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Date of Award

Summer 8-15-2013

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The Wnt/β-catenin (β-cat) signaling cascade is essential to many fundamental processes. Perturbation of this pathway results in various diseases, including colorectal cancer (CRC). Previously, our group identified VEGFR-1 as a positive regulator of β-cat signaling. As such, this study aims to further explore VEGFR-1-mediated regulation of β-cat signaling in CRC, specifically examining Tyr phosphorylation of β-cat. We monitored changes in a β-cat/TCF transcriptional reporter, TOPflash, in CRC cells after pharmacological inhibition and genetic knockdown of VEGFR-1 activity. A proteomic approach, in conjunction with mutagenesis analysis, was employed to map novel β-cat phosphorylation sites targeted by VEGFR-1 tyrosine kinase activity. Additionally, a β-cat firefly luciferase reporter (β-cat-FLuc) was used to evaluate changes in β-cat stabilization in response to inhibition of VEGFR-1 activity. Finally, we monitored phenotypic changes in CRC cells subjected to VEGFR-1 knockdown. The data confirmed that VEGFR-1 is a positive modulator of β-cat signaling by showing attenuation of TF reporter signals after abrogating VEGFR-1 activity. An in vitro kinase (IVK) assay demonstrated that β-cat could be a direct substrate of VEGFR-1 and physical association of the two proteins was confirmed by co-immunoprecipitation. Collectively, proteomics, mutagenesis analysis and functional assays identified pTyr709 and pTyr716 as phospho-specific targets of VEGFR-1. Phosphorylations at these sites were critical for β-cat signaling activities and might affect stabilization of β-cat. Finally, VEGFR-1 knockdown induced reductions in cell migration rates and cell viability due to an increase in apoptosis in CRC cells. In summary, our data suggest that VEGFR-1 impacts stabilization of β-cat in a Tyr phosphorylation-dependent manner, which involves, at least, phosphorylation of Tyr709 and Tyr716 of β-cat. Importantly, VEGFR-1 per se could be a potential therapeutic target for refractory CRC.

Language

English (en)

Chair and Committee

David Piwnica-Worms

Committee Members

Ron Bose, Sergej Djuranovic, Fanxin Long, Jason Weber, Zhongsheng You

Comments

Permanent URL: https://doi.org/10.7936/K72N50QQ

Available for download on Tuesday, August 15, 2113

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