Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/26158 |
| Date | 15 February 2011 |
| Creators | Charames, George Shawn |
| Contributors | Bapat, Bharati |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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