�.00;က292 U/ml423.0 (1.7 to 5.3)1.7 (0.9 to 3.4)Cytoplasmic p-ATR 0.888protein expressionHigh 38ReferenceReference Low622.2 (1.2 to 4.2)1.1 (0.5 to 2.4)p-ATR, Phosphorylated ataxia-telangiectasia and tfiih/ner complex atp-dependent 5-3 dna helicase subunit rad3 (RAD3)-related protein; FIGO, poorer survival (Tables I, III, and IV; Figure 2). Thesefindings demonstrate that ATR expression is negativelycorrelated with the severity of ovarian cancer. It has been. It has beenTransforming Sequence 2 (Ect2)] increase in advancedEOC and play a role in its transformation, even thoughcytoplasmic proteins do not (26). However, our study ofEOC showed that the incidence of nuclear p-ATR proteinexpression was very low, and that the incidence of nuclearp-ATM protein expression was similar in both the high andlow expression groups (Table II). Expression patterns ofnuclear p-ATR and p-ATM proteins were also notassociated with the severity of clinicopathologicalcharacteristics in cases of EOC. These findings suggestthat nuclear ATR and ATM proteins do not play criticalIn conclusion, this study demonstrates that ATR andATM pathways are not associated with the occurrence ofplatinum resistance. Our results also demonstrate that lowexpression of cytoplasmic ATR protein may havepotential as a prognostic factor of EOC. Indeed, ourfindings suggest that cytoplasmic ATR protein isnegatively correlated with prognosis of EOC. Overall, ourresults significantly improve the understanding over therole of DDR in ovarian cancer, especially for ATR andATM pathways. AcknowledgementsThis study was supported by grant no. 02-2010-036 from the SeoulNational University Bundang Hospital Research Fund.Conflicts of InterestThe Authors declare that they have no conflict of interest withregard to this study.References1Liang Y, Lin SY, Brunicardi FC, Goss J and Li K: DNA damageresponse pathways in tumor suppression and cancer treatment.World J Surg 2Connell CM, Shibata A, Tookman LA, Archibald KM, Flak MB,Pirlo KJ, Lockley M, Wheatley SP and McNeish IA: GenomicDNA damage and ATR-CHK1 signaling determine oncolyticadenoviral efficacy in human ovarian cancer cells. J Clin Invest3Cimprich KA and Cortez D: ATR: an essential regulator ofgenome integrity. Nat Rev Mol Cell Biol 4Murdoch WJ and McDonnel AC: Roles of the ovarian surfaceepithelium in ovulation and carcinogenesis. Reproduction 5Rosen EM: BRCA1 in the DNA damage response and at6Ouyang G, Yao L, Ruan K, Song G, Mao Y and Bao S:Genistein induces G2/M cell-cycle arrest and apoptosis ofhuman ovarian cancer cellsactivation of DNA damagecheckpoint pathways. Cell Biol Int 7Zuco V, Benedetti V and Zunino F: ATM- and ATR-mediatedresponse to DNA damage induced by a novel camptothecin,8Rai R, Dai H, Multani AS, Li K, Chin K, Gray J, Lahad JP,Lia

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