Display Full Text | Download Full Text
3.91 MB PDF file

Degree

Doctor of Philosophy, Ohio State University, Biochemistry, 2007.

Abstract

Follicular cell derived thyroid cancer is the most common endocrine malignancy. While patients with diagnosed with early stage disease have an excellent prognosis, patients with invasive or metastatic thyroid cancer have poor survival rates. Because progressive thyroid cancer is unresponsive to chemotherapy, there is a critical need to identify novel therapeutic targets. Genetic alterations that result in enhanced activation of the RAS-RAF and PI3K-AKT pathways occur in more than 50% of thyroid cancers. However, the key regulators of thyroid cancer invasion and metastases are less certain. We report here that OSU-03012 (OSU) inhibited proliferation and induced cytotoxicity at doses sufficient to inhibit PDK-1 mediated AKT phosphorylation in NPA, WRO, and ARO. OSU inhibited cell motility in NPA cells at doses below its IC50 for PDK-1 and below those sufficient to reduce AKT phosphorylation, suggesting another mechanism for the inhibition of migration. p21 activated kinases (PAK) are regulators of cell motility that are regulated by PDK1. Phospho-PAK levels were reduced with 1 µM of OSU in motile NPA cells and a reduced phospho-vimentin was detected at similar doses. Further studies demonstrated that OSU competitively inhibits ATP binding to PAK. Finally, overexpression of constitutively active-PAK1 rescued the anti-migratory effects of OSU. We determine if PAK expression or activity were altered in thyroid cancer. Protein and total RNA was isolated from ten PTC tissue samples and from the normal tissue in the opposite lobes for Western blot and quantitative RT-PCR of all PAK isoforms. PAK2 and PAK4 mRNA and protein expression levels were increased in the majority of PTCs. In addition, the levels of phosphorylated PAK were higher in 8 of 10 PTCs. PAK is known to functionally phosphorylate C-RAF at serine 338. In B-RAF, nine potential PAK phosphorylation sites were found. One site aligned to serine 338 in C-RAF. Subsequent experiments demonstrated that B-RAF and PAK co-immunoprecipitate. Further studies are planned to clarify if the BRAF-PAK interactions are functional and to identify specific phosphorylation sites. Taken together, these data support a role for PAK activity in thyroid cancer and suggest it might be an appropriate therapeutic target for this disease in patients.

Subject Headings

Chemistry, Biochemistry

Keywords

Thyroid Cancer; p21-activated kinase; B-RAF; OSU03012; HDAC42; migrtaion

Advisor

Matthew D Ringel

Pages

191p.

Document number: osu1189790066
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1189790066

This ETD has been downloaded 506 times (through March 2013)