This gene encodes a DNA topoisomerase, an enzyme that controls and alters the topologic states of DNA during transcription. This nuclear enzyme is involved in processes such as chromosome condensation, chromatid separation, and the relief of torsional stress that occurs during DNA transcription and replication. It catalyzes the transient breaking and rejoining of two strands of duplex DNA which allows the strands to pass through one another, thus altering the topology of DNA. Two forms of this enzyme exist as likely products of a gene duplication event. The gene encoding this form, beta, is localized to chromosome 3 and the alpha form is localized to chromosome 17. The gene encoding this enzyme functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. Reduced activity of this enzyme may also play a role in ataxia-telangiectasia. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2016]
Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/β-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through β-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/β-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear β-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.
DNA topoisomerases are essential to modulate DNA topology during various cellular genetic processes. The expression and distinct prognostic value of topoisomerase isoforms in non-small-cell lung cancer (NSCLC) is not well established. In the current study, we have examined the mRNA expression of topoisomerase isoforms by using Oncomine analysis and investigated their prognostic value via the Kaplan-Meier plotter database in NSCLC patients. Our analysis indicated that the expression level of topoisomerases in lung cancer was higher compared with normal tissues. Especially, high expression of two topoisomerase isoforms, TOP2A and TOP3A, was found to be correlated to worse overall survival (OS) in all NSCLC and lung adenocarcinoma (Ade) patients, but not in lung squamous cell carcinoma (SCC) patients. In a contrast, high expression of isoforms TOP1 and TOP2B indicated better OS in all NSCLC and Ade, but not in SCC patients. Meanwhile, high expression of TOP1MT and TOP3B was not correlated with OS in NSCLC patients. Furthermore, we also demonstrated a relationship between topoisomerase isoforms and the clinicopathological features for the NSCLC patients, such as grades, clinical stages, lymph node status, smoking status, gender, chemotherapy and radiotherapy. These results support that TOP2A and TOP3A are associated with worse prognosis in NSCLC patients. In addition, our study also shows that TOP1 and TOP2B contribute to favorable prognosis in NSCLC patients. The exact prognostic significance of TOP1MT and TOP3B need to be further elucidated. Comprehensive evaluation of expression and prognosis of topoisomerase isoforms will be a benefit for the better understanding of heterogeneity and complexity in the molecular biology of NSCLC, paving a way for more accurate prediction of prognosis and discovery of potential drug targets for NSCLC patients.
Chattopadhyay I, Wang J, Qin M, et al. Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures. Oncotarget. 2017; 8(6):10324-10347 [PubMed] Free Access to Full ArticleRelated Publications
Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.
BACKGROUD: As magicians of the DNA world, topoisomerases resolve all of the topological problems in relation to DNA during a variety of genetic processes. While the prognostic value of topoisomerase isoenzymes in epithelial ovarian carcinoma (EOC) is still elusive. In current study, we investigated the prognostic value of topoisomerase isoenzymes in the EOC patients. Kaplan Meier plotter (KM plotter) database were used to assess the relevance of individual topoisomerase isoenzyme mRNA expression to EOC patients overall survival (OS), in which updated survival information and gene expression data were from a total of 1,648 EOC patients. RESULTS: High expression of TOP1 and TOP2A were found to be correlated to worse OS in all patients and serous patients, but not in endometrioid patients. Contrary to TOP1 and TOP2A, TOP3A and TOP3B expression were associated with better OS in all patients and serous patients, but not in endometrioid patients. While TOP2B were not found any significant prognostic value for EOC patients. From the Oncomine database, we also found widespread upregulation in the expression of TOP1 and TOP2A genes in primary tumor tissues. Albeit limited in number, all datasets exhibiting differential expression showed TOP3A and TOP3B under-regulated. CONCLUSION: These results strongly supported that TOP1 and TOP2A were potential biomarkers for predicting poor survival of EOC patients, while TOP3A and TOP3B were expected to be further exploited as tumor suppressors. Comprehensive understanding of the topoisomerase isoforms may have guiding significance for the diagnosis treatment and prognosis in EOC patients.
Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA)-sensitive acute promyelocytic leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrant interaction among PML/RARA, nucleophosmin (NPM), and topoisomerase II beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML); therefore, our model may be applicable to other more common leukemias driven by NPM.
Small cell lung cancer (SCLC) is an extremely aggressive cancer that frequently recurs. Twenty-three human SCLC lines were selected representing varied Myc status. Gene expression of lung cancer, stem-like, hedgehog pathway, and notch pathway genes were determined by RT(2)-PCR array and Exon 1.0 ST array. Etoposide and topotecan concentration response was examined. The IC50's for etoposide and topotecan ranged over nearly 3 logs upon 96 hrs exposure to the drugs. Myc status, TOP2A, TOP2B and TOP1 mRNA expression or topoisomerase 1 and topoisomerase 2 protein did not account for the range in the sensitivity to the drugs. γ-secretase inhibitors, RO429097 and PF-03084014, had little activity in the SCLC lines over ranges covering the clinical Cmax concentrations. MYC amplified lines tended to be more sensitive to the bromodomain inhibitor JQ1. The Smo antagonists, erismodegib and vismodegib and the Gli antagonists, HPI1 and SEN-450 had a trend toward greater sensitivity of the MYC amplified line. Recurrent SCLC is among the most recalcitrant cancers and drug development efforts in this cancer are a high priority.
Bhullar KS, Jha A, Rupasinghe HP Novel carbocyclic curcumin analog CUR3d modulates genes involved in multiple apoptosis pathways in human hepatocellular carcinoma cells. Chem Biol Interact. 2015; 242:107-22 [PubMed] Related Publications
Anticancer activity of a novel curcumin analog (E)-2-(4-hydroxy-3-methoxybenzylidene)-5-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)cyclopentanone (CUR3d) was studied using a human hepatocellular carcinoma cell line (HepG2). The results showed that CUR3d completely inhibits the tumor cell proliferation in a dose- and time-dependent manner. CUR3d at 100 μmol/L activated the pro-apoptotic caspase-3 along with downregulation of anti-apoptotic BIRC5 and Bcl2. CUR3d treatment controlled the cancer cell growth by downregulating the expression of PI3K/Akt (Akt1, Akt2) pathway along with NF-κB. CUR3d down-regulated the members of epidermal growth receptor family (EGFR, ERBB3, ERBB2) and insulin like growth receptors (IGF1, IGF-1R, IGF2). This correlated with the downregulation of G-protein (RHOA, RHOB) and RAS (ATF2, HRAS, KRAS, NRAS) pathway signaling. CUR3d also arrested cell cycle via inhibition of CDK2, CDK4, CDK5, CDK9, MDM2, MDM4 and TERT genes. Cell cycle essential aurora kinases (AURKα, AURKβ) and polo-like kinases (PLK1, PLK2, PLK3) were also modulated by CUR3d. Topoisomerases (TOP2α, TOP2β), important factors in cancer cell immortality, as well as HIF-1α were downregulated following CUR3d treatment. The expression of protein kinase-C family (PRKC-A, PRKC-D, PRKC-E) was also attenuated by CUR3d. The downregulation of histone deacetylases (Class I, II, IV) and PARP I further strengthened the anticancer efficacy of CUR3d. Downregulation of carcinogenic cathepsins (CTSB, CTSD) and heat shock proteins exhibited CUR3d's potency as a potential immunological adjuvant. Finally, the non-toxic manifestation of CUR3d in healthy liver and lung cells along with downregulation of drug resistant gene ABCC1 further warrant need for advance investigations.
Anthracyclines are used in over 50% of childhood cancer treatment protocols, but their clinical usefulness is limited by anthracycline-induced cardiotoxicity (ACT) manifesting as asymptomatic cardiac dysfunction and congestive heart failure in up to 57% and 16% of patients, respectively. Candidate gene studies have reported genetic associations with ACT, but these studies have in general lacked robust patient numbers, independent replication or functional validation. Thus, the individual variability in ACT susceptibility remains largely unexplained. We performed a genome-wide association study in 280 patients of European ancestry treated for childhood cancer, with independent replication in similarly treated cohorts of 96 European and 80 non-European patients. We identified a nonsynonymous variant (rs2229774, p.Ser427Leu) in RARG highly associated with ACT (P = 5.9 × 10(-8), odds ratio (95% confidence interval) = 4.7 (2.7-8.3)). This variant alters RARG function, leading to derepression of the key ACT genetic determinant Top2b, and provides new insight into the pathophysiology of this severe adverse drug reaction.
Beltrame L, Di Marino M, Fruscio R, et al. Profiling cancer gene mutations in longitudinal epithelial ovarian cancer biopsies by targeted next-generation sequencing: a retrospective study. Ann Oncol. 2015; 26(7):1363-71 [PubMed] Related Publications
BACKGROUND: The majority of patients with stage III-IV epithelial ovarian cancer (EOC) relapse after initially responding to platinum-based chemotherapy, and develop resistance. The genomic features involved in drug resistance are unknown. To unravel some of these features, we investigated the mutational profile of genes involved in pathways related to drug sensitivity in a cohort of matched tumors obtained at first surgery (Ft-S) and second surgery (Sd-S). PATIENTS AND METHODS: Matched biopsies (33) taken at Ft-S and Sd-S were selected from the 'Pandora' tumor tissue collection. DNA libraries for 65 genes were generated using the TruSeq Custom Amplicon kit and sequenced on MiSeq (Illumina). Data were analyzed using a high-performance cluster computing platform (Cloud4CARE project) and independently validated. RESULTS: A total of 2270 somatic mutations were identified (89.85% base substitutions 8.19% indels, and 1.92% unknown). Homologous recombination (HR) genes and TP53 were mutated in the majority of Ft-S, while ATM, ATR, TOP2A and TOP2B were mutated in the entire dataset. Only 2% of mutations were conserved between matched Ft-S and Sd-S. Mutations detected at second surgery clustered patients in two groups characterized by different mutational profiles in genes associated with HR, PI3K, miRNA biogenesis and signal transduction. CONCLUSIONS: There was a low level of concordance between Ft-S and Sd-S in terms of mutations in genes involved in key processes of tumor growth and drug resistance. This result suggests the importance of future longitudinal analyses to improve the clinical management of relapsed EOC.
Gaba RC, Groth JV, Parvinian A, et al. Gene expression in hepatocellular carcinoma: pilot study of potential transarterial chemoembolization response biomarkers. J Vasc Interv Radiol. 2015; 26(5):723-32 [PubMed] Related Publications
PURPOSE: To perform a feasibility study to explore the relationship between hepatocellular carcinoma genetics and transarterial chemoembolization treatment response to identify potential biomarkers associated with enhanced treatment efficacy. MATERIALS AND METHODS: In this single-institution study, pretreatment hepatocellular carcinoma biopsy specimens for tumors in 19 patients (14 men, five women; mean age, 59 y) treated with chemoembolization between 2007 and 2013 were analyzed for a panel of 60 chemotherapy-sensitivity, hypoxia, mitosis, and inflammatory genes with the QuantiGene Plex 2.0 mRNA detection assay. Demographic, disease, and procedure data and tumor response outcomes were collected. Quantitative mRNA levels were compared based on radiologic response between tumors exhibiting complete response (CR) versus partial response (PR). RESULTS: The study sample included 19 biopsy specimens from tumors (mean size, 3.0 cm; grade 1, n = 6; grade 2, n = 9; grade 3, n = 4) in patients treated with a mean of two conventional chemoembolization sessions. Thirteen and six tumors exhibited CR and PR, respectively, at a mean of 116 days after treatment. Tumors with CR showed a significant increase in (P < .05) or trend toward (P < .1) greater (range, 1.49-3.50 fold) pretreatment chemotherapy-sensitivity and mitosis (ATF4, BAX, CCNE1, KIF11, NFX1, PPP3CA, SNX1, TOP2A, and TOP2B) gene mRNA expression compared with tumors with PR, in addition to lower CXCL10 levels (0.48-fold), and had significantly (P < .05) higher (1.65-fold) baseline VEGFA levels. CONCLUSIONS: Genetic signatures may allow prechemoembolization stratification of tumor response probability, and gene analysis may therefore offer an opportunity to personalize locoregional therapy by enhancing treatment modality allocation. Further corroboration of identified markers and exploration of their respective predictive capacity thresholds is necessary.
Hsieh MH, Tsai CH, Lin CC, et al. Topoisomerase II inhibition suppresses the proliferation of telomerase-negative cancers. Cell Mol Life Sci. 2015; 72(9):1825-37 [PubMed] Related Publications
Telomere maintenance is required for chromosome stability, and telomeres are typically elongated by telomerase following DNA replication. In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. Previous studies have indicated that yeast Sgs1 and Top3 may work together to remove highly negative supercoils that are generated from recombination. However, the mechanism by which cells eradicate highly positive supercoils during recombination remains unclear. In the present study, we demonstrate that TOP2 is involved in telomere-telomere recombination. Disturbance of telomeric structure by RIF1 or RIF2 deletion alleviates the requirement for TOP2 in telomere-telomere recombination. In human telomerase-negative alternative lengthening of telomere (ALT) cells, TOP2α or TOP2β knockdown decreases ALT-associated PML bodies, increases telomere dysfunction-induced foci and triggers telomere shortening. Similar results were observed when ALT cells were treated with ICRF-193, a TOP2 inhibitor. Importantly, ICRF-193 treatment blocks ALT-associated phenotypes in vitro, causes telomere shortening, and inhibits ALT cell proliferation in mice. Taken together, these findings imply that TOP2 is involved in the ALT pathway, perhaps by resolving the highly positive supercoil structure at the front of the helicase. Inhibition of topoisomerase II may be a promising therapeutic approach that can be used to prevent cell proliferation in ALT-type cancer cells.
Phloridzin (phlorizin or phloretin 2'-O-glucoside) is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin) using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA) ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2), growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR) and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK), cell cycle machinery (CDKs, TERT, TOP2A, TOP2B) as well as epigenetics regulators (HDACs). These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects mediated through the attenuated expression of several key proteins involved in cell cycle regulation, DNA topoisomerases IIα activity and epigenetic mechanisms followed by cell cycle arrest and apoptosis.
AIM: To investigate the effect of Girdin knockdown on the chemosensitivity of colorectal cancer cells to oxaliplatin and the possible mechanisms involved. METHODS: Four siRNAs targeting Girdin were transfected into the chemoresistant colorectal cancer cell line DLD1. Real-time polymerase chain reaction (PCR) was employed to assess Girdin mRNA expression and the most effective siRNA was chosen for conversion into shRNA. Then, DLD1 cells were infected with lentiviruses expressing the Girdin shRNA and a scramble control, respectively, and Girdin mRNA and protein expression levels were assessed by real-time PCR and Western blotting. Furthermore, microarray experiments were used to assess global gene expression profile after Girdin suppression in DLD1 cells. Finally, the cytotoxic effect of simultaneous treatment with oxaliplatin and adriamycin (an inhibitor of a significantly downregulated gene after Girdin suppression in DLD1 cells) was examined by MTT assay. RESULTS: The most effective siRNA suppressed Girdin expression with an inhibition efficiency of 57%. Compared with the scramble control, DLD1 cells infected with the Girdin shRNA displayed decreased Girdin mRNA and protein levels (P < 0.05), and Girdin knockdown significantly enhanced chemosensitivity to oxaliplatin in colorectal cancer cells (P < 0.05). Microarray data revealed that 381 and 162 genes were upregulated and downregulated in response to Girdin reduction, respectively, with ratios > 1.2 or < 0.8 (P < 0.01). Interestingly, TOP2B (DNA topoisomerase 2-β) was downregulated (ratio = 0.78, P = 0.0001) and oxaliplatin/adriamycin combination resulted in increased cell death compared with treatments with individual agents (P < 0.05). CONCLUSION: Girdin knockdown enhances chemosensitivity of colorectal cancer cells to oxaliplatin via TOP2B down-regulation. These findings provide a promising approach to overcome the chemoresistance of colorectal cancer cells.
BACKGROUND: Glioblastoma subtypes have been defined based on transcriptional profiling, yet personalized care based on molecular classification remains unexploited. Topoisomerase II (TOP2) contributes to the transcriptional signature of the proneural glioma subtype. Thus, we targeted TOP2 pharmacologically with etoposide in proneural glioma models. METHODS: TOP2 gene expression was evaluated in mouse platelet derived growth factor (PDGF)(+)phosphatase and tensin homolog (PTEN)(-/-)p53(-/-) and PDGF(+)PTEN(-/-) proneural gliomas and cell lines, as well as human glioblastoma from The Cancer Genome Atlas. Correlation between TOP2 transcript levels and etoposide susceptibility was investigated in 139 human cancer cell lines from the Cancer Cell Line Encyclopedia public dataset and in mouse proneural glioma cell lines. Convection-enhanced delivery (CED) of etoposide was tested on cell-based PDGF(+)PTEN(-/-)p53(-/-) and retroviral-based PDGF(+)PTEN(-/-) mouse proneural glioma models. RESULTS: TOP2 expression was significantly higher in human proneural glioblastoma and in mouse proneural tumors at early as well as late stages of development compared with normal brain. TOP2B transcript correlated with susceptibility to etoposide in mouse proneural cell lines and in 139 human cancer cell lines from the Cancer Cell Line Encyclopedia. Intracranial etoposide CED treatment (680 μM) was well tolerated by mice and led to a significant survival benefit in the PDGF(+)PTEN(-/-)p53(-/-) glioma model. Moreover, etoposide CED treatment at 80 μM but not 4 μM led to a significant survival advantage in the PDGF(+)PTEN(-/-) glioma model. CONCLUSIONS: TOP2 is highly expressed in proneural gliomas, rendering its pharmacological targeting by intratumoral administration of etoposide by CED effective on murine proneural gliomas. We provide evidence supporting clinical testing of CED of etoposide with a molecular-based patient selection approach.
Smith KA, Cowell IG, Zhang Y, et al. The role of topoisomerase II beta on breakage and proximity of RUNX1 to partner alleles RUNX1T1 and EVI1. Genes Chromosomes Cancer. 2014; 53(2):117-28 [PubMed] Related Publications
Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1- RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment.
Kolar Z, Burdova A, Jamaspishvili T, et al. Relation of ETS transcription factor family member ERG, androgen receptor and topoisomerase 2β expression to TMPRSS2-ERG fusion status in prostate cancer. Neoplasma. 2014; 61(1):9-16 [PubMed] Related Publications
Fusion of TMPRSS2 with ERG in prostate cells is determined by double-strand DNA breaks induced by androgen signaling and transcription stress. The enzyme topoisomerase 2β (TOP2B) mediating DNA processing, plays an important role in DNA cleavage. The aim of this study was to analyse expression of AR, TOP2B and ERG in relation to TMPRSS2-ERG gene rearrangement and relevant clinicopathological characteristics in prostate cancer (CaP). Immunohistochemical staining and FISH were used for investigation. ERG expression in prostate cell lesions positively correlated with levels of TMPRSS2-ERG fusion gene (p<0.0001). The most significant co-expression of ERG was found with AR in CaP (p=0.001). Significantly more frequent co-expression of ERG was also revealed with TOP2B (p=0.028). ERG protein expression did not correlate with CaP differentiation status as we found no significant differences in ERG expression for different Gleason categories. We demonstrated a statistically significant positive correlation between the percentage of cells with fusion gene TMPRSS2-ERG in CaP and metastatic potential of tumors (p=0.011). Besides these positive corelations of AR with ERG (p=0.001) and TOP2B with ERG (p=0.028), we also demonstrated a significant co-expression of AR with TOP2B (p=0.007) in CaP. There was a statistically significant increase in the TOP2B H-index in locally advanced CaP in comparison with localized tumors (p=0.046). ERG expression correlates with occurrence of TMPRSS2-ERG fusion and with AR-driven malignant transformation. The results indicate that detection of the TMPRSS2-ERG fusion gene and parallel immunohistochemical examination of AR, TOP2B and ERG has diagnostic significance and may be useful in assessing the biological character of the prostate cancer as well as selecting the best treatment.
The Eighth International Biennial Conference on RNA polymerases I and III (the 'Odd Pols') was held June 7-11, 2012 at The Airlie Center in Warrenton Virginia, USA. It was sponsored by the Universite Laval and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, and organized by Rich Maraia and Tom Moss. The meeting honored the memory of Pierre Thuriaux (Jan 1, 1950-March 18, 2012) and David Schneider reminisced on the important accomplishments his mentor Masayasu Nomura (1927-2011). The goal of the conference was to bring together the world's experts on RNA polymerase I and RNA polymerase III to highlight and share their latest results and varied experimental approaches. The meeting drew attendees from twelve countries and most contributed through oral and poster presentations. The talks were organized into several sessions subdivided into 10 distinct topics. The keynote speaker, Ian Willis, opened the meeting with his presentation entitled "New Regulators of Signaling to Odd Pols" and the closing presentation was given by Patrick Cramer with his presentation "Conservation of the RNA polymerase I, II and III transcription initiation machineries". Here we present some of the highlights from the meeting using summaries provided by the participants.
BACKGROUND: Cytosine arabinoside-based chemotherapy coupled with anthracycline is currently the first-line treatment for acute myeloid leukaemia (AML), but diverse responses to the regimen constitute obstacles to successful treatment. Therefore, outcome prediction to chemotherapy at diagnosis is believed to be a critical consideration. METHODS: The mRNA expression of 12 genes closely involved in the actions of cytosine arabinoside and anthracycline was evaluated by real-time reverse transcriptase PCR (RT-PCR), in 54 diagnostic bone marrow specimens of M2-subtype AML. RESULTS: Low expression levels of ribonucleotide reductase M2 (RRM2) and high expression levels of topoisomerase 2 beta (TOP2B) were correlated with longer survival in a univariate analysis. Another interesting finding is that high ratios of TOP2B/RRM2 and TOP2B/TOP2 alpha (TOP2A) in a combined analysis were also shown to have a prognostic impact for longer survival with improved accuracy. Among the four markers, when adjusted for the influence of other clinical factors in multivariate analysis, the TOP2B/TOP2A ratio was significantly correlated with treatment outcomes; patients with high ratios trended toward longer disease-free survival (HR, 0.24; P=0.002) and overall survival (HR, 0.29; P=0.005). CONCLUSION: Genes with distinct expression profiles such as TOP2B/TOP2A expression ratio at diagnosis can be employed for outcome prediction after the treatment with standard regimens in AML patients with M2 subtype.
Arivazhagan A, Kumar DM, Sagar V, et al. Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor. J Neurooncol. 2012; 107(2):289-97 [PubMed] Related Publications
The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.
Wang C, Yan L, Wang Y, et al. Overexpression of Lewis(y) antigen protects ovarian cancer RMG-1 cells from carboplatin-induced apoptosis by the upregulation of Topo-I and Topo-II β. Anat Rec (Hoboken). 2011; 294(6):961-9 [PubMed] Related Publications
Lewis (y) antigen, a difucosylated oligosaccharide, has been shown to be associated with malignant properties of ovarian carcinomas. In this study, we have investigated the potential role of Lewis (y) antigen, which was stably transfected into ovarian cancer RMG-1 cells, on carboplatin-induced apoptosis. Overexpression of Lewis (y) antigen effectively protected vitronectin-adherent RMG-1 cells from carboplatin-induced apoptosis as assessed by Hoechst 33258 staining and flow cytometry. Treatment with anti-Lewis (y) antigen, anti-integrin αv, or anti-integrin β3 antibody partially abolished the protective effect on apoptosis and markedly inhibited the expression of Topo-II β in cells overexpressing Lewis (y) antigen (all P < 0.01). Moreover, elevated expression of Topo-I and Topo-II β was found in Lewis (y) antigen-overexpressing cells (P < 0.01). However, no obvious changes in Topo-II α were observed throughout the study (P > 0.05). Taken together, these data suggest that the overexpression of Lewis (y) antigen confers cell adhesion-mediated drug resistance to apoptosis in ovarian cancer cells by the upregulation of Topo-I and Topo-II β. Therefore, the inhibition of Lewis (y) antigen may be a novel strategy of cancer chemotherapy.
An emerging model of transcriptional activation suggests that induction of transcriptional programs, for instance by stimulating prostate or breast cells with androgens or estrogens, respectively, involves the formation of DNA damage, including DNA double strand breaks (DSB), recruitment of DSB repair proteins, and movement of newly activated genes to transcription hubs. The DSB can be mediated by the class II topoisomerase TOP2B, which is recruited with the androgen receptor and estrogen receptor to regulatory sites on target genes and is apparently required for efficient transcriptional activation of these genes. These DSBs are recognized by the DNA repair machinery triggering the recruitment of repair proteins such as poly(ADP-ribose) polymerase 1 (PARP1), ATM, and DNA-dependent protein kinase (DNA-PK). If illegitimately repaired, such DSBs can seed the formation of genomic rearrangements like the TMPRSS2-ERG fusion oncogene in prostate cancer. Here, we hypothesize that these transcription-induced, TOP2B-mediated DSBs can also be exploited therapeutically and propose that, in hormone-dependent tumors like breast and prostate cancers, a hormone-cycling therapy, in combination with topoisomerase II poisons or inhibitors of the DNA repair components PARP1 and DNA-PK, could overwhelm cancer cells with transcription-associated DSBs. Such strategies may find particular utility in cancers, like prostate cancer, which show low proliferation rates, in which other chemotherapeutic strategies that target rapidly proliferating cells have had limited success.
Azarova AM, Lin RK, Tsai YC, et al. Genistein induces topoisomerase IIbeta- and proteasome-mediated DNA sequence rearrangements: Implications in infant leukemia. Biochem Biophys Res Commun. 2010; 399(1):66-71 [PubMed] Free Access to Full ArticleRelated Publications
Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2beta knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2alpha and Top2beta cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2beta in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, gamma-H2AX, was dependent on the Top2beta isozyme and proteasome activity. Fourth, the requirement for Top2beta and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2beta cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2beta-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2beta and proteasome in genistein-induced infant leukemia.
DNA double-strand breaks (DSBs) can lead to the development of genomic rearrangements, which are hallmarks of cancer. Fusions between TMPRSS2, encoding the transmembrane serine protease isoform 2, and ERG, encoding the v-ets erythroblastosis virus E26 oncogene homolog, are among the most common oncogenic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSBs. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process that required TOP2B and components of the DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia cells showed strong coexpression of androgen receptor and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSBs in generating TMPRSS2-ERG rearrangements.
Yu ZW, Zhong LP, Ji T, et al. MicroRNAs contribute to the chemoresistance of cisplatin in tongue squamous cell carcinoma lines. Oral Oncol. 2010; 46(4):317-22 [PubMed] Related Publications
MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. They are strongly implicated in human cancers, including oral squamous cell carcinoma (OSCC). Evidence for the involvement of miRNAs as important regulators of chemosensitivity and chemoresistance in OSCC is not well understood. In this study, miRNA microarray was firstly used to compare the differential miRNAs levels between the cisplatin-sensitive tongue squamous cell carcinoma line (Tca8113) and its cisplatin-resistant subline (Tca/cisplatin). Three miRNAs of miR-21, -214, and -23a were validated by miRNAs real-time PCR, and intervened by anti-miRNA oligonucleotides (miR-214 and -23a) and pre-miRNA plasmid transfection (miR-21). Further relationship between miR-23a and DNA topoisomerase II beta (TOP2B) on the chemoresistance against cisplatin was studied. There were 19 out of 480 differential miRNAs between the Tca8113 and Tca/cisplatin cells. miR-214 and -23a were found increased as with chemoresistance against cisplatin in the Tca/cisplatin cells while miR-21 was found decreased as with chemosensitivity for cisplatin in the Tca/cisplatin cells. Intervention of these three miRNAs could decrease the chemoresistance against cisplatin in Tca/cisplatin cells. Transfection of anti-miR-23a into the Tca/cisplatin cells could increase the TOP2B protein expression. Our results suggest the existence of differential miRNAs with chemosensitivity and chemoresistance between the cisplatin-sensitive and resistant tongue squamous cell carcinoma lines. miR-21 serves as a chemosensitive miRNA, while miR-214 and -23a serve as chemoresistant miRNAs in the tongue squamous cell carcinoma lines. miR-23a is an up-stream regulator of TOP2B to realize the chemoresistance of cisplatin.
McNamara S, Nichol JN, Wang H, Miller WH Targeting PKC delta-mediated topoisomerase II beta overexpression subverts the differentiation block in a retinoic acid-resistant APL cell line. Leukemia. 2010; 24(4):729-39 [PubMed] Related Publications
Retinoic acid (RA) relieves the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. However, RA treatment alone invariably results in RA resistance, both in vivo and in vitro. RA-resistant cell lines have been shown to serve as useful models for elucidation of mechanisms of resistance. Previously, we identified topoisomerase II beta (TOP2B) as a novel mediator of RA-resistance in APL cell lines. In this study, we show that both TOP2B protein stability and activity are regulated by a member of the protein kinase C (PRKC) family, PRKC delta (PRKCD). Co-treatment with a pharmacologic inhibitor of PRKCD and RA resulted in the induction of an RA responsive reporter construct, as well as the endogenous RA target genes, CEBPE, CYP26A1 and RIG-I. Furthermore, the co-treatment overcame the differentiation block in RA-resistant cells, as assessed by morphological analysis, restoration of promyelocytic leukemia nuclear bodies, induction of CD11c cell surface expression and an increase in nitro-blue-tetrazolium reduction. Cumulatively, our data suggest a model whereby inhibition of PRKCD decreases TOP2B protein levels, leading to a loss of TOP2B-mediated repressive effects on RA-induced transcription and granulocytic differentiation.
Györffy B, Lanczky A, Eklund AC, et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat. 2010; 123(3):725-31 [PubMed] Related Publications
Validating prognostic or predictive candidate genes in appropriately powered breast cancer cohorts are of utmost interest. Our aim was to develop an online tool to draw survival plots, which can be used to assess the relevance of the expression levels of various genes on the clinical outcome both in untreated and treated breast cancer patients. A background database was established using gene expression data and survival information of 1,809 patients downloaded from GEO (Affymetrix HGU133A and HGU133+2 microarrays). The median relapse free survival is 6.43 years, 968/1,231 patients are estrogen-receptor (ER) positive, and 190/1,369 are lymph-node positive. After quality control and normalization only probes present on both Affymetrix platforms were retained (n = 22,277). In order to analyze the prognostic value of a particular gene, the cohorts are divided into two groups according to the median (or upper/lower quartile) expression of the gene. The two groups can be compared in terms of relapse free survival, overall survival, and distant metastasis free survival. A survival curve is displayed, and the hazard ratio with 95% confidence intervals and logrank P value are calculated and displayed. Additionally, three subgroups of patients can be assessed: systematically untreated patients, endocrine-treated ER positive patients, and patients with a distribution of clinical characteristics representative of those seen in general clinical practice in the US. Web address: www.kmplot.com . We used this integrative data analysis tool to confirm the prognostic power of the proliferation-related genes TOP2A and TOP2B, MKI67, CCND2, CCND3, CCNDE2, as well as CDKN1A, and TK2. We also validated the capability of microarrays to determine estrogen receptor status in 1,231 patients. The tool is highly valuable for the preliminary assessment of biomarkers, especially for research groups with limited bioinformatic resources.
Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2alpha is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2beta is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2alpha-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2alpha and not Top2beta in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an alpha isoform-specific manner. Heterozygous disruption of the human TOP2alpha gene confers increased NK314 resistance, whereas TOP2beta homozygous knock-out cells display increased NK314 sensitivity, indicating that the alpha isoform is the cellular target. We further show that the absence of Top2beta does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2alpha-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents.
Nebral K, Schmidt HH, Haas OA, Strehl S NUP98 is fused to topoisomerase (DNA) IIbeta 180 kDa (TOP2B) in a patient with acute myeloid leukemia with a new t(3;11)(p24;p15). Clin Cancer Res. 2005; 11(18):6489-94 [PubMed] Related Publications
PURPOSE: The nucleoporin 98 kDa (NUP98) gene has been reported to be fused to 17 different partner genes in various hematologic malignancies with 11p15 aberrations. Cytogenetic analysis of an adult de novo acute myelogenous leukemia (M5a) revealed a t(3;11)(p24;p15), suggesting rearrangement of NUP98 with a novel partner gene. EXPERIMENTAL DESIGN: Fluorescence in situ hybridization (FISH) was used to confirm the involvement of NUP98 in the t(3;11)(p24;p15). Selection of possible NUP98 partner genes was done by computer-aided analysis of the 3p24 region using the University of California Santa Cruz genome browser. Fusion gene-specific FISH and reverse transcription-PCR analyses were done to verify the presence of the new NUP98 fusion. RESULTS: FISH analysis using a NUP98-specific clone showed a split signal, indicating that the NUP98 gene was affected by the translocation. Of the genes localized at 3p24, TOP2B was selected as a possible fusion partner candidate gene. Dual-color fusion gene-specific FISH and reverse transcription-PCR analysis verified that NUP98 was indeed fused to TOP2B. In addition to reciprocal NUP98-TOP2B and TOP2B-NUP98 in-frame fusion transcripts, an alternatively spliced out-of-frame TOP2B-NUP98 transcript that resulted in a premature stop codon was detected. Analysis of the genomic breakpoints revealed typical signs of nonhomologous end joining resulting from error-prone DNA repair. CONCLUSIONS: TOP2B encodes a type II topoisomerase, which is involved in DNA transcription, replication, recombination, and mitosis, and besides TOP1, represents the second NUP98 fusion partner gene that belongs to the topoisomerase gene family. This finding emphasizes the important role of topoisomerases in malignant transformation processes.
Vey N, Mozziconacci MJ, Groulet-Martinec A, et al. Identification of new classes among acute myelogenous leukaemias with normal karyotype using gene expression profiling. Oncogene. 2004; 23(58):9381-91 [PubMed] Related Publications
Conventional cytogenetic analysis currently stratifies acute myelogenous leukaemia (AML) into prognostically relevant groups. However, approximately 50% of adult AMLs have normal cytogenetics (NC-AMLs), and represent a heterogeneous and poorly understood group. We analysed gene expression in 55 AML samples including 53 cases from adult patients with NC-AML (n = 36), trisomy 8, t(15;17), t(8;21), t(11;19), 7q deletion, and two cell lines using 9000-gene DNA microarrays. Global hierarchical clustering showed that NC-AMLs are a heterogeneous group. Supervised analysis distinguished two subgroups of NC-AML: one subgroup constituted a homogeneous NC cluster ('pure NC-AML'), and the other NC-AMLs were close to the AML cases with translocations ('translocation like'). Gene expression signatures were also derived for patients with trisomy 8, as well as FLT3 and MLL gene duplications. Importantly, samples from 24 NC-AML patients who could be evaluated for clinical outcome were analysed. In all, 43 genes that discriminated two classes of patients with significantly different prognosis were identified. The poor prognosis class contained a majority of 'pure NC-AMLs', whereas the 'translocation-like' AMLs were in the good prognosis class. Discriminator genes included genes involved in drug resistance (TOP2B), protein transport (MTX2, SLC35A2), and cell signalling (MAPK1, PRKAB2). Our results demonstrate the transcriptional heterogeneity of NC-AMLs, and suggest the existence of 'translocation-like' NC-AMLs and of a gene expression signature that may predict response to chemotherapy.