CDKN2A and CDKN2B Co-deletion Enhances Ammonia Metabolism in PDAC Liver Metastasis

Background:
Liver metastases are often the direct cause of death of pancreatic ductal adenocarcinoma (PDAC) patients. We recently found, through analysis of 17,000 primary and metastatic PDAC samples, that deletion of CDKN2A and CDKN2B (encoding p15/p16 respectively) was more prevalent in liver metastasis compared to primary or other metastases sites. We also found that p15/p16 deletion enabled growth in liver environment. The liver environment is characterized by high ammonia levels, and we hypothesized that p15/p16-deletion confers growth advantage under these conditions.

Aim:
Reveal the mechanism p15/p16-deletion enables PDAC growth in liver conditions.

Methods:
PDAC cells, COLO357, PANC-1 and MIA PaCa-2, were either p15/p16 silenced or co-overexpressed. Transcriptomic was conducted with RNAseq. cJun pathway was studied by qRT-PCR, western-blot and gene-reported assay.

Results:
Transcriptomic analysis revealed enrichment in ammonia-associated gene pathway, especially GLUL up-regulation, leading to increased ammonia assimilation. Consequently, p15/p16 deleted cells grow better in high ammonia levels. GLUL promoter contains cJun binding sites, and transcriptomic analysis revealed elevated cJun expression in p15/p16-deleted cells. p15/p16 deletion led to activation of the cJUN pathway evidenced by JNK and cJun phosphorylation, cJun expression, and cJun transactional activity. We studied expression of GLUL following JNK inhibition and found that JNK inhibition decreased GLUL expression specifically in p15/p16-deleted cells. The opposite was observed upon p15/p16 overexpression.

Conclusions:
These data indicate that p15/p16 deletion leads to altered ammonia metabolism through GLUL upregulation, mediated by the JNK-cJUN pathway. This may affect adaptation of PDAC cells in the liver. This may lead to novel therapeutic strategies for PDAC patients.