DNA fragile site breakage as a measure of chemical exposure and predictor of individual susceptibility to form oncogenic rearrangements

Carcinogenesis, Mar 2017

Lehman, Christine E., Dillon, Laura W., Nikiforov, Yuri E., Wang, Yuh-Hwa

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DNA fragile site breakage as a measure of chemical exposure and predictor of individual susceptibility to form oncogenic rearrangements

Carcinogenesis DNA fragile site breakage as a measure of chemical exposure and predictor of individual susceptibility to form oncogenic rearrangements Christine E.Lehman 1 Laura W.Dillo n 1 Yuri E.Nikiforov 0 Yuh-Hwa Wang 1 0 Department of Pathology and Laboratory Medicine, University of Pittsburgh , Pittsburgh, PA 15261 , USA 1 Department of Biochemistry and Molecular Genetics, University of Virginia , 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0733 , USA Chromosomal rearrangements induced by non-radiation causes contribution to the majority of oncogenic fusions found in cancer. Treatment of human thyroid cells with fragile site-inducing laboratory chemicals can cause preferential DNA breakage at theRET gene and generate theRET/PTC1 rearrangement, a common driver mutation in papillary thyroid carcinomas (PTC). Here, we demonstrate that treatment with non-cytotoxic levels of environmental chemicals (benzene and diethylnitrosamine) or chemotherapeutic agents (etoposide and doxorubicin) generates significant DNA breakage withinRET at levels similar to those generated by fragile site-inducing laboratory chemicals. This suggests that chronic exposure to these chemicals plays a role in the formation of non-radiation associatReEdT/PTC rearrangements. We also investigated whether the sensitivity of the fragRilEeT region could predict the likelihood of rearrangement formation using normal thyroid tissues from patients with and withouRtET/PTC rearrangements. We found that normal cells of patients with thyroid cancer driven byRET/PTC rearrangements have significantly higher blunt-ended, double-stranded DNA breaks atRET than those of patients withouRtET/PTC rearrangements. This sensitivity of a cancer driver gene suggests for the first time that a DNA breakage test at thReET region could be utilized to evaluate susceptibilityRtEoT/PTC formation. Further, the significant increase of blunt-ended, double-stranded DNA breaks, but not other types of DNA breaks, in normal cells from patients withRET/PTC-driven tumors suggests that blunt-ended double-stranded DNA breaks are a preferred substrate for rearrangement formation, and implicate involvement of the non-homologous end joining pathway in the formation of RET/PTC rearrangements. - Introduction Chromosomal rearrangements are a common genetic abno-r (PTC) ( 3 ) accounting for approximately 20% of adult3)( and mality involved in the initiation of cancer development. These approximately 45% of childhood sporadic PTC cases 4(). PTC rearrangements result in the disruption of genetic material, is responsible for the rising incidence of thyroid cancer in the which can lead to the expression of oncogenic fusion proteins USA ( 5 ), where thyroid cancer is now the most rapidly incre-as or the disruption of processes involved in tumor suppression1(). ing cancer type 5(,6). Although increases in thyroid cancer have DNA strand breaks must occur in participating gene regions to been well documented following exposure to high doses of ra-di initiate all chromosomal rearrangements. We have shown that ation ( 7,8 ), many tumors are sporadic in nature 4( ,9 ), suggestfragile site-inducing conditions can create DNA breaks within ing other factors are involved in initiating DNA breaksRiEnT/ RET/PTC participating genes and ultimately lead to the for-ma PTC genes. RET, an oncogene involved in recurrent chromosomal tion ofRET/PTC rearrangements2(). RET/PTC rearrangements are rearrangements found in thyroid3( ,4,9,10 ) and lungs (11,12) is a common mutation observed in papillary thyroid carcinoma located within fragile site FRA10G. Our previous study offers Abbreviations we found that patients with thyroid cancer driven bRyET/PTC rearrangements have significantly more blunt-ended doubleDEN diethylnitrosamine stranded breaks atRET intron 11 compared to patients without PTC papillary thyroid carcinoma the rearrangements, suggesting that this increased breakage leads NHEJ non-homologous end joining to the formation ofRET/PTC rearrangements. The results support the hypothesis that chronic low-dose exposure to environmental direct evidence for the role of fragile sites in cancer-specific fragile site-inducing chemicals or residual chemotherapy agents rearrangements 2(). contributes to the formation of non-radiation induceRdET/PTC DNA fragile sites are sensitive to a variety of chemicals and rearrangements, which are more common than radiation-induced have been identified in regions with deletions and chromosomal RET/PTC in thyroid cancer. Also, the significant increase in bluntrearrangements 1( 3,14 ). Environmental agents such as benzene ended DNA breaks withinRET in normal cells ofRET/PTC patients (in cigarette smoke and automobile exhaust) and diethylnit-ro implicate blunt-ended DNA breaks in the formation oRfET/PTC samine (DEN) (in cigarette smoke, pesticides and cured meat), rearrangements. To our knowledge, this is the first study to d-em and chemotherapeutic drugs, can significantly increase fragile onstrate the sensitivity of a cancer driver gene in normal cells, site breakage 1( 3,14 ) and are positively associated with the risk suggesting that a test to detect DNA breakageREaTtcould provide of thyroid cancer1( 5–26 ). Benzene has been associated with an an early indication of susceptibility to PTC. increased risk of thyroid cancer after exposure to volcanic ac-tiv ity at Mount Etna1(9), and an increased risk of thyroid cancer Materials and methods was observed in female textile workers with 10 or more years of benzene exposure (25). Specific nitrosamines, including DEN, Cell line and culture conditions have been shown to cause thyroid tumors in animal studies2 7(). Human thyroid epithelial cells immortalized by SV-40 (HTori-3) were p-ur Increasing dietary nitrate intake (which generates nitrosamine chased from the European Collection of Authenticated Cell Culture and by interacting with aminesin vivo, or by cooking) is positively were grown in RPMI1640 medium (Gibco) supplemented with 10% fetal associated with thyroid cancer risk in older wome2n4() and men bovine serum and penicillin/streptomycin. (17). Chemotherapy regimens which contain the topoisomerase II inhibitors etoposide or doxorubicin have been linked to s-ec Tissue sample procurement ondary PTC following treatment for primary osteosarcom1a8(), Tissue samples were obtained from the University of Pittsburgh Health rhabdomyosarcoma (22), Ewing’s sarcoma and Wilms tumor Sciences Tissue Bank using a protocol approved by the University of (15). We have found that DNA topoisomerases I  and II partic-i Pittsburgh Institutional Review Board. Two paired samples (tumor and pate in initiating fragile site breakage at tRhEeT oncogene (28). normal surrounding tissue) were obtained from each of six patients with Therefore, investigating the sensitivity of tRhEeT region to these an RET/PTC+ tumor. Seven samples of normal thyroid tissue were also chemicals is critical for understanding whether these agents are obtained from patients with benign nodules but without PTC (PTC−). Prior to likely to contribute to the formation of non-radiation induced procurement for this study, tissue samples were histologically analyzed to RET/PTC rearrangements, which are more common than radi-a tdreatnersmcrinipetatsuem-poorlytimsesuraesaencdhsauinrrroeuancdtiinogn n(RoTr-mPaClR)maasrgdienssc. rFiubretdhperre,vrieovuesrlsye tion-inducedRET/PTC in thyroid cancer. (2), was used to verify the presence ofRET/PTC rearrangements in each While common fragile sites are found in all individuals, the tumor sample and to confirm lack ofRET/PTC rearrangements in the su-r extent of DNA breaks at particular fragile sites varies among rounding normal tissue or in the normal tissue provided by PTC− patients. individuals (29). Double-stranded DNA breaks are commonly repaired through homologous recombination or non-homol-o Cell treatments and analyses gous end joining (NHEJ) (30), but dysfunction in these pathways Cell viability was assessed as previously described28() using propidium can contribute to increased DNA breakage. Alternatively, an iodide staining and quantification by flow cytometry. Based on known accumulation of chemical exposures could increase the number fragile site-inducing conditions13(), HTori-3 cells were treated for 24  h of DNA breaks and overwhelm cellular DNA repair pathways, with 0.4 µM aphidicolin (APH) and various concentrations of benzene resulting in the formation of chromosomal rearrangement1s).( (0.5–1  mg/ml; Sigma-Aldrich), DEN (3.5–7  mg/ml; Sigma-Aldrich), etopoTherefore, the DNA fragility at rearrangement-participating and side (0.3–0.5 µM; Sigma-Aldrich) or doxorubicin (5–10 nM; Sigma-Aldrich), breakage-sensitive gene regions may be indicative of an ind-i to dTeotaernmalinyzeetahcetiovpetaimpoapltdoosissa, gHeTowrhi-i3cchedllids wnoerteatlrteeartceedlwlivtihabtihleitcyh. emicals vidual’s exposure to environmental factors and/or an unfavo-ra described above for 24 h, harvested and resuspended in 1X Annexin V bin-d ble genotype for DNA stability, and thus predict a potential for ing buffer containing Annexin V (BD Biosciences). Early apoptotic cells were the formation of cancer-causing chromosomal rearrangements. then quantified using a FACSCalibur flow cytometer and FlowJo software. Both canonical NHEJ and microhomology-mediated end-joining To analyze cell growth, the number of viable HTori-3 cells was de-ter (MMEJ or alt-NHEJ) are implicated in the formation of inter- mined by trypan blue exclusion using a hemocytometer. Cells were plated and intra-chromosomal rearrangements 3( 1–35 ). Studies by at 1 ×  105 viable cells and treated with the indicated chemical con-cen Ghezraoui et al. (36) demonstrated that chromosomal translo-ca trations. After 24 h of chemical treatment, viability was determined and tions depend on canonical NHEJ in human cells, and the ends of the cells were washed with PBS and re-plated in chemical-free media to DNA breaks, such as blunt or long staggered, can influence the recover for an additional 24 h before being quantified again. characteristics of rearrangement fusion points. platFeosrabnrdeatkrpeoaintetddaetppercotxioimna,HteTloyri1-83 chellalste×(1r 1 0w5)itwhe0rµ.e4M plAaPtHed,0in.5 smixg-/wmelll In this study, we first demonstrated thRaEtT intron 11, the major benzene, 3.5 mg/ml DEN, 0.3  µM etoposide or 5 nM doxorubicin for 24 h. patient break point region found in PTC patients37(–40) is sensi- Genomic DNA was then isolated from treated and untreated cells in pa-ral tive to DNA breakage when exposed to non-cytotoxic, low doses lel for DNA break analyses. of environmental chemicals (benzene or DEN) or chemother-a peutic agents (etoposide or doxorubicin). This sensitivity was also Detection and quantification of all DNA breaks by detected in the form of blunt-ended DNA double-stranded breaks ligation-mediated PCR (LM-PCR) generated by these exposures. Using normal human thyroid tissue The total number of DNA breaks including single-stranded nicks and samples from patients with or withoutRET/PTC rearrangements, double-stranded DNA breaks was detected and quantified as described previously (2,28) (Supplementary Figure  1A, left panel). Briefly, genomic as benzene and DEN are encountered daily and are associated DNA was isolated from HTori-3 cells or normal thyroid tissues of patients, with an increased risk of thyroid cancer17( ,24,25 ). In order to followed by primer extension using a ′5-biotinylated primer matched mimic daily, low level exposure to environmental chemicals, we to the regions of interest. DNA breaks were ligated to the asymm-etri used a cell viability assay to determine optimal, non-cytotoxic cAamlpldifuipclaetxioLnLo3f/LtPh2e lDinNkAerbraenakds twhaesnaicshoileavteeddthursoinugghstnreesptteadviPdCinR obfetahdes. dosages of benzene and DEN to ensure that significant levels extension-ligation products for each region of interest. Linker and primer of cell death were not induced relative to the untreated control sequences have been described previously 2(). PCR products were resolved (Figure 1A). Treatment of human thyroid epithelial cells (HToriby gel electrophoresis and sequenced to verify their identity. Each band 3) with 0.5  mg/ml benzene or 3.5  mg/ml DEN for 24  h did not visualized on the gel represents one break isolated within the gene region induce apoptosis, as measured by flow cytometry analysis of of interest. DNA breaks were then quantified as the number of DNA breaks annexin V staining F(igure 1B). Cell growth, measured by trypan per 100 cells. blue exclusion over a period of 48 h, was not perturbed following the same chemical treatment conditionsF(igure 1C). Detection and quantification of double-stranded Therefore, these conditions were used to determine whether DNA breaks by LM-PCR RET intron 11 was sensitive to DNA breakage upon exposure To detect only blunt-ended double-stranded DNA breaks, genomic DNA to non-cytotoxic levels of benzene or DEN. HTori-3 cells were isolated from HTori-3 cells or normal thyroid tissues of patients was -sub treated with 0.5 mg/ml benzene, 3.5 mg/ml DEN or 0.4µ M APH jected to a protocol described previously41() (Supplementary Figure 1A, (as a positive control) for 24 h before being harvested for LM-PCR right panel). Genomic DNA was directly ligated to the asymmetrical analysis of all types of DNA breaks, including single-stranded SdeupphleaxdexLLG3-/1L0P02 cloilnukmenr,s parniodr ltioganteisotnedmPiCxRt.uPrCeRs pwroedreuctpusrwifeiered retshorlovuegdh nicks and double-stranded DNA breaks with ′-5overhangs, by gel electrophoresis, sequenced and quantified as described above. 3′-overhangs and blunt ends (Supplementary Figure 1A, available To detect all double-stranded DNA breaks, genomic DNA was first i n-cu at Carcinogenesis Online, left panel2();). Both benzene and DEN bated withE. coli DNA polymerase I large (Klenow) fragment (New England induced significantly higher DNA breakage aRtET compared to Biolabs) followed by heat inactivation at 75°C. The Klenow fragment -con untreated cellsFi(gure 2A and Supplementary Figure 1B, availaverts double-stranded DNA breaks with eithe′r- 5or 3′-overhangs to blunt ble atCarcinogenesis Online). Also, the frequencies of DNA brea-k ends through its polymerase and ′3-5′-exonuclease activities. The blunt- age induced by either of these chemicals were similar to that ended DNAs were then ligated directly to the LL3/LP2 linker. Detection induced by APH, which we have shown leads to the formation of and quantification of the DNA breaks was performed as described above. RET/PTC rearrangements 2(). FHIT, encompassing the fragile site To detect the combination of ′5-overhang and blunt-ended double- FRA3B (42), also showed a significant increase in breakage upon sFtrraagnmdeendt D(3N′–A5′ berxeoa-k)sl,agcekninogmic3′-5D′-NeAxonwuacsleiansceubaactteidvitwyit(hNetwheEnKglelannodw treatment with either chemical, while the non-common fragile Biolabs), which creates blunt-ended DNA only from′-5overhang DNA site, G6PD region (2), was insensitive to fragile site induction by breaks. Detection and quantification of the DNA breaks was performed as these environmental chemicals as well as APHFi(gure 2A). described previously. Next, we investigated the induction of blunt-ended d-ou To verify the ability of the assay to detect the specified type of DNA ble-stranded breaks withinRET intron 11 by these chemicals, break, genomic DNA from HTori-3 cells was digested with various rest-ric since this type of break could be an immediate precursor in the tion enzymes (New England Biolabs) to generate either single-stranded formation of rearrangements. Genomic DNA from treated or nicks (by Nt.BstNBI digestion), ′5-overhangs (BbvI digestion), 3′-overhangs untreated cells was subjected to a modified LM-PCR procedure (BanII digestion) or blunt-ended (HaeIII digestion) double-stranded DNA detecting only blunt-ended breaks (Supplementary Figure  1A, Fbirgeuarkes ,1Aa,nadvasiulabbjelcetaetCdartcoinotgheenesfiosuOrnplrinoet.ocols listed in Supplementary available atCarcinogenesis Online, right panel). Treatment with either benzene or DEN significantly increased the number of Analysis of individual types of DNA breaks blunt-ended double-stranded DNA breaks in this region as Normal thyroid tissue from patients withRET/PTC rearrangements or compared to untreated cellsFi(gure  2B). Further, blunt-ended patients with benign nodules but without PTC, was evaluated indiv-idu double-stranded DNA breaks withinFHIT were also significantly ally for each types of breaks as follows. To determine the amount of -sin increased compared to untreated cells, but with no difference gle-stranded nicks only, the number of all double-stranded DNA breaks in the G6PD region. This result suggests that benzene and DEN (from Klenow fragment-treated DNAs) was subtracted from the number induce fragile site-specific breakage within human thyroid e-pi of all DNA breaks. To quantify ′3-overhang DNA breaks only, the num- thelial cells and demonstrate the sensitivity of human thyroid ber of 5′-overhang and blunt-ended double-stranded DNA breaks (from epithelial cells to low-dose chemical exposures. This induction 3′ to 5′ exo-treated DNA) was subtracted from the number of all double- of DNA breakage atRET further suggests a role for these longstranded DNA breaks (from Klenow fragment-treated DNAs). The amount term, low-dose chemical exposures in the formation of nonof 5′-overhang DNA breaks was quantified by subtracting the number of radiation induced RET/PTC rearrangements, which are more balnudnbtl-eunndte-ednddoeudbldeo-usbtlrea-nsdteradnDdNeAd bDrNeAakbsrweaitkhst(hfreonmu′3mtobe5r′ oe′xf-oo5v-terrehaatnegd common than radiation-inducedRET/PTC in thyroid cancer. DNA). Treatment of primary tumors with chemotherapy regimens including topoisomerase II inhibitors such as etoposide or dox-o rubicin has been previously linked to secondary cancers incl-ud Results ing PTC ( 15,18,22 ). Using previous pharmacokinetic studies to mimic residual low doses following chemotherapy administr-a Environmental chemicals and chemotherapeutic tion (43,44), we treated HTori-3 cells with various concentrations drugs induce significant DNA breakage within of each drug for 24 hours and analyzed cell viability, apoptosis fragile site genes including RET intron 11 and cell growthF.igure 1 shows that 0.3µ M etoposide or 5 nM Intron 11 of theRET gene, the major patient break point region doxorubicin did not induce significant cell death or apoptosis, (37–40), is sensitive to low doses of APH, a classic fragile site- and cell growth was not hindered. inducing condition 2(). DNA breakage at this region, under fra-g Next, we examined the overall DNA breaks in HTori-3 cells ile site-inducing conditions, leads to the formation RoEfT/PTC after 24 h treatment with 0.µ3M  etoposide or 5 nM doxorubicin. rearrangements 2(). Many fragile site-inducing chemicals such Both chemicals significantly increased breakage within intron 11 of RET as well as inFHIT (Figure  2A and Supplementary as a direct substrate to generate cancer-specific rearrangements. Figure  1B, available atCarcinogenesis Online) even at these low Therefore, DNA breakage at these sensitive fragile regions may non-cytotoxic doses. Treatment with either drug, however, did be a useful metric for patient prognosis and the risk of devel-op not induce significant breakage withinG6PD, again suggest- ing therapy-related secondary cancers. ing the specific sensitivity of DNA fragile sites to these drugs. Importantly, these low dose concentrations are approximately Frequency of RET breakage in RET/PTC patient 300–1000-fold lower than the concentrations found in the normal tissues is predictive of RET/PTC plasma of patients immediately after chemotherapy treatment rearrangements ( 43,44 ), demonstrating the deleterious consequences to cells The presence of RET/PTC rearrangements in patients withRET/ that are not killed by treatment with chemotherapeutic agents. PTC-driven thyroid cancer suggests prior chemical and en-vi Further, we specifically examined blunt-ended double- ronmental agent exposures and/or unfavorable genotypes for stranded DNA breakage induced after exposure to either che-mi DNA stability, which cause breakage in the intron 11 region cal. The results show that these low-dose chemotherapy drugs of RET. Therefore, we hypothesize that the sensitivity oRfET also induce significant blunt-ended DNA breakage within intron intron 11 signals the net balance of an individual’s ex-po 11 of RET and withinFHIT (Figure 2B). This suggests that cells are sure to environmental factors and unfavorable genotypes, still susceptible to DNA damage in these regions when exposed and can predict the likelihood ofRET/PTC rearrangements. to a residual dose of chemotherapy treatment, and the sign-ifi If indeed RET breakage can indicate a potential to form the cant amount of blunt-ended double-stranded breaks could serve rearrangements, normal cells ofRET/PTC patients should compared to PTC− patient tissues (Supplementary Figure  2, available atCarcinogenesis Online). Also, no difference in DNA breaks was observed between these two sample groups at the FHIT/FRA3B region or theALK/FRA2N, indicating that individ-u als susceptible toRET/PTC rearrangements might not have glo-b ally susceptible fragile sites, but have fragile site breaks specific to the individual’s translocation. These results suggest that DNA breakage at specific fragile sites could be a valuable indicator for the potential formation of chromosomal rearrangements at these sites. Next, we chose to focus only on DNA breakage aRtET as it is the common partner gene in 19 rearrangements known to cause thyroid cancer9(). By analyzing all types of DNA breakage within RET in each of the normal tissues from the two patient groups, we found that there was no significant difference in the -fre quency of breakage between these two groupsFi(gure 3B). There was also no significant difference in total DNA breakage between groups, when analyzingFHIT, 12p12.3 or G6PD. This indicates that blunt-ended DNA breaks are increased specifically in the normal cells of patients withRET/PTC-driven tumors, and suggests that blunt-ended, double-stranded DNA breaks contribute to the formation ofRET/PTC rearrangements. Blunt-ended DNA breaks are the only type of double-stranded DNA breaks increased in normal cells of RET/PTC+ patients compared to RET/PTC− patients Figure  2. Frequency of DNA breakage withinRET, FHIT and G6PD regions in HTori-3 cells after 24 h treatment with APH, benzene, DEN, etoposide or do-xo rubicin. (A) The breakage frequency is represented as all DNA breaks per 100 cells which includes single-stranded nicks′,-o5verhang, 3′ overhang and blunt- In addition to single-stranded DNA nicks, there are three types ended double-stranded DNA breaks. B() The frequency of only blunt-ended double-stranded DNA breaks per 100 cells after 24-h treatment. Both assays are of DNA double-stranded break ends: staggered ends with either represented by the mean ± SEM of at least three replicated experiments. As-ter 5′- or 3′-overhangs and blunt ends. We observed that when co-m isks indicateP < 0.05 as compared to the respective untreated sample following paring the amount of overall DNA breaks aRtET intron 11 to analysis by one-way Anova and Dunnett’s multiple comparison test. that of blunt-ended double-stranded breaks, the vast majority of DNA breaks in each patient tissue were not blunt-ended doublehave higher DNA breakage atRET than patients without the stranded DNA breaks F(igure  3A compared to B). DNA doublerearrangements. stranded breaks must occur for the formation of chromosomal To test this, normal thyroid tissue surroundinRgET/PTC+ rearrangements, therefore, this prompted us to examine the d-is tumors was compared with normal thyroid tissue from patients tribution of DNA breaks among the four types of break ends, and with benign nodules. First, total RNA was isolated from each to shed light on the possible substrate required for the for-ma tumor tissue to verify the presence ofRET/PTC rearrangement tion ofRET/PTC rearrangements. in the tumor. To ensure a lack of significant tumor cell conta-mi We utilized the activity of the Klenow fragment Eo.f Coli nation within the normal tissues, total RNA was isolated from DNA polymerase I to convert both ′5- and 3′-overhang to bluntnormal tissue of six patients withRET/PTC rearrangements and ended double-stranded breaks, and the Klenow fragment seven patients with benign nodules, and a lack oRfET/PTC rear- mutant (3′–5′ exo-) lacking 3′–5′ exonuclease activity to co-n rangement in these thyroid tissues was verified by RT-PCR (data vert only 5′-overhang ends to blunt ends. The number of DNA not shown). Genomic DNA was also isolated from each normal breaks detected from treatment of genomic DNA with these tissue, and DNA breakage withinRET was evaluated. enzymes followed by the blunt-end double-stranded detection Using the procedure to detect only blunt-ended, double- protocol (Supplementary Figure  1A, available aCtarcinogenesis stranded DNA breakage, we found that normal cells surrou-nd Online, right panel) allows us to detect all four types of DNA ing RET/PTC+ tumors have a significantly increased frequency breaks. To test the feasibility of our methods, genomic DNA from of blunt-ended DNA breaks withinRET intron 11, as compared HTori-3 cells was digested with various restriction enzymes to to the normal thyroid cells from patients withouRtET/PTC generate either single-stranded nicks (by Nt.BstNBI digestion), (P < 0.001) (Figure 3A and Supplementary Figure 1C, available at 5′-overhang (BbvI digestion), 3′-overhang (BanII digestion) or Carcinogenesis Online). This significant increase in DNA breakage blunt-ended (HaeIII digestion) double-stranded DNA breaks. was specific toRET intron 11, as there were no significant diff-er The digested DNA was subjected to the four protocols listed ences in the breakage frequency withinFHIT, ALK or two non- in Supplementary Figure 1A, available atCarcinogenesis Online. CFS control regions, 12p12.3 orG6PD (45). ALK, located within In the RET intron 11 region, all three double-stranded breaks the fragile site FRA2N, is involved in rearrangements found in up were detected as expected in Klenow fragment-treated DNA to 10% of thyroid cancer cases4(6), therefore, the lack of a si-g (Supplementary Figure 3, available atCarcinogenesis Online, lane nificant difference in theALK region between our patient groups 2), and only 5′ overhang and blunt-ended breaks were mea-s demonstrates the specificity ofRET/PTC rearrangement forma- ured in Klenow (3′–5′exo-)-treated samples (lane 3). Lanes 1 tion following increased DNA breakage aRtET. Interestingly, and 4 showed the expected products for all four types of breaks normal cells from patients withRET/PTC1 driven tumors have a and blunt-ended breaks, respectively, when using the all break significant increase in DNA breakage at the partner genCeC,DC6, detection method and the blunt end detection method. All four products were verified by Sanger sequencing to be located at the Discussion expected restriction enzyme cut sites inRET intron 11. These results demonstrate the specificity of our assay to detect single- In this study, we examined whether fragile site breakage could stranded nicks, 3′-overhang, 5′-overhang and blunt-ended do-u be used to assess the consequence of long-term, low-dose ble-stranded DNA breaks. chemical exposure and predict susceptibility to chromosomal Applying these procedures to measure DNA breaks within rearrangement formation. We found that low-dose, non-cy-to RET intron 11 from the normal thyroid cells surroundinRgET/ toxic exposure to two common environmental chemicals, be-n PTC-driven tumors and from patients without PTC, no significant zene and DEN, generate significantly more DNA breaks within difference between the two patient groups was observed in the RET intron 11 compared to untreated cells. We also found that amount of all three types of double-stranded DNA breaks co-m both benzene and DEN induced significantly more blunt-ended bined (Figure 4A, left panel). Similarly, the combination of′-5over- double-stranded DNA breaks withinRET intron 11 at a rate sim-i hang and blunt-ended double-stranded DNA breaks showed no lar to that of APH treatment, which we have shown can lead significant difference between the two groupsFi(gure  4A, right to RET/PTC rearrangements 2(). Another study demonstrated panel). When individual types of DNA break were evaluated, that H2O2 can induce DNA double-stranded breaks and contr-ib blunt-ended, double-stranded DNA breaks were the only type ute to RET/PTC rearrangements 4(7), also suggesting the role of DNA break increased in normal thyroid cells of patients with of chemical exposures on DNA breakage atRET. Therefore, our RET/PTC-driven tumors compared to PTC− patients, and no si-g study demonstrates the importance of investigating these and nificant difference was observed when comparing single-strand other low-dose environmental chemicals and their role in the DNA breaks, 3′-overhang or 5′-overhang DNA breaks between the formation of non-radiation associatedRET/PTC rearrangements two patient groups F(igure  4B). This observation suggests that in thyroid cancer. While the HTori-3 cells used for this an-aly blunt-ended double-stranded DNA breaks could be the preferred sis were immortalized by SV40 and therefore may have altered substrate for rearrangement formation, and that a test for blunt-DNA damage checkpoint and repair, we previously showed 4( 1 ) ended double-stranded DNA breakage aRtET could offer an early that in normal human hematopoetic stem and progenitor CD3+4 indication of susceptibility to PTC. cells, similar, non-cytotoxic levels of benzene, DEN, etoposide biomarkers to predict cancer susceptibility. Our results dem-on strate that normal thyroid cells of patients wRitEhT/PTC-driven tumors have significantly increased blunt-ended, doublestranded DNA breaks withinRET, and suggest that this diffe-r ence could be utilized to predict the propensity to forRmET/ PTC rearrangements. This is the first study to show significantly higher double-stranded DNA breakage at a cancer driver gene in normal cells of patients with cancer driven by the respective gene. A test to detect DNA breakage aRtET could offer an early indication of susceptibility to PTC, and facilitate prompt prev-en tion and timely treatments. Our data suggests that an ind-ivid ual’s susceptibility to fragile site breakage and rearrangement formation is due to the combined effects of both chemical exp-o sures and their susceptibility to these exposures. Therefore, this test could also be used to evaluate individuals at high risk for PTC, either due to environmental exposures and/or unfavorable genotypes. A diagnostic tool to measure individual susceptib-il ity to PTC could improve early detection and possible long-term clinical outcomes. Further, more than half of cancer-specific recurrent chromosomal rearrangements possess DNA brea-k points located within at least one fragile si4t9e).(Therefore, the use of a test for DNA breakage within fragile regions could be expanded to other breakage-sensitive regions in the genome to predict the propensity to form a variety of cancer-specific rearrangements involving these genes. This susceptibility test would be widely helpful to tailor chemotherapy regimens, to monitor high-risk populations and patients in remission, and Figure 4. Detection of single-stranded,′-3overhang, 5′-overhang and blunt-ended to improve screening tests for other environmental toxins and double-stranded DNA breaks at thReET intron 11 region in normal thyroid cells stress factors. from RET/PTC+ or PTC− patients. A() The breakage frequency is represented for all Various studies have demonstrated that NHEJ and MMEJ are double-stranded DNA breaks or the combination of′-5overhang and blunt-ended the DNA repair pathways required to generate chromosomal bDlNuAntb-reenadkesd. ADlNlAdboruebalkes-s.Bt(r)aTnhdeebdrebarkeaagkse finrecqluudenes′c-yo3voefrehaacnhgD,5N′-Aovberrehaakntgyapendis translocations in human cancers 31(,32,34–36). The types shown and represented as DNA breaks/100 cells. Diamonds represent the average of DNA break ends can influence the characteristics of r-ear DNA breaks from at least three experimental replicates of each theRE6T/PTC+ rangement fusion points (36). Sequences at the fusion points patient tissues and 7 PTC− tissues analyzed. The horizontal lines represent the of RET/PTC rearrangements often appear as short deletions mean of each patient group. Asterisk representsP < 0.001 as compared to PTC−. or duplications 3(7,38,40). The significant amount of bluntended double-stranded DNA breaks found in normal cells of and doxorubicin can induce significant DNA breakage at fr-ag patients with RET/PTC rearrangements could represent the ile site genes includingRET in the presence of optimal DNA immediate substrates for the NHEJ pathway to form rearran-ge repair, indicating the relevance of DNA fragility induced by these ments. Ghezraoui et  al. (36) demonstrated the formation of chemicals. Further, our results suggest the need for more se-nsi chromosomal translocations following TALEN-induced bre-ak tive tests to screen environmental chemicals, as genes within age (with 5′-overhang ends); however, the frequency of these fragile sites are particularly susceptible to damage. translocations was 3-fold lower than that of Cas9-induced Secondary cancers following chemotherapy and radiation breakage (with blunt ends), which further suggests the imp-or treatment are a known risk4( 8 ) and the ability to better mo-ni tant role of blunt-ended double-stranded DNA breaks in the tor and predict deleterious consequences following treatment of formation of chromosomal translocations. Knockdown of co-m primary cancer is needed. Here, we found that low dose etop-o ponents within the NHEJ pathway decreased the formation side and doxorubicin, at levels 300–1000-fold lower than those of TMPRSS2 fusion transcripts in human prostate cells, which found in the plasma of patients following chemotherapy4( 3,44 ), demonstrates NHEJ as the major repair mechanism required to can cause a significant increase in DNA breakage at genes in generate fusion genes 3( 3 ). fragile sites such asRET and FHIT (Figure 2). The low concentr-a Our study provides important insight into the potential for tions used in our study mimic the residual conditions in which low-dose exposure to environmental chemicals to induce fr-ag cells survive chemotherapeutic treatment but are still exposed ile sites and promote the formation of DNA breaks at genes to these drugs. These results indicate that DNA breakage at involved in cancer specific chromosomal rearrangements. genes within fragile sites can occur in surviving cells, with the Further, it provides a foundation for using DNA fragile site potential to form cancer-causing gene rearrangements found in breakage to predict susceptibility to chromosomal rearran-ge secondary cancers. Therefore, the capability of detecting DNA ments, which would be valuable to monitor high-risk patient breakage at cancer-specific rearrangement-participating gene groups, tailor chemotherapy regimens, and increase the se-n regions will be important for patients about to undergo ch-em sitivity of screening for carcinogenic chemicals. These results otherapy. Identifying those at high risk before administering also further implicate NHEJ as the major repair mechanism chemotherapy would help guide treatment decisions toward required to generate chromosomal translocations founds in those that are less likely to affect DNA breakage. cancers such at PTC. To recapitulate these results in per-iph While diagnostic techniques for detecting cancer formation eral blood mononuclear cells of patients witRhET/PTC-driven have greatly improved, there is still an urgent need for additional tumors compared to healthy individuals would advance these results to the development of a DNA diagnostic test . Paz-Elizur 16 . de Vathaire , F. et  al. ( 1999 ) Second malignant neoplasms after a first et  al. (50) has demonstrated that the activity of DNA repair cancer in childhood: temporal pattern of risk according to type of enzymes such as OGG in PBMCs is a sufficient surrogate for treatment . Br. J. Cancer , 79 , 1884 - 1893 . DNA repair activity in lung tissues. As fragile site breakage is 17 . Kilfoy , B.A. et al. ( 2011 ) Dietary nitrate and nitrite and the risk of- thy the result of the combination of detrimental exposures and roid cancer in the NIH-AARP diet and health study . Int. J. Cancer , 129 , 160 - 172 . insufficient DNA repair processes, this suggests that PBMCs 18 . Kim , M.S. et al. ( 2008 ) Secondary thyroid papillary carcinoma in ost-eo could serve as a sufficient surrogate for the analysis of fragile sarcoma patients: report of two cases . J. Korean Med . Sci., 23 , 149 - 152 . site breakage in a variety of tissues. To expand on these results 19 . Pellegriti , G. et al. ( 2009 ) Papillary thyroid cancer incidence in the- vol and use DNA fragile site breakage as a tool to predict chro-mo canic area of Sicily . J. Natl. Cancer Inst. , 101 , 1575 - 1583 . somal rearrangements in a variety of solid tumors would be 20 . Vane , D. et  al. ( 1984 ) Secondary thyroid neoplasms in pediatric ca-n immensely valuable in advancing the field of clinical oncology cer patients: increased risk with improved survival . J. Pediatr. Surg. , 19 , diagnostics. 855 - 860 . 21. Veiga , L.H. et al. ( 2012 ) Chemotherapy and thyroid cancer risk: a report from the childhood cancer survivor study . Cancer Epidemiol . Biomar-k Supplementary material ers Prev ., 21 , 92 - 101 . Supplementary data are available aCtarcinogenesis online . 22. Venkitaraman , R. et  al. ( 2008 ) Childhood papillary thyroid cancer as second malignancy after successful treatment of rhabdomyosarcoma . Acta Oncol ., 47 , 469 - 472 . Funding 23 . Verneris , M. et al. ( 2001 ) Thyroid carcinoma after successful treatment National Institute of Health (RO1GM101192) and National Cancer of osteosarcoma: a report of three patients . J. Pediatr. Hematol . Oncol., Institute (RO1CA113863) . 23 , 312 - 315 . 24. Ward , M.H. et al. ( 2010 ) Nitrate intake and the risk of thyroid cancer and thyroid disease . Epidemiology , 21 , 389 - 395 . Acknowledgements 25 . Wong , E.Y. et al. ( 2006 ) Reproductive history, occupational exposures, and thyroid cancer risk among women textile workers in Shanghai, We thank Dr. Maggie Ng for providing statistical guidance and China . Int. Arch. Occup. Environ. Health , 79 , 251 - 258 . analysis. In addition, we thank members of the Wang laboratory 26 . Yen , B.C. et  al. ( 1993 ) Multiple hamartoma syndrome with osteos-ar for helpful discussions . coma. Arch. Pathol. Lab. Med ., 117 , 1252 - 1254 . Conflict of Interest Statement: None declared. 27 . Lijinsky , W. ( 1995 ) Metastasizing tumors in rats treated with alkylating carcinogens . Carcinogenesis , 16 , 675 - 681 . References 28 . Dillon , L.W. et al. ( 2013 ) DNA topoisomerases participate in fragility of the oncogene RET . PLoS One , 8 , e75741 . 1. Gasparini , P. et al. ( 2007 ) The role of chromosomal alterations in human 29 . Denison , S.R. et  al. ( 2003 ) How common are common fragile sites in cancer development . J. Cell Biochem ., 102 , 320 - 331 . humans: interindividual variation in the distribution of aphidicolin2 . Gandhi, M. et  al. ( 2010 ) DNA breaks at fragile sites generate on-co induced fragile sites . Cytogenet. Genome Res. , 101 , 8 - 16 . genic RET / PTC rearrangements in human thyroid cells . Oncogene , 29 , 30 . Shrivastav , M. et  al. ( 2008 ) Regulation of DNA double-strand break 2272-2280. repair pathway choice . Cell Res ., 18 , 134 - 147 . 3. Nikiforov , Y.E. et al. ( 2011 ) Molecular genetics and diagnosis of thyroid 31 . Bennardo , N. et al. ( 2008 ) Alternative-NHEJ is a mechanistically distinct cancer . Nat. Rev. Endocrinol. , 7 , 569 - 580 . pathway of mammalian chromosome break repair . PLoS Genet ., 4 , 4 . Fenton , C.L. et  al. ( 2000 ) The ret/PTC mutations are common in sp-o e1000110. radic papillary thyroid carcinoma of children and young adults . J. Clin. 32 . Deriano , L. et al. ( 2013 ) Modernizing the nonhomologous end-joining Endocrinol . Metab., 85 , 1170 - 1175 . repertoire: alternative and classical NHEJ share the stage . Annu. Rev. 5. Davies , L. et  al. ( 2006 ) Increasing incidence of thyroid cancer in the Genet ., 47 , 433 - 455 . United States , 1973 - 2002 . JAMA, 295 , 2164 - 2167 . 33 . Lin , C. et al. ( 2009 ) Nuclear receptor-induced chromosomal proximity 6 . Enewold , L. et al. ( 2009 ) Rising thyroid cancer incidence in the United and DNA breaks underlie specific translocations in cancer . Cell , 139 , States by demographic and tumor characteristics, 1980 - 2005 . Cancer 1069- 1083 . Epidemiol. Biomarkers Prev ., 18 , 784 - 791 . 34. McVey , M. et al. ( 2008 ) MMEJ repair of double-strand breaks (director's 7 . Ron , E. et al. ( 1995 ) Thyroid cancer after exposure to external radiation: cut): deleted sequences and alternative endings . Trends Genet ., 24 , a pooled analysis of seven studies . Radiat. Res. , 141 , 259 - 277 . 529 - 538 . 8. Wartofsky , L. ( 2010 ) Increasing world incidence of thyroid cancer: 35 . Schwer , B. et al. ( 2016 ) Transcription-associated processes cause DNA increased detection or higher radiation exposure? Hormones (Athens), double-strand breaks and translocations in neural stem/progenitor 9 , 103 - 108 . cells. Proc. Natl. Acad. Sci. USA , 113 , 2258 - 2263 . 9. Romei , C. et  al. ( 2016 ) A comprehensive overview of the role of the 36 . Ghezraoui , H. et al. ( 2014 ) Chromosomal translocations in human cells RET proto-oncogene in thyroid carcinoma . Nat. Rev. Endocrinol. , 12 , are generated by canonical nonhomologous end-joining . Mol. Cell , 55 , 192 - 202 . 829 - 842 . 10. Grieco , M. et al. ( 1990 ) PTC is a novel rearranged form of the ret proto- 37. Bongarzone , I. et  al. ( 1997 ) Comparison of the breakpoint regions of oncogene and is frequently detecteidn vivo in human thyroid papillary ELE1 and RET genes involved in the generation of RET/PTC3 oncogene carcinomas . Cell , 60 , 557 - 563 . in sporadic and in radiation-associated papillary thyroid carcinomas . 11. Kohno , T. et al. ( 2012 ) KIF5B-RET fusions in lung adenocarcinoma . Nat. Genomics , 42 , 252 - 259 . Med., 18 , 375 - 377 . 38 . Jhiang , S.M. et  al. ( 1992 ) Detection of the PTC/retTPC oncogene in 12 . Takeuchi, K. et al. ( 2012 ) RET, ROS1 and ALK fusions in lung cancer. Nat. human thyroid cancers . Oncogene , 7 , 1331 - 1337 . Med., 18 , 378 - 381 . 39 . Nikiforov , Y.E. et  al. ( 1999 ) Chromosomal breakpoint positions su-g 13 . Yunis , J.J. et al. ( 1987 ) Fragile sites are targets of diverse mutagens and gest a direct role for radiation in inducing illegitimate recombination carcinogens . Oncogene , 1 , 59 - 69 . between the ELE1 and RET genes in radiation-induced thyroid carci-no 14 . Dillon , L.W. et al. ( 2012 ) The role of fragile sites in sporadic papillary mas . Oncogene , 18 , 6330 - 6334 . thyroid carcinoma . J. Thyroid Res. , 2012 , 927683 . 40. Smanik , P.A. et  al. ( 1995 ) Breakpoint characterization of the ret/PTC 15 . Black , P. et al. ( 1998 ) Secondary thyroid carcinoma after treatment for oncogene in human papillary thyroid carcinoma . Hum. Mol. Genet ., 4 , childhood cancer. Med . Pediatr. Oncol., 31 , 91 - 95 . 2313 - 2318 . 41. Thys , R.G. et  al. ( 2015 ) Environmental and chemotherapeutic agents 45 . Zlotorynski , E. et al. ( 2003 ) Molecular basis for expression of common induce breakage at genes involved in leukemia-causing gene re-ar and rare fragile sites . Mol. Cell Biol ., 23 , 7143 - 7151 . rangements in human hematopoietic stem/progenitor cells . Mutat . 46 . Kelly , L.M. et  al. ( 2014 ) Identification of the transforming STRN-ALK Res ., 779 , 86 - 95 . fusion as a potential therapeutic target in the aggressive forms of-thy 42 . Corbin , S. et al. ( 2002 ) Identification of unstable sequences within the roid cancer . Proc. Natl. Acad. Sci. USA , 111 , 4233 - 4238 . common fragile site at 3p14 . 2: implications for the mechanism of dele- 47. Ameziane-El- Hassani , R. , et al. ( 2010 ) Role of H2O2 in RET/PTC1 chr-o tions within fragile histidine triad gene/common fragile site at 3p14.2 mosomal rearrangement produced by ionizing radiation in human in tumors . Cancer Res. , 62 , 3477 - 3484 . thyroid cells. Cancer Res. , 70 , 4123 - 4132 . 43. Kersting , G. et al. ( 2012 ) Physiologically based pharmacokinetic mod-el 48 . Meadows , A.T. ( 2001 ) Second tumours . Eur. J. Cancer , 37 , 2074 - 2079 . ling of high- and low-dose etoposide: from adults to children . Cancer 49 . Burrow , A.A. et al. ( 2009 ) Over half of breakpoints in gene pairs involved Chemother . Pharmacol., 69 , 397 - 405 . in cancer-specific recurrent translocations are mapped to human 44 . Kontny , N.E. et al. ( 2013 ) Population pharmacokinetics of doxorubicin: chromosomal fragile sites . BMC Genomics , 10 , 59 . establishment of a NONMEM model for adults and children older than 50 . Paz-Elizur , T. et al. ( 2003 ) DNA repair activity for oxidative damage and 3 years . Cancer Chemother. Pharmacol., 71 , 749 - 763 . risk of lung cancer . J. Natl. Cancer Inst. , 95 , 1312 - 1319 .

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Lehman, Christine E., Dillon, Laura W., Nikiforov, Yuri E., Wang, Yuh-Hwa. DNA fragile site breakage as a measure of chemical exposure and predictor of individual susceptibility to form oncogenic rearrangements, Carcinogenesis, 2017, 293-301, DOI: 10.1093/carcin/bgw210