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BMC Genomics

, 14:624

Human and rodent genomics

Abstract

BackgroundPancreatic cancer is a deadly disease with a five-year survival of less than 5%. A better understanding of the underlying biology may suggest novel therapeutic targets. Recent surveys of the pancreatic cancer genome have uncovered numerous new alterations; yet systematic functional characterization of candidate cancer genes has lagged behind. To address this challenge, here we have devised a highly-parallel RNA interference-based functional screen to evaluate many genomically-nominated candidate pancreatic cancer genes simultaneously.

ResultsFor 185 candidate pancreatic cancer genes, selected from recurrently altered genomic loci, we performed a pooled shRNA library screen of cell growth-viability across 10 different cell lines. Knockdown-associated effects on cell growth were assessed by enrichment or depletion of shRNA hairpins, by hybridization to barcode microarrays. A novel analytical approach COrrelated Phenotypes for On-Target Effects; COPOTE was used to discern probable on-target knockdown, based on identifying different shRNAs targeting the same gene and displaying concordant phenotypes across cell lines. Knockdown data were integrated with genomic architecture and gene-expression profiles, and selected findings validated using individual shRNAs and-or independent siRNAs. The pooled shRNA library design delivered reproducible data. In all, COPOTE analysis identified 52 probable on-target gene-knockdowns. Knockdown of known oncogenes KRAS, MYC, SMURF1 and CCNE1 and a tumor suppressor CDKN2A showed the expected contrasting effects on cell growth. In addition, the screen corroborated purported roles of PLEKHG2 and MED29 as 19q13 amplicon drivers. Most notably, the analysis also revealed novel possible oncogenic functions of nucleoporin NUP153 ostensibly by modulating TGFβ signaling and Kruppel-like transcription factor KLF5 in pancreatic cancer.

ConclusionsBy integrating physical and functional genomic data, we were able to simultaneously evaluate many candidate pancreatic cancer genes. Our findings uncover new facets of pancreatic cancer biology, with possible therapeutic implications. More broadly, our study provides a general strategy for the efficient characterization of candidate genes emerging from cancer genome studies.

KeywordsPancreatic cancer Functional genomics RNAi screen shRNA screen NUP153 KLF5 AbbreviationsaCGHArray-based comparative genomic hybridization

COPOTECOrrelated phenotypes for on-target effects

FDRFalse discovery rate

KDKnockdown

PCRPolymerase chain reaction

RNAiRNA interference

RT-PCRReverse transcription PCR

Q-RT-PCRQuantitative RT-PCR

shRNAShort hairpin RNA

siRNAShort interfering RNA.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-14-624 contains supplementary material, which is available to authorized users.

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Author: A Hunter Shain - Keyan Salari - Craig P Giacomini - Jonathan R Pollack

Source: https://link.springer.com/







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