摘要:實驗室長遠的目標在鑑定抗DNA拓樸異構酶藥物的選擇性殺腫瘤特性(tumor-selective killing)之分子機制及研究拓樸異構酶相關的生理功能。在研究人類DNA拓樸異構酶III同功酶(hTOP3 and hTOP3細胞功能時意外發現hTOP3可能具有腫瘤壓制因子(tumor-suppressor-like)的特性;在前計劃執行期間,我們利用RNA干擾技術(RNAi)成功地在細胞中將hTOP3的表達降低,建立hTOP3缺失的細胞株;在細胞功能的測試實驗中我們觀察到hTOP3缺失的細胞株會有細胞檢控點的缺失及染色體數目異常的表現型發生,而其腫瘤與轉移特性卻大大地提昇;另外,過量表達hTOP3則造成癌細胞腫瘤發生表現型之喪失,此腫瘤壓制能力與已知的腫瘤壓制因子p53主導之途徑有關;而且我們的實驗結果也指出在大部分腫瘤細胞中的hTOP3表現量相對於正常細胞的表現量較少;因此我們假設hTOP3為腫瘤壓制因子。本計劃的目標便將專注在研究人hTOP3在癌化過程或腫瘤發生(tumorigenesis)上扮演之角色及其壓制腫瘤發生的作用機制;其中將特別研究p53在DNA拓樸異構酶IIIalpha抑制細胞癌化過程之重要性。在一九九九年時,人類腫瘤生物學上產生了重大突破,科學家們利用特定的一組基因,包括端粒酶(hTERT telomerase)及致癌因子(oncogenic alleles, e.g. H-RASV12) ,可以將人類正常細胞轉型成腫瘤細胞,建立了in vitro的腫瘤發生實驗系統(in vitro neoplastic transformation experimental system);我們計畫利用基因取代法(gene transfer)和RNAi分子技術來建立與人類癌化過程有直接相關的細胞老化(cellular senescence)和in vitro的腫瘤發生實驗系統,再配合腫瘤發生分析實驗來分析hTOP3在癌化過程或腫瘤發生上扮演之角色及其作用機制;我們亦會此腫瘤發生實驗系統來初步鑑定抗拓樸酶藥物的殺腫瘤選擇性與致癌因子的相關性。本計劃主要的目標有下列三點:(1) 提供hTOP3為腫瘤壓制因子的實驗證據;(2) 探討p53在DNA拓樸異構酶IIIalpha抑制細胞癌化參與的角色;(3)研究hTOP3在腫瘤抑制上之分子機制及其運用。 此計劃預期將開啟腫瘤學研究的新視野,所得相關資訊亦可提供腫瘤藥品設計開發的前瞻性觀念。
Abstract: The long-term objective for our lab is to understand the molecular basis for the action mechanism of and potential tumor selectivity for DNA topoisomerase-targeting drugs as well as to investigate cellular and physiological functions of topoisomerases. In the previous studies, we have revealed the involvement of hTOP3 in DNA damage checkpoints and potentially in the tumorigenic process. We showed that ectopic hTOP3 expression and RNA interference (RNAi)-mediated knockdown of hTOP3 expression in cancer cells conferred reduction and enhancement of tumorigenic propensity, respectively, suggesting hTOP3 might act as an anti-cancer block. Consistent with the direct involvement of hTOP3 in tumorigenesis, knockdown of hTOP3 expression in cell lines with ectopic hTOP3 expression regained cellular tumorigenic ability. Most strikingly, hTOP3 deficiency, like oncogenic RAS, converted immortalized RHEK cells into tumor cells. Toward understanding of the underlying mechanism(s) responsible for the tumor-suppressive activity of hTOP3, we have found the potential contribution of a known tumor suppressor p53 and hTOP3 deficiency, like p53 in-activation, resulted in great reduction in cellular senescence. Our results also showed a novel protein-protein interaction between hTOP3 and p53. Therefore, it would be of our great interest to investigate the potential contribution of p53 to the tumor-suppressive activity of hTOP3. Recent advances in genetic manipulations, coupled with primary human cells, had allowed the construction of several experimental in vitro tumorigenesis models. With substitution of other genes, siRNAs and microRNAs for the established SV40ER, hTERT and H-RAS, these experimental models have led to the identification of novel molecules and pathways during human cell transformation. Therefore, in this grant application, we also proposed to construct the functional genetic and experimental models of human cancers in primary human epithelial cells and fibroblast with serial introductions of cooperative oncogenes and/or RNAi-mediated inactivation of tumor suppressors. The established in vitro oncogenic models will be used not only to further explore the molecular mechanism(s) underlying the undefined tumor-blocking role of hTOP3, but also to dissect the oncogenic selectivity for topoisomerase-targeting drugs based on defined tumor genetic makeup. The specific aims are as following: 1. To firmly establish hTOP3 as an anti-cancer block: The states of oncogenic pathways and DNA damage checkpoints will be first examined in established cell lines with different levels of hTOP3. The cooperative relationship between hTOP3, p53 and known oncogenic alleles (e.g. H-RASV12) will also be examined. Moreover, the truncated and anti-sense hTOP3 constructs, which have been previously shown to complement phenotypes of ATM kinase-defected cells, will also be generated to provide further supports for functions of hTOP3 in checkpoint and tumor suppression. The requirement of enzymatic activity and functional domains of hTOP3 will also be determined. 2. To investigate the roles of p53 in the tumor-suppressive activity of hTOP3: The potential roles of hTOP3 and its dependence on ARF-p53 and/or RB-p16 will first be evaluated with different senescence models. Specifically, models with expression of oncogenes (e.g. H-RASV12, mos, BRAF600) and replication proteins (e.g. Cdc6, cyclin E) will be established in both primary human fibroblast and epithelial cells. In addition, recombinant proteins will be used to validate the direction interaction between p53 and hTOP3 and their activity impacts on each other. 3. To explore the underlying mechanism(s) of hTOP3 as a tumor suppressor using in vitro tumorigenesis model: Here, we focus on molecularly dissecting the oncogenic pathways responsible for the tumor-suppressive activity hTOP3 by taking advantage of the recent developed in vitro neoplastic transformation model. Both senescence and tumorigenesis assays will be performed. Moreover, the genotypic selectivity associated with defined human oncogenic alterations specific for the cytotoxic action of topoisomerase-targeting drugs will also be explored. Previous reports have shown that the Bloom's syndrome gene product, BLM, forms a direct physical association with the human topoisomerase III isozyme. the phenotype of RecQ helicase mutants may be at least partially the consequence of a functional impairment of topoisomerase III. The fact that mutations in human BLM helicases give rise to cancer-prone disorders therefore raises the possibility that the gene encoding hTOPO III may also be a tumor suppressor gene. Specifically, t Consistent with this notion, the BLM tumor suppressor. Genome instability, through an increased rate of genomic alterations, enables the accumulation of sufficient ‘hits’ for the multi-step tumorigenesis. It not only underlies many malignant properties of tumors and the ability to escape therapy, but also it might itself serve as an important contributing factor during cancer development. The understanding of human cancer and the ability to treat and prevent it depends largely on our knowledge of the mechanisms and pathways utilized during tumor development/tumorigenesis. In the previous grant period (01/01/06 – 12/31/08), we studied the potential role(s) of human DNA topoisomerase III isozymes (hTOP3 and 3) in the regulation of cell cycle checkpoint and chromatin structure. Unexpectedly, hTOP3-deficiency led to not only chromosome instability (CIN) but enhanced tumorigenic propensity, suggesting hTOP3 might serve as an anticancer block possibly through maintenance of genome integrity. Interestingly, both genomic instability and cytidine deamination have been shown to be abnormally high in pre-cancerous tissues and cancer cells. Our group has also recently demonstrated the involvement of topoisomerases in regulating the activation-induced cytidine deaminase (AID)-mediated mutagenesis. However, whether genomic instability and/or other mechanism(s) enhanced by forced-expression of AID or hTOP3-deficiency are responsible for their roles in tumorigenesis remains to be determined. Recent progress in combining genetic manipulations and primary human cells has allowed the construction of experimental in vitro cell models for human tumorigenesis, which subsequently led to identify novel molecules as well as pathways during human cell transformation. Therefore, in this grant application, we proposed to construct the functional genetic and experimental models of human cancers with serial introductions of cooperative oncogenes and/or downregulated tumor suppressor genes into primary human cells. Specifically, the established in vitro models will be used to investigate the underlying molecular mechanism(s) that might be responsible for the undefined tumorigeneic role of hTOP3. The contribution of the enhanced genomic instability mediated by forced expression of activation-induced cytidine deaminase (AID) to tumorigenesis will also be evaluated in these experimental models. Moreover, these engineered, paired cancer cell lines will also be utilized to investigate the tumor selectivity for anti-topoisomerase drugs based on defined tumor genetic makeup. The specific aims are as following: 1. To tailor the drug selectivity into different oncogenic settings during tumorigenesis. The oncogene-induced senescence and in vitro tumorigenesis models with defined genetic elements will be established. The gene expression of various DNA topoisomerase, AID as well as tumor-selectivity-associated molecules (e.g. ISG15) in these established paired cancer cell lines will be determined. In addition, the genotype selectivity of oncogenic alterations for cytotoxic action of topoisomerase-targeting drugs can be similarly resolved. 2. To explore the underlying molecular mechanism(s) of hTOP3 as a tumor suppressor using gene expression profile analysis and in vitro experimental tumorigenesis model. Here, we will try to identify the potential hTOP3-targeting genes and explore their relationship with the tumorigenic role of hTOP3. Specific experimental designs with the recent developed in vitro neoplastic transformation model will be employed to investigate the potential tumor-suppressive role of hTOP3. Both senescence and tumorigenesis assays will be performed. 3. To examine whether unscheduled AID-mediated mutagenesis leads to cancer development. The contribution of potential mutagenic activity of AID in oncogenic transformation will be specifically examined in vitro. Specific focus will be first placed on mutations conferring loss-of-function of p53 oncogenic pathways. In addition, the molecular pathways involved in abnormal expression of AID in different engineered oncogenic settings and pathological viral-related conditions will also be investigated.