Integrative genomic and functional profiling of the pancreatic cancer genome

A Hunter Shain,Craig P Giacomini,Jonathan R Pollack,Keyan Salari

doi:10.1186/1471-2164-14-624

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.

Highlights

Pancreatic cancer is a deadly disease with a five-year survival of less than 5%
In an effort to accelerate this process, we developed a pooled short hairpin RNA (shRNA) screening strategy to simultaneously evaluate 185 candidate pancreatic cancer genes that together represent 104 different loci of recurrent DNA amplification or deletion; the genes selected included both candidate oncogenes and tumor suppressors
Pancreatic cancer cell lines were infected with a pooled shRNA lentiviral library comprising 558 shRNAs targeting the 185 genes (Additional file 1)

Summary

Introduction

Pancreatic 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. A better understanding of the underlying disease biology is needed to develop new and successful treatment strategies to manage this deadly disease. TGFBR2, its upstream receptor, is deleted in 20% of tumors, underscoring a central importance of this signaling pathway in pancreatic cancer. Deletions and mutations in five different subunits of the SWI/SNF chromatin remodeling complex have been found to occur in about a third of cases [6]. Despite what is already known, recent surveys of the pancreatic cancer genome have identified scores of additional candidate cancer genes that merit further investigation [7,8]

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Conclusion