Studies of amplification in rhabdomyosarcoma

Division Of Basic Sciences - Nci

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Abstract

To further study amplification of the 12q13-q14 chromosomal region in FP RMS, we previously used data from The Cancer Genome Atlas (TCGA) to compare our findings in RMS with amplification of this region in glioblastoma multiforme, dedifferentiated liposarcoma, and lung adenocarcinoma. Our analysis of the 12q13-q14 amplicons in these four cancer categories revealed high confidence regions of amplification ranging from 0.5 Mb in glioblastoma multiforme to 1.6 Mb in lung adenocarcinoma. Comparison of these amplified regions identified an overlap of 0.2 Mb across the four cancer categories as well as the presence of a 0.5 Mb amplified region that was specific to FP RMS. The commonly amplified 0.2 Mb region contained 15 genes, of which seven genes (including CDK4) were overexpressed in amplicon-positive samples in all four cancer categories. In addition, the 0.5 Mb region specific to FP RMS contained 18 genes, four of which were overexpressed in amplified FP RMS (NEMP1, NAB2, SHMT2 and R3HDM2). To further study this amplified segment that is specific to FP RMS, we analyzed expression of NEMP1, NAB2, SHMT2 and R3HDM2 in a panel of 8 FP RMS cell lines, consisting of 7 lines without and one line (RH30) with the 12q13-q14 amplicon. Expression studies revealed higher levels of NAB2 and SHMT2 mRNA in RH30 cells compared to the other seven FP RMS lines, and subsequent western blot studies revealed that only SHMT2 was overexpressed at the protein level in RH30 cells. As confirmation of this latter finding, our study of 13 FP RMS PDXs also showed higher SHMT2 RNA and protein expression in the 3 PDXs with SHMT2 amplification compared to the 10 PDXs without amplification. To examine the consequences of changes in SHMT2 expression in FP RMS cells, several lentiviral shRNA constructs targeting SHMT2 were transduced into RH30 and resulted in decreased SHMT2 expression. This decrease in SHMT2 expression was accompanied by decreased growth in bulk culture and decreased growth of colonies when cells were plated at low density. Furthermore, analysis of in vitro transforming activity revealed that the shRNA-mediated decrease in expression results in a substantial decrease in focus formation of RH30 cells. Finally, injection into immunocompromised mice also revealed a substantial decrease in tumorigenic activity. Therefore the loss of SHMT2 expression in RH30 cells causes a substantial decrease in oncogenic activity that is related to changes in cellular proliferation and/or survival. In a complementary set of experiments, we transduced a cDNA expression construct for SHMT2 into two FP RMS lines (Rh5 and RH41) without 12q13-q14 amplification to increase SHMT2 expression to levels similar to those seen in amplified RH30 cells. In these studies, the increased SHMT2 expression was associated with increased growth in bulk culture and increased growth of colonies when the cells were plated at low density. Furthermore, analysis of oncogenic activity revealed that increased SHMT2 expression resulted in an increase in focus formation in vitro and tumorigenic activity in vivo. In a final set of experiments, we examined drugs that directly or indirectly inhibit SHMT2-associated signaling. Studies of SHIN1, a direct inhibitor of SHMT2, in our native and genetically-modified FP RMS lines demonstrated that SHIN1 sensitivity is inversely proportional to SHMT2 amplification and expression. We attribute the decreased potency of this direct-acting agent in the presence of high SHMT2 expression to the ability of high levels of SHMT2 protein to bind and sequester the drug, thereby decreasing drug potency. We next moved further down the signaling pathway to find a drug that inhibits a downstream protein that is activated without changes in protein expression. Since the folate pathway is downstream of SHMT2, we selected the folate inhibitor pemetrexed. Similar analysis of pemetrexed in our native and genetically-modified FP RMS lines revealed that pemetrexed sensitivity is directly proportional to SHMT2 amplification and expression, thus suggesting that pemetrexed may provide a targeted therapy for the subset of FP RMS with 12q13-q14 amplification. In conclusion, our studies demonstrate that the 12q13-q14 amplicon in FP RMS contains at least two driver oncogenes. In addition to functioning as oncogenic drivers of the amplicon, at least one of these genes may be a useful target for molecular-directed therapy of tumors with the amplicon.