studies

330A AASLD ABSTRACTS HEPATOLOGY, October, 2015
Disclosures:
The following authors have nothing to disclose: Takumi Kawaguchi, Eitaro Taniguchi,
Minoru Itou, Tetsuharu Oriishi, Takuji Torimura
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Two-step forward genetic screens of transposon mutagenesis
and pooled shRNA library identify novel tumor
suppressor genes and potential therapeutic target in
HCC
Takahiro Kodama, Nancy Jenkins, Neal Copeland; Cancer Biology,
Houston Methodist Research Institute, Houston, TX
Background: The use of emerging sequencing and genomics
technologies has recently identified many mutated and/or differentially
expressed genes in HCC. However, it is still difficult
to identify true driver genes for HCC because of the large number
of passenger mutations and other gene-regulatory mechanism
such as epigenetic regulation, SNP and non-coding RNA.
To overcome this difficulty, we performed two-step forward
genetic screens and aimed to discover novel tumor suppressor
genes (TSGs) in HCC. Methods: A transposon mutagenesis
screen was performed using mice that harbor the conditional
SB system (loxP-STOP-loxP(LSL)-SB11 transposase and a T2/
Onc2 or T2/Onc3 concatemer) and Alb-cre with or without
sensitizing mutation of HBs-Ag transgene (Liver-SB/HBV or Liver-SB/noHBV).
To validate candidate TSGs in a high-throughput
manner, we subsequently performed in vivo small-scale
pooled shRNA library screen using mouse immortalized liver
progenitor cells (iLPCs). Results: We collected 334 and 146
tumors from the Liver-SB/HBV and Liver-SB/noHBV mice,
respectively, and identified 3523 and 2177 candidate cancer
genes, respectively. List of 1917 candidate genes common
in two transposon screening were narrowed down into 250
genes that had non-synonymous mutation or mRNA downregulation
reported in human HCC. Fifteen lentiviral pooled shRNA
libraries (50 shRNAs/pool) targeting these 250 genes were
introduced into iLPCs individually and assessed for their effects
on subcutaneous tumor growth of transduced iLPCs in Nude
mice. Eleven pooled libraries showed significant increase in
tumor formation rate of iLPCs compared to negative control
shRNA (16.7% - 32.1% vs 4.8%, P