To identify Gli dependent downstream target genes, we performed gene expression profiling on Gli3T expressing Panc1 cells and vector controls. We transfected Panc1 cells by using a Gli3TIRES nuclearGFP expression construct, and 24 h posttransfection we isolated GFP optimistic cells by FACS and carried out expression profiling by using Affymetrix chips. As anticipated, we detected upregulation of Gli3 that probably reflects the expression with the ectopic Gli3T transgene. We identified 265 genes that have been considerably down regulated by Gli3T ; amid them, PTCH1 and FOXA2 are recognized transcriptional targets in the Hh Gli pathway . Interestingly, we also recognized a few genes concerned in regulating Ras intracellular signal transduction, such as SOS2 , RASA1 , RIN2, and RASSF4 5 , suggesting feasible feedback regulation of Kras signaling in cancer cells influenced by Gli activity.
The PI3K AKT and MEK ERK pathways are Kras stimulated signaling pathways which have been implicated in tumorigenesis price PHA-767491 . In our transcriptional profiling, we uncovered that two subunits of PI3K, PIK3R1 and PIK3C2B, have been amid the genes whose expression was down regulated significantly by Gli3T , indicating a potential interaction between Gli and PI3K AKT signaling. Thus, we additional examined the status of those two critical Kras downstream pathways in PDAC cells. We observed that Akt phosphorylation was down regulated markedly in Gli3T expressing Panc1 cells but was elevated in mouse pancreatic tumors with each Kras and Gli1 activation . In contrast, ERK phosphorylation was not altered significantly in mouse PanIN lesions and human cancer cells upon Gli regulation .
These outcomes are in agreement with a earlier report that showed activation of AKT, but not ERK, when an energetic form of Gli2 was expressed in mouse PD0325901 pancreas . Hh Gli regulates Wnt signaling in a number of developmental, tissue regeneration and tumorigenic contexts . Interestingly, a latest examine suggested that Wnt signaling also may perhaps be regulated by Hh signaling in pancreatic tumors . So, we examined the canonical Wnt activity in Panc1 cells wherever Gli transcription activity was inhibited by Gli3T. Quantitative RTPCR examination showed the expression of a wellestablished Wnt target gene, AXIN2, was not inhibited by Gli3T expression in Panc1 and MiaPaCa2 cells .
This result is consistent with the absence of Wnt pathway target genes in the checklist of differentially expressed genes recognized by our microarray experiments and with the absence of catenin nuclear accumulation from the PanIN lesions with the two Kras and Gli1 activation . These information also are steady with our genetic result that Gli3T expression did not influence embryonic pancreatic growth , wherever canonical Wnt signaling plays a prominent role .