Polr1a Promotes Melanoma Metastasis: Noncanonical NF-κB Regulation and Potential as a Therapeutic Target
Background
A study reports that lowering Polr1a expression in melanoma cells reduces noncanonical NF-κB signaling, migration, and invasion, and decreases lung metastatic burden in mice. Published in Oncogene, the study combines cell and animal experiments with patient-data analyses to suggest a role for Polr1a in regulating metastasis. It is not a clinical trial testing treatment efficacy in patients with melanoma.
Melanoma treatments include immunotherapy and BRAF and MEK inhibitors, but not all patients respond. To identify new genes that regulate metastasis, the researchers focused on genes whose expression was associated with poor prognosis. Genome-wide screening in animals also presents a challenge: maintaining enough cells edited for each gene. The team therefore narrowed the candidates to 348 genes and designed an in vivo CRISPR knockout screen.
Key findings
The researchers introduced a pooled library containing six single-guide RNAs (sgRNAs) per target gene into mouse melanoma SW1 cells. They implanted these cells into C3H mice and compared sgRNA distributions in primary tumors and lung metastatic lesions 38 days later. This approach evaluated pooled cell populations targeting different genes; it did not remove all 348 genes from each cell. Polr1a was selected from the leading candidates for follow-up validation.
An association was also observed in patient data. Analyses of 458 patients with both clinical and gene-expression data from The Cancer Genome Atlas (TCGA), and of the subset of 353 patients with metastatic melanoma, found that high POLR1A expression was associated with poorer overall survival. These analyses alone cannot establish causality in patients or determine treatment response.
In follow-up experiments, the researchers used short hairpin RNA (shRNA) to knock down Polr1a expression. This had no marked effect on the survival or proliferation of mouse melanoma cells, but reduced their migration and invasion. Invasion by SW1 cells fell to less than half the control level, and reduced migration and invasion were also observed in human melanoma A375 and 451Lu cells.
Mechanistic analyses showed selective changes in the synthesis of certain proteins. Ribosome profiling (Ribo-seq) in SW1 cells found no change in overall translation efficiency, and separately measured amounts of 28S and 18S ribosomal RNA were maintained. By contrast, polysome analysis showed reduced translation of NF-κB2 and RelB. RelB and p52 protein levels and NF-κB transcriptional activity decreased in mouse cells. Re-expressing RelB restored the reduced cell migration, supporting the interpretation that the noncanonical NF-κB pathway contributes to Polr1a-mediated regulation of migration.
The experiments also need to be distinguished by drug. BMH-21 and CX-5461 inhibited migration in cell experiments, while CX-5461 was used in the animal antimetastatic experiments. CX-5461 reduced tumor growth and lung metastases, and a reduction in lung metastases was also observed in a model in which the primary tumor was surgically removed. This supports an antimetastatic effect that cannot be explained solely by shrinkage of the primary tumor.
Significance and outlook
The researchers propose Polr1a, a component of RNA polymerase I, as a target for regulating melanoma metastasis and discuss the possibility of using inhibitors as preoperative treatment. However, the precise molecular mechanism behind selective regulation of translation remains unresolved, and tumor size and metastatic counts varied substantially among animals. The authors note that CX-5461 may have a narrow therapeutic window—the range between doses that produce an effect and those that cause toxicity.
Neither immune activation nor the effects of combining treatment with immune checkpoint inhibitors were demonstrated in this study. The actual observations were a trend toward reduced CD8⁺ immune-cell infiltration and lower PD-L1 expression, making it difficult to conclude that immune responses were enhanced. Clinical experience in other cancer types also does not guarantee efficacy in melanoma patients or a timeline for starting clinical trials. The study design and findings are available in the original paper.
Identification and characterization of novel mechanisms driving melanoma metastases and ways to target them are paramount for the development of effective treatment modalities. Here, we employed in vivo CRISPR knockout screening targeting the genes associated with poor prognosis to identify Polr1a as a potent driver of melanoma metastasis. High Polr1a levels correlate with increased metastasis and reduced survival in patients. Polr1a inhibition suppressed migration, invasion, and the ability of melanoma cells to colonize lungs. Ribo-seq analysis revealed that Polr1a is involved in regulating the non-canonical NF-κB pathway. Indeed, targeting Polr1a decreased levels of RelB and p52 and suppressed non-canonical NF-κB transcriptional activity; this suppression was responsible for the effects of Polr1a on melanoma cell migration. Accordingly, pharmacological inhibition of Polr1/Polr1a suppressed cell migration, tumor growth, and metastases. We discuss the potential utilization of Polr1 inhibitors for neoadjuvant treatment of melanoma.
This study provides a basis for evaluating metastatic potential separately alongside tumor size when assessing melanoma treatment candidates. The reduction in lung metastases in CX-5461-treated mice even when the primary tumor was removed could inform further preclinical evaluation of preoperative treatment candidates. For example, measuring primary-tumor response separately from postoperative metastatic burden could help assess a drug’s antimetastatic effects more clearly. Before clinical application, however, the therapeutic window, toxicity, long-term safety, and combination effects require further investigation. It also remains unconfirmed whether the association between high POLR1A expression and poor survival alone can be used to select patients for treatment.