Abstract A23: Characterizing ORCTL3: An anticancer gene isolated in a novel genetic screen.

Abstract

Abstract In previous studies by our group a high-throughput screening for pro-apoptotic genes resulted in the isolation of a cancer-specific apoptosis gene known as organic cation transporter like-3 (ORCTL3). It was found that ORCTL3 induces apoptosis specifically in transformed tumor cells but not in normal cells through an endoplasmic reticulum (ER) stress signal. However, the protein's exact mechanism of action, its functional domain/s, and its interactors remained unknown. The aim of this study is to characterize ORCTL3 by defining the functional parts of the protein and studying its tumor-specific apoptosis signaling pathway. The main experimental procedures used in this study were cell culture of various cancer and non-transformed cell lines, transient and stable transfection using different transfection methods, a number of apoptosis read-out assays such as DiOC6/PI staining for FACS analysis, shRNA downregulation, immunostaining and western blotting. Our results show that the first four transmembrane domains of the protein, which is made of 12 transmembrane domains, are sufficient to induce apoptosis in cancer cell lines if expressed to a critical level. This part of the protein induces apoptosis even more efficiently if specifically retained at the ER. Overexpressing this protein in vitro leads to an increase in ATF4 protein level and the splicing of XBP-1, both outcomes are markers of an ER stress signal. Moreover, the cell death signal induced by ORCTL3 is significantly reduced in cells with downregulated ATF4. In addition to its involvement in the conventional ER stress signal, other findings strongly suggest that ORCTL3 triggers an apoptosis signal that is mediated by the ARCosome, a novel caspase 8-activation complex. When ORCTL3 was overexpressed in cells with Bap31 and Caspase 8 downregulation, the cell death signal was significantly reduced compared to the control cells. Furthermore, ORCTL3 is found to induce cleavage of Bap31 at a very early stage of apoptosis. Our results also show that ORCTL3 does not induce apoptosis in CV-1 cells (primary monkey kidney cells) and some preliminary findings indicate that it induces cell death once these cells are transformed with H-ras. The mouse ORCTL3 that has been used in all above experiments is shown to be more stable than the human homolog, which did not show any toxicity when overexpressed in cancer cells, indicating a specific signal induced only by the mouse homolog of the gene solely in cancer cells. The conclusions drawn from the work carried out so far is that a small part of mouse ORCTL3 protein induces apoptosis specifically in cancer cells and not in normal cells through an ER stress signal that involves conventional ER stress pathways through the activation of ATF4 and XBP-1 in addition to a signal that involves the novel and recently described ARCosome platform. The on-going study describes a potential novel anti-cancer gene that, so far, overcomes the lack of specificity that most of the other cancer targeting agents display. 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 A23.