Abstract 2041: Large-scale RNAi screening identifies PCTK3/CDK18 as a putative cancer-related molecular target

RNAi screening is a powerful approach for the identification of proteins that have the potential to be developed as anti-cancer molecular targets. In this study we used an unbiased, large-scale, synthetic siRNA-mediated RNAi screen to identify vulnerable targets in breast cancer cells with the hope of finding novel therapeutic targets. Using cell viability as the read-out, we first screened a whole human kinome library plus 350 additional genes (four siRNAs per gene) in a cell line representative of triple negative (ER -ve, PR -ve, Her2/Neu -ve) breast cancer, MDA-MB-468. The results of two screens conducted in MDA-MB-468 cells were highly reproducible with good correlation (0.82) between duplicate screens performed separately. These screens identified several well-defined kinases whose loss-of-function reduced the viability in MDA-MB-468 cells, including PLK1, AURKB, and BIRC5. Some less well-characterized kinases also were identified to be required for the viability of MDA-MB-468 cells including PIK3R1, HUNK, CKB, and PCTK3/CDK18. The PCTK3/CDK18 gene encodes a member of the PCTAIRE protein kinase subfamily of CDC2-related serine/threonine-specific protein kinases. Most of the limited functional studies of PCTK3/CDK18 have focused its biological role in neuronal cells, but one study has reported an increase in the expression of PCTK3/CDK18 in breast cancer (Valladares et al., Cancer Genet Cytogenet. 2006 170:147). We observed that with multiple siRNAs, we induced an 80% decrease in the viability of MDA-MB-468 cells 96 hours following siRNA transfection. Over time we saw a 10%, 20% and 65% decrease in viability at 24h, 48h and 72h, respectively. As little is known of the function of the PCTK3/CDK18 protein, we are using multiple approaches to elucidate the cellular functions of PCTK3/CDK18 in the context of breast cancer. For example, we are investigating the cellular responses seen following loss of function of PCTK3 that could lead to the reduction in the viability phenotype observed. We have found that silencing PCTK3/CDK18 induces apoptosis as demonstrated by PARP cleavage and caspase activation, and cell cycle arrest in the S-phase. To further elucidate the role of PCTK3 in cell growth, we generated concordant gene expression signatures using different RNAi effectors targeting PCTK3/CDK18. We found these altered expressions of genes are involved mostly in DNA damage pathway and cell cycle pathway. While PCTK3 inhibition was lethal to MDA-MB-468 triple negative breast cancer, in contrast, silencing of PCTK3 in the non-transformed breast epithelial cell line MCF10A was well tolerated. This study further highlights the potential of high-throughput siRNA screens to identify novel cancer related molecular targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2041. doi:10.1158/1538-7445.AM2011-2041