Abstract 1571: Decrypting the transcriptome profile of Pak4 using next generation sequencing

Abstract

Abstract The PAK4 (p-21 Activated Kinase 4) protein kinase has long been associated with cancer because of its key roles in regulating cell proliferation, cell cycle progression and cell morphology. The PAK4 gene is amplified in different types of cancers, including breast cancer. PAK4 overexpression is associated with oncogenic transformation in several breast cancer cell lines, while PAK4 inhibition has been shown to significantly reduce their tumorigenic potential. However, there is limited information available on PAK4’s mechanism of action in promoting tumorigenesis. To gain an insight into PAK4 downstream signaling pathways, we performed Next Generation Sequencing (NGS) on RNA samples collected from non-transformed immortalized mouse mammary epithelial cells (WT iMMECs) and iMMECs overexpressing PAK4. Research from our lab has shown that unlike WT iMMECs, iMMECs overexpressing PAK4 formed tumors when injected into mammary fat pads of mice, suggesting a central role for PAK4 in tumor formation. Previous studies to delineate PAK4 signaling pathways have mostly focused on substrates phosphorylated by PAK4. This study, however, takes into account genes which may act downstream of PAK4 and play an important role in mediating long term PAK4 function. RNA-seq analysis offers the ability to discover new genes and splice variants and measure transcript expression. mRNA was isolated in triplicates from WT iMMECs and iMMECs overexpressing PAK4; mRNA from each condition was then sequenced using Illumina NextSeq platform. The RNA-seq data generated was analyzed using Top Hat and Cufflinks software, which created a list of genes whose expression was significantly different in these two cell types, and previously unknown to be regulated by PAK4 (26 genes were up regulated and 51 genes were down regulated by more than a log2 fold change of 3). A qPCR analysis of 8 of these genes validated the sequencing data. Among the different genes we identified, we chose to focus on ParvB, which is consistently down regulated by almost seven fold in the iMMECs overexpressing PAK4. Previous studies have suggested a tumor suppressor role for ParvB in regulating breast tumorigenicity. We hypothesize that PAK4 mediates mammary tumor formation through ParvB downregulation. Additional studies investigating the role of ParvB in the PAK4 signaling pathway are under investigation. Future studies will also involve studying the other genes we found to be regulated by PAK4 overexpression and determining their role in tumorigenesis. Citation Format: Chetan Rane, Misaal Patel, Li Cai, Audrey Minden. Decrypting the transcriptome profile of Pak4 using next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2017-1571