Detection of Tumor Suppressor Genes in Cancer Development by a Novel shRNA-Based Method

Pancreatic cancer is one of the deadliest cancers with poor survival rates and limited therapeutic options. To improve the understanding of this disease's biology, a prerequisite for the generation of novel therapeutics, new platforms for rapid and efficient genetic and therapeutic screening are needed. Therefore, a combined in vitro/in vivo hybrid shRNA assay was developed using isolated murine primary pancreatic ductal cells (PDCs), in which oncogenic Kras(G12D) could be activated in vitro by genomic recombination through 4OH-tamoxifen-induced nuclear translocation of Cre-ERT2 expressed under control of the ROSA26 promoter. Further genetic manipulation was achieved through selective and stable RNAi against the tumor suppressors p16(Ink4a) (CDKN2A) or Trp53 (TP53) using lentiviral gene delivery. Treatment of PDCs with 4OH-tamoxifen increased phosphorylation of ERK downstream of KRAS, and subsequent lentiviral transduction resulted in sustained target gene repression. Double-mutant PDCs were then reintroduced into the pancreata of NOD-SCID-gamma (NSG) mice and monitored for tumor growth. Orthotopic implantation of PDCs carrying the activated Kras(G12D)-allele and shRNA against p16(Ink4a) or Trp53 resulted in tumor growth, metastasis, and reduced survival of NSG mice. In contrast, Kras(G12D) alone was not sufficient to induce tumor growth.

The combinatory in vitro/in vivo approach described in this study allows for rapid and efficient identification of genes involved in carcinogenesis and opens new avenues for the development of therapeutic strategies to improve cancer treatment.