Transcription-Induced DNA Double Strand Breaks: Both Oncogenic Force and Potential Therapeutic Target?

Published OnlineFirst March 8, 2011; DOI: 10.1158/1078-0432.CCR-10-2044

Clinical Cancer Molecular Pathways Research

Michael C. Haffner, Angelo M. De Marzo, Alan K. Meeker, William G. Nelson, and Srinivasan Yegnasubramanian

Abstract An emerging model of transcriptional activation suggests that induction of transcriptional programs, for instance by stimulating prostate or breast cells with androgens or estrogens, respectively, involves the formation of DNA damage, including DNA double strand breaks (DSB), recruitment of DSB repair proteins, and movement of newly activated genes to transcription hubs. The DSB can be mediated by the class II topoisomerase TOP2B, which is recruited with the androgen receptor and estrogen receptor to regulatory sites on target genes and is apparently required for efficient transcriptional activation of these genes. These DSBs are recognized by the DNA repair machinery triggering the recruitment of repair proteins such as poly(ADP-ribose) polymerase 1 (PARP1), ATM, and DNA-dependent protein kinase (DNA-PK). If illegitimately repaired, such DSBs can seed the formation of genomic rearrangements like the TMPRSS2- ERG fusion oncogene in prostate cancer. Here, we hypothesize that these transcription-induced, TOP2B- mediated DSBs can also be exploited therapeutically and propose that, in hormone-dependent tumors like breast and prostate cancers, a hormone-cycling therapy, in combination with topoisomerase II poisons or inhibitors of the DNA repair components PARP1 and DNA-PK, could overwhelm cancer cells with transcription-associated DSBs. Such strategies may find particular utility in cancers, like prostate cancer, which show low proliferation rates, in which other chemotherapeutic strategies that target rapidly proliferating cells have had limited success. Clin Cancer Res; 17(12); 1–7. 2011 AACR.

Background damage and repair (5–12). For instance, unbiased proteo- mics approaches as well as candidate protein analyses have The prevailing reductionist view of transcription has shown that proteins such as Ku70, Ku80, poly(ADP-ribose) been challenged by mounting recent evidence suggesting polymerase 1 (PARP1), DNA-dependent protein kinase that induction of transcription involves profound and (DNA-PK), and class II topoisomerase (TOP2B) can be dynamic changes in genome organization and structure. associated with transcription factors including androgen In this emerging view, hundreds of transcription factors receptor (AR) and upstream stimulating factor 1 and can and cofactors are present at locally high concentrations in bind to regulatory elements of target genes upon activation "transcription factories" in the nucleus (1). Induction of of transcription (9, 12). Inhibition of the catalytic function widespread transcriptional changes, for example during of several of these proteins results in an impaired induction hormonal stimulation of hormone-responsive cells, can of transcriptional programs, suggesting a crucial role for result in large-scale alterations in genome organization DNA repair in transcriptional regulation. Among these, arising from movement of different target gene loci and TOP2B is especially interesting because it has been shown distal regulatory elements to these transcriptional hubs, by numerous recent reports to be associated with multiple establishing cell type–specific expression patterns (1–4). nuclear hormone receptors and transcription factors and In addition, recent reports have shown that efficient has also been shown to be bound to promoters and induction of transcriptional programs by a wide array of enhancers of newly transcribed genes (5, 6, 11, 12). Inter- nuclear hormone receptors and other transcription factors estingly, TOP2B catalytic activity appears to be required for involves the recruitment of several factors involved in DNA efficient transcriptional initiation and may be involved in the establishment of complex chromosomal conforma- tional changes during activation of transcription (5, 6, Authors' Affiliation: Sidney Kimmel Comprehensive Cancer Center, 12, 13). Johns Hopkins University School of Medicine, Baltimore, Maryland The two human class II topoisomerases, TOP2A and Corresponding Author: Srinivasan Yegnasubramanian, Johns Hopkins University School of Medicine, 1650 Orleans Street, CRB2, Rm 145, TOP2B, can resolve DNA topologic constraints by passing Baltimore, MD 21231. Phone: 410-502-3425; Fax: 410-502-9817; E-mail: one DNA segment through a transient DNA double strand [email protected] break (DSB) in another segment, in an ATP-dependent doi: 10.1158/1078-0432.CCR-10-2044 fashion (14). In addition to being able to resolve super- 2011 American Association for Cancer Research. helical tension introduced by under- or overwinding of the

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Haffner et al

DHT DNA double strand break

DHT AR TOP2B AR

DHT

TOP2B DHT

AR POLII Ku70 Ku80 DNA-PK

PARP1 ATM Other Repair Proteins?? ROS Fork collision Dietary carcinogens Repair, movement to Repair infidelity transcription hubs, and active transcription Persistent DSBs Senescence Illegitimate repair Apoptosis Formation of structural rearrangements Genomic instability

Figure 1. An emerging model suggests that efficient induction of transcriptional programs can involve the generation of DSBs and perhaps other DNA damage, repair of these breaks via recruitment of DNA repair proteins, and large-scale movement of genes and regulatory elements to transcriptional hubs, in which transcription (co)factors are present at high local concentration. These events are illustrated for induction of transcriptional programs by AR. Upon transcriptional activation, AR associates with TOP2B and binds to AR target sites. DNA repair proteins including Ku70, Ku80, PARP1, ATM, DNA-PK, and possibly other DNA repair enzymes also get recruited to these sites. TOP2B can mediate DSBs in associated DNA. Reactive oxygen species (ROS), perhaps generated during AR signaling, dietary carcinogens, and collision with the replication or transcription forks can transform these transient breaks into frank DSBs. Persistence of these breaks can result in induction of cellular senescence and apoptosis. Illegitimate repair of these breaks can lead to the formation of structural rearrangements. Such pathways are likely at work for other induced transcriptional programs, including those mediated by ER, RAR, and AP1.

DNA double helix, the class II topoisomerases are uniquely that TOP2B may be involved in resolving topologic con- able to resolve higher order topologic constraints, such as straints arising from movement of chromosomal segments tangles and knots. TOP2A and TOP2B are encoded by during transcriptional induction. Such a role for TOP2B in separate genes, and although they show a high degree of transcriptional initiation would be analogous to the sequence homology, they differ significantly in their expres- requirement of TOP2A in resolving topologic constraints sion patterns and cellular functions (14, 15). Whereas associated with DNA replication and chromosomal segre- TOP2A is almost exclusively expressed in proliferating cells, gation. where it is required for resolution of topologic constraints Using an elegant assay that allows site-specific labeling of arising during replication and chromosomal segregation, DSBs, Ju and colleagues showed that, during transcriptional TOP2B can be expressed throughout the cell cycle in divid- activation by the estrogen receptor (ER), transient DSBs are ing and nondividing cells (14, 16, 17). Given the recent generated at regulatory elements of ER-regulated genes (6). findings of its association with transcription factors and its This finding was further independently corroborated for catalytic activity at gene regulatory sites, we can speculate other signaling pathways including the AR (Fig. 1; refs. 5,

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Targeting Transcription-Induced DSBs for Cancer Therapy

6, 12). Induction of these transient DSBs, tracked with the Regardless of their role in transcriptional regulation, it is recruitment of TOP2B to these sites and targeted depletion of clear that the generation of DNA damage, particularly the TOP2B, dramatically reduced DSB generation, suggesting generation of DSBs, during induction of transcriptional that these transcription-associated DSBs were mediated by programs, may represent a critical vulnerability for cells. TOP2B. Interestingly, these DSBs were recognized by the DSB DSBs play a major role in carcinogenesis: On one hand, repair machinery. The DSB recognition proteins Ku70 and unresolved DSBs can lead to cell cycle arrest, senescence, Ku80, for instance, have been shown to bind together with and apoptosis; and on the other hand, if illegitimately nuclear hormone receptors at target sites (6, 8–10, 12). repaired, such transcription-induced DSBs can seed the Furthermore, the DSB repair–associated protein PARP1 formation of genomic rearrangements, amplifications, and the kinases DNA-PK and ATM were also found to be and deletions (Fig. 1; refs. 30–32). associated with newly transcribed genes in different model Structural genomic alterations can be found in the systems (Fig. 1; refs. 5, 6, 9, 12). It is likely that other DNA majority of tumors, and certain genomic rearrangements repair proteins are also associated with transcriptional com- are pathognomonic for specific malignancies (32–34). For plexes. Future efforts to comprehensively characterize the instance, in the case of prostate cancer, androgen-regulated repair proteins and pathways involved in transcriptional genes are frequently fused to oncogenic transcription fac- initiation are critically needed. tors of the ETS family. The most prominent of these According to the conventional view, the transient DSBs prostate cancer–specific recurrent rearrangements involves induced by TOP2 enzymes during their catalytic cycle are the androgen-regulated gene TMPRSS2 and the ETS family extremely fleeting and are not easily isolatable, unless the member ERG, occurring in >50% of prostate cancer cases TOP2-DNA cleavage complex is stabilized by TOP2 poi- (35, 36). These rearrangements bring ETS transcription sons such as etoposide (18, 19). The TOP2 transient DSB is factors under the transcriptional control of the AR, result- thought to be masked, evading recognition, recruitment, ing in an androgen-driven and prostate cancer–specific and activity of the DSB repair machinery. However, in these overexpression of an ETS fusion oncogene. The nonran- recent reports, the TOP2B-mediated DSBs associated with dom and often lineage-specific nature of recurrent rearran- transcriptional induction were persistent, on the order of gements suggests a controlled underlying mechanism one to several hours, and were apparently recognized by involved in their formation (5, 7, 37). the DSB repair machinery (5, 6, 20). The exact biochemical For such a rearrangement to occur, the DNA loci mechanisms by which transient DSBs that are induced involved would likely need to (i) be subject to DSB for- during the catalytic cycle of TOP2B can be converted into mation, (ii) at least transiently be in close spatial proximity, frank and persistent breaks is not well understood. How- and (iii) become subject to illegitimate repair abnormally ever, several potential hypotheses can be generated on the joining the two gene loci together. As described above, the basis of accumulating evidence. Reactive oxygen species induction of transcriptional programs could facilitate each (ROS), such as those generated by intrinsic nuclear hor- of these steps, and therefore stimulation of transcriptional mone signaling (11, 21) or other transcription factor processes could be involved in generating these structural signaling or by inflammatory cells often seen associated rearrangements (5, 7, 38). For instance, stimulation of AR with cancer lesions in vivo (22), can covalently trap TOP2 signaling in prostate cells by dihydrotestosterone (DHT) enzymes on DNA, stabilizing the TOP2-DNA cleavable can cause the TMPRSS2 and ERG gene loci to come in close complex (23). Furthermore, several dietary constituents, proximity (7, 37), perhaps as a part of larger-scale genome especially bioflavonoids, have been shown to mimic TOP2 reorganization to facilitate movement of induced genes poisons in stabilizing the TOP2-cleavage complex (14, 24). (and possibly bystanders such as ERG) to transcriptional Collision of these DNA-TOP2 complexes with the replica- hubs (1, 2, 4). Concurrently, induction of androgen signal- tion fork or with transcription complexes (25, 26), fol- ing induces DSBs at precise TMPRSS2-ERG rearrangement lowed by proteasomal degradation of TOP2B (27), can junction sites. These DSBs at the TMPRSS2 and ERG genes result in the conversion of the covalent TOP2B-DNA clea- can be mediated by TOP2B as a consequence of DHT- vage complex into frank DSBs (Fig. 1). induced AR signaling in prostate cells or by other nuclease More generally, it is becoming evident that efficient activities, which can be induced by severe exogenous gen- induction of transcriptional programs may involve, and otoxic insults applied during DHT stimulation of prostate perhaps require, generation of many forms of DNA cells (5, 7). Broken DNA ends can become illegitimately damage, including DNA cytosine deamination (28, 29), repaired by DSB repair machinery, including the error- base damage by local ROS produced by hormone stimula- prone nonhomologous end-joining pathway, to create de tion (11), LINE1-ORF2 endonuclease activity (7), and novo TMPRSS2-ERG gene fusions (5, 7). Therefore, at least TOP2B-mediated DSB (5, 6), at transcriptional regulatory in the case of recurrent rearrangements in prostate cancer, sites. These DNA damage events apparently recruit the dysregulation of events that would normally occur during appropriate classes of DNA repair proteins, including those induction of transcriptional programs might predispose involved in base-excision repair (11, 28, 29), and nonho- prostate cells to develop prostate cancer–specific recurrent mologous end-joining (6, 7, 12). The precise role of DNA gene fusions involving AR target genes. Whether dysregula- damage and DNA repair proteins in transcriptional regulation of transcriptional mechanisms may be involved in tion is largely unknown. creating lineage-specific recurrent rearrangements in other

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cancers is still unknown. This hypothesis is certainly worth of genomic deletions, amplifications, and rearrangements investigating further given the recent findings showing a observed in many cancers. general involvement of DNA damage and repair in lineage- specific transcriptional programs induced by ER, RAR, AP1, Clinical–Translational Advances and insulin (6, 11, 12). The development of these transcription-associated DSBs Generation of DSBs is one of the major mechanisms of may be exacerbated in the setting of actively replicating action for cancer chemotherapeutic drugs. Most of the neoplastic cells compared with terminally differentiating existing DSB-inducing chemotherapeutics in clinical use normal cells. To illustrate this potentially generalizable require cell division and, therefore, only efficiently target principle, we can review the recent evidence in prostate highly proliferative cells (44). Furthermore, breaks are not neoplasia. It is interesting that the prostate cancer only induced specifically in tumor cells of the target tissue TMPRSS2-ERG gene rearrangements seem to appear first, but also nonspecifically at other organ systems, resulting in often subclonally, in luminal cells of premalignant lesions many treatment-associated side effects. called prostatic intraepithelial neoplasia (PIN) and are The above-described finding that DSBs can be gener- apparently selected for during subsequent progression to ated during transcription opens several new avenues for invasive carcinoma (5, 39). Given that they are almost developing more targeted therapeutic approaches, which never seen in normal prostate cells (5, 39), we can raise the will be illustrated here using breast and prostate cancer as question of whether any special factors create a predilection primary examples. Breast and prostate carcinoma are both for PIN cells and not normal prostate cells to initially typically hormone-dependent neoplasias and, for the develop these rearrangements. PIN luminal cells may har- most part, respond well to initial hormone ablative bor somewhat dysregulated androgen signaling, which is therapies (45). Unfortunately, a majority of tumors even- known to promote both caricaturized differentiation path- tually escape hormone ablation, resulting in clinical ways as well as proliferation and/or survival pathways in relapse of the disease. It is, however, interesting that neoplastic cells (40, 41). The proliferating cells in PIN the resistant cancer cells can still express the AR and/or lesions likely need to reestablish AR transcriptional pro- ER, suggesting that these cells still depend on AR and ER grams, involving DNA damage, chromosomal reorganiza- signaling (45). The hormone dependence of most pri- tion, and DNA repair, after every cell division, increasing mary tumors can be recapitulated in many cell line the chances that these cells generate gene rearrangements models of breast and prostate cancer, which require the that are then subject to selection. In contrast, androgen presence of estrogen or androgen for proliferation. Long- signaling in normal cells induces terminal differentiation term culture of these cell lines under hormone-depleted with suppression of proliferation, so that de novo transcrip- conditions results in the developmentofclonesthatare tional programs only need to be established once, thus resistant to hormone deprivation, which can grow in the limiting the chance of creating transcription-related gen- absence of estrogen or androgens. ome damage. In this regard, in human prostate tissues, AR Paradoxically, such preclinical models of castrate-resis- and TOP2B, which were rarely both present in normal tant prostate cancer cells respond to low-dose androgen prostate cells at high levels, often showed robust coexpres- treatment with significant growth inhibition (46). The sion in PIN luminal cells (5), possibly because of an clinical relevance of this finding is supported by anecdo- ongoing requirement for TOP2B in reestablishing AR tran- tal case reports in the literature indicating that substantial scriptional programs after cell division in PIN, but not in and prolonged responses can be achieved by testosterone normal cells. This coexpression of AR and TOP2B in the replacement therapy in patients with castrate-resistant PIN cells may predispose these cells to formation of tran- disease (47–49). Similarly, hormone-deprived breast can- scription-related TOP2B-mediated DSBs and formation of cer cells can respond to estrogen with growth cessation rearrangements. Of course, because they can lose check- and apoptosis, and high-dose diethylstilbestrol can delay point pathways that would normally induce senescence or disease progression in patients with metastatic breast cell death in response to persistent genome insults, neo- cancer (50, 51). plastic cells are also more prone than are normal cells to Although the precise mechanisms behind this paradox- surviving and proliferating despite persistent genome ical antitumor response of hormone-ablated breast and damage. prostate cancers to hormone replacement therapy are Because such processes are likely ongoing in cancer cells, unclear, we can formulate some hypotheses on the basis the mechanisms described here may be more generally of the recent findings about AR- and ER-induced tran- involved in generating an ongoing TOP2B or transcrip- scriptional programs. Long-term hormone withdrawal tion-induced genomic instability (TIN). This transcription- from hormone ablative therapy of prostate and breast associated TIN model would be somewhat distinct and cancers may reset transcriptional programs that are complementary to other forms of genomic instability that mediated by nuclear hormone receptors. Upon hormone are typically thought to be connected to errors in DNA stimulation, these programs may become reinitiated, a replication (31, 42, 43). Indeed, a combination of both TIN process that could result in the induction of TOP2B- and replication-related genome damage may create a "per- mediated DSBs (5, 6), as well as DNA damage from other fect storm" of genomic instability, leading to the high rate pathways (7, 12). To exploit this process, we would

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propose a cyclic administration of either androgens or somerase bond that is generated during the TOP2 cleavage estrogens in patients who have undergone hormone process and, thereby, restores 50-phosphate termini at ablative therapies for prostate and breast cancer, respec- TOP2 cleavage sites, allowing subsequent repair of tively. In such a hormone-cycling protocol for breast TOP2-mediated DSBs (60). Loss of TDP2 renders cells cancer, for instance, patients undergoing aromatase inhi- highly sensitive to the cytotoxic effects of TOP2 poisons. bition therapy would be treated with short intermittent Therefore, inhibition of TDP2 could synergize with hor- doses of estrogen. In the case of prostate cancer, phar- mone-cycling strategies in inducing apoptosis owing to macologic castration using a gonadotropin-releasing overwhelming TOP2B-induced DSBs (61). hormone agonist or a CYP17 antagonist could be com- Given the evidence that activation of transcriptional bined with cyclic administration of androgens. It is worth programs involves TOP2B-mediated DSBs, a combination noting that a recent clinical trial of rapid androgen cycling of a topoisomerase-targeting drug together with hormone- in men with prostate cancer established the feasibility of cycling therapy may be particularly effective. Class II topoi- such an approach (52). We hypothesize that during such somerases have been at the center of oncology drug devel- a hormone-cycling regime, DSBs would be specifically opment for a long time; topoisomerase II–targeting drugs generated in the prostate and breast or other androgen have been successfully used clinically in a variety of cancers and estrogen target tissues, respectively. (62). TOP2 poisons, such as etoposide, have long been Repair of these transcription-induced DNA lesions thought to be effective only in highly proliferative tumors, seems, at least partly, to depend on the activity of well- because only tissues with a high proliferation fraction characterized repair enzymes like PARP1 and DNA-PK (5, would express high levels of TOP2A (14, 62). The new 6, 12). This opens up exciting therapeutic opportunities in insights into the role of TOP2B in transcriptional regula- which these repair proteins can be targeted by inhibitors tion, however, suggest that TOP2-targeted drugs may be that are already in clinical development in order to over- effective even in cancers showing low proliferation frac- whelm the cell with DSBs that are specifically induced by tions if transcriptional programs can be cyclically induced. reactivating nuclear hormone signaling. An ongoing clinical trial (ClinicalTrials.gov identifier PARP1 participates in DNA damage repair and has been NCT01084759) is currently testing the safety and efficacy shown to be specifically recruited to sites of active tran- of testosterone cycling in combination with oral etoposide scription(Fig.1;refs.6,9,12).Geneticaswellasphar- in prostate cancer patients with rising prostate-specific macologic experiments crippling PARP1 function have antigen undergoing androgen ablative therapy (63). provided strong evidence for the important role of PARP1 in the repair of DSBs (53). PARP inhibitors are currently Concluding Remarks being tested in clinical trials in combination with a variety of genotoxic chemotherapeutics but also show great pro- As shown by recent reports, efficient activation of tran- mise as single agents in tumors carrying BRCA1 and scriptional programs in response to stimuli, such as hor- BRCA2 mutations (53–55). Novel preclinical findings mone exposure, involves the generation of DNA damage showing that PTEN-deficient cells are especially sensitive and/or DSBs, recruitment of DNA repair proteins, and to PARP1 inhibition raised further interest in other syn- large-scale genome reorganization to allow movement of thetic lethal combination therapies (56). A variety of activated genes and regulatory loci to transcriptional hubs. highly potent PARP inhibitors are currently under clinical The DSBs, if illegitimately repaired, can seed formation of investigation and show favorable side effect profiles (53). genomic rearrangements and perhaps contribute to A combination therapy of hormone-cycling with PARP1 ongoing genomic instability. Although these transcrip- inhibition could be advantageous in patients who have tion-related DSBs pose a risk for generation of oncogenic failed conventional hormone ablative therapies and genomic rearrangements and perhaps ongoing genomic could be of particular relevance in tumors that show instability, we may be able to take advantage of these genetic alterations that induce synthetic lethality in com- processes to target certain cancers. For instance, for breast bination with PARP inhibition. and prostate cancer, it may be possible to use a hormone- DNA-PK plays a critical role in the cellular response to cycling strategy to induce these DSBs repetitively and DSBs because it associates with them and initiates repair perhaps couple such a strategy with pharmacologic agents, (57). Inhibition of DNA-PKs has been shown to sensitize such as TOP2 poisons or PARP and/or DNA-PK inhibitors, cancer cells to chemotherapy- and radiation-induced in order to overwhelm the cancer cell with breaks, ulti- DSBs (57, 58). Although clinical use of most of the mately leading to cell death. Such a strategy would be existing DNA-PK inhibitors (59) has been limited by analogous to the largely successful approach of using general safety and specificity concerns (57), new com- TOP1 and TOP2 poisons to overwhelm cancer cells pounds with more favorable risk profiles could be used in with replication-associated DSBs mediated by TOP1 and future trials testing such inhibitors in the context of TOP2A. hormone cycling. A pharmacologically unexplored, but potentially inter- Disclosure of Potential Conflicts of Interest esting target is tyrosyl-DNA phosphodiesterase (TDP2/ 0 TTRAP). TDP2 cleaves the 5 phosphotyrosyl-DNA topoi- No potential conflicts of interest were disclosed.

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Grant Support 0049), the Prostate Cancer Foundation, and the Patrick C. Walsh Prostate Cancer Research Fund/Dr. and Mrs. Peter S. Bing Scholarship (to S. Yegnasubramanian). This work was supported in part by funding from the NIH/National Cancer Institute (CA58236), the Department of Defense Congressionally Directed Medical Received December 23, 2010; revised February 17, 2011; accepted Research Program's Prostate Cancer Research Program (PC073533/W81XH-08–1- February 17, 2011; published OnlineFirst March 8, 2011.

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www.aacrjournals.org Clin Cancer Res; 17(12) June 15, 2011 7

Transcription-Induced DNA Double Strand Breaks: Both Oncogenic Force and Potential Therapeutic Target?

Michael C. Haffner, Angelo M. De Marzo, Alan K. Meeker, et al.

Clin Cancer Res Published OnlineFirst March 8, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-2044

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