Abstract 1017: Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines
Authors: Fook Alves Veruska Lia , Zanatta Daniela B. , oliveira mariana bleker , eugenio angela isabel pereira , fernando rodrigo carlini , Strauss Bryan E. , Colleoni Gisele Wally Braga 
Published: August 2015

Abstract
Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, providing a selective advantage for tumor cell survival, due in part, to its ability to inhibit cell death via APAF1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM). Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis. Methods: The expression of HSP70 and TRIAP1 genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1) - three independent transductions, in triplicate. Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry with annexin V and propidium iodide (PI) staining. We also evaluated APAF1 and Caspase9 genes expression by qPCR and Caspase 9 and Caspase 3/7 protein activity. Results: The cell lines RPMI8226 (without deletion of TP53 by FISH) and U266 (deletion of one allele of TP53 by FISH) were chosen for the transduction experiments because they showed significant levels of expression of HSP70 and TRIAP1. The efficiency of transduction, with GFP reporter gene, was 70% for both cell lines. Silencing of both genes was confirmed by qPCR and Western blotting. Inhibition of TRIAP1 increased the percentage of cells in late apoptosis (p&lt;0.001 for RPMI8226 and P&lt;0.01 for U266) and was accompanied by increased expression of Caspase9 in both MM cell lines (p&lt;0.001 for RPMI8226 and p&lt;0.05 for U266). Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines (p&lt;0.01 for RPMI8226 and p&lt;0.05 for U266) and these results were confirmed by increased activity of Caspase3/7 (p&lt;0.01 for RPMI8226 and p&lt;0.05 for U266). Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact of this method on the induction of late apoptosis, through APAF1/Caspase9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever FISH status for TP53 (Support by FAPESP 2010/17668-6).
Citation Format: Veruska Lia Fook Alves, Daniela B. Zanatta, mariana bleker oliveira, angela isabel pereira eugenio, rodrigo carlini fernando, Bryan E. Strauss, Gisele Wally Braga Colleoni. Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2015-1017