Identification of novel genes critical to survival of malignant peripheral nerve sheath tumor cells via medium throughput screening using lenti-viral shRNA

Ami .V. Patel, Ph.D.,Nancy Ratner, Ph.D., Cincinnati Childrens Hospital Medical Center, Dept. of Experimental Hematology and Cancer Biology, Cincinnati, OH.

Malignant peripheral nerve sheath tumor (MPNST) is a soft tissue sarcoma that is highly invasive and eventually lethal. About 50% of MPNSTs have mutation and/or loss of the NF1 gene, while the rest are spontaneous. Many MPNSTs also show mutations in p53 and/or homozygous deletion of CDKN2. In an effort to identify novel genes that play key roles in MPNST tumorigenesis, we performed a genome wide micro-array analysis using human neurofibroma and MPNST (Miller et al., 2009). This analysis identified 130 genes that are up-regulated >3 fold in MPNSTs as compared to normal human Schwann cells. Over expression of these genes in tumors as compared to normal cells suggested that some might be critical for survival of MPNST cells. This hypothesis was directly tested by studying effects on survival of MPNST cells invitro post down regulation of the 130 genes using lenti-viral short hairpin (sh) RNA. For each shRNA experiment a shnon-targeting lenti-virus was used as a negative control and shSOX9, a construct previously shown to kill MPNST cells in vitro, was used as a positive control for an effect on cell survival. Screening was performed sequentially allowing for selection of genes with significant effects on survival.  In screen I, T265 MPNST cells were exposed to each shRNA virus.  In screen II, shRNAs were tested for effects on adenocarcinoma cells (A549) vs T265 cells. These two rounds of screens identified 8/130 genes specific for survival of T265 cells. Screen III wasperformed on four different MPNST cell lines, including T265 (NF1-/-), 8814 (NF1-/-), S462TY (NF1-/-) and SSTS26T (sporadic MPNST wild-type for NF1), to identify shRNA’s with similar or different effects on cell survival in the presence or absence of wild-type NF1gene.  No significant difference in survival was observed for NF null MPNST cells vs sporadic MPNST cell line for any gene. This approach facilitates a streamlined pathway from genome wide gene expression analysis to identification of 8 novel genes critical to survival of cancer cells in vitro and potential therapeutic targets toward a cure for MPNSTs.

Supported by NIH28840 (to NR).  A.P. is the recipient of a DOD NF Postdoctoral Fellow Award.