The online version of this article (doi:10.1186/s12943-017-0619-6) contains supplementary material, which is available to authorized users.
Vasohibin 2 (VASH2) has previously been identified as an agiogenenic factor and a cancer related protein. Here we investigated the association of VASH2 expression and chemoresistance in pancreatic cancer.
Immunohistochemical staining for VASH2 was performed on 102 human pancreatic cancer samples. Pancreatic cancer cell line models exhibiting overexpression or knockdown of VASH2 were generated. Gene expression analyses were carried out to determine genes differentially regulated by VASH2. Putative transcription factors that are downstream mediators of gene expression regulated by VASH2 were queried bioinformatically. Dual-luciferase reporter assays and ChIP assays were performed to confirm transactivation of target genes following VASH2 overexpression or knockdown.
VASH2 protein expression was higher in human pancreatic cancer than in paired adjacent tissues and elevated VASH2 levels were associated with gemcitabine chemoresistance. In cell line models of pancreatic cancer, VASH2 expression induced gemcitabine chemoresistance in vitro and in vivo. It was discovered that expression of ribonucleotide reductase regulatory subunit M2 (RRM2) is regulated by VASH2; immunohistochemical analysis demonstrated a positive association of VASH2 expression and RRM2 expression in human pancreatic cancer tissues. Bioinformatics analyses revealed that induction of the Jun proto-oncogene (JUN) by VASH2 is responsible for upregulation of RRM2 expression; this JUN-dependent regulation of RRM2 by VASH2 was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays, which demonstrated that JUN directly binds with the RRM2 promoter to activate transcription.
These data suggest that VASH2 reduces the chemosensitivity to gemcitabine in pancreatic cancer cells via JUN-dependent transactivation of RRM2.
Additional file 2: Representation of immunostaining pictures for weak and strong VASH2 staining in pancreatic cancer tissues. (TIF 3090 kb)
Additional file 3: Expression of VASH2 in stably transfected PANC-1 and SW1990 pancreatic cancer cells. PANC-1 cells were transfected with a vector expressing EGFP (PANC-1-EGFP) or VASH2 (PANC-1-VASH2), and SW1990 cells were transduced with a scrambled shRNA (SW1990-scramble) or a shRNA targeting VASH2 (SW1990-shVASH2). VASH2 protein expression was assessed by immunoblotting. (TIF 294 kb)
Additional file 4: Human gene expression analysis results using NimbleGen 12x135K microarrays. Differentially expressed genes were identified through Fold Change filtering. Fold change cutoffs of ≥ 3 (upregulation, 211 genes) or ≤ 0.33 (downregulation, 192 genes) were used to identify genes that were significantly differentially expressed. (XLS 69 kb)
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- Vasohibin 2 reduces chemosensitivity to gemcitabine in pancreatic cancer cells via Jun proto-oncogene dependent transactivation of ribonucleotide reductase regulatory subunit M2
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