Abstract
Gemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal. Here, we utilize a genome-wide CRISPR screen to identify that loss of ELP5 reduces the gemcitabine-induced apoptosis in GBC cells in a P53-dependent manner through the Elongator complex and other uridine 34 (U34) tRNA-modifying enzymes. Mechanistically, loss of ELP5 impairs the integrity and stability of the Elongator complex to abrogate wobble U34 tRNA modification, and directly impedes the wobble U34 modification-dependent translation of hnRNPQ mRNA, a validated P53 internal ribosomal entry site (IRES) trans-acting factor. Downregulated hnRNPQ is unable to drive P53 IRES-dependent translation, but rescuing a U34 modification-independent hnRNPQ mutant could restore P53 translation and gemcitabine sensitivity in ELP5-depleted GBC cells. GBC patients with lower ELP5, hnRNPQ, or P53 expression have poor survival outcomes after gemcitabine chemotherapy. These results indicate that the Elongator/hnRNPQ/P53 axis controls gemcitabine sensitivity in GBC cells.
Highlights
Gemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal
We found that the protein levels but not mRNA levels of these genes were downregulated in hnRNPQdepleted cells (Supplementary Fig. 8h, i), suggesting that these heterogeneous nuclear ribonucleoprotein Q (hnRNPQ) targets might be associated with gemcitabineinduced cytotoxic effects in tumor cells
In the present study, we employ a genome-wide clustered regularly interspaced short palindrome repeat (CRISPR) screen to identify determinate genes that are essential for gemcitabine sensitivity in GBC
Summary
Gemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal. We utilize a genome-wide CRISPR screen to identify that loss of ELP5 reduces the gemcitabine-induced apoptosis in GBC cells in a P53-dependent manner through the Elongator complex and other uridine 34 (U34) tRNAmodifying enzymes. GBC patients with lower ELP5, hnRNPQ, or P53 expression have poor survival outcomes after gemcitabine chemotherapy These results indicate that the Elongator/hnRNPQ/P53 axis controls gemcitabine sensitivity in GBC cells. RNA interference (RNAi), via short hairpin RNA (shRNA) that can inactivate gene function in a sequence-specific manner, has been a predominant approach for loss-of-function screens in previous decades, but the high off-target effects and inherently incomplete protein depletion are major limitations for RNAi screens on a genome-wide scale[11]. We uncover the key role and mechanism of the aberrant Elongator/hnRNPQ/P53 axis in gemcitabine resistance of GBC cells and provide biomarkers to predict gemcitabine sensitivities and survival outcomes in GBC patients
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