体内CRISPR敲除筛选揭示核糖体生物发生是黑色素瘤转移的驱动因素和潜在治疗靶点

Metastatic melanoma is a highly aggressive cancer that spreads rapidly, with a five-year survival rate below 15%. Despite advancements in treatment options, patient outcomes remain poor, highlighting the urgent need for novel therapeutic approaches. Here, we have applied in vivo CRISPR KO screening to uncover new genes involved in metastasis and identified Polr1a as one of the drivers of melanoma metastasis. Polr1a is a major subunit of RNA polymerase I (Polr1), which is responsible for the transcription of 47S rDNA, resulting in the generation of 5.8S, 18S, and 28S rRNA needed for ribosome formation. Pharmacological and genetic inhibition of Polr1a impaired migration of melanoma cells, leading to the hypothesis that moderate inhibition of Polr1a preferentially suppresses the translation of genes needed for the metastatic spread. Indeed, genetic inhibition of Polr1a significantly decreased the invasion ability of melanoma cells. Importantly, pharmacological inhibition with CX-5461 (Polr1 inhibitor) significantly slowed down the primary tumor growth and dramatically decreased the number of lung metastases. Also, CX-5461 treatment significantly suppressed metastasis when used in combination with primary tumor resection, demonstrating an impressive metastasis-specific effect independent of primary tumor growth inhibition. To identify the potential mechanism by which Polr1a regulates melanoma metastasis, we have conducted in parallel RNA seq and Ribo seq analysis in Polr1a KD melanoma cells. Inhibition of Polr1a did not affect global translation; however, KEGG enrichment analysis has revealed the non-canonical NF-κB signaling pathway as one of the most strongly impacted on the translational level. Indeed, western blot analysis of the cells with Polr1a KD confirmed that a decrease in Polr1a level is associated with the decrease of p52 and ReIB, which are the key proteins responsible for the activation of the non-canonical NF-κB pathway. Re-expression of RelB in the cells with Polr1a KD rescued the migration phenotype, demonstrating that Polr1a regulates melanoma migration ability in an NF-κB-dependent manner.Overall, these results suggest Polr1a as a prospective new target for the treatment of metastatic melanoma and shed light on a potential mechanism responsible for the pro-metastatic effect of this gene.