Abstract 267: Large-scale RNAi screening of human kinome identifies putative breast cancer related molecular targets
Authors: Oliver Ou, Konrad Huppi, Kristen Gehlhaus, Tamara Jones, Natasha Caplen
Journal: Cancer Research (2012)

Abstract To identify vulnerable genes in breast cancer cells that may be therapeutic candidates for treatment of breast cancer, we conducted an unbiased, large-scale, synthetic siRNA-mediated RNAi screen. 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. We identified approximately 40 kinases whose loss-of-function reduced the viability of MDA-MB-468 cells based on the criteria of at least two of four siRNAs per gene generating a Z score of ≤ −1.5. To validate the top candidates from our screen, PLK1, AURKB and PCTK3/CDK18), we tested additional siRNAs (from multiple vendors) in different breast cancer cell lines including ER positive cell lines, CAMA-1 and MCF-7, HER-2 amplified cell lines, HCC-1954 and AU565, triple-Negative (Basal A) cell lines HCC-1937 and MDA-MB-468, and triple-Negative (Basal B) cell lines BT549 and MDA-MB-231. We found silencing of PLK1, AURKB or PCTK3/CDK18 reduced the cell viability in majority of these cell lines to a certain degree and also induced cell cycle arrest and apoptosis. PLK1 and AURKB are being actively pursued as molecular targets in cancer but little is known of the function of the PCTK3/CDK18 protein, with most of the limited functional studies of PCTK3/CDK18 having focused on a role in neuronal cell signaling. One study has though reported an increase in the expression of PCTK3/CDK18 in breast cancer (Valladares et al., Cancer Genet Cytogenet. 2006 170:147). We found silencing of PCTK3/CDK18 is lethal to most breast cancer cells, but it is well tolerated in the untransformed breast epithelial cell line MCF10A. The PCTK3/CDK18 gene encodes a member of the PCTAIRE protein kinase subfamily of CDC2-related serine/threonine-specific protein kinases. We observed that with multiple siRNAs, we induced an 80% decrease in the viability of MDA-MB-468 cells 96 hours following siRNA transfection. To ensure that the observed inhibitory effect is indeed due to silencing of PCTK3/CDK18 we performed an RNAi rescue experiment, by creating a silent third-codon point mutation within the region targeted by one of the PCTK3 siRNAs. We found the RNAi-induced inhibition and cell cycle arrest is countered by expression of a functional version of the target gene that is resistant to the silencing siRNA. No specific inhibitor of PCTK3/CDK18 is available, but a proteome-wide CDK/CRK-specific kinase inhibitor RGB-286147 does decrease cell viability, and induce cell cycle arrest and apoptosis of MDA-MB-468 cells. We are currently further investigating the normal and cancer-related functional roles of PCTK3/CDK18 and are pursuing strategies for the identification of PCTK3/CDK18 specific inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 267. doi:1538-7445.AM2012-267