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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 partic- ular, 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. Ó2013 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- Ó2013 American Association for Cancer Research. dent manner (6, 21). As it has been evidenced that www.aacrjournals.org 239 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst November 19, 2013; DOI: 10.1158/1535-7163.MCT-13-0729 Bertozzi et al. 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 1Â 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 mLcontains1Â TaqMan Universal Master Materials and Methods PCR Mix, 1Â 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.

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