Date of Graduation
Doctor of Philosophy (PhD)
Gary E. Gallick, Ph.D.
Sue-Hwa Lin, Ph.D.
David J. McConkey, Ph.D.
Bradley W. McIntyre, Ph.D.
Jeffrey N. Myers, M.D., Ph.D.
Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind and activate integrins but the mechanism by which this occurs is unknown. As integrin activation and overexpression of talins promote prostate cancer metastasis, understanding the mechanism by which talins activate integrins will better elucidate their roles in Prostate cancer metastasis. Phosphorylation of talins on serine 425 has been associated with β1 integrin functions. Work in this dissertation tested the hypothesis that increased talin1 S425 phosphorylation was required for β1 integrin activation and promotion of prostate cancer metastasis.
I first used shRNA to knockdown talins and demonstrated that talin1, but not talin2, is required for β1 integrin activation. Furthermore, talin1 S425 phosphorylation, but not total talin1 expression, correlated with metastatic potential of cultured prostate cancer cells. To directly test whether talin1 phosphorylation activates β1 integrins, I generated talin1 S425 mutants. Expressing a non-phosphorylatable mutant, talin1S425A in talin1-silenced PC3-MM2 and C4-2B4 prostate cancer cells decreased β1 integrin activation, adhesion, motility, and increased the sensitivity of cells to anoikis. In contrast, re-expression of the phosphorylation-mimicking mutant, talin1S425D led to increased β1 integrin activation and generated biologic effects opposite to talin1S425A expression. Mechanistically, I demonstrated that increased Cdk5 kinase activity correlates with expression of its activator p35, and is responsible for talin1 phosphorylation and β1 integrin activation.
Next, I demonstrated that talin1 phosphorylation is required for Prostate cancer bone metastasis in vivo. In the metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1S425A, in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in bone following intracardiac injection, while re-expression of phosphorylation-mimicking mutant, talin1S425D restored their ability to metastasize to bone. I further demonstrated by immunohistochemical staining that talin phosphorylation is significantly increased in human bone metastases when compared to normal tissues, primary tumors, or lymph node metastases, demonstrating the clinical relevance of talin S425 phosphorylation.
In summary, I have identified a novel signaling pathway whereby overexpression of p35 leads to increased Cdk5 activity, resulting in talin phosphorylation and β1 integrin activation. This signaling axis is responsible for increased metastatic potential of prostate cancer cells. Therefore, talin1, Cdk5 and p35 may be new targets for development of therapies for prostate cancer bone metastasis.
Cdk5, integrin, metastasis, p35, prostate cancer, talin