Abstract 1623: The spliceosome as a new therapeutic target for anticancer treatment
Authors: Quidville Virginie , Alsafadi Samar , Goubar Aïcha , Durieu Catherine , Baconnais Sonia , LeCam Eric , Dessen Philippe , Vagner Stephan , Andre Fabrice 
Published: June 2012

Background: Bioinformatics analyses of pathways that are differentially expressed between malignant and benign lesions could allow discovering new therapeutic targets. In the present study, we identified spliceosome assembly components as candidate target for cancer treatment. The spliceosome, an intracellular ribonucleoprotein complex, is involved in eukaryotic pre-mRNA processing by splicing out intronic nucleic acids.

Experimental procedures: Pathway analyses were done on exon and gene expression arrays datasets. Differential gene expression between malignant and benign lesions was applied to four public datasets. Small nuclear ribonucleoprotein E (SNRPE) downregulation was achieved by short interfering RNA technology in SKBr-3 and MDA-MB-231 human breast cancer cells. Functional effects of siRNA-induced knockdown of SNRPE expression on cell viability, cell cycle distribution and apoptosis were respectively determined using WST-1 assay, FACS analysis and annexin-V/7-AAD double-staining assay. Autophagy was investigated by electron microscopy (EM). We also evaluated alternative RNA splicing at the exon level using SpliceArray microarray.

Results: Pathway analyses with BioCarta database identified spliceosome assembly component (SAC) as the most differentially expressed pathway between breast malignant and benign lesions (permutation p=0.002). The differential expression of SAC was confirmed in four different public dataset on breast, ovarian, lung and skin cancers. Based on these data we investigated whether SNRPE, a component of spliceosome, could be a candidate target. Knockdown of SNRPE protein expression resulted in a high inhibition of SKBr-3 (70%) and MDA-MB-231 (48%) cell proliferation as compared to non-targeting siRNA-transfected cells. Splice array analyses suggested a deregulation of alternative splicing, together with a dramatic reduction of mTOR transcripts. mTOR downregulation, together with a decrease in phosphorylation of downstream protein in the mTOR pathway (i.e 4E-BP1) was confirmed by western blot analyses. We then investigated the mechanisms of cell death. siRNA-mediatd SNRPE knockdown was not found to induce typical apoptosis as annexin-V-positive cells were not detected. However, the presence of numerous vesicular organelle of autophagy revealed by EM is strongly amplified in SNRPE-transfected cells. This observed deregulation of the mTOR pathway in SNRPE-depleted cells might in part explain the induction of autophagy following SNRPE silencing.

Conclusion : Our data report that specific inactivation of SNRPE protein expression induces mTOR downregulation and autophagy in cancer cell lines, suggesting that the spliceosome could represent a new potential target that may open new perspectives in cancer therapy. Molecular mechanisms that lead to mTOR dowregulation by SNRPE extinction are being investigated.

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 1623. doi:10.1158/1538-7445.AM2011-1623