Mechanisms of Ect2 Regulation in Cytokinesis and Oncogenesis
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Cook, Danielle. Mechanisms of Ect2 Regulation In Cytokinesis and Oncogenesis. University of North Carolina at Chapel Hill, 2013. https://doi.org/10.17615/74w5-s310APA
Cook, D. (2013). Mechanisms of Ect2 Regulation in Cytokinesis and Oncogenesis. University of North Carolina at Chapel Hill. https://doi.org/10.17615/74w5-s310Chicago
Cook, Danielle. 2013. Mechanisms of Ect2 Regulation In Cytokinesis and Oncogenesis. University of North Carolina at Chapel Hill. https://doi.org/10.17615/74w5-s310- Last Modified
- March 22, 2019
- Creator
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Cook, Danielle
- Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
- Abstract
- Ect2 is a member of the human Dbl family of guanine nucleotide exchange factors and are activators of Rho family small GTPases (RhoA, Rac1 and Cdc42). Ect2 was identified originally as an oncogene. However, the truncated and activated Ect2 variant identified was due to a DNA manipulation artifact and to date has not been found in human cancers. Ect2 has also been shown to be essential for mammalian cell cytokinesis in established cell culture models. My studies involve further evaluation of Ect2 in normal and neoplastic cell biology. First, to further evaluate a role for Ect2 in cytokinesis, we evaluated the consequences of an Ect2 deficiency in normal cells. Our studies showed that an Ect2 deficiency in primary mouse embryo fibroblasts (MEFs) caused a defect in cytokinesis, migration, and altered cell morphology in vitro. In vivo loss of Ect2 in a mouse caused embryonic lethality. Thus, Ect2 is essential for normal development; a characteristic that has only been found in two other Dbl family RhoGEFs of 25 members evaluated. Second, in a gene array analysis of colorectal cancer (CRC), we identified ECT2 transcriptional overexpression and further analysis of oncomine database also confirmed the transcriptional overexpression of Ect but was not due gene amplification. We then found an increase in Ect2 protein expression in primary and metastatic CRC tumors and cell lines. Depletion of endogenous Ect2 by shRNA in CRC cell lines caused a reduction in anchorage-independent growth and Matrigel invasion without a corresponding defect in cytokinesis. Analyses of Ect2 protein expression in a CRC tumor microarray (N=149) found Ect2 protein overexpression in tumor tissue, but surprisingly, the ratio of cytoplasmic to nuclear Ect2 correlated with improved patient survival. This finding contrasted with earlier studies that suggested that the mislocalization of the normally nuclear restricted Ect2 to the cytoplasm was important for Ect2 to function as an oncogene. With this unexpected finding, I then utilizing my validated MEF and CRC cell line models to assess the structural and functional requirements for Ect2 support of normal cell cytokinesis and cancer cell growth. Ect2 is a large multidomain protein. In addition to tandem Dbl homology RhoGEF catalytic and pleckstrin homology regulatory domains found in all Dbl RhoGEFs (>70 human members), Ect2 contains N-terminal tandem BRCT domains not found in any other RhoGEFs and two nuclear localization sequences that promote Ect2 nuclear localization. Using missense mutants of full length Ect2 with impaired domain/motif function, I found that the BRCT domains are dispensable for Ect2-dependent CRC growth but necessary for normal cell cytokinesis. In contrast, disruption of the Ect2 nuclear localization sequences abolished the ability of Ect2 to support both normal cell cytokinesis and CRC tumor cell growth. The former observation was unexpected since there is no nuclear envelope during cytokinesis. The latter finding was also surprising, contrasting with previous findings in lung cancer, but consistent with our findings in CRC tumor tissue microarray. In particular, we found that the cytoplasmic localized, constitutively activated Ect2 that potently causes growth transformation of NIH 3T3 mouse fibroblasts was deleterious for CRC growth. In summary, my studies may resolve a paradox in the field, to explain why the originally truncated and activated Ect2 identified in mouse fibroblasts have not been found in human cancer.
- Date of publication
- May 2013
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- In Copyright
- Advisor
- Der, Channing
- Degree
- Doctor of Philosophy
- Graduation year
- 2013
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