The Natural Inhibitor of DNA Topoisomerase I, Camptothecin, Modulates HIF-1A Activity by Changing Mir Expression Patterns in Human Cancer Cells

Published OnlineFirst November 19, 2013; DOI: 10.1158/1535-7163.MCT-13-0729

Molecular Cancer Cancer Biology and Signal Transduction Therapeutics

The Natural Inhibitor of DNA Topoisomerase I, Camptothecin, Modulates HIF-1a Activity by Changing miR Expression Patterns in Human Cancer Cells

Davide Bertozzi1, Jessica Marinello1, Stefano G. Manzo1, Francesca Fornari2, Laura Gramantieri2, and Giovanni Capranico1

Abstract DNA topoisomerase I (Top1) inhibition by camptothecin derivatives can impair the hypoxia-induced cell transcriptional response. In the present work, we determined molecular aspects of the mechanism of camptothecin’s effects on hypoxia-inducible factor-1a (HIF-1a) activity in human cancer cells. In particular, we provide evidence that low concentrations of camptothecin, without interfering with HIF-1a mRNA levels, can reduce HIF-1a proteinexpressionandactivity.Asluciferase assays demonstrated the involvement of the HIF-1a mRNA 30 untranslated region in camptothecin-induced impairment of HIF-1a protein regulation, we performed microarray analysis to identify camptothecin-induced modiﬁcation of microRNAs (miRNA) targeting HIF-1a mRNA under hypoxic-mimetic conditions. The selected miRNAs were then further analyzed, demonstrating a role for miR-17-5p and miR-155 in HIF-1a protein expression after camptothecin treatments. The present ﬁndings establish miRNAs as key factors in a molecular pathway connecting Top1 inhibition and human HIF-1a protein regulation and activity, widening the biologic and molecular activity of camptothecin derivatives and the perspective for novel clinical inter- ventions. Mol Cancer Ther; 13(1); 239–48. 2013 AACR.

Introduction Interestingly, the response to hypoxia can be effectively Hypoxia-inducible factor-1(HIF-1)isatranscription modulated by three DNA topoisomerase I (Top1) poisons factor that acts as the main regulator of the cellular (topotecan, camtothecin-20-ester(S), and 9-glycineamido- response to low oxygen tension. It is a dimer consti- 20(S)-camptothecin HCl) in U251 human glioma cells (6); tuted by HIF-1a and HIF-1b subunits, and its cellular however, the molecular mechanism has not been identi- level is finely governed by the interplay of enzymatic fied yet. Top1 is a nuclear enzyme that can relax negative processes that regulate the ubiquitination and pro- and positive DNA supercoils (7), and is the specific target teasomal degradation of the HIF-1a subunit, primar- of the natural product camptothecin and its synthetic ily mediated by the von Hippel–Lindau protein (1, 2). derivatives, which are effective antitumor drugs (8–10). Under cellular oxygen deprivation, HIF-1a accumu- The pharmacologic activity of camptothecin analogs is lates, translocates into the nucleus, and associates with due to the drug’s ability to stabilize Top1-DNA cleavage its b subunits to activate the transcription of several complexes (Top1cc) at replication forks thus leading to target genes, ultimately leading to hypoxia adaptation collision with DNA polymerase and irreversible DNA and survival response. The association of HIF-1 with damage and apoptosis (11–13). Besides the main interfer- pathologic events such as cancer, cardiovascular dis- ence with the replication process, in recent years it has orders, and inflammation leads to an increased scien- been studied as the cellular response to Top1ccs formation tific interest in the development of new therapeutic at transcribed genomic regions (9, 14, 15), as the bulk of strategies (3–5). cellular Top1 is localized at transcription sites in mam- malian cells (16–19). Nevertheless, camptothecin analogs are also known to be endowed with other biologic activ- Authors' Affiliations: 1Department of Pharmacy and Biotechnology, Uni- versity of Bologna; and 2Center for Applied Biomedical Research, ities, including antiangiogenic effects (20), and there- S. Orsola-Malpighi University Hospital, Bologna, Italy fore it has been proposed that modulation of hypoxia D. Bertozzi and J. Marinello share first authorship for this article. response cascade by camptothecin could be relevant for the drug’s antitumor activity (21, 22). Corresponding Author: G. Capranico, Department of Pharmacy HIF-1a and Biotechnology, University of Bologna, via Irnerio 48, 40126 The effects of topotecan on the human gene Bologna, Italy. Phone: 39-051-2091209; Fax: 39-051-2091224; E-mail: have been previously investigated demonstrating that [email protected] low doses of drug (500 nmol/L or below) inhibit HIF- doi: 10.1158/1535-7163.MCT-13-0729 1a protein accumulation during hypoxia in a time-depen- 2013 American Association for Cancer Research. dent manner (6, 21). As it has been evidenced that

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topotecan does not affect the level of mRNA transcrip- pellet was resuspended in 3.6 mL of Acetate-EDTA tion, it has been suggested a posttranscriptional level Buffer [50 mmol/L NaOAc (pH 5.2), 10 mmol/L EDTA] of regulation by the drug (15). The compound does not containing 240 mL of SDS 25% and 3.6 mL of acid phenol have any effect on the HIF-1b subunit. In addition to (pH 4.5) and mixed vigorously every minute for 10 min- this evidence, we previously demonstrated that campto- utes at 65C. After a short incubation on ice, samples thecin increases the levels of two antisense transcripts were centrifuged for 15 minutes at 12,000 g. The upper at the 50 and 30 ends of the HIF-1a gene in human can- phase was collected and added to 3.9 mL of chloro- cer cells with a cell type and stress specificity (23–25). form/isoamyl alcohol then mixed and centrifuged for These antisense RNAs may be involved in mRNA regu- 10 minutes at 5,000 g. The resulting upper phase was lation, providing circumstantial evidence of the inter- then isopropanol precipitated. DNA was digested with play between Top1 inhibition and response to hypoxia. DNase I, and RNA was phenol-extracted and ethanol Moreover, the transcriptional response to Top1ccs in precipitated. After verifying its quality on a 1% agarose human cancer cell lines includes hyperphosphorylation gel, 1 mg of total RNA was used to prepare cDNA using and enhanced escape from pausing of RNA Pol II SuperScript III (Life Technologies) following the man- (14, 15, 26), activation of cyclin-dependent kinase (CDK; ufacturer’s instruction. Random (N6) and poly(T) pri- refs. 14, 23, 27), induction of transcription-dependent mers were used for total RNA retrotranscription. Reac- DNA double-strand breaks (DSB; refs. 28, 29), chromatin tions included a 25C preannealing step for 5 minutes, remodeling (9, 23), and increased antisense ncRNAs at and then retrotranscription was performed at 50Cfor divergent CpG promoters (30). Consequently, the inhibi- 50 minutes. tion of Top1 activity can lead to several alterations of transcription regulation that directly or indirectly may Quantitative real-time PCR impact on the hypoxia pathways. Real-time PCR (RT-PCR) was performed using the In this work, we aimed at the definition of molecu- LightCycler and the FastStart DNA Master SYBR Green lar factors connecting Top1ccs with HIF-1a protein I kit (Roche Diagnostics). Quantification and melting regulation and activity. In particular, we focused on curveanalyseswereperformedusingtheRocheLight- posttranscriptional molecular mechanisms involving Cycler software as indicated by the supplier. PCR reac- microRNAs (miRNAs) and the 30 untranslated region tions contained 1 FastStart DNA SYBR Green I Master 0 a (3 UTR) of the HIF-1 mRNA.Assomecamptothecin Mix, 2.08 mmol/L MgCl2 and 350 nmol/L of each derivatives have been approved for cancer therapy, the primer. Specificity of PCR products was routinely con- additional evidence that these compounds are able trolled by melting analysis and agarose gel electropho- to overcome the effects of HIF-1a accumulation in resis. Differently from the above-specified protocol, miR hypoxic cells may lead to a better understanding of the expression was determined retrotranscribing with the antiangiogenic and antitumor activity of camptothecin TaqMan MicroRNA RT Kit (Applied Biosystem) and analogs. The new data may lead to different approaches quantifying by the StepOne Real-Time PCR System to develop novel therapeutics for the treatment of hu- (Applied Biosystem) with the TaqMan microRNA man cancer and other diseases. Assays Kit (Applied Biosystem). In particular, every reaction of 20 mLcontains1 TaqMan Universal Master Materials and Methods PCR Mix, 1 TaqMan MicroRNA Assay Mix, and 1.33 Cell lines and treatments mL of cDNA. For primers sequences, see Bertozzi and The cancer cell lines HeLa and HEK293 were purchased colleagues (24). from American Type Culture Collection (LGC Standards S.r.l.) 4 years before this publication and were grown in Western blot analyses Dulbecco’s Modified Eagle Medium (DMEM; HeLa) or Cells were washed with PBS and lysed for 15 minutes Minimum Essential Medium (MEM; HEK293) with 10% at 4C with 50% radioimmunoprecipitation assay FBS (M-Medical S.r.l.). Cells were maintained at 37Cin (RIPA) buffer (Tris-HCl pH 7.4 50 mmol/L, NaCl 150 a humidified incubator containing 20% O2 and 5% CO2. mmol/L, EDTA 1 mmol/L, NaF 1 mmol/L, sodium Cell line identity was periodically certified with the Cell deoxycholate 1%, Triton X-100 1%, SDS 0.1%) and 50% ID System (Promega) by BMR Genomics S.r.l.. Exponen- HNTG Buffer (HEPES pH 7.4 50 mmol/L, NaCl 150 tially growing cells were exposed to 0.5 mmol/L of camp- mmol/L, Triton X-100 0.1%, glycerol 10%) in the pres- tothecin for the indicated times at 37C, unless specified ence of protease inhibitors. The lysate was transferred in otherwise. In case of cotreatments, cells were incubated a tube and centrifuged for 20 minutes, maximum speed. with desferrioxamine (250 mmol/L) in the presence of The supernatant was recovered and quantified by Brad- camptothecin (0.5 mmol/L) for the indicated time. Drugs ford Protein Assay. Western blotting analyses were were purchased from Sigma. conducted using appropriate antibodies and the levels of cellular proteins were visualized with peroxidase- RNA extraction and reverse transcription coupled secondary antibodies using Pierce ECL Plus After treatment, cells were washed twice with ice- Western Blotting Substrate (Thermo Scientific). b-Actin cold PBS and collected through centrifugation. The antibody was from Santa Cruz Biotechnology and

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HIF-1b antibody was from Novus Biologicals. For the Feature Extraction 10.5 software (Agilent Techno- HIF-1a detection, BD Biosciences antibody was used. logies) was used to obtain the microarray raw data. Raw data from one-color miRNA microarrays were normal- Cells transfection and luciferase assay ized and analyzed by the GeneSpring GX software Functional assay for 30UTR-HIF-1a. Twenty-four hours version 11.5.1 (Agilent Technologies). The value of each after seeding (80,000 cells in each well of a 24-wells miRNA was obtained doing the average of replicates. plate), cells were transfected with plasmid pRL (renilla Differentially expressed miRNAs were identified by luciferase control reporter, 0.5 mg) and pGL3-30UTR- using a filter based on a fold change of 1.45 combined HIF-1a or pGL3-stiI (0.5 mg), using Lipofectamine with an ANOVA (P < 0.05) with Benjamini and Hoch- 2000 (Life Technologies) following the manufacturer’s berg correction for false-positive reduction. instructions. Five hours after transfection, the medium Raw and normalized data have been submitted to was replaced with fresh one and, after additional 19 ArrayExpress with the accession number E-MTAB-2022. hours, the drug treatment was started. Finally, cells were lysed with 100 mL of lysis buffer 1 (Promega) for 15 minutes under agitation. Results Functional assay for HIF-1a activity. Twenty-four Camptothecin inhibition of HIF-1a accumulation hours after seeding (60,000 cells in each well of a 24- under hypoxic-mimetic conditions depends on the wells plate), cells were transfected with pRL (0.04 30UTR of HIF-1a mRNA mmol/L), pGL3-HRE-Luc (0.2 mmol/L) and, if indicated, To elucidate the mechanism of the camptothecin- 55 pmol of specific anti-miR (TaqMan anti-miR inhib- induced reduction of HIF-1a protein levels and activity, itor; Life Technologies). Transfection was performed we first performed time course experiments of HIF-1a with RNAiMAX (Life Technologies) following manu- protein accumulation triggered by desferrioxamine, an facturer’s instructions. Twenty-four hours after trans- iron chelator that mimics the response to hypoxia, in fection, medium was replaced with fresh medium com- HeLa and HEK293 cells. Western Blot analyses of cell pleted with drugs. Finally, cells were lysed with 100 mL lysates demonstrated that a low concentration of camp- of lysis buffer 1 (Promega) for 15 minutes under tothecin (0.5 mmol/L) strongly prevents the desferriox- agitation. For the luminometer reading, 100 mLofLucif- amine-dependent HIF-1a accumulation after 8 to 24 erase Assay Reagent II (Promega) were added to 20 mL hours (Fig. 1A), whereas it does not affect HIF-1b levels of cellular lysate. After the first reading, 100 mLofStop (Fig. 1A, left). The results are thus in agreement with & Glo Reagent (Promega) were added and renilla lumi- the specific effect of camptothecin on the a subunit of nescence was determined. the HIF-1 factor (6, 21). We then measured camptothecin effects on HIF-1a miRNA microarray and data analysis activity with a reporter assay by using a vector expres- RNAs were hybridized on a Agilent Human miRNA singaluciferasegeneunderthecontrolofhypoxia microarray (#G4470B; Agilent Technologies). This responsive elements (HRE). Cells were transfected with microarray consists of 60-mer DNA probes synthesiz- the vector, and after 24 hours treated with desferriox- ed in situ and contains 15,000 features that represent amine and camptothecin for additional 24 hours. 723 human miRNAs, sourced from the Sanger miRBASE Desferrioxamine treatment induces a strong increase public database (Release 10.1). One-color miRNA ex- in luciferase activity that is reversed by 0.5 mmol/L pression was performed according to the manufacturer’s camptothecin up to 50% and 20% in HEK293 and HeLa procedure, as described in Ferracin and colleagues cells, respectively (Fig. 1B). To assess the activity of (31). Briefly, total RNA fraction was obtained from HIF-1a, we also determined by quantitative RT-PCR samples by using the acid phenol method as previously (qRT-PCR) the transcriptional activation of the endog- described in this section. RNA quality was assessed enous VEGF gene following cell exposure to desferri- by using the Agilent 2100 Bioanalyzer (Agilent Techno- oxamine. Camptothecin reduces desferrioxamine- logies). Low-quality RNAs (RNA integrity number activatedVEGFexpressioninbothcelllinesafter6and below 7) were excluded from microarray analyses. 24 hours of treatment (Fig. 1C), whereas in normoxic Labeled miRNAs were obtained from 500 ng of total condition camptothecin does not affect the VEGF RNA through the ligation of a 50-cytidine bisphosphate- mRNA level. In contrast, levels of HIF-1a mRNA Cy3 (pCp-Cy3; Agilent Technologies) group at the 30 end in HeLa cells are not reduced significantly under hyp- of each miRNA. To enhance the T4 RNA-ligase (Pro- oxic-mimetic conditions in the presence of campto- mega) efficiency, total RNA was previously treated with thecin (Fig. 1D). HEK293 cells show instead a different alkaline phosphatase (Amersham) at 37C for 30 min- kinetics, with a decrease at short times of treatment utes. Labeled miRNAs were purified by chromatogra- and an increase at longer times. The latter effect could phy columns (Micro Biospin 6; Biorad) and then hybrid- be due to a homeostatic cellular response to the campto- ized on a microarray. Hybridizations were performed at thecin-induced protein reduction. Thus, the campto- 55C for 17 hours in a rotating oven. Images at 5-mm thecin-dependent decrease of HIF-1a protein activity, resolution were generated by the Agilent scanner and confirmed by VEGF mRNA reduction in both cell lines,

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A HeLa HEK293 120 120 kDa HIF-1α kDa HIF-1α Figure 1. Camptothecin (CPT) 86 42 activity under hypoxic-mimetic kDa HIF-1β kDa β-Actin conditions in human cancer cells. A, HIF-1a content was determined CPT in HeLa and HEK 293 cells by DFX Western blotting in the presence or B absence of desferrioxamine (DFX) HeLa HEK293 and camptothecin (250 and 0.5 mmol/L, respectively). HIF-1b and 100 100 b-actin are loading controls. B, 80 80 HIF-1a activity was determined in 60 60 HeLa and HEK 293 cells transfected with a plasmid 40 40 expressing the luciferase gene

activity (%) 20 20 under the control of HREs. Twenty- 0 0 four hours after transfection, cells Relative luciferase Relative CPT were treated either with DFX desferrioxamine alone or desferrioxamine and camptothecin C (see above concentrations) for 24 HeLa HEK293 hours. Values are normalized 2.5 2.5 against a control renilla-expressing vector, and over desferrioxamine- 2 2 treated samples. Values, means SD of four determinations from at 1.5 1.5 least three independent experiments. Levels of VEGF (C) 1 1 (VEGF) and HIF-1a (D) mRNAs in HeLa and 0.5 0.5 HEK 293 cells following 6 and 24 hours of 0.5 mmol/L camptothecin Relative RNA levels Relative 0 0 treatments (independent CPT experiments). Values are DFX normalized to cytochrome B mRNA levels. Camptothecin-treated D samples are normalized to HeLa HEK293 untreated samples, whereas 2.5 2.5 camptothecin þ desferrioxamine cotreated samples are normalized 2 2 to desferrioxamine-treated samples. Values, means SD of 1.5 1.5 four determinations from two 1 1 independent experiments. , data ﬁ P < (HIF-1 α ) that are statistically signi cant ( 0.5 0.5 0.05; t test). Lack of genomic DNA contamination was conﬁrmed by Relative RNA levels Relative 0 0 a-sat DNA quantitation in each CPT experiment. DFX 6 h 24 h

cannot be attributed to a marked reduction of HIF-1a ic-mimetic conditions (Fig. 2A). A similar situation has mRNA levels in HeLa and in HEK293 cell lines. been evidenced for 30aHIF-1a in HeLa cells (Fig. 2B), To further investigate if this modulation could be whereas the transcript level in HEK293 cells is even in some way influenced by the previously demon- significantly reduced in hypoxia. Therefore, camptothe- strated camptothecin stimulation of 30 or 50-antisense cin modulation of HIF-1a protein levels seems unlikely transcripts (30 and 50aHIF-1a) at the human HIF-1a to be under the control of the two long noncoding gene locus (23, 24), we evaluated the level of the two antisense RNAs at the HIF-1a gene locus. antisense RNAs in the absence and presence of desfer- As the above results suggested that camptothecin rioxamine in both cell lines (Fig. 2). The results dem- inhibition of HIF-1a accumulation and activity may onstrated that no significant increase of the 50aHIF-1a likely be due to posttranscriptional events, we then level is detected after 6 and 24 hours of treatment assessed whether the 30UTR of HIF-1a mRNA is with a low camptothecin concentration under hypox- involved in the mechanism. We transfected cells with

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A HeLa HEK293 3 3 2.5 2.5 Figure 2. Transcription levels of 50 0 2 2 (A) or 3 -antisense RNAs (B) at the HIF-1a gene locus in HeLa and 1.5 1.5 HEK293 cells treated with 1 1 desferrioxamine and/or (5 ′ aHIF-1 α ) camptothecin for the indicated 0.5 0.5 times. Values are normalized to RNA levels Relative 0 0 cytochrome B mRNA levels. CPT Camptothecin-treated samples are normalized to untreated samples, DFX whereas camptothecin þ B desferrioxamine cotreated HeLa HEK293 samples are normalized to 3 3 desferrioxamine-treated 2.5 samples. Values, means SD 2.5 of four determinations from two 2 2 independent experiments. , data that are statistically 1.5 1.5 signiﬁcant (P < 0.05; t test). Lack 1 1 of genomic DNA contamination (3 ′ aHIF-1 α ) was conﬁrmed by a-sat DNA 0.5 0.5 quantitation in each experiment. RNA levels Relative 0 0 CPT DFX

6 h 24 h

a construct in which the HIF-1a 30UTR was linked to a HEK293 cells (Fig. 3). The results overall show that the luciferase gene under the control of the early SV40 30UTR plays an important role in the reduction of the promoter, and then cells were treated for 16 hours with luciferase activity and likely of HIF-1a protein regula- desferrioxamine and/or camptothecin. Interestingly, in tion under hypoxic-mimetic conditions. hypoxic-mimetic conditions, camptothecin treatment markedly reduces luciferase activity in both cell lines Low camptothecin concentrations modulate global 0 (Fig. 3), suggesting a 3 UTR-dependent mechanism for patterns of miR expression in human HeLa cells the camptothecin effect. In addition, camptothecin sig- As camptothecin-stabilized Top1ccs have been dem- niﬁcantly reduces luciferase activity in normoxia in onstrated to widely interfere with transcriptional mecha- HEK293 but not in Hela cells, indicating that the mech- nisms at active promoters (23, 30), it is likely that anism is also operative under normoxia at least in this interference may modulate gene expression and

HeLa HEK293 150 150

100 100

50 50 activity (%) Relative luciferase Relative 0 0 DFX Control CPT

Figure 3. HIF-1a 30UTR is involved in camptothecin-mediated decrease of HIF-1a proteininhypoxic-mimeticconditions. HeLa and HEK 293 cell lines have been transfected with a luciferase plasmid carrying the 30UTR of HIF-1a. Twenty-four hours after transfection, cells were treated either with desferrioxamine alone (250 mmol/L) or desferrioxamine and camptothecin (250 and 0.5 mmol/L respectively) for 16 hours. Values are normalized to a control renilla-expressing vector and untreated samples. Values, means SD of four determinations from at least three independent experiments. , data that are statistically signiﬁcant (P < 0.05; t test).

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Table 1. miRNA modulation by camptothecin in normoxic and hypoxic-mimetic condition (fold change >1.45).

Normoxia Hypoxia

Upregulated Downregulated Upregulated Downregulated Total 68 (5) 6 (2) 33 (5) 12 (1) Targeting HIF-1a 5a — 2b 1c

NOTE: Together with the total number of regulated miRNAs, in parentheses are reported the number of miRNAs emerged as differentially regulated by camptothecin after the ANOVA test (P 0.05) with Benjamini and Hochberg correction. ahsa-miR-15b; hsa-miR-376c; hsa-miR-18a; hsa-miR-155; hsa-miR-18b. bhsa-miR-376c; hsa-miR-155. chsa-miR-582-5p.

determine several cascade events. The above evidence the microarray experimental data (not corrected for that HIF-1a 30UTR mediates the camptothecin-induced false-positive reduction, as the ANOVA test resulted reduction of luciferase (Fig. 3) suggested an involvement in an extremely shorter list) with the in silico predicted of miRNAs in downregulation of mRNA translation HIF-1a–targeted miRNAs, revealing that five and two upon camptothecin treatments of cells. camptothecin upregulated miRNAs target HIF-1a To study whether Top1ccs may affect miR expression, in normoxia and hypoxic-mimetic conditions, respec- we conducted a genome-wide analysis of miR expression tively (Table 1). The most significantly upregulated patterns with the Agilent miRNA microarray platform in miR was miR-155, which was therefore selected for HeLa cells treated with a low dose of camptothecin for 6 further analyses. In addition, microarray data suggested hours. The drug effect was evaluated both in normoxic that miR-155 could not be the only player in the mod- and hypoxic-mimetic conditions. To identify the miRNAs ulation of HIF-1a levels as several other HIF-1a–tar- that are differentially expressed between camptothecin- geted miRNAs showed interesting behavior. In fact, two treated and control cells, we did a statistical comparison miRNAs derived from the same cluster (miR-18a and between the two groups of samples using a filter based on miR-18b) were upregulated by camptothecin (Table 1). a fold change of 1.45 (for the complete list of miRNAs We therefore selected miR-18a for further validation. emerged by the analysis refer to Supplementary Table S1). Finally, we decided to select one miRNA (miR-17-5p), Sixty-eight and six miRNAs emerged as up- and down- which is upregulated by camptothecin even if the level regulated, respectively, by camptothecin in normoxia of upregulation was not above the 1.45 fold-change (Table 1). Similar analysis was performed for desferriox- threshold, as miR-17-5p targets HIF-1a 30UTR at 1.1 kb amine and camptothecin cotreated samples, and in this upstream to the 30 end of mRNA, and has a basal case 33 and 12 miRNAs resulted up- and downregulated, expression level higher that the other selected miRNAs. respectively (Table 1). To highlight the differentially expressed miRNAs in both oxygen conditions, we ana- miRNAs are involved in the camptothecin-induced lyzed the two categories and we revealed that 22 and 2 HIF-1a protein reduction in desferrioxamine-treated of the upregulated and downregulated transcripts, re- cells spectively, are in common between the normoxic and Next,wehaveinvestigatedtheselectedmiR-155, hypoxic-mimetic conditions (Supplementary Fig. S1). miR-18a, and miR-17-5p by qRT-PCR in the two human An ANOVA test (P 0.05) with Benjamini and Hochberg cell lines. Camptothecin can readily increase the levels correction for false-positive reduction was performed of miR-155, miR-17-5p, and miR-18a in HeLa cells in on the miRNAs emerged as differentially regulated both hypoxic-mimetic and normoxic conditions (Fig. by camptothecin. The ANOVA test left fewer miRNAs 4A). In the presence of desferrioxamine, the effects significantly altered in each category (Table 1, values of camptothecin are dependent on the treatment time, between bracket). Thus, the analyses show that camp- withastrongerincreaseat24hoursthan6hours.Simi- tothecin is able to modify specifically the miRNA pool lar analyses in HEK293 cells demonstrated some dif- in HeLa cells, with a tendency to upregulate their expres- ferences in the kinetics of the miRNAs increase. sion levels. miR-155 transcription is stimulated by camptothecin in Then, we predicted all the HIF-1a–targeting miRNAs by normoxic condition and not significantly in the pre- using miRanda (http://www.microrna.org/microrna/ sence of desferrioxamine. On the contrary, miR-17-5p home.do), TargetScan (http://www.targetscan.org/), slightly increases after 24 hours but not after 6 hours of PicTar (http://pictar.mdc-berlin.de/), and Diana (http:// camptothecin treatment under both conditions. miR-18a diana.cslab.ece.ntua.gr/) algorithms. We next intersected shows a pattern similar to miR-155, with a stimulation

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Camptothecin Interferes with HIF-1a via microRNAs

A HeLa miR-155 miR-17-5p miR-18a 4 4 4 3 3 3 Figure 4. Expression levels of selected miRNAs in HeLa (A) and 2 2 2 HEK 293 (B) cell lines in normoxic 1 1 1 and hypoxic-mimetic conditions expression Relative miR Relative after 6 and 24 hours of 0 0 0 camptothecin treatments. Values –+–+ –+–+ –+–+CPT are normalized to U6RNA levels. ––++ ––++ ––++DFX Camptothecin-treated samples are also normalized to untreated B HEK293 þ samples, whereas camptothecin miR-155 miR-17-5p miR-18a desferrioxamine–treated samples are normalized to desferrioxamine- 4 4 4 treated samples. Values, means SD of four determinations from two 3 3 3 independent experiments. , data that are statistically signiﬁcant 2 2 2 (P < 0.05; t test). 1 1 1 expression Relative miR Relative 0 0 0 –+–+ –+–+ –+–+CPT ––++ ––++ ––++DFX 6 h 24 h

in normoxia and no effects in the presence of desfer- bars, no anti-miR, Fig. 5). Then, we determined the rioxamine (Fig. 4B). The effect of desferrioxamine on the effect of the selected miRs by transfecting the corre- expression of the studied miRNAs has also been sponding anti-miR. The results show that anti-miR17-5p evaluated highlighting the fact that transcription of is able to suppress the camptothecin-reduced luci- miR-18a is induced by 6 hours of hypoxia in both cell ferase activity in both cell lines, whereas anti-miR-155 lines but not at longer times (Supplementary Fig. S2), is effective to a lesser extent (Fig. 5). In contrast, the whereas miR-17-5p is not modified or partially reduc- anti-miR-18a was ineffective, whereas the combination ed in HeLa and HEK293 cells, respectively (Supple- of all the tested anti-miRs gave a full suppression of mentary Fig. S2). Differently, the miR-155 transcript camptothecin effects (Fig. 5). level increases after 24 hours of desferrioxamine treat- Taken together, the present results show that, in ment in HeLa cells. The results are therefore in agree- hypoxic-mimetic conditions, camptothecin can inhibit ment with the microarray data in HeLa cells, suggesting HIF-1a protein accumulation by the modulation of that camptothecin may modulate the HIF-1a protein miRNAs targeting the HIF-1a mRNA. Here, we have level through the action of different miRNAs. shown that miR-17-5p and miR-155 can be among If the selected and analyzed miRNAs are involved in those HIF-1a–targeted miRNAs altered by low doses the observed camptothecin-dependent reduction of of camptothecin. HIF-1a, then specific anti-miR expression should suppress this effect. Thus, we next performed experiments Discussion in which cells were transfected with a plasmid with the The present report defines the molecular players luciferase gene under the control of HRE, together connecting Top1ccs with HIF-1a protein regulation with an anti-miR against one of the selected miR-155, and activity, demonstrating that Top1 inhibitors can be miR-18a, or miR17-5p. Then, 24 hours after transfection, sharp regulators of gene activity. Camptothecin and its cells were treated either with desferrioxamine alone derivatives have been deeply studied since their dis- or with desferrioxamine and camptothecin for 24 hours. covery, and for several decades their cytotoxic activity As previously reported in Fig. 1B, first of all, we can was strictly associated only with irreversible replica- observe that the treatment of desferrioxamine alone tive DNA damage leading to S-phase checkpoint acti- significantly increases the luciferase activity compared vation, G2 arrest, and cell death. In the past few years, with control cells, confirming the activation of hypoxia the attention has been oriented back on these chemi- response pathways (Fig. 5). After cotreatment with cal inhibitors as several additional effects have been desferrioxamine and camptothecin, we observe a reduc- highlighted, mostly reflected on the interference with tion of the luciferase activity in both cell lines (black transcription. We evidenced that Top1 inhibition favors

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HeLa HEK293 160 160 140 140 120 120 100 100 80 80 60 60

activity (%) 40 40 20 20 Relative luciferase Relative 0 0 ––+ ––+CPT –++ –++DFX

No anti-miR Anti-scrbl Anti-17-5p Anti-18a Anti-155 Anti-miR all

Figure 5. Anti-miR-17-5p reverts camptothecin-induced HIF-1a activity reduction in hypoxic-mimetic conditions both in HeLa and HEK 293 cell lines. Cells have been cotransfected with a luciferase plasmid under the control of HREs and with the indicated anti-miR. Twenty-four hours after transfection, cells were treated either with desferrioxamine alone (250 mmol/L) or desferrioxamine and camptothecin (250 and 0.5 mmol/L, respectively) for further 24 hours. Values are normalized to control renilla-expressing vector and desferrioxamine-treated samples. Values, means SD of four determinations from at least three independent experiments. , data that are statistically signiﬁcant (P < 0.05; t test).

RNA Pol II escape from the promoter-proximal pausing based mechanism of HIF-1a reduction. Interestingly, site of several genes, enhances histone acetylation camptothecin can significantly alter miR expression and reduces nucleosome density in a CDK-dependent profiles, and in particular can increase miR-17-5p and manner, and finally induces alterations of splicing miR-155, therefore affecting HIF-1a activity in human events (9, 14, 23, 28, 29, 32). It is noteworthy that cancer cells. camptothecin can increase genome-wide the level of In recent years, many miRNAs targeting comple- antisense RNAs at CpG island promoters of divergent mentary sequences in 30UTR have been discovered, and genes (30) and, specifically for the present work, it evidence has much increased showing their import- is able to induce the transcription of long noncod- ance in regulating physiologic and pathologic cellular ing antisense RNAs at the 50 and 30 end of the HIF-1a pathways (36). As camptothecin treatment determines gene (23, 24), which may regulate gene expression strong modification of transcription of several mRNAs posttranscriptionally (24). Camptothecin can also acti- (30, 37, 38), we questioned if the drug could modulate vate an ataxia telangiectasia mutated (ATM)- and DNA- the miRNAs expression profiles in two human cancer dependent protein kinase (DNA-PK)-dependent tran- cell lines and, if that was the case, if miRNAs modi- scriptional response in quiescent cells, such as neurons fications reflect in the HIF-1a protein expression. (28, 29), likely leading to DNA breaks and transcription- Microarray results pushed us to further investigate associated DNA rearrangements (33). Finally, Top1 theroleofthreeselectedmiRNAsinthemodulation inhibitors can unsilence the paternal Ube3a allele in a of HIF-1a protein accumulation in the presence of mouse model, giving the possibility to restore function- camptothecin. qRT-PCR confirmed an increased levels al UBE3A protein and treat Angelman syndrome of the miRs under the experimental conditions. Inter- (34, 35). Thus, several findings on camptothecin activity estingly, luciferase experiments performed after anti- not related to replicative DNA damage and cell killing miR transfections demonstrated that miR-17-5p and of S-phase cells prompted us to define transcriptional miR-155 can be two important players in the reduction molecular response triggered by Top1ccs and affecting of HIF-1a protein accumulation and activity by low HIF-1a gene activity. camptothecin concentrations. Western blot experiments Here,wehaveshowntheroleofselectedmiRNAsin showed modest alterations of camptothecin effects in the molecular interference of low camptothecin concen- the presence of anti-miRs (not shown). This could be trations with HIF-1a proteinaccumulationunderhyp- due to different sensitivity between methodologies in oxic-mimetic conditions. The drug treatment strongly assessing HIF-1a protein levels, but also to the involve- prevents the protein accumulation as detected by west- ment of different posttranscriptional mechanisms ern blotting and luciferase assay, even though the of HIF-1a regulation. For example, it has been demon- two methods show different sensitivity. The campto- strated that HuR and PTB RNA-binding proteins can thecin-induced impairment of HIF-1a accumulation also cooperate and enhance HIF-1a translation by bind- impacts the hypoxia-mediated cascade of events, with ing to the 50UTR of HIF-1a mRNA(39);therefore,they adecreaseofHIF-1a activity and VEGF mRNA tran- may overcome the effect of the studied anti-miRs in the scription. Interestingly, the studied low concentrations case of HIF-1a mRNA.Inaddition,theresultsoverall of camptothecin do not interfere with HIF-1a transcript indicate that other miRNAs, besides the ones studied in levels, in agreement with a posttranscriptional miR- the present work, can likely be involved in the

246 Mol Cancer Ther; 13(1) January 2014 Molecular Cancer Therapeutics

Camptothecin Interferes with HIF-1a via microRNAs

mechanism of HIF-1a interference by camptothecin ity can be due to a more specific transcription response with a cell-type specificity. Here, we have demonstrated involving small noncoding RNA, such as miR-17-5p and for the first time that Top1ccs can modulate HIF-1a via miR-155. miRNAs in human cancer cell lines. Recent data (40) demonstrated that higher concen- Disclosure of Potential Conflicts of Interest trations of camptothecin affect few miRNAs in nor- No potential conflicts of interest were disclosed. moxic condition in HCT116 cells, and between them, miR-142-3p was the most significantly upregulated Authors' Contributions with a consequent downregulation of hundreds of pre- Conception and design: D. Bertozzi, J. Marinello, S.G. Manzo, dicted target genes, as those of "ubiquitin-mediated G. Capranico Development of methodology: D. Bertozzi, G. Capranico proteolysis" gene family. Interestingly, modifications Acquisition of data (provided animals, acquired and managed patients, of levels of several miRNAs after camptothecin treat- provided facilities, etc.): D. Bertozzi, J. Marinello, S.G. Manzo Analysis and interpretation of data (e.g., statistical analysis, biostatis- ments in normoxic cancer cells have been related to tics, computational analysis): D. Bertozzi, J. Marinello, S.G. Manzo, resistance or sensitivity to Top1 inhibitors (41–44). F. Fornari, L. Gramantieri, G. Capranico Overall, these studies confirm that different concentra- Writing, review, and/or revision of the manuscript: D. Bertozzi, J.Marinello,G.Capranico tions of Top1 inhibitors finely modulate the balance Study supervision: D. Bertozzi, G. Capranico of cellular miRNAs levels. In addition to mentioned data, the present report shows that Top1 inhibition Acknowledgments impairs the normal level of miRNAs not only in normal The authors thank the Centro Interdipartimentale per le Ricerche but also in hypoxic-mimetic conditions. The reported Biotecnologie (CIRB) of Bologna University for equipment facilities. They miRNA expression profile alterations could impact dif- also thank M. Ferracin and the Ferrara Functional Genomics (Microarray Facility at Dipartimento Medicina Sperimentale e Diagnostica—Univer- ferent cellular pathways besides the cellular response sity of Ferrara). to hypoxia. In conclusion, the present study aimed at clarifying Grant Support the molecular factors connecting the Top1 inhibition This work was supported by grants from the Associazione Italiana per with HIF-1a protein regulation and activity. The find- la Ricerca sul Cancro (AIRC), Milan, Italy (grant IG10184; to G. Capranico), and the University of Bologna PhD Program in Cellular and Molecular ings demonstrate that camptothecin cellular effects are Biology (to D. Bertozzi and S.G. Manzo). associated not only with replicative DNA damage, The costs of publication of this article were defrayed in part by the G2 arrest, and programmed cell death, but also with payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate the transcription-specific miR-based response. Our find- this fact. ings, showing that low camptothecin concentrations can finely modulate miR expression profiles, indicate Received August 30, 2013; revised October 28, 2013; accepted November that camptothecin anticancer and antiangiogenic activ- 11, 2013; published OnlineFirst November 19, 2013.

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248 Mol Cancer Ther; 13(1) January 2014 Molecular Cancer Therapeutics

The Natural Inhibitor of DNA Topoisomerase I, Camptothecin, Modulates HIF-1 α Activity by Changing miR Expression Patterns in Human Cancer Cells

Davide Bertozzi, Jessica Marinello, Stefano G. Manzo, et al.

Mol Cancer Ther 2014;13:239-248. Published OnlineFirst November 19, 2013.

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