Abstract 1908: Search for mediators of the lethal effect of COPZ1 depletion in thyroid tumor cells

Abstract

Abstract We previously identified COPZ1 as an example of "non-oncogene" addiction for thyroid cancer cells, since its silencing drastically impairs the viability. Notably, the inhibition of COPZ1 has no effect on normal cells such as primary and normal immortalized thyrocytes. COPZ1 belongs to the coatomer protein complex I, involved in assembly of coated vesicles on Golgi membranes, the retrograde transport of proteins in the ER-Golgi secretory pathway, endosome maturation, autophagy and lipid homeostasis. We found that COPZ1 depletion leads to abortive autophagy, ER stress, unfolded protein response (UPR) and apoptosis. To better identify intracellular pathways that are activated upon COPZ1 silencing and that contribute to cell death, we performed a gene expression profiling of COPZ1-depleted 8505C cells (anaplastic thyroid tumor cells), 72h after siRNA transfection, by using Affymetrix Gene ST 2.0 array. Hierarchical clustering was applied on most variable genes in the entire dataset and specific gene expression patterns were identified in COPZ1-silenced cells compared to the control (non targeting siRNA-transfected cells), by means of SAM software (q-value 0). Functional annotation analyses by means of DAVID 6.8 bioinformatic tool and Gene Set Enrichment Analysis were performed. We found 321 genes specifically modulated in COPZ1-depleted 8505C cells; the main altered functional categories dealt with cell cycle regulation, chromosome organization and protein metabolism. Specifically, gene sets associated to the UPR, interferon signalling, vesicular transport and the translation machinery were found upregulated, whereas those related to DNA metabolism, telomere maintenance, cell cycle check points, cytoskeleton and cell adhesion, non-coding RNA and mRNA metabolism were found downregulated. Furthermore, we performed a proteomic profiling of 8505C cellular extracts (at 48h after COPZ1 depletion) by a nLC-ESI-MS/MS analysis. Bioinformatic analysis identified 271 down-regulated and 291 up-regulated proteins compared to the control. Overall, GeneTrail2 analysis revealed that the most changing pathways are related to RNA metabolism involved in splicesome and RNA processing, thus likely explaining the extensive transcriptional remodelling observed in the later time point. More interestingly, the gene expression profiling was also carried out in Nthy-ori 3-1 cells (immortalized normal human thyroid cells) that, unlike tumor cells, are resistant to cell death after COPZ1 depletion. We found no significant differentially expressed genes in COPZ1-depleted cells in comparison to the control, in keeping with the absence of a lethal effect, despite COPZ1 silencing. In conclusion, we identified novel functions of COPZ1 and further pathway interactions, in addition to that already known, that can be considered other critical nodes in the oncogenic network whose inhibition will result in cell collapse. Citation Format: Maria Chiara Anania, Katia Todoerti, Italia Bongarzone, Elena Cetti, Tiziana Di Marco, Antonino Neri, Angela Greco. Search for mediators of the lethal effect of COPZ1 depletion in thyroid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1908.