DNA Topoisomerase III Alpha Regulates P53-Mediated Tumor Suppression
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Published OnlineFirst February 13, 2014; DOI: 10.1158/1078-0432.CCR-13-1997 Clinical Cancer Human Cancer Biology Research DNA Topoisomerase III Alpha Regulates p53-Mediated Tumor Suppression Mei-Yi Hsieh1, Jia-Rong Fan1, Han-Wen Chang1, Hsiang-Chin Chen1, Tang-Long Shen2,3, Shu-Chun Teng1, Yen-Hsiu Yeh1, and Tsai-Kun Li1,3 Abstract Purpose: Human DNA topoisomerase III alpha (hTOP3a) is involved in DNA repair surveillance and cell-cycle checkpoints possibly through formatting complex with tumor suppressors. However, its role in cancer development remained unsolved. Experimental Design: Coimmunoprecipitation, sucrose gradient, chromatin immunoprecipitation (ChIP), real time PCR, and immunoblotting analyses were performed to determine interactions of hTOP3a with p53. Paired cell lines with different hTOP3a levels were generated via ectopic expression and short hairpin RNA (shRNA)-mediated knockdown approaches. Cellular tumorigenic properties were analyzed using cell counting, colony formation, senescence, soft agar assays, and mouse xenograft models. Results: The hTOP3a isozyme binds to p53 and cofractionizes with p53 in gradients differing from fractions containing hTOP3a and BLM. Knockdown of hTOP3a expression (sh-hTOP3a) caused a higher anchorage-independent growth of nontumorigenic RHEK-1 cells. Similarly, sh-hTOP3a and ectopic expression of hTOP3a in cancer cell lines caused increased and reduced tumorigenic abilities, respectively. Genetic and mutation experiments revealed that functional hTOP3a, p53, and p21 are required for this tumor-suppressive activity. Mechanism-wise, ChIP data revealed that hTOP3a binds to the p53 and p21 promoters and positively regulates their expression. Two proteins affect promoter recruitments of each other and collaborate in p21 expression. Moreover, sh-hTOP3a and sh-p53 in AGS cells caused a similar reduction in senescence and hTOP3a mRNA levels were lower in gastric and renal tumor samples. Conclusion: We concluded that hTOP3a interacts with p53, regulates p53 and p21 expression, and contributes to the p53-mediated tumor suppression. Clin Cancer Res; 20(6); 1489–501. Ó2014 AACR. Introduction have evolved to prohibit neoplastic transformation (3), and It is generally believed that cancer results when cells thus the function and/or gene expression of the regulatory acquire genetic alterations that cause uncontrolled prolif- factors of senescence and apoptosis safeguards must be eration. Consequently, genetic errors resulting in loss-of- altered in a normal cell to become cancerous. TP53 function of tumor-suppressor genes and/or gain-of-func- , the most frequently mutated gene in cancers tion of oncogenes are critically involved in cancer develop- ( 50%), encodes tumor suppressor p53, which functions ment (1). Supportively, inherited diseases with genomic as a transcription factor that drives the senescent (via p21), instability, such as Bloom syndrome, are prone to cancer apoptotic (via BAX), and DNA repair (via GADD45) path- development (2). In addition, genomic instability is recog- ways (4). In addition to genetic alterations, other p53 nized recently as an enabling characteristic of cancer. Two inactivation mechanisms via BRD7 (bromodomain-con- safeguard mechanisms, namely senescence and apoptosis, taining 7) dysfunction or elevated microRNA expression have been reported in cancers (5–7). Chromatin modifying and remodeling complexes have also shown to regulate Authors' Affiliations: 1Department and Graduate Institute of Microbiology, p53-dependent p21 expression (8, 9). Notably, recent College of Medicine, 2Department of Plan Pathology and Microbiology, reports on cancer genomic analyses revealed the potential College of Bioresources and Agriculture, and 3Center for Biotechnology, National Taiwan University, Taipei, Taiwan contribution of chromatin factors to tumorigenesis (10). Together, these results suggest that cellular factors could Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). contribute to tumorigenesis via directly modulating p53 expression and/or functionally cooperating with p53-reg- Corresponding Author: Tsai-Kun Li, Department and Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Room 711, ulated gene expression. Section 1, Jen-Ai Road, Taipei 10051, Taiwan. Phone: 886-2231-23456, Two epigenetic mechanisms, chromatin modification and ext. 88287/88294; Fax: 886-2239-15293; E-mail: [email protected] remodeling, are known to influence both chromatin struc- doi: 10.1158/1078-0432.CCR-13-1997 ture and gene expression. Nevertheless, functional roles of Ó2014 American Association for Cancer Research. the other epigenetic regulators of chromatin DNA topology www.aacrjournals.org 1489 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst February 13, 2014; DOI: 10.1158/1078-0432.CCR-13-1997 Hsieh et al. hTOP3a caused S-phase impairment and rescued some Translational Relevance phenotypes of Ataxia telangiectasia cells (29). It is unclear Transcription factor p53 plays a central role in tumor how TOP3s carries out above specialized essential func- suppression and stress response, thus its expression and tions. Two explanations are offered: (i) TOP3 is present in function must be tightly regulated. Recent studies have multiprotein complexes that perform above functions; or also highlighted the collaborative importance of chro- (ii) both the enzymatic activities and properties of TOP3 are matin and transcription factors in regulated gene expres- greatly modulated by its interacting proteins. Together, sion. Here, we demonstrated that hTOP3a interacts through physical interaction with specific DNA-binding physically and functions genetically with p53 and thus factors, chromatin regulators such as TOP3a participate in contributing to p53/p21-mediated tumor suppression. the specialized biologic functions. Regarding the tumori- Our observations of reduced hTOP3a expression in genic involvement, TOP3s have been shown to exist in tumor samples and its contribution to p53 expression different complexes whose component proteins function and functions further suggest a novel notion that down- in genome stability, checkpoints, and possibly cancer devel- regulation of hTOP3a level is a new way of creating p53 opment (16, 30, 31). Our group also reported that TOP3 dysfunction during tumorigenesis. Because p53 and functions in the alternative lengthening of telomeres via the DNA topoisomerases are clinically useful targets for recombination pathway and hTOP3a knockdown (sh- cancer intervention, our mechanism-based study might hTOP3a) using shRNA technology caused an elevated lead to important therapeutic strategies. telomerase expression (32). Our result resembles telome- rase reactivation during cancer progression thus consistent with a potential role for hTOP3a in tumorigenesis. Here, we report a novel role for hTOP3a in tumorigen- in transcription programming remain to be elucidated. esis and describe its mechanism of action via physically Through their enzymatic activity, DNA topoisomerases reg- and genetically interacting with p53 to regulate p53/p21- ulate various biological functions (11–13), and torsional mediated transcription program. Our results demonstrat- stress accompanied with the separation of DNA strands ed that hTOP3a binds to p53 through p530sDNA-binding during transcription can only be relieved by DNA topoi- region. Similar to the phenotypes caused by p53 deficien- somerases. Recent studies revealed that the TOP2b isozyme cy, sh-hTOP3a not only promoted anchorage-indepen- is involved in regulated gene expression through specifically dent growth but also reduced b-galactosidase (b-gal) binding to transcription factors, for example, human TOP2b senescence staining of cells. Through modulating hTOP3a (hTOP2b) binds to the androgen receptor (AR) and subse- expression, we revealed that the cellular levels of hTOP3a quently contributing to AR-directed transcription program- are inversely correlated to tumorigenic growth in vitro ming and to DNA sequence rearrangements during the and in vivo. Molecularly, hTOP3a regulates tumorigenic development of prostate cancer (14). Consistently, our growth in a p53- and p21-dependent manner. Moreover, recent study has demonstrated a specific involvement of the hTOP3a binds to both the p53 and p21 promoter regions nitric oxide-activated TOP2b cleavable complexes in the and positively regulates their expression. Together, our inflammation-associated DNA damage, mutagenesis, and results suggest that hTOP3a acts through and/or together skin melanoma formation (15). Interestingly, hTOP3a plays with p53 as an anticancer block and subsequently pro- roles in maintaining genomic stability by forming DNA vides the first example of how a general chromatin enzyme repair surveillance complexes with BLM, FANCD2, and collaborates with a DNA-binding transcription factor to BRCA1 tumor-suppressor proteins (16–18) and/or acting act on specific transcription programming and subsequent in combination with BLM, RMI1, and RMI2 to promote the cellular functions. dissolution of double Holliday junctions (19, 20). Consid- ering the importance of genomic instability in tumorigenesis Materials and Methods and the specific interactions of hTOP3a with tumor sup- Chemicals, plasmids, and antibodies pressors, above reports suggest that hTOP3a might function All chemicals and