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theses

PAK4, belonging to Group II PAKs, is an important signaling protein with key roles in regulating cell growth, cell survival and cell morphology. PAK4 protein levels are higher in several subtypes of breast cancer cells and PAK4 gene is amplified in basal like breast cancer. PAK4 overexpression in a mouse mammary epithelial cell line (iMMEC) results in oncogenic transformation of these cells, while siRNA mediated PAK4 down regulation in a human triple negative breast cancer cell line, MDA-MB-231, resulted in significant inhibition of its tumorigenic potential. These results point to a central role for PAK4 in mammary tumorigenesis. PAK4 expression pattern makes it an attractive drug target, however, it is important to note that PAK4 has both kinase-dependent and – independent functions in regulating tumor formation. Hence, blocking its kinase activity alone is insufficient in blocking its tumorigenic potential. Our lab collaborated with Karyopharm Therapeutics to investigate a novel PAK4 inhibitor, KPT-9274. I observed that treatment with KPT-9274 and KPT-8752 (isoform of KPT-9274) inhibited the cell growth, cell survival and cell motility of breast cancer cell lines, with this effect most significantly observed in triple negative breast cancer cell lines. I observed that treatment with KPT inhibitors inhibited PAK4 protein levels along with PAK4 associated downstream signaling pathways in triple negative breast cancer cell lines. Most importantly, I observed that KPT- 9274 significantly inhibited tumor growth in mouse xenograft models of 3 human triple negative breast cancer cell lines. KPT-9274 was capable of reducing steady state PAK4 protein levels in vivo, without significantly affecting PAK1 protein levels, indicating that KPT-9274 exhibits in vivo PAK4 specificity. This study shows that PAK4 can serve as a novel drug target in triple negative breast cancer therapy and KPT-9274 can have clinical benefits for the triple negative breast cancer population. Next, I analyzed RNA-seq data of samples collected from non-transformed WT iMMECs and iMMECs overexpressing PAK4, that form tumors in mice. Sequencing analysis identified several genes previously unknown to be regulated by PAK4. Using q-PCR, I was able to validate the RNAseq data, further suggesting that RNA-seq is a promising and reproducible tool to study PAK4 induced transcriptional changes. This study reveals the PAK4 transcriptome profile in mammary tumor forming cells, and can provide translational utility in other types of cancers as well. Delineating the varied effectors of PAK4 signaling cascade will help uncover novel biomarkers for cancer, with some serving as potential therapeutic targets.

ETD_8258

Ph.D.

Includes bibliographical references

by Chetan K. Rane

doi:10.7282/T3V127WH

ETD doctoral

The author owns the copyright to this work.