CSIR Central

Studies on the regulation of expression of CDC20 and search for its new molecular interactors

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata


Field	Value

Title	Studies on the regulation of expression of CDC20 and search for its new molecular interactors

Creator	Das, Tania

Subject	Molecular & Human Genetics

Description	Advancement in the understanding of the molecular basis of cancer has led to the emergence of tumor-specific, molecularly targeted agents that specifically kill malignant cells more efficiently than ever. A number of cell signaling molecules, cellcycle regulators, metabolic enzymes and cell surface macromolecules are currently considered as potential targets for the development of novel therapeutic agents for cancer treatment. Cdc20 a key spindle assembly checkpoint (SAC) protein, activates APC/C during mitosis. Therefore, the major function of Cdc20 is to coordinate mitotic progression by facilitating the orderly degradation of mitotic APC/Cb substrates. Deregulation of Cdc20 causes erroneous mitotic division followed by chromosomal instability (CIN). CIN is the hallmark of cancer. It is observed that the overexpression of CDC20 occurs in several oral cell lines and primary head and neck tumors and provide evidence that such overexpression of CDC20 is associated with premature anaphase promotion, resulting in chromosome abnormalities in the cell lines. More over invitro experiments have also shown that mitotic instability may be a mechanism for developing resistance to cytotoxic drugs. Thus proper Cdc20 expression and function plays a crucial role in the context of the disease like cancer. In this study we investigated first, the transcriptional regulation of Cdc20, second Cdc20 mediated regulation of other cell cycle genes and finally we tried to establish the Cdc20–Mad2 complex as a potential therapeutic target. As a result we proposed for the first time, an oncogenic regulation of Cdc20. We showed that the protooncogene Myb transctivates CDC20 in colorectal cancer cells and also in tumour patient samples. We also reported that Cdc20 may negatively regulate its own expression when the endogenous Cdc20 level goes up. We hypothesized that this occurs probably due to maintain its own balance, since the proper level of Cdc20 is crucial for maintaining the genomic stability of the daughter cells. To validate our hypothesis we rescued the Myb mediated trans-activation of CDC20 from the Cdc20 auto inhibition, by lowering the endogenous Cdc20 level in the cell. More over we showed that the Myb trans-activation response site and the Cdc20 auto-inhibition response site resides very close to each other within 71 bp upstream of CDC20 transcription start site. All these evidences confers that the transcription of CDC20 is a dual regulated phenomena operated by Myb and Cdc20 itself and the mode of Summary - VIII - operation depends upon the endogenous level of the Cdc20 protein. We also found the involvement of p300 in Myb mediated trans-activation. As well as HDAC1 take part in Cdc20-auto-inhibition. Additionally ubiquitination has a role in the autoregulation of Cdc20. Unubiquitinated Cdc20 protein was found to be more prone for its own inhibition. Finally to assess the fuctional significance of this oncogenic regulation of Cdc20, we showed that over-expression of Myb leads to premature anaphase by over expressing CDC20, followed by deregulation of SAC. Cdc20 is reported to participate in the transcriptional regulation of mitotic genes by CBP/p300 complex. Further, we have dissected the transcriptional role of Cdc20 protein in a global aspect. We performed the whole genome microarray analysis in the altered Cdc20 expressing condition in HepG2 cells and found 252 differentially expressed genes. The altered genes mainly belong to cell cycle and apoptotic pathways. We found Rb, an important tumor suppressor gene is one of the major downstream target of Cdc20. We have validated our observation in human cancer cell line. Finally we established Withaferin A (WA) as an inhibitor of Cdc20-Mad2 complex in colorectal cancer cells. It causes spindle assembly checkpoint (SAC) arrest in HCT116 and SW480 colorectal adenocarcinoma cell lines by stabilizing Cyclin B1 and its downstream molecules. Further we dissected the mechanism of action of WA in these cell lines. We found that WA leads to proteasomal degradation of the Cdc20- Mad2 complex that actually causes SAC arrest and apoptosis, as consequences. We also rescued the Cyclin B1 degradation and the cell proliferation by over expression of Mad2 in presence of WA. Together the experimental results suggests that WA treated cells are arrested at the metaphase allowing sufficient time to activate the apoptotic machinery leading to mitotic death of the cells. Collectively this continuing effort is being made to identify Cdc20 as a new oncogenic lead based on the knowledge drawn from the molecular analysis of cancer cell lines and primary tumor that will enrich the repertoire of new molecular targets for cancer management. Part of this work has been published in Biochemical Pharmacology, 91 (2014) 31–39

Date	2015

Type	Thesis NonPeerReviewed

Format	application/pdf

Identifier	http://www.eprints.iicb.res.in/2108/1/Thesis_Complete__Tania_Das.pdf Das, Tania (2015) Studies on the regulation of expression of CDC20 and search for its new molecular interactors. PhD thesis, JU.

Relation	http://www.eprints.iicb.res.in/2108/