satellite rna increases dna damage and accelerates tumor formation … · dna damage and repair...

DNA Damage and Repair

Satellite RNA Increases DNA Damage andAccelerates Tumor Formation in Mouse Models ofPancreatic CancerTakahiro Kishikawa, Motoyuki Otsuka, Tatsunori Suzuki, Takahiro Seimiya,Kazuma Sekiba, Rei Ishibashi, Eri Tanaka, Motoko Ohno, Mari Yamagami, andKazuhiko Koike

Abstract

Highly repetitive tandem arrays such as satellitesequences in the centromeric and pericentromeric regionsof chromosomes, which were previously considered tobe silent, are actively transcribed in various biologicalprocesses, including cancers. In the pancreas, this aberrantexpression occurs even in Kras-mutated pancreatic intrae-pithelial neoplasia (PanIN) tissues, which are precancerouslesions. To determine the biological role of satelliteRNAs in carcinogenesis in vivo, we constructed mousemajor satellite (MajSAT) RNA-expressing transgenicmice. However, these transgenic mice did not show spon-taneous malignant tumor formation under normal breed-ing. Importantly, however, DNA damage was increased inpancreatic tissues induced by caerulein treatment or high-

fat diet, which may be due to impaired nuclear localizationof Y-Box Binding Protein 1 (YBX1), a component ofthe DNA damage repair machinery. In addition, whencrossed with pancreas-specific Kras-mutant mice, MajSATRNA expression resulted in an earlier increase in PanINformation. These results suggest that aberrant MajSATRNA expression accelerates oncogenesis by increasing theprobability of a second driver mutation, thus acceleratingcells to exit from the breakthrough phase to the expansionphase.

Implications: Aberrant expression of satellite RNAs acceler-ates oncogenesis through a mechanism involving increasedDNA damage. Mol Cancer Res; 16(8); 1255–62. �2018 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is one of the most

intractable diseases and is ranked the third leading cause ofcancer-related death in the United States (1). The genetic muta-tion profile of PDAC is relatively simple. So-called "driver genemutations" occur mostly in four major genes: KRAS, TP53,CDKN2A, and SMAD4 (2–5). The mutations occur sequentiallyduring pancreatic carcinogenesis (6, 7). However, a constitutivelyactive KRAS mutation is likely required for initiation of carcino-genesis, because it is detected at an extremely high probability(over 95%) in PDAC and in 36%–87% of pancreatic intraepithe-lial neoplasia (PanIN) tissues, which are precancerous lesions forPDAC (5, 8, 9). In addition, genetically engineeredmousemodelsthat harbor mutated Kras genes in the pancreas develop PanINtumors, whereas mice with other mutations do not (10–12).

Satellite sequences, highly repetitive noncoding arrays mostlyin the centromeric and pericentromeric regions of the chromo-

somes (13),were believed to be transcriptionally silenced throughcontinuous heterochromatin formation in the normal state.However, recent studies have demonstrated that these sequencescan be transcribed to yield satellite noncoding RNAs with impor-tant roles in the organization and regulation of genomes (14).Also, aberrant expression and high levels of heterogenous tran-scripts from satellite regions were recently found in variousepithelial cancers, especially in pancreatic cancer tissues (15).This deregulated expression begins during PanIN formation inKras-mutated mice through the development of invasive carci-noma (15, 16). Moreover, satellite RNA is released into thebloodstream and detected in the serum in both pancreatic cancerpatients and patients with intraductal papillary mucinous neo-plasm, another type of precancerous lesion in the pancreas (17).We initially hypothesized that the expression of these satelliteRNAs at such an early stage of carcinogenesis may play a rolein the subsequent oncogenic processes. In our previous study,we showed that overexpression of mouse major satellite RNA(MajSAT RNA) increases the number of genomic and mitochon-drial DNA mutations in mouse primary cell lines derived fromKras-mutated PanIN tumors, and that this leads to the cellulartransformation of precancerous cells (16). We also found thatMajSAT RNA specifically binds to YBX1, one of which functions isan enhancer of several DNA damage repair pathways (18). Innormal cells, YBX1 translocates into the nucleus from the cyto-plasmunder various genotoxic stresses, including oxidative stress,to repair the induced DNA damage. However, in MajSAT RNA–expressing cells, YBX1 is trapped in the cytoplasm by MajSATRNA, resulting in delayed recovery from DNA damage and anincrease in the random gene mutation rate (16).

Department of Gastroenterology, Graduate School of Medicine, The Universityof Tokyo, Tokyo, Japan.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Motoyuki Otsuka, Department of Gastroenterology,Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo 113-8655, Japan. Phone: 813-3815-5411, ext. 37966; Fax: 813-3814-0021;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0139

�2018 American Association for Cancer Research.

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Althoughwe previously showed a contribution ofMajSAT RNAexpression in carcinogenesis in vitro, additional investigation wasneeded to determine the role of MajSAT RNA in DNA damagerepair and tumorigenesis in vivo. Therefore, in this study, weexamined whether the oncogenic functions of MajSAT RNAdetected in vitro were similarly observed in vivo using a novelMajSAT RNA–expressing transgenic mouse model.

MethodsGeneration of MajSAT RNA–expressing transgenic mice

To generateMajSAT RNA–expressing transgenicmice, plasmidsexpressing MajSAT RNA were modified as follows: the pLVSIN-EF1a-MajSAT vector was constructed as described previously(16). The linearized transgene was excised from this plasmid bydigestion at the AccI andNotI sites, which weremicroinjected intoC57BL/6J mouse embryos (Center for Disease Biology and Inter-active Medicine of the University of Tokyo, Tokyo, Japan). Geno-typing was performed by PCR using DNA isolated from tail snips.Two different mouse lines were maintained. Pdx1cre/þ (19),and LSL-KrasG12D/þ (20) were intercrossed to generate Pdx1cre/þ;LSL-KrasG12D/þ(PK) mice on a >95% C57BL/6 background (19).

Detection of MajSAT RNA expressionTo confirm the expression of MajSAT RNA in MajSAT RNA–

expressing transgenic mice, mouse tissues were immediatelyfrozen in liquid nitrogen after resection and stored�80�C. Frozentissues were crushed without thawing using SKmill (Tokken) andimmediately immersed in ice-cold Isogen II reagent (Nippongene). The expression of MajSAT RNA was quantified using theTRAP-ddPCR method as described previously (17). Briefly, totalRNA was hybridized with biotin-labeled RNA oligonucleotideprobes, which were complementary to the core MajSAT RNAsequence. The sequence of the probe was 50-CCUUCAGUGUG-CAUUUCUCAUU-30. Consequently, nonhybridized RNAs weredigested by RNase A/T1, which selectively targets single-strandedRNA. The protected double-stranded core sequence was reversetranscribed using the TaqMan MicroRNA RT Kit (Thermo FisherScientific) and quantified using the QX200 Droplet Digital PCRsystem (Bio-Rad Laboratories).

Caerulein treatment and high-fat diet feedingTo induce acute pancreatitis, 50 mg/kg/body weight caerulein

(Sigma-Aldrich) was injected intraperitoneally every 8 hours for 2consecutive days (21). Mice were 7–11 weeks old and weighed20–30 g. The final day of caerulein injectionwas considered day 0.For high-fat diet feeding, special forage with 60 kcal% fat(D12492, Research Diets) was fed to each mouse for 12 weeks.All mice were 9–13 weeks old and weighed 22–30 g.

IHCIHC was performed as described previously (22). Antigen

retrieval was performed by incubating the slides in a microwaveoven in 10mmol/L sodium citrate buffer (pH 6.0) for 15minutesfollowing deparaffinization. Endogenous peroxidase activity wasblocked by incubation in 3% hydrogen peroxide buffer for 13minutes other than when dying 8-OHdG. To minimize nonspe-cific background staining, slides were blocked in 5% normal goatserum (Dako Corporation, Carpinteria) for 15 minutes at roomtemperature. Tissues were incubated overnight at 4�C with pri-mary antibodies diluted with Can Get Signal Immunostain

Immunoreaction Enhancer Solution (Toyobo Life Science). Sig-nals were enhanced by Vectastain ABC kit (Vector Laboratories)according to themanufacturer's protocol and visualizedwith 3,30-diaminobenzidine in buffered substrate (Nichirei Bioscience).For the primary antibodies derived from mice, Histofine MouseStain Kit (Nichirei Bioscience) was used to block endogenousimmunoglobulin in the tissue. The following antibodies wereused for the assay: anti-8-OHdG (#MOG-202P, JaICA), anti-YB1(#ab12148; Abcam), anti-phospho-Histone H2A.X (#2577; CellSignaling Technology). The number of positive cells was deter-mined by counting 100 nuclei in every eighth field of view fromthree mice in each group.

Mitochondrial DNA copy number measurementMitochondrial DNA copy number was measured as described

previously (16). Briefly, 100 ng isolated total DNAwere subjectedto quantitative PCR using SYBR green (StepOnePlus RealtimePCR system; Thermo Fisher Scientific), and the relative copynumber was calculated using the DDCt method. H19 gene levels,as nuclear DNA copy numbers, were used for normalization (23).Each quantification was performed in triplicate. The primers usedwere as follows:mt-Co1, Fw: 50-CCCAATCTCTACCAGCATC-30

and Rv: 50-GGC TCA TAG TAT AGC TGG AG-30, nuclear-H19, Fw:50-GTA CCC ACC TGT CGT CC-30 and Rv: 50-GTC CACGAG ACCAAT GAC TG-30.

Statistical analysisStatistically significant differences between two groups

were identified using Student t test when the variances wereequal and Welch t test when variances were unequal. P valuesless than 0.05 were considered to indicate statistical signifi-cance. A survival curve was created using Kaplan–Meier meth-od. All analyses were performed using JMP Pro software(SAS Institute Inc).

Study approvalAll of the experimental protocols were approved by the internal

ethics committee for animal experimentation (approval number:#H17-042) and conducted in accordance with the Guidelines forthe Care andUse of Laboratory Animals of theGraduate School ofMedicine, the University of Tokyo (Tokyo, Japan).

ResultsInflammation in MajSAT RNA–expressing transgenic mice

On the basis of our recent results showing thatMajSAT RNAhasan oncogenic role in vitro (16), we constructed MajSAT RNA–expressing mice to determine the role of these RNAs in vivo.Endogenous MajSAT RNAs are expressed as heterogenous tran-scripts ranging from 200 to 8,000 bases in length with numeroussequence variations, including a number of tandem repeats(15, 16). However, it is technically difficult to reproduce suchdiversity via external overexpression. Therefore, we adopted atransgene approach that included approximately three tandemrepeats of basic MajSAT repetitive sequences, which are 855 bp inlength, to cover junctional parts at least twice, similar to our in vitroanalyses (Fig. 1A) (16).

MajSAT RNA expression in transgenic mice was confirmedusing the TRAPmethod followed by droplet digital PCR (ddPCR),which enables precise quantification of heterogenous MajSATtranscripts. We originally developed this method to measure

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Mol Cancer Res; 16(8) August 2018 Molecular Cancer Research1256




serumhuman satellite RNAswith ultrahigh sensitivity (17).Usingthis method, only core sequences are concentrated and aligned ina uniform length fromheterogeneousMajSATRNAs, and they canbe quantified using quantitative PCR. In pancreas and liver tissuesfrom the transgenic mice, MajSAT RNA levels were approximatelydouble those of control mice, indicating that the transgene wasindeed expressed (Fig. 1B).

After 1 year of breeding, we did not observe any tumorigenicchanges in any organs in these transgenic mice (SupplementaryFig. S1A), except for spontaneous lymphomas in the liver, spleen,thymus, and thyroid in 5 cases out of 11 mice (SupplementaryFig. S1B), which were rarely observed in the wild-type mice. Inaddition, greater inflammatory cell infiltration and thickening

of vascular walls were reproducibly detected in the pancreas,liver, and kidneys in 2-year-old MajSAT RNA–expressing mice(Fig. 1C). This was not observed in the 1-year-old mice definitely(Supplementary Fig. S1A). Consistently, biochemical tests ofthe sera of 2-year-old MajSAT RNA–expressing mice showed asignificant increase in aspartate transaminase levels (Supplemen-tary Fig. S1C). These results suggest that MajSAT RNA expressionleads to spontaneous chronic inflammation during long-termbreeding.

MajSAT RNA–expressing mice show increased DNA damageNext, we determined the effects of inflammation on carcino-

genic processes. As we previously reported that MajSAT RNA–

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Establishment of MajSAT RNA–expressing mice. A, A schematic of the transgene construct. Approximately three tandem repeats of MajSAT RNA core sequenceswere inserted between the EF1a promotor and SV40 polyA signal sequences. B, Confirmation of MajSAT RNA expression by the TRAP-ddPCR method.MajSAT RNA levels were measured from 100 ng total RNA extracted from the pancreatic tissues and livers of wild-type and MajSAT RNA–expressing mice. Theabsolute copy number ofMajSATRNA represents themean� SEof fourmice each. � ,P<0.05.C,Pathologic images ofMajSATRNA–expressingmice. RepresentativeH&E images are shown. The left six panels show the images of the pancreas, liver, and kidneys from wild-type mice, and the right panels show images fromMajSAT RNA–expressing mice. All mice were 2 years old (n ¼ 5 for wild and n ¼ 8 for MajSAT). Scale bar, 100 mm.

MST RNA Increases DNA Damage and Tumor Formation

www.aacrjournals.org Mol Cancer Res; 16(8) August 2018 1257




expressing pancreatic cells show impaired DNA damage repairand increased DNA mutation rates (16), acute pancreatitis wasinduced by constitutive hourly intraperitoneal injections of caer-ulein (21, 24). The degree of inflammation was not significantlydifferent between wild-type and MajSAT RNA–expressing mice(Supplementary Fig. S2A). In addition, serum amylase and lipaselevels, which reflect pancreatic damage, did not differ significantlybetween wild-type and MajSAT RNA–expressing mice (Supple-mentary Fig. S2B), while alanine aminotransferase, alkaline phos-phatase, triglyceride, and glucose levels were occasionally differ-ent. Therefore, we determined DNA damage levels in the pancre-atic tissues of MajSAT RNA–expressing mice by evaluating theexpression of 8-hydroxy-20-deoxyguanosine (8-OHdG), a repre-sentative oxidative DNA damage marker, using IHC. 8-OHdGstained was seen in the nucleus in MajSAT RNA–expressingpancreatic tissues, in a dot pattern that was similar to the positionof chromatin 7 days after caerulein treatment (Fig. 2A). Thepercentage of 8-OHdG–positive cells was gradually decreased inwild-type mice as time passed after caerulein treatment (Fig. 2B),

which reflects repair of the DNA damage. In contrast, recoverywas significantly prolonged in MajSAT RNA–expressing mice(Fig. 2B).

As the copy number of mitochondrial DNA is correlatedinversely with the degree of mitochondrial DNA damage andmutation (25), wemeasured the gene copy number of mitochon-drial cytochrome c oxidase I (mt-Co1), normalized to genomicH19 gene levels. Themt-Co1 copynumberdecreased immediatelyafter caerulein treatment and gradually recovered over 7 days inwild-type mice, while restoration of the copy number was signif-icantly impaired in MajSAT RNA–expressing mice (Fig. 2C). Thissuggests greater accumulation of mitochondrial DNA damage inMajSATRNA–expressingmice,which is consistentwithour in vitroresults (16).

MajSAT RNA impairs YBX1 nuclear localization, leading toaccumulation of DNA damage

We previously reported that MajSAT RNA binds to YBX1,which functions as an enhancer of the DNA damage repair

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DNAdamagewas increased inMajSATRNA–expressingpancreatic tissue after caerulein-inducedpancreatitis.A,Representative IHC images of 8-OHdG (in brown) atday 7 after caerulein treatment (n ¼ 3 in each group). The right panels are magnified views of the rectangles in the left panels. Scale bar, 30 mm. Arrows showspots of strong indicating the colocalization of 8-OHdG with chromatin in the nucleus. B, Percentage of 8-OHdG–positive cells after caerulein treatment.8-OHdG–positive cells were visually counted in every eighth field of view. Data represent the mean � SE of three mice each. � , P < 0.05. C, Mitochondria encodedgene (mt-Co1) levels in mouse pancreas were quantified by quantitative PCR. The decreased levels indicate an increase in mitochondrial DNA damage andmutations. Values from the nontreated control were set as 1.0. Data represent the mean � SE of three mice each. � , P < 0.05. D, Representative IHC imagesof YBX1 protein at day 0 just after caerulein treatment. The right panels aremagnified views of the rectangles in the left panels (n¼ 3 in each group). Scale bar, 30 mm.YBX1 is stained brown and the nucleus blue. Arrows show the colocalization of YBX1 with chromatin in the nucleus. E, Percentage of nuclear YBX1-positivecells after caerulein treatment. Cells in which YBX1 was detected in the nucleus were visually counted in every eighth field of view. Data represent the mean� SE ofthree mice each. � , P < 0.05.

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pathway. In addition, we found that MajSAT RNA inhibitsthe nuclear translocation of YBX1 after oxidative stress, result-ing in suppression of the YBX1 DNA repair function (16).Therefore, we evaluated YBX1 in tissues from wild-typeand MajSAT RNA–expressing mice after caerulein treatmentusing IHC. Nuclear YBX1 was detected as strong spots in thenucleus, suggesting that YBX1 coexisted with chromatin where8-OHdG was also strongly stained (Fig. 2D). Consistent withthe in vitro data, the percentage of cells with nuclear YBX1staining was significantly increased in wild-type mice imme-diately after caerulein treatment. In contrast, MajSAT RNA–expressing mice had a relatively poor response (Fig. 2D and E),suggesting that spatial depletion of YBX1 at DNA damagesites by MajSAT RNA expression caused a delay in DNAdamage repair.

We further examined the rate of gH2AX-positive cells aftercaerulein treatment, to determine the degree of DNA double-stranded breaks. Although we expected that these rates were alsohigher in MajSAT RNA–expressing mice, there were no significantdifferences between wild-type and MajSAT RNA–expressing cells

(Supplementary Fig. S2C and D), suggesting that damaged DNAaccumulation was probably induced by impairment of the rela-tively mild DNA damage repair system, which includes baseexcision repair (BER). As YBX1 interacts mainly with proteinsinvolved in BER to enhance its activities (18), the inhibitionof YBX1 by MajSAT RNA could cause an increase in 8-OHdG,but not gH2AX.

To confirm these results in other models, we examinedDNA damage and YBX1 localization using a high-fat diet-induced inflammation model, which induces relatively low-grade inflammation in the pancreas (26–28). Although therewere no significant differences in increase of body weightduring the 12-week treatment (Supplementary Fig. S3A), Maj-SAT RNA–expressing mice showed more inflammatory changesin the pancreas compared with wild-type mice (SupplementaryFig. S3B). Consistent with the caerulein treatment protocol,more 8-OHdG–positive cells were detected, and the nucleartranslocation of YBX1 was inhibited in MajSAT RNA–expres-sing mice compared with wild-type mice (SupplementaryFig. S3C and S3D).

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MajSAT RNA–expressing mice show more, and earlier, PanIN foci in Kras-mutated pancreatic tissues. A, Representative pathologic images of 14-week-old PK andPKM mouse pancreatic tissues (n ¼ 3 in each group). The top panels show the pancreas of PK mice and the lower panels the pancreas of PKM mice. PanINfoci are circled in white. Right panels are magnified views of the rectangles in the left panels. Scale bar, 200 mm. B, The number of PanIN foci is higher in PKMmice atearlier ages. Data represent the mean � SE of three independent mice each. � , P < 0.05. C, Representative H&E-stained image of 1-year-old PK and PKM mice(n ¼ 3 in each group). Scale bar, 400 mm. D, Kaplan–Meier survival curve of PK (n ¼ 21) and PKM mice (n ¼ 21). The blue and red lines represent PK andPKM mice, respectively. The P value was calculated using a log-rank test.






MajSAT RNA accelerates PanIN formation in pancreaticKras-mutant mice

As spontaneous tumorigenesis was not observed in the pan-creatic tissues of MajSAT RNA–expressing mice, we crossedthe MajSAT RNA–expressing mice with mice that have a consti-tutively active Kras mutation in their pancreatic cells (Pdx1cre/þ;LSL-KrasG12D/þ mice). The Kras-mutant mice spontaneouslydevelop adenomatous tumors in the pancreas, which resemblehuman PanIN tissues, but they rarely develop adenocarcinomaeven aftermore than 1 year of breeding (29, 30).We compared thepancreatic phenotypes between Pdx1cre/þ; LSL-KrasG12D/þ mice(PK) and Pdx1cre/þ; LSL-KrasG12D/þ; MajSATþ/� mice (PKM). Inthe PK model, small PanIN foci began to develop sporadically atapproximately 6 weeks of age, and almost all normal acinarregions were replaced by benign tumors and fibrous tissues at40 weeks of age. As shown in Fig. 3, the number of PanIN foci wassignificantly increased in PKMmice compared with PKmice at anearly age (within 24 weeks; Fig. 3A and B). These foci increasedand expanded, eventuallymerging, andbecameuncountable after30weeks of age. At this point, there were no significant differencesin the number of PanIN foci between PK and PKM mice. Nomorphologic changes were observed, including malignant trans-formation such as vessel invasion andmetastasis, even though themicewere bred for over a year (Fig. 3C).However, the survival rate

of PKM mice was significantly lower compared with PK mice(Fig. 3D). PKM mice tended to have more severe chronic pancre-atitis with a greater depletion of acinar and islet cells than PKmiceat 30–40weeks of age (Supplementary Fig. S4A). Furthermore, PKmice developed various skin tumors in neck, eyes, whisker roots,anal canal, and vulvo-vaginal skin, where Pdx1 is reported to beexpressed (31). Although these tumors were pathologically local-ized papillomas and did not show carcinogenetic changes, theirsize and number were significantly larger in PKM than PK mice(Supplementary Fig. S4B).

DiscussionIn this report, we showed that MajSAT RNA expression delays

the repair of mild DNA damage by inhibiting nuclear transloca-tion of YBX1, thus increasing the development of PanIN in Kras-mutated mice (Fig. 4). These results were consistent with ourprevious in vitro results using Kras-mutated primary cell lines.

Although MajSAT RNA expression without Kras mutation didnot result in tumor development in the pancreas, the inflamma-tion induced by caerulein or high-fat diet led to the accumulationof DNA damage and the absence of YBX1 nuclear localization.This suggests that DNA repair mechanisms that are executedby YBX1, such as BER, were impaired after the induction of

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MajSAT RNA increases DNA damageand accelerates tumorigenesis byinhibiting DNA repair via YBX1. Aschematic of the proposed MajSATRNA function in vivo. Kras mutationcauses oxidative stress and low levelsof continuous DNA damage. YBX1,which is normally translocated to thenucleus where it is involved in DNAdamage repair, is trapped by MajSATRNA in the cytoplasm. Unrepairedoxidized DNAs cause accumulation ofrandom point mutations and increasethe probability of accidental drivergene mutations, which trigger cells toenter the expansion phase from thebreakthrough phase during theoncogenesis.

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genotoxic stress in MajSAT RNA–expressing mice. As wedescribed previously, these phenomena led to an increase inrandom mutations in vitro (16). We were not able to clearlydemonstrate the colocalization of MajSAT RNA and YBX1 invivo, because costaining of RNA and protein is technicallydifficult. This is particularly true in formalin-fixed, paraffin-embedded tissue due to the incompatibility of in situ hybrid-ization and IHC techniques. However, we speculate that Maj-SAT RNA expression impaired the nuclear localization of YBX1after DNA damage, similar to what we observed in vitro (16). Infact, 8-OHdG expression was reduced in the chromatin of wild-type mice, where YBX1 was concentrated. Conversely, 8-OHdGexpression was significantly observed in the chromatin, wherelocalization of YBX1 was not detected, in MajSAT RNA–expres-sing mice. These results suggest that MajSAT RNA expressioninhibits the nuclear translocation of YBX-1, which theoreticallylocalizes in DNA-damaged regions for their repair (18).

We also showed that the emergence of PanIN foci in Kras-mutated pancreatic tissues occurred significantly earlier by ectopicMajSAT RNA expression compared with canonical PK mice.Generally, Kras mutations increase oxidative DNA damage spon-taneously through abnormal production of reactive oxygen spe-cies (32). This suggests that in PK and PKM mice, mutated Krasitself may function in a similar manner as the inflammationinduced by caerulein or a high calorie diet, thus leading toaccumulation of genomicmutations and earlier PanIN formationin PKM mice.

Carcinogenesis is divided into three phases: the breakthrough,expansion, and invasion phase (8). In the breakthrough phase, acell acquires a driver gene mutation and begins to proliferateabnormally. After many years, these cells randomly obtain asecond driver gene mutation that enables the cells to thrive intheir local environment, thus entering the expansion phase. Thisphase is followed by the acquisition of a greater number of drivergenemutations, which allows the cells to enter the invasive phase.In human tissues, there is usually a long period between thebreakthrough and expansion phases. On the basis of our obser-vation thatMajSAT RNA expression inmice results in significantlyearlier PanIN formation, we speculate that aberrant MajSAT RNAexpression may accelerate the expansion phase by increasing theprobability of developing a second driver gene mutation (Fig. 4).

Although earlier PanIN formation was observed in PKM mice,we did not observe any significant differences in tumor formationbetween PK and PKM mice after 1 year of breeding. This may bebecause after PanIN formation in transgenic mice, endogenousMajSAT RNA is expressed at much higher levels than those of thetransgene. In fact,we expressedMajSATRNAswith three repeats ofthe consensus sequence. EndogenousMajSAT RNAs, however, aremore heterogeneous and have additional repeats. While there is

currently no adequate method to express all heterogeneousMajSAT RNAs, we speculate this is the reason why the impact ofthe artificial overexpression was lost after PanIN formation.

While significant differences in malignant transformationbetween PK and PKMmice were not observed, the prognosis wasworse for PKM than PK mice. Although the precise reason for thedifferences in prognosis remains to be elucidated, PKM miceshowed worse chronic pancreatitis, which may have resulted inpancreatic endocrine and exocrine dysfunction. In addition, skintumors were more severe in PKM mice, which may have affectedtheir physical strength.

In summary, this study confirmed that aberrant expression ofMajSATRNA is one of the oncogenic promotors through impairedDNAdamage repair, at least partly through YBX1mislocalization.Further elucidating the molecular mechanisms of deregulatedMajSAT RNA expression as well as the downstream events mayresult in additional preventative strategies against pancreaticcancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Kishikawa, M. Otsuka, K. SekibaDevelopment of methodology: T. Kishikawa, K. SekibaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Kishikawa, T. Suzuki, T. Seimiya, K. Sekiba,R. Ishibashi, E. Tanaka, M. Ohno, M. YamagamiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Kishikawa, M. Otsuka, K. SekibaWriting, review, and/or revision of the manuscript: T. Kishikawa, M. Otsuka,K. SekibaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Kishikawa, K. SekibaStudy supervision: K. Sekiba, K. Koike

AcknowledgmentsThis work was supported by Grants-in-Aid from the Ministry of Education,

Culture, Sports, Science and Technology, Japan (#16H05149, #16KT0109,#26860492, #17K15923, and #15H04807; to M. Otsuka, T. Kishikawa,andK.Koike), andby the Project forCancer ResearchAndTherapeutic Evolution(P-CREATE) from Japan Agency for Medical Research and Development(AMED; #JP17cm0106419, to T. Kishikawa; #JP19cm0106602, to M. Otsuka),and by Pancreatic disease research promotion award from Pancreas ResearchFoundation of Japan (to T. Kishikawa).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 7, 2018; revised March 15, 2018; accepted April 24, 2018;published first May 10, 2018.

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Kishikawa et al.




2018;16:1255-1262. Published OnlineFirst May 10, 2018.Mol Cancer Res Takahiro Kishikawa, Motoyuki Otsuka, Tatsunori Suzuki, et al. Formation in Mouse Models of Pancreatic CancerSatellite RNA Increases DNA Damage and Accelerates Tumor

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satellite rna increases dna damage and accelerates tumor formation … · dna damage and repair...

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