Abstract A22: A genome-wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance.

Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells are a non-malignant prostate cell line that undergo cell death upon detachment from extracellular matrix. To identify genetic regulators of anoikis, RWPE-1 cells were infected with a pooled lentiviral hairpin shRNA library with 54,021 hairpins targeting 11,255 genes. After infection, cells were cultured in suspension conditions for three weeks and an anoikis-resistant cell population was selected. From this population, genomic DNA was isolated and shRNA sequences were amplified and sequenced. Thirty four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions. We selected α/β hydrolase domain containing 4 (ABHD4) for further analysis as it conferred the greatest protection to anoikis in our screening assays. To validate the effects of ABHD4 knockdown on anoikis resistance, we infected RWPE-1 with 2 independent shRNA targeting ABHD4 or control sequences. We also over-expressed ABHD4 in wild type cells. Finally, we co-infected cells with ABDH4 cDNA and shRNA as a rescue experiment to demonstrate on-target activity. Target knockdown or over-expression after infection was confirmed by Q-RTPCR or immunoblotting. Using two independent shRNA, knockdown of ABHD4 inhibited anoikis as evidence by increased clonogenic growth compared to cells infected with control sequences. Demonstrating an on-target effect, rescue of ABHD4 expression returned levels of clonogenic growth to wild type levels. Finally, over-expression of ABHD4 increased sensitivity to anoikis and less clonogenic growth was observed in these cells compared to control cells. Resistance to anoikis after ABHD4 knockdown was associated with decreased cleavage of PARP and decreased activation of caspases-3, 8 and 9, but was independent in changes of FLIP expression. Interesting, resistance to anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, reductions in the levels of ABHD4 confer resistance to anoikis while over-expression of the target enhances anoikis in the anoikis-sensitive cell line RWPE-1. As such, we have identified a novel genetic regulator of anoikis sensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A22.