Studying the integrated stress response in tumor progression and drug resistance (Doctoral thesis)
Development and growth of a tumor as well as its ability to metastasize involves a complex relationship with the tissue microenvironment. A proliferating tumor encounters multiple stress conditions from the microenvironment such as hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells have co-opt elaborate cytoprotective mechanisms which provide them with distinct advantages to thrive. These mechanisms constitute a complex of homeostatic signaling pathways and are collectively known as the Integrated Stress Response (ISR). Thus, deciphering the signaling pathways which get activated in the tumor microenvironment has been paramount to develop new therapeutic strategies for treatment. The Unfolded Protein Response (UPR) (which is a more specialized form of ISR), is activated in response to unfolded proteins in the Endoplasmic Reticulum (ER) and involves translational and transcriptional activation of regulated signaling pathways designed to relieve cellular stress and attenuate cancer cell death. Intriguingly, in the case of unresolved or acute ER stress, the UPR can promote cell death. However, the mechanisms by which the UPR influences cell fate in the presence of ER stress are poorly understood. As a result, a comprehensive analysis to determine critical regulators of UPR is of utmost importance. To address the abovementioned question we have used a functional CRISPR mediated genetic knockout screen to determine novel regulators of UPR and to investigate the mechanisms by which these regulators control cellular fate following chronic ER stress. More specifically, we delivered a lentiviral genome-scale CRISPR-Cas9 knockout (GeCKO_V2) library to Sq20B cells (human squamous head and neck carcinoma) and A375 (human melanoma) cells. The library is targeting 18,080 genes with 64,751 unique guide sequences and enables both negative and positive selection screening, according to which sgRNAs are over-represented (pro-apoptotic genes), or underrepresented (pro-survival genes) following ER stress induced by thapsigargin and tunicamycin, known activators of the ER stress through distinct mechanisms. Our highest negative ranking gene candidates included Survivin/BIRC5, a well-studied protein which is overexpressed in tumor cells compared normal tissues. Survivin has been reported to act as an inhibitor of apoptosis; however, its main function is to promote proper regulation of mitosis and cytokinesis as part of the Chromosomal Passenger Complex. Our results show that genetic knock down or chemical inhibition of Survivin led to sensitization to ER stress. Additionally, its ablation caused formation of micronuclei and multinucleated cells due to failure of cytokinesis and as a result promotion of aneuploidy and chromosomal instability. Intriguingly, Survivindeficient cells had also an expanded ER and activated UPR. We hypothesize that these cells that have significantly increased DNA content which imposes higher transcriptional and translational demands on the cells, thereby causing ER expansion in order to support them. This, in turn, creates a reliance of these cells on a functioning UPR, whose inhibition results in cell death. Our results demonstrate that polyploidy, and perhaps more generally, genomic instability represents cellular stresses during which a hyperactive UPR may be required for survival. Thereby this study reveals new vulnerabilities for therapeutic intervention in malignancy as well as new therapeutic schemes by combinational targeting of Survivin and inhibition of UPR activation.
|Institution and School/Department of submitter:||Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής|
|Appears in Collections:||Διδακτορικές Διατριβές|
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|Δ.Δ. ΛΕΛΗ ΝΕΚΤΑΡΙΑ-ΜΑΡΙΑ 2019.pdf||4.26 MB||Adobe PDF||View/Open|
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