Hypoxia but not normoxia promotes Smoothened transcription through upregulation of RBPJ and Mastermind-like 3 in pancreatic cancer

We previously demonstrated that Hedgehog (Hh) signaling is activated under hypoxia through upregulation of transcription of Smoothened (SMO) gene. However, the mechanism of hypoxia-induced activation of SMO transcription remains unclear. In the analysis of altered expressions of genes related to Hh signaling between under normoxia and hypoxia by DNA microarray analysis, we picked up 2 genes, a transcriptional regulator, recombination signal binding protein for immunoglobulin-kappa-J region (RBPJ) and a transcriptional co-activator, Mastermind-like 3 (MAML3). Expressions of SMO, MAML3 and RBPJ were increased under hypoxia in pancreatic ductal adenocarcinoma cells (PDAC). RBPJ and MAML3 inhibition under hypoxia led to decreased SMO and GLI1 expressions, whereas SMO expression in MAML3-inhibited and RBPJ-inhibited cells under normoxia showed no change. However, overexpression of RBPJ under normoxia led to increased SMO expression. Additionally, cells knocked down for MAML3 and RBPJ inhibition under hypoxia showed decreased invasiveness through matrix metalloproteinase-2 suppression and decreased proliferation. Xenograft mouse models showed that MAML3 and RBPJ knockdown inhibited tumorigenicity and tumor volume. Our results suggest that hypoxia promotes SMO transcription through upregulation of MAML3 and RBPJ to induce proliferation, invasiveness and tumorigenesis in pancreatic cancer.