Cell cycle regulation targets of MYCN identified by gene expression microarrays

Background: We have previously shown that MYCN knockdown causes a G(1) arrest in MYCN amplified (MNA), p53 wild type (wt) and p53 mutant MNA neuroblastoma cell lines, with increases in p21(WAF1) and hypo RB in p53 wt cell lines. 1 Hypothesis: MYCN acts by inhibiting p21(WAF1), and also by p21(WAF1) independent mechanisms to override the G1 checkpoint in exponentially growing cells. Methods: Genes potentially regulated by MYCN were identified using gene expression microarrays in p53 wt MNA IMR - 32 and p53 mutant MNA SKNBE(2c) neuroblastoma cell lines treated with MYCN or scrambled siRNA. Results were validated using qRT - PCR and confirmed using the regulatable MYCN expression system (SHEP Tet21N). Results: MYCN knockdown altered the expression of several cell cycle related genes. SKP2 was down regulated in both cell lines, and up regulated in MYCN+Tet21N cells. Expression of the WNT antagonist DKK3 increased in both cell lines and decreased in MYCN+ Tet21N cells. Expression of CDKN1C (p57(cip2)) and TP53INP1 also increased after MYCN knockdown. Conclusions: MYCN may override the G1 checkpoint through down - regulation of SKP2 and TP53INP1 resulting in reduced p21WAF1 expression in p53 wt cell lines, and in addition may act through the WNT signaling pathway in a p53 independent manner.