J. Mazar1, A. Rosado1, J. Shelley2, J. Marchica2, T. Westmoreland1 1Nemours Children Hospital,Surgery,Orlando, FL, USA 2Sanford Burnham Prebys Medical Discovery Institute,Orlando, FL, USA
Introduction: MYCN amplification is among the most significant biomarkers associated with the diagnosis and poor prognosis of human childhood neuroblastoma. Over the last 20 years, there has been only limited improvement in the overall survival rate of children with this gene amplification. As a result, we are investigating novel molecular events surrounding neuroblastoma, with an emphasis on long non-coding RNAs (lncRNAs). We recently identified the lncRNA SPRY4-IT1, a regulator of tumor growth in various human cancers, as differentially expressed in MYCN-amplified versus MYCC-expressing neuroblastomas. It is our hypothesis that MYCN amplification controls the regulation of SPRY4-IT1 expression through the regulation of MYCC.
Methods: In order to test this hypothesis, we prepared total cellular RNA, isolated from the human MYCN-amplified neuroblastoma cell lines CHLA-122, IMR-32, SK-N-Be(1), and SMS-KAN, as well as the non-amplified cell lines SK-N-AS, LAN-6, SK-N-SH. Knockdown of MYCN was performed by transfection of MYCN-specific siRNA into the IMR-32, SK-N-Be(1), and SMS-KAN cell lines. Exogenous expression of MYCC was performed by transfection of a MYCC expression plasmid (pcDNA6/MYCC) into the IMR-32 and SMS-KAN cell lines. Total cellular RNA was isolated 48 hours post-transfection. The expression of MYCN, MYCC, and the lncRNA SPRY4-IT1 were measured using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). GAPDH was measured as an internal load control.
Results:The expression of MYCN was confirmed as highly expressed in all the MYCN-amplified cell lines as compared to the MYCN non-amplified cells. Interestingly, MYCC expression inversely correlated with MYCN expression, showing dramatically higher expression in the MYCN non-amplified cells. This expression pattern also positively correlated with that of the lncRNA SPRY4-IT1. Knockdown of MYCN in all three MYCN-amplified cell lines resulted in dramatic increases in both MYCC and SPRY4-IT1. Surprisingly, exogenous expression of MYCC in the MYCN-amplified cells had no effect on MYCN expression, but highly up-regulated SPRY4-IT1.
Conclusion:The inverse correlation of MYCN and MYCC suggests competitive inhibition. Since the loss of MYCN leads to significant increases in both MYCC and SPRY4-IT1, and exogenous expression of MYCC had no effect on MYCN, but dramatically increased SPRY4-IT1 expression, we can conclude that MYCN is an upstream negative regulator of MYCC, and MYCC is an upstream positive regulator of SPRY4-IT1. This offers the promising possibility of SPRY4-IT1 as a prognostic biomarker of MYCN down-regulation. Together, these data identify a promising network of regulation in MYCN-amplified cells, allowing for the possibility of circumventing MYCN activity in these difficult to treat childhood cancers.