TARGETING PROTEIN KINASE D BY NOVEL SMALL MOLECULE INHIBITORS AND RNA INTERFERENCE IN PROSTATE CANCER

LAVALLE, COURTNEY TARGETING PROTEIN KINASE D BY NOVEL SMALL MOLECULE INHIBITORS AND RNA INTERFERENCE IN PROSTATE CANCER. Doctoral Dissertation, University of Pittsburgh.

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Abstract

Protein kinase D (PKD) has been implicated in a variety of cellular processes and pathological conditions including cancer. However, targeting PKD therapeutically and dissecting PKD-mediated cellular responses remains difficult due to lack of a potent and selective inhibitor. Here, we report the discovery of a novel class of pan-PKD inhibitors, CID755673 and its analogs. Subsequently, we use these inhibitors in conjunction with RNA interference technology to show that PKD regulates prostate cancer cell growth and motility. CID755673 was discovered in collaboration with our colleagues at the University of Pittsburgh as a compound demonstrating nanomolar potency and high selectivity for PKD inhibition. To enhance its selectivity and potency for potential in vivo application, several analogs of CID755673 were generated. After initial activity screening, 5 analogs having equal or greater in vitro potencies as CID755673 were chosen for further analysis. Our data showed that modifications to the aromatic core structure significantly increased potency while retaining high specificity for PKD. In prostate cancer cells, all compounds inhibited phorbol 12-myristate 13-acetate (PMA)-induced autophosphorylation of PKD1, with kb-NB142-70 being most active. Importantly, these inhibitors caused a dramatic arrest in cancer cell proliferation. Migration and invasion were also inhibited by this class of compounds, with varying potencies that correlated to their cellular activity, suggesting an active role for PKD in these processes. To confirm PKD involvement in prostate cancer biology, we used short hairpin RNA (shRNA)- and small interfering RNA (siRNA)-mediated knockdown of specific PKD isoforms, demonstrating that knockdown of PKD2 and/or PKD3 significantly reduces proliferation, migration, and invasion in metastatic PC3 prostate cancer cells. We also found that inhibition of PKD expression or activity decreases secretion of several key tumor-promoting factors including matrix metalloproteinase (MMP)-9, interleukin (IL)-6, IL-8, and growth-regulated oncogene α (GROα). Finally, we demonstrated that inducible knockdown of PKD3 in both subcutaneous and orthotopic xenograft models leads to reduced prostate tumor growth. Taken together, these data provide much-needed pharmacological tools for the study of PKD function, validate PKD as a promising therapeutic target in prostate cancer treatment, and broaden our understanding of the molecular mechanisms of PKD function in prostate cancer progression.

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