Original ArticlesSKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma
Introduction
MYCN amplification is found in 25% of neuroblastoma and is strongly associated with advanced-stage and aggressive disease [1], [2]. Although different strategies for inhibiting MYCN have been investigated, direct therapeutic targeting of the oncoprotein presents a significant challenge [3], [4]. We have previously identified a number of genes involved in the regulation of the G1 checkpoint whose expression changes in relation to MYCN status [5]. One potential MYCN target gene is the F-box protein S-phase kinase-associated protein 2 (SKP2) which is the substrate recognition subunit for the SCFSKP2 E3 ligase complex. Implicated primarily in G1/S progression, SKP2 targets cell cycle regulators (e.g. p27KIP1, p21CIP1 and p57KIP2) for ubiquitination and degradation, thereby promoting cell cycle progression. Overexpressed in multiple cancer types, the main oncogenic contribution of SKP2 is attributed to targeting the CDK inhibitor p27KIP1 (p27 hereafter) for degradation, with the inverse relationship of high SKP2 and low p27 level often correlating with poor patient prognosis [6], [7], [8].
SKP2 overexpression has been identified as an indicator of high-risk neuroblastoma and found to correlate with low p27 protein levels and high E2F activity, suggesting SKP2 contributes to disease development and progression [9]. Moreover, RNA studies have previously demonstrated a relationship between MYCN and SKP2 expression in primary neuroblastoma tumours and cell lines [5], [9], suggesting SKP2 may be a potential MYCN target gene.
SKP2 has recently been identified as a direct c-MYC target gene [10]. Due to the homology within the Myc family and functional overlap often observed between c-MYC and MYCN, established c-MYC targets are often automatically presumed to be regulated by MYCN. However, different expression patterns are observed for these transcription factors in normal development and as oncogenes they are involved in distinct tumour types (e.g. c-MYC in Burkitt's lymphoma and MYCN in neuroblastoma). Furthermore not all c-MYC gene targets are found to be expressed in MYCN-expressing neuroblastoma cells most probably due to the reported differences in E-box selection between MYCN and c-MYC [11], as reviewed in Refs. 12 and 13. We therefore hypothesise that SKP2 is a direct MYCN target gene and contributes to the aggressive phenotype of MYCN-amplified tumours.
To investigate this hypothesis, we assessed the correlation between MYCN and SKP2 protein, and SKP2 mRNA expression in the conditional MYCN-expressing SHEP Tet21N cells and a panel of MYCN amplified and non-amplified neuroblastoma cell lines. Using chromatin immunoprecipitation (ChIP) and reporter gene assays, this is the first study to show that SKP2 is a direct MYCN transcriptional target. In addition, using SKP2 siRNA knockdown we highlight the therapeutic potential of targeting SKP2 in neuroblastoma.
Section snippets
Cell culture and reagents
MYCN-amplified neuroblastoma cell lines used were LAN5 [14], IMR32 [15] and p53 mutant IMR-KAT100 [16] and SK-N-BE(2c) [17] cells. Non-MYCN amplified cell lines used were SHSY5Y [17], GIMEN [18] and p53 mutant SKNAS [19]. Tet21N cells were cultured in media containing 1 µg/ml tetracycline (Sigma-Aldrich) for 24 hrs prior to experiments to switch off MYCN expression [20]. The identity of all cell lines was confirmed by karyotyping and was consistent with published reports. Cells were cultured in
SKP2 protein expression correlates with MYCN expression in MYCN-regulatable Tet21N cells
Tet21N MYCN- cells showed an ~3-fold reduction in SKP2 mRNA 24 hr after tetracycline addition (Fig. 1A), which corresponded with decreased SKP2 protein expression over the 5 day tetracycline exposure period (Fig. 1B). Reduced SKP2 expression was associated with increased protein levels of the SKP2 targets p27 and p21CIP1 (p21 hereafter), suggesting that stabilisation of these CDK inhibitors induced the growth inhibition observed in the exponentially proliferating Tet21N MYCN− cells (Fig. 1C).
To
Discussion
The oncogenic potential of SKP2 overexpression has been identified in multiple human cancers and although often associated with accelerated p27 proteolysis, SKP2 has been demonstrated to contribute to malignancy independently of the F-box [26], [27]. We previously identified SKP2 as a potential MYCN target in neuroblastoma and others have shown SKP2 transcript and protein levels to correlate with MYCN amplification, and to independently indicate a poor outcome in neuroblastoma patients [5], [9]
Conflicts of interest
None declared.
Acknowledgements
We would like to thank the following for providing cell lines: Manfred Schwab (SHEP-Tet21N), Penny Lovat (SHSY5Y, IMR32), Patrick Reynolds (LAN5), Barbara Spengler (SK-N-Be(2C)), Michelle Haber (IMR-KAT100), Mirco Ponzoni (GIMEN) and Jean Bénard (SKNAS). We would also like to thank Javier Lèon for the SKP2-reporter construct and Raymond Stallings (Royal College of Surgeons in Ireland, Dublin, Ireland) for the MYCN ChIP-chip analysis. This study was supported by Cancer Research UK (1089464).
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