To further study amplification of the 12q13-q14 chromosomal region in RMS, we previously used data from The Cancer Genome Atlas to compare our findings in RMS with amplification of this region in glioblastoma multiforme (GBM), dedifferentiated (DD) liposarcoma (LPS), and lung adenocarcinoma (LUAD). To statistically define a high confidence region of amplification, we divided the chromosomal regions of interest into multiple segments, determined the copy number of each segment and assessed whether there were statistically relevant differences in copy number between amplicon-positive and amplicon-negative samples within each segment. Our analysis of 12q13-q14 amplification in FP RMS, GBM, LUAD, and DDLPS revealed high confidence regions of amplification ranging from 0.5 Mb in GBM to 1.6 Mb in LUAD. In our analysis of these amplified regions, we observed an overlap of 0.2 Mb across the four tumor categories, which contained 15 genes, including CDK4. In addition, there was a 0.5 Mb amplified region specific to FP RMS. We analyzed RNAseq data to assess if genes in the amplified region are overexpressed in these cases. For the 12q13-q14 amplicons, 18 genes in GBM, 15 genes in LUAD, 15 genes in LPS, and 14 genes in RMS were significantly overexpressed in amplified samples. Of note, CDK4 and six nearby genes within the 0.2 Mb overlapping amplified region were overexpressed in amplicon-positive samples in all four cancer categories. Furthermore, four genes were differentially expressed only in amplified FP RMS and map to the 0.5 Mb FP RMS-specific amplified region. To further study this 12q13-q14 amplification event that is relatively specific to fusion-positive RMS, we analyzed expression of the four genes from the 0.5 Mb FP RMS-specific amplified region in a panel of 8 FP RMS cell lines, consisting of 7 lines without and one line (RH30) with the 12q13-q14 amplicon. RNA expression studies revealed higher RNA expression of two of these genes in RH30 cells compared to the other seven FP RMS lines, and subsequent Western blot studies of showed higher expression of one of these gene products at the protein level in RH30 cells. Several lentiviral shRNA constructs targeting this gene were transduced into RH30 and resulted in decreased expression. This decrease in expression was accompanied by decreased growth in bulk culture and decreased growth of colonies when the cells were plated at low density. Furthermore, analysis of transforming activity by the focus formation assay revealed a substantial decrease in foci of RH30 cells within a lawn of confluent NIH 3T3 cells. Therefore the loss of expression of this gene in RH30 cells is associated with a substantial decrease in cellular proliferation and/or survival under several conditions. In a complementary experiment, we transduced a cDNA expression construct for this gene into two FP RMS lines (Rh5 and RH41) without 12q13-q14 amplification to increase RNA expression to levels similar to those seen in amplified RH30 cells. In these studies, the increase in expression was associated with a substantial increase in cellular proliferation and/or survival under several conditions. In particular, the increased expression was accompanied by increased growth in bulk culture and increased growth of colonies when the cells were plated at low density. Furthermore, analysis of transforming activity by the focus formation assay revealed a substantial increase in foci of RH5 and RH41 cells within a lawn of confluent NIH 3T3 cells. These studies demonstrate that the 12q13-q14 amplicon in FP RMS contains at least two driver oncogenes and we predict that there may be additional overexpressed genes in these amplicons that also affect the oncogenic properties of these tumor cells. Furthermore, these studies reveal that there are important functional differences in the composition of the amplified genes between tumor types that initially appeared to amplify the same general region.
