TDP2 suppresses genomic instability induced by androgens in the epithelial cells of prostate glands

450 | Genes Cells. 2020;25:450–465. wileyonlinelibrary.com/journal/gtc Genes to Cells Received: 28 February 2020 | Revised: 30 March 2020 | Accepted: 30 March 2020 DOI: 10.1111/gtc.12770 ORIGINAL ARTICLE TDP2 suppresses genomic instability induced by androgens in the epithelial cells of prostate glands Md. RaselAl Mahmud1 | KenichiroIshii2 | CristinaBernal-Lozano3 | IreneDelgado-Sainz3 | MasakazuToi4 | ShusukeAkamatsu5 | ManabuFukumoto6 | MasatoshiWatanabe2 | ShunichiTakeda1 | FelipeCortés-Ledesma3,7 | HiroyukiSasanuma1 1Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan 2Department of Oncologic Pathology, Mie University Graduate School of Medicine, Tsu, Japan 3Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC–Universidad de Sevilla Universidad Pablo de Olavide, Sevilla, Spain 4Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 5Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan 6RIKEN Center for Advanced Intelligence Project, Tokyo, Japan 7Topology and DNA Breaks Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd. Correspondence Felipe Cortés-Ledesma, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC– Universidad de Sevilla Universidad Pablo de Olavide, Sevilla 41092, Spain. Email: [email protected] Hiroyuki Sasanuma, Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Email: [email protected] Funding information Takeda Medical Research Foundation; Mitsubishi Foundation, Grant/Award Number: ID30123 Communicated by: Akira Shinohara Abstract Androgens stimulate the proliferation of epithelial cells in the prostate by activating topoisomerase 2 (TOP2) and regulating the transcription of target genes. TOP2 resolves the entanglement of genomic DNA by transiently generating double-strand breaks (DSBs), where TOP2 homodimers covalently bind to 5′ DSB ends, called TOP2-DNA cleavage complexes (TOP2ccs). When TOP2 fails to rejoin TOP2ccs generating stalled TOP2ccs, tyrosyl DNA phosphodiesterase-2 (TDP2) removes 5′ TOP2 adducts from stalled TOP2ccs prior to the ligation of the DSBs by nonhomologous end joining (NHEJ), the dominant DSB repair pathway in G0/G1 phases. We previously showed that estrogens frequently generate stalled TOP2ccs in G0/ G1 phases. Here, we show that physiological concentrations of androgens induce several DSBs in individual human prostate cancer cells during G1 phase, and loss of TDP2 causes a five times higher number of androgen-induced chromosome breaks in mitotic chromosome spreads. Intraperitoneally injected androgens induce several DSBs in individual epithelial cells of the prostate in TDP2-deficient mice, even at 20hr postinjection. In conclusion, physiological concentrations of androgens have very strong genotoxicity, most likely by generating stalled TOP2ccs. KEYWORDS androgen, atypical epithelial hyperplasia, DNA double-strand break, prostatic intraepithelial neoplasia, TDP2, topoisomerase 2 | 451 Genes to Cells AL MAHMUD etAL. 1 | INTRODUCTION Sex hormones, estrogens and androgens, strongly stimulate the proliferation of epithelial cells in the mammary glands and prostate, respectively (La Vignera, Condorelli, Russo, Morgia, & Calogero,2016; Liang & Shang,2013). Activated estrogen receptors α/β (ERs) and androgen receptor (AR) quickly induce transcription of ERand AR-responsive genes as transcription factors (Kokontis, Takakura, Hay, & Liao, 1994; Shang, Hu, DiRenzo, Lazar, & Brown, 2000; Wang etal.,2011; Yang etal.,2016). The antagonists against these receptors are widely used as first-line therapies for breast and prostate cancer patients (Horwich et al., 2013; Musgrove & Sutherland,2009). There are two major mechanisms for chemical cancerogenesis, the stimulation of cellular proliferation and the induction of mutagenesis (Loeb & Harris,2008). It has been widely believed that sex hormones enhance oncogenesis through the former mechanism but not the latter (Henderson & Feigelson,2000). However, recent studies have suggested that androgens can drive oncogenesis by activating topoisomerase II (TOP2), which generates DSBs during its physiological catalysis and can generate chromosome translocation (Gómez-Herreros etal.,2017; Haffner etal.,2010; Nelson, Haffner, & Yegnasubramanian,2018). It remains unclear how many breaks are actually generated by the physiological concentration of androgens. Activated ERs and AR trigger signal-dependent early transcriptional responses by recruiting TOP2 to their promoter and enhancer segments (Manville et al., 2015; Pommier, Sun, Huang, & Nitiss,2016). TOP2 has been reported to be involved in RNA polymerase II promoter-pause release upon physiological signals by androgens, insulin, glucocorticoids, N-methyl-d-aspartate (NMDA), retinoic acid, heat shock and serum (reviewed in refs. Austin etal.,2018; Madabhushi,2018). TOP2 forms a homodimer and resolves DNA catenanes by catalyzing the transient formation of gated DSBs, which is followed by the enzymatic rejoining of the broken strands through intrinsic intramolecular ligation activity (Gale & Osheroff,1992; Nitiss,2009). TOP2 becomes covalently bound to the 5′ DNA end of the transiently formed gated DSBs, generating TOP2-DNA cleavage complexes (TOP2ccs). The catalysis by TOP2 occasionally becomes “abortive” and remains unsealed, leading to the generation of stalled TOP2ccs (Gómez-Herreros etal.,2014; Hoa etal.,2016). Thus, the sex hormones enhance carcinogenesis, possibly through an increase in the number of stalled TOP2ccs and generation of mutations. Indeed, we previously showed that physiological concentrations of estrogens frequently generate stalled TOP2ccs in G0/G1 phases (Sasanuma etal.,2018). Stalled TOP2ccs are repaired by the two-step process, the removal of 5′ TOP2 adducts followed by the direct ligation of DSBs by nonhomologous end joining (NHEJ). Tyrosyl DNA phosphodiesterase-2 (TDP2) is the only known enzyme that is capable of accurately removing 5′ TOP2 adducts from stalled TOP2ccs (Ledesma, Khamisy, Zuma, Osborn, & Caldecott,2009; Schellenberg etal., 2017). NHEJ requires preceding removal of 5′ TOP2 adducts for its direct ligation of the DSBs in G0/G1 phase (Gómez-Herreros etal.,2013). The loss of TDP2 causes attenuated transcriptional responses to androgens in prostate cells, suggesting that exposure to androgens may frequently cause the abortive catalysis of TOP2. However, it remains elusive how many stalled TOP2ccs are actually generated by physiological concentrations of androgens. Another unresolved question is the role played by TDP2 in the prevention of prostatic hyperplasia and oncogenesis. The potential causal relationship between the loss of TDP2 and the oncogenesis is suggested by TCGA database, which shows that homozygous deep deletion of the TDP2 gene is seen in 0.4% and 0.8% of the cancers arising in the breast and prostate tissues, respectively, but not in other cancer types (Sasanuma et al., 2018). Considering the fact that TDP2 is ubiquitously expressed in the human tissues (Fagerberg etal.,2014; Yue etal.,2014), an unresolved question is why are defects in TDP2 seen specifically in the tissues where cellular proliferation is stimulated by the sex hormones, androgens as well as estrogens. In this study, we examined the genotoxic effect of androgens on TDP2-deficient human prostate cancer LNCaP cell line and mouse prostate. Physiological concentrations of androgens induced 4.7 and 16 DSBs in individual wild-type and TDP2−/− cells at G0/G1 phase, respectively. This genotoxicity depends on both activated AR and TOP2. These data indicate that androgens have strong genotoxicity, the efficient induction of stalled TOP2ccs in G1 phase. We also demonstrate the strong genotoxicity of androgen in the prostate epithelial cells of TDP2-deficient mice. The loss of TDP2 caused the abnormal proliferation of epithelial cells following three times daily injection of androgens into 2-month-old mice and also resulted in progressive prostate hyperplasia in 2and 6-month-old mice. We propose that TDP2 suppresses abnormal proliferation of the epithelial cells in the prostate gland by promoting the repair of androgen-induced DSBs and ensuring proper transcriptional responses to androgens. 2 | RESULTS 2.1 | Androgens induce DSBs in serumstarved LNCaP cells To investigate the genotoxic effect of androgens on LNCaP human prostate cancer cells, which express functional AR (Horoszewicz et al., 1983), we enriched G1-phase cells more than 90% by serum starvation for 48hr (Figure S1a) and analyzed only cyclin-A-negative, G1-phase cells. We 452 | Genes to Cells AL MAHMUD etAl. pulse-exposed the cells to 1nM of R1881, a synthetic and orally active androgenic steroid (Tran etal., 2009), for two hours and subsequently removed R1881 from the medium (Figure1a). The potency of R1881 at 1nM is comparable to that of 10nM androgens, a physiological serum concentration after puberty (Corsini etal.,2016; Kemppainen etal.,1999). We then analyzed γH2AX foci 2hr after the removal of R1881. Remarkably, R1881-treated LNCaP cells displayed 5.2±0.6 (SD; standard deviation) γH2AX foci per cell and 17-fold induction of γH2AX foci over the baseline (0.3±0.06 (SD)) in G0/G1-phase LNCaP cells (Figure1b,c, Figure S1b), indicating the strong genotoxicity of androgens. 2.2 | Androgen-induced DNA damage is dependent on a functional androgen receptor We next investigated whether functional AR is required for androgen-induced γH2AX-focus formation in serumstarved LNCaP cells. We exposed the LNCaP cells to R1881 together with a clinically relevant concentration of an inhibitor against the androgen receptor, flutamide (Schellhammer etal.,1997). The exposure to the antagonist completely repressed R1881-induced γH2AX-focus formation in serum-starved LNCaP cells (Figure 1d,e, Figure S1c). This observation indicates that the activation of the androgen receptor is required for androgen-dependent DSB formation. 2.3 | Androgen-induced DNA damage depends on both TOP2α and TOP2β We hypothesized that androgen-induced γH2AX foci were caused by TOP2. There are two isoforms of TOP2, TOP2α and TOP2β (Austin et al., 2018; Madabhushi, 2018), which have an overlapping role in transcription (Sasanuma et al., 2018). To explore this hypothesis, we generated TOP2β−/− LNCaP cells (Figure S1d) and depleted TOP2α more than 20-fold using shRNA compared with shControltreated cells (Figure S1e,f). The loss of TOP2β reduced the number of γH2AX foci at 2hr from 5.2±0.6 (SD) to 2.7±0.6 (Figure1g, Figure S1g), suggesting that at least 50% of the androgen-induced γH2AX foci represent stalled TOP2βccs. We previously showed that TOP2α compensates for the lack of TOP2β in the induction of DSBs by estrogens (Sasanuma etal.,2018). We depleted TOP2α by using shRNA and found the resulting TOP2β−/−/shTOP2α cells completely suppressed the induction of γH2AX foci by R1881 during G1 phase (Figure1f,g, Figure S1g). These results indicate that androgen-induced DSBs depend on TOP2α and TOP2β. 2.4 | Loss of TDP2 causes prolonged γH2AX-focus accumulation after a pulse exposure of prostate cancer cells to androgens To investigate the role of TDP2 in the repair of androgeninduced DSBs, we disrupted the TDP2 gene in LNCaP cells (Figure S2a). We pulse-exposed serum-starved cells to 1nM of R1881 for 2hr and subsequently monitored the resolution kinetics of γH2AX foci at 4hr, 12hr and 24hr after the addition of R1881 (Figure2a). The pulse exposure to R1881 induced 5.2±0.6 (SD) γH2AX foci in wild-type cells and 18±2 (SD) γH2AX foci in TDP2−/− cells at 4hr (Figure2b,c, Figure S2b). The number of γH2AX foci reduced to a background level in wild-type cells by 24hr (Figure2b,c, Figure S2b). In marked contrast, TDP2−/− cells showed only a ~20% decrease in the number of γH2AX foci from 4 to 24hr (from 18±2 to 14±2 foci). This result clearly indicates a significant contribution of TDP2 to the repair of androgen-induced DSBs. To assess a functional interaction between TDP2 and NHEJ, we inactivated NHEJ by treating serum-starved TDP2−/− cells with an inhibitor, NU7441 against DNA-PKdependent protein kinase catalytic subunit (DNA-PKcsi), which enzyme is a central component of NHEJ (Blackford & Jackson,2017). The inhibition of NHEJ in wild-type cells increased the number of androgen-induced γH2AX foci from 5.2±0.6 to 21±2 per cell (Figure2b,c, Figure S2b). Wildtype cells treated with DNA-PKcsi displayed virtually no decrease in the number of androgen-induced γH2AX foci from 4 to 24hr after the addition of androgens (Figure2b,c, Figure S2b). The data indicate that NHEJ plays an essential role in the repair of androgen-induced DSBs during the G1 phase. Strikingly, the number of γH2AX foci at 24 hr was very similar between TDP2−/− cells and the DNA-PKcsi-treated cells (14±2 foci in TDP2−/− versus 14±1 foci in DNAPKcsi-treated wild-type in Figure2c). Moreover, the loss of TDP2 did not further increase the number of γH2AX foci in DNA-PKcsi-treated cells (14±2 foci in TDP2−/− versus 16±6 foci in DNA-PKcsi-treated TDP2−/− in Figure2c). Considering the role of TDP2 in the removal of 5′ TOP2 adducts from stalled TOP2ccs, this epistatic relationship between TDP2 and NHEJ indicates that TDP2 contributes to NHEJ-mediated repair of androgen-induced DSBs. In summary, androgens have a very strong genotoxic potential, and collaboration between TDP2 and NHEJ plays a key role in preventing genome instability caused by androgens. To verify that androgen-induced DSBs seen in TDP2−/− cells represent stalled TOP2ccs, we created TDP2−/−/ TOP2β−/−/shTOP2α cells (Figure S2c). The inactivation of both TOP2α and TOP2β completely suppressed androgen-induced DSBs at 4hr in TDP2−/− cells (Figure2d,e). Collectively, TDP2 is required for the repair of TOP2dependent DSBs induced by androgens via NHEJ-mediated | 453 Genes to Cells AL MAHMUD etAL. (a) (b) (d) (f) (g) (c) (e) hr hr hr hr hr hr 454 | Genes to Cells AL MAHMUD etAl. repair in G1 phase. A physiological concentration of androgens generates ~20 DSBs in individual cells at G1 phase, and these DSBs are most likely reflected stalled TOP2ccs. 2.5 | Androgens induce chromosome breaks in mitotic chromosome spreads To confirm the genotoxicity of androgens in cycling cells, we quantified the numbers of aberrant chromosomes in mitotic spreads following 69-hr continuous exposure of cycling cells to 1nM R1881. R1881 treatment increased chromosome aberrations by 3.0-fold (from 2.0±0.4 to 6.0±0.8 foci) in wild-type and 11-fold (from 2.0±1 to 22±2 foci) in TDP2−/− cells, respectively (Figure2f). In conclusion, a physiological concentration of androgens produces a few aberrant chromosomes even in wild-type cells, and TDP2 plays an important role in the faithful repair of androgen-induced chromosome aberrations. 2.6 | Androgens induce prominent γH2AX foci in prostate epithelial cells of TDP2−/− mice To verify the genotoxicity of androgens in vivo, we administrated R1881 by intraperitoneal injection (ip) into wild-type and TDP2−/− mice at 2months of age and monitored the number of γH2AX foci in the prostate, where the expression level of AR is relatively higher than other tissues in both humans and mouse (Figure S3a,b) (El-Alfy etal.,1999; Mirosevich etal.,1999). The selective expression of AR is detected in the prostate epithelial cells, which highly express cytokeratin 8/18 (Ishii, Imanaka-Yoshida, Yoshida, & Sugimura,2008; Wang, Hayward, Cao, Thayer, & Cunha,2001). R1881 injection caused 1.3±1 and 6.3±3 γH2AX foci per cell at 6hr after ip in the prostate epithelial cells (cytokeratin 8/18-positive) of wild-type and TDP2−/− mice, respectively (Figure3a,b, Figure S4a) (Toivanen & Shen,2017). Remarkably, TDP2−/− mice still displayed 7.0±2 γH2AX foci at 12hr after ip, while wild-type mice displayed a background level of foci at 12hr (Figure3a,b, Figure S4a). We also analyzed the spleen, which poorly expresses AR in both humans and mice (Figure S3a,b) (Fagerberg etal.,2014; Yue etal.,2014). As expected, essentially no γH2AX foci were induced by androgens in the spleen of wild-type or TDP2−/− mice (Figure S4b,c). Taken together, androgens are highly genotoxic in both human prostate cancer cells and the epithelial cells of the mouse prostate particularly in the absence of TDP2. We previously reported that intraperitoneal injection of estrogens (17β-estradiol) induces a several times higher numbers of γH2AX foci at 6hr after ip in the epithelial cells of the mammary glands in BRCA1-deficient mice in comparison with wild-type controls (Sasanuma etal.,2018). In this study, we injected estrogens into wild-type and TDP2−/− mice. The number of γH2AX foci in mammary epithelial cells was only slightly higher in TDP2−/− mice than wild-type at 6hr after ip (Figure3c,d). This result indicates that TDP2 does not play a very important role in the repair of estrogen-induced DSBs in the mouse mammary gland. 2.7 | Androgens stimulate the proliferation of prostate epithelial cells in TDP2−/− mice to a greater extent than in wildtype mice Activated AR stimulates cellular proliferation by controlling transcription of AR-target genes (Wang etal.,2009). A recent study indicates the role for TDP2 in ensuring proper transcriptional response to androgens. We here examined the effect of androgens on the proliferation of the epithelial cells in the mouse ventral prostates, which are very sensitive to androgens (Kerr & Searle,1973; Sandford, Searle, & Kerr, 1984; Sugimura, Cunha, & Donjacour, 1986), of FIGURE 1 A synthetic androgenic steroid, R1881-induced DSBs in wild-type LNCaP cells is dependent on both functional AR and TOP2. (a) Schematic diagram of experimental design to examine R1881-induced γH2AX-focus formation in human prostate cancer LNCaP cells. After 48-hr serum starvation, we incubated the wild-type LNCaP cells with 1nM R1881 for 2hr, washed-out R1881, and further incubated the cells without R1881 for 2hr. γH2AX foci were analyzed at 4hr after the addition of R1881 (time 52hr). (b, c) Representative images (b) and median numbers of R1881-induced γH2AX foci (c). The nuclei are outlined. Error bars show standard deviation (SD) of three independent experiments. The number of the counted cells is described in Table S4. A Single asterisk indicates p=2.0×10–4, which was calculated by an unpaired two-tailed t test. The box plots of γH2AX foci in “c” are shown in Figure S1b. Scale bar represents 25μm. (d, e) Representative image (d) and median numbers of R1881-induced γH2AX foci (e). The wild-type LNCaP cells were exposed to R1881 together with flutamide for 2hr (time 48–50hr in “a”), followed by incubation in drug-free media for an additional 2hr. The nuclei are outlined. Error bars show standard deviation (SD) of three independent experiments. The number of the counted cells is described in Table S4. A single asterisk indicates p=6.5×10–6, which was calculated by an unpaired two-tailed t test. The box plots of γH2AX foci in “e” are shown in Figure S1c. Scale bar represents 25μm. (f, g) Representative images (f) and median numbers of R1881-induced γH2AX foci (g) in the indicated genotypes. We analyzed γH2AX foci with the same experimental procedure in “a”. The nuclei are outlined. The generation of the TOP2β−/− mutant cells is depicted in Figure S1d. Error bars show standard deviation (SD) of three independent experiments. The number of the counted cells is described in Table S4. Single, double and triple asterisks indicate p=2.6×10–3, p=5.5×10–2 (no significant difference) and p=5.2×10–1 (no significant difference), respectively, which were calculated by an unpaired two-tailed t test. The box plots of γH2AX foci in “g” are shown in Figure S1g. Scale bar represents 25μm | 455 Genes to Cells AL MAHMUD etAL. (a) (b) (c) (d) (f) (e) 456 | Genes to Cells AL MAHMUD etAl. 2-month-old mice. To this end, we daily injected R1881 for three days and examined the prostate at day 4 (Figure4a). To visualize cycling cells, we injected R1881 together with 5-ethynyl-2'-deoxyuridine (EdU), which is incorporated into newly synthesized DNA during S phase. As expected, substantial numbers of EdU-positive (EdU+) cells were detectable in the epithelial cells of the intestine and splenocytes (Figure S5a) due to the rapid turnover of these cells (CREAMER, Shorter, & Bamforth, 1961; Gelberg, 2007; Kamath etal.,2000). The 3days’ injection of R1881 showed only a subtle but significant increase in the proliferation of the epithelial cells in wild-type mice (from 1.4±0.1 at day 0 to 1.8±0.1 at day 3) (Figure4b,c). In contrast, this injection increased the percentage of EdU+ epithelial cells by three times in TDP2−/− mice (from 2.1±0.3 at day 0 to 5.2±0.2 on day 3). Likewise, the 3days’ injection of R1881 dramatically increased the percentage of the cytokeratin 8/18-positive epithelial cells (Figure S5b) that expressed the proliferating cell nuclear antigen (PCNA), a conventional biomarker for cycling cells, from 3.0±0.3% to 8.8±0.3% in TDP2−/− mice, compared with those of wild-type mice (Figure4d). These results consistently indicate that TDP2 prevents the abnormal proliferation of prostate epithelial cells in response to injected R1881. Considering the role of TDP2 in the repair of stalled TOP2ccs, a defect in their rejoining might change transcriptional responses to androgens and abnormally stimulate the proliferation of the epithelial cells. 2.8 | Development of hyperplasia of prostate epithelial cells in TDP2−/− mice The above data indicated that androgens induced several DSBs in individual prostate epithelial cells and strongly stimulated their proliferation in the absence of TDP2. To evaluate the proliferation rate of the epithelial cells, we stained PCNA in the ventral prostate, comparing between 2-month and 6-month-old mice (Figure 5a). The percentage of PCNA+ epithelial cells was 2.5±0.1% and 3.0±0.3% in wild-type and TDP2−/− mice at the age of 2months, respectively. The percentage did not increase in wild-type mice (2.8±0.3%), while the percentage increased to 11±5% in TDP2−/− mice at the age of 6months. One possible scenario is that the accumulation of mutations with aging in the absence of TDP2 might increase the sensitivity of the prostate epithelial cells to androgens leading to their enhanced proliferation (Figure5b). We next investigated the consequence of the enhanced proliferation, the hyperplasia of epithelial cells in the prostate. We analyzed the ventral prostate of wild-type and TDP2−/− mice at 2months and 6months of age. We counted the number of epithelial cells along the fixed distance of the lumen surface in 10 ducts of each mouse. Wild-type mice did not exhibit morphological abnormality of ventral prostate (Figure5c). In contrast, TDP2−/− mice at both 2 and 6months of age exhibited hyperplasia in epithelial cells of ventral prostate. The number of epithelial cells was twofold higher in 2-month-old TDP2−/− mice compared with 2-month-old wild-type mice (Figure5c,d). The hyperplasia of the ventral prostate was twofold more frequently observed in 6-monthold TDP2−/− mice than 2-month-old ones. Epithelial layers were folded as a consequence of the increase in the number of epithelial cells. Interestingly, 6-month-old TDP2−/− mice exhibited the accumulation of epithelial cells having a disordered, multilayered organization in more than 75% of the examined ducts. Nonetheless, we did not detect malignant tumors. Collectively, TDP2 prevents the abnormal proliferation of epithelial cells in the ventral prostate. 3 | DISCUSSION We here reveal previously unappreciated very strong genotoxicity of a physiological concentration of androgens equivalent to a serum concentration in males after puberty. A 2-hr pulse exposure to androgens induced approximately five TOP2-dependent DSBs per cell in the G1 phase in an FIGURE 2 TDP2 functions in the repair of R1881-induced DSBs in LNCaP cells. (a) Schematic diagram of experimental design to examine the repair kinetics of R1881-induced γH2AX foci in LNCaP cells. R1881 and DNA-PKcsi (NU7441) were simultaneously added into the medium at time 48hr. R1881 was only removed from the medium at time 50hr, but DNA-PKcsi exposure was continued until cells were harvested to inactivate NHEJ. γH2AX foci were analyzed at the indicated time (0, +4, +12 and +24hr after addition of R1881). (b, c) Representative images (b) and median numbers of R1881-induced γH2AX foci (c) in the indicated genotypes. The generation of TDP2−/− mutant cells is depicted in Figure S2a. The nuclei are outlined. Error bars show standard deviation (SD) of three independent experiments. The number of the counted cells is described in Table S4. Single, double, triple and quadruple asterisks indicate p=1.2×10–4, p=3.3×10–5, p=5.7×10–1 (no significant difference) and p=6.4×10–1 (no significant difference), respectively, which were calculated by an unpaired two-tailed t test. The box plots of γH2AX foci are shown in Figure S2b. Scale bar represents 25μm. (d, e) Representative images (d) and median numbers of R1881-induced γH2AX foci (e) in the indicated genotypes. Error bars show standard deviation (SD) of three independent experiments. The nuclei are outlined. The number of the counted cells is described in Table S4. Single and double asterisks indicate p=3.4×10–4 and p=7.0×10–3, respectively, which were calculated by an unpaired two-tailed t test. Scale bar represents 25μm. (f) R1881-induced chromosome aberrations in mitotic chromosome spread. Following 48-hr incubation with media containing charcoal-filtrated serum, cells were further incubated in media containing charcoal-filtrated serum in the absence (“–”) or presence (“+”) of R1881 (1nM) for 72hr. Error bars are standard deviation (SD) of three independent analyses. The number of the counted cells is described in Table S4. A Single asterisk indicates p=3.1×10–3, which were calculated by an unpaired two-tailed t test | 457 Genes to Cells AL MAHMUD etAL. FIGURE 3 Genotoxicity of R1881 and 17β-estradiol in the prostate and mammary tissues, respectively, of TDP2−/− mice. (a, b) Representative images (a) and median numbers of γH2AX-focus-positive (b) cells in luminal epithelial cells of the prostate at the indicated time after ip with R1881. We killed three 2-month-old mice of each genotype and counted only luminal cells stained with cytokeratin-8/18 (CK-8/18), a marker of epithelial cells. The box plot of R1881-induced γH2AX foci is shown in Figure S4a. The number of the counted cells is described in Table S4. Single and double asterisks indicate p=4.9×10–2 and p=1.3×10–2, which were calculated by an unpaired two-tailed t test. Scale bar represents 25μm. (c, d) Representative images (c) and the average number of γH2AX foci (d) cells in the epithelial cells of the mammary gland at the indicated time after ip with 17β-estradiol (E2). We subtracted the average number of foci in E2-treated epithelial cells at 6hr from the average number of foci in E2-untreated epithelial cells. Actual numbers of foci per cell are shown in Figure S4d. The data of wild-type #1, 53BP1−/− and 53BP1−/−/BRCA1−/− mice were re-calculated as the number of γH2AX foci per cell from the data that has been previously published (Sasanuma etal.,2018). We counted only cells stained with cytokeratin-8/18 (CK-8/18). We killed three mice each carrying either the wild-type #2 or TDP2−/− genotype at two months of age and analyzed more than 100 epithelial cells for each mouse. The number of the counted cells is described in Table S4. Single, double and triple asterisks indicate p=1.0×10–3, p=1.1×10–2 and p=2.0×10–1 (no significant difference), respectively, which were calculated by an unpaired two-tailed t test. Scale bar represents 25μm (a) (b) (c) (d) (hr) (hr) (hr) h r 458 | Genes to Cells AL MAHMUD etAl. AR-receptor-dependent manner. The loss of either NHEJ or TDP2 caused an increase in androgen-induced DSBs to ~20 per cell. Strikingly, this loss resulted in 14 to 16 DSBs left unrepaired even at 24hr after a 2-hr pulse exposure to androgens, which is in marked contrast with the complete rejoining of androgen-induced DSBs in wild-type cells at 24hr (Figure2c). A few androgen-induced DSBs were left unrepaired in individual prostate epithelial cells of TDP2deficient mice even at 20hr after the injection of androgen when androgen-induced DSBs were completely repaired in wild-type mice (Figure 3b). These data highlight the key role in the TDP2-NHEJ axis (Gómez-Herreros etal.,2013) in protecting prostate epithelial cells from androgen-induced genome instability. The genotoxicity of androgens as well as estrogens can be mediated by the following two mechanisms. A physiological concentration (1 nM) of 17β-estradiol generates DSBs in wild-type MCF-7 breast cancer cells by generating stable RNA:DNA hybrid structures known as R-loops and their collision with DNA replication forks causes DSBs (Stork etal., 2016). Hence, androgens can generate DSBs through the collision between R-loops and DNA replication forks. Since R-loops are not a serious threat to genome instability in G0/G1 phases (reviewed in ref. Aguilera & GarcíaMuse,2012), androgen-induced DSBs in G0/G1 phases are generated by a mechanism other than R-loops. Recent studies have shown the second mechanism for androgen-induced DSBs, TOP2-dependent DSBs (Haffner et al., 2010) (reviewed in refs. Madabhushi,2018; Nelson etal.,2018). We here showed TDP2 plays a vital role in the efficient repair of androgen-induced DSBs (Figures2e and 3b), suggesting that they include 5′ TOP2 adducts at their ends. Indeed, FIGURE 4 R1881 facilitates the cellular proliferation in the prostate gland of TDP2−/− mice. (a) Schematic diagram of experimental design for a daily injection of R1881 in 2-month-old mice. (b, c) R1881-induced cellular proliferation by EdU incorporation in the mouse prostate. Representative images (b) of the EdU-positive (EdU+) cells and the average numbers of R1881-induced EdU+ (c) luminal epithelial (CK-8/18positive) cells in the prostate gland of mice. The number of the counted cells is described in Table S4. EdU (30mg/kg body weight) and R1881 (15mg/kg body weight) together were intraperitoneally (ip) injected into the mice (days 0, 1 and 2 in “a”). The indicated tissues were isolated 24hr after the last ip (day 3 in “a”). Small intestine and spleen were analyzed as positive controls in Figure S5a. Single and double asterisks indicate p=2.9×10–3 and p=5.3×10–5, respectively, which were calculated by an unpaired two-tailed t test. Scale bar represents 75μm. (d) R1881induced cellular proliferation by PCNA immunostaining in the mouse prostate. The graph shows the average numbers of R1881-induced PCNApositive (PCNA+) cells. We analyzed PCNA+ cells with the same experimental procedure in “a”. The number of the counted cells is described in Table S4 Single and double asterisks indicate p=3.1×10–2 and p=1.1×10–5, respectively, which were calculated by an unpaired two-tailed t test. Representative images of PCNA+ cells in the prostate are shown in Figures S5b (a) (c) (d) (b) | 465 Genes to Cells AL MAHMUD etAL. Yang, S., Jiang, M., Grabowska, M. M., Li, J., Connelly, Z. M., Zhang, J., … Yu, X. (2016). Androgen receptor differentially regulates the proliferation of prostatic epithelial cells in vitro and in vivo. Oncotarget, 7, 70404–70419. Yue, F., Cheng, Y., Breschi, A., Vierstra, J., Wu, W., Ryba, T., … Ren, B. (2014). A comparative encyclopedia of DNA elements in the mouse genome. Nature, 515, 355–364. https://doi.org/10.1038/natur e13992 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Al Mahmud MR, Ishii K, Bernal-Lozano C, et al. TDP2 suppresses genomic instability induced by androgens in the epithelial cells of prostate glands. Genes Cells. 2020;25:450–465. https:// doi.org/10.1111/gtc.12770