GSE131222
Transcriptional control of subtype switching ensures adaptation and growth of pancreatic cancer
Purpose: The goal of this study is to determine whether ectopic expression of the GLI2 transcription factor in the human pancreatic cancer cell line, YAPC is sufficient to cause gene expression changes associated with a EMT switch. Methods: RNA was isolated from YAPC cells engineered to express a doxycycline inducible cassette for ectopic expression of GLI2 following treatment with 1ug/ml of Dox for 6 days. Control YAPC cells expressing an "empty vector" dox inducible cassette were similarly treated for 6 days with 1ug/u Dox and RNA was collected. Three biologically destinct replicates were submitted for library preparation and RNA-sequencing on an Illumina hiseq 2000. The sequence reads that passed quality filters were analyzed at the transcript level using TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays Results: RNA-seq data confirmed stable over-expression of GLI2 in the YAPC-rtta-GLI2 cells and not in the EV control cells treated with Dox. Target genes of interest were validated by qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for all target genes tested.  Gene set enrichment analysis of differentially expressed genes showed enrichment of EMT associated pathways which was further validated using functional assays. In addition a statistically significant alteration in SPP1 transcript was discovered in GLI2 overexpressing cells which formed the basis of ongoing experiments in the study. Conclusions: Our data support a role for GLI2 in regulation of genes associated with basal-like subtype switching including SPP1 Overall design: mRNA profiles from human pancreatic cancer cell lines YAPC-rtta-GLI2 and YAPC-rtta-EV treatment with doxycyline for 6 days were compared, in triplicate.