Breast cancer is a heterogeneous disease and remains the most common malignancy among women worldwide. The majority of deaths from breast cancer are due to the metastasis of tumor cells into other organs in the body. RhoGTPases are a family of hydrolase enzymes that play an essential role in breast cancer cell metastasis. Group II PAKs (PAK4, PAK5, PAK6) are effector proteins downstream of the Rho GTPases. They have been implicated in many cellular processes, including cytoskeleton organization, cell proliferation, survival, and metastasis. PAK6 is the less studied member of group II PAKs. To investigate the role of PAK6 in breast cancer cell morphology and migration, three complementary approaches were adopted; CRISPR technology, mutational studies, and 14- 3-3 interaction studies. Despite extensive experimental methods, PAK6 knockout clones were not derived; however, the expression of PAK6 was depleted using CRISPR technology. Data presented here suggest that knockout of PAK6 expression is deleterious to cell survival. Nevertheless, the CRISPR approach did yield a decrease in the expression of PAK6, which demonstrated that depletion of PAK6 expression induced MDA-MB-231 cell elongation and migration. To further explore the role of PAK6 in breast cancer progression, patient mutations were exploited. The modeling of PAK6 mutations taken from patient samples found that PAK6 patient mutation (R457G) decreased MDA-MB-231 cell migration and increased cell circularity. In addition, the overexpression of active PAK6 (S531N) induced MDA-MB-231 cell migration and rounding through RhoA/ROCK, and it was associated with the phosphorylation of FilaminA at Ser2152. FilaminA was identified to interact with PAK6, and therefore it could be a substrate for PAK6. PAK6 was also determined to interact with 14-3-3 Gamma and 14-3-3 Epsilon through amino acids Thr99 and Ser113. A PAK6 (T99A/S113A) mutant induced MDA-MB-231 cell rounding through the RhoA/ROCK pathway. This was associated with decreasing the phosphorylation of ezrin at Thr566 and thus inactivates it, a novel pathway in breast cancer. Taken together, these results suggest that PAK6 may affect cell morphology and migration via manipulation of Rho GTPase signaling.
Date of Award | 1 Aug 2022 |
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Original language | English |
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Awarding Institution | |
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Supervisor | Claire Wells (Supervisor) & Jeremy Carlton (Supervisor) |
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The Role of PAK6 in Breast Cancer Cell Morphology and Migration
Kuwair, L. (Author). 1 Aug 2022
Student thesis: Doctoral Thesis › Doctor of Philosophy