WT1 expression is inversely correlated with MYCN amplification or expression and associated with poor survival in non-MYCN-amplified neuroblastoma

Abstract

Neuroblastoma (NB) is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. A striking feature of this tumor is its clinical heterogeneity. Several tumor progression markers have been delineated so far, among which MYCN amplification, which occurs in about 25% of total NB cases, with the percentage increasing to 30% in advanced stage NB. Although MYCN amplification is strongly correlated with NB of poor outcome, the MYCN status cannot alone predict all cases of poor survival in NB. Indeed NB without MYCN amplification (about 70-80% of NB) are not always favorable. WT1 was initially identified as a tumor suppressor gene involved in the development of a pediatric renal tumor (Wilms' tumor). Here, we describe an inverse correlation between WT1 expression and MYCN amplification and expression. However and most notably, our results show that WT1 gene expression is associated with a poor outcome for patients showing non-MYCN-amplified tumors. Thus WT1 expression is clinically significant in NB and may be a prognostic marker for better risk stratification and for an optimized therapeutic management of NB.

Keywords: Differentiation; MYCN; Neuroblastoma; Wilms' tumors.

Figure 1

Scatter plots showing mean of WT1 mRNA expression versus MYCN amplification (A) or expression (B) in Neuroblastoma. The relative WT1 mRNA expression, MYCN copy number and relative MYCN mRNA expression was monitored by qPCR and RT‐qPCR, respectively, in a set of 67 primary neuroblastoma, a subset of 51 primary stage 4 neuroblastoma, and a subset of 43 primary stage 4 neuroblastoma in children over 18 months. Expression values were normalized to GAPDH housekeeping gene. Two‐tailed Mann–Whitney tests were used. An expression cut‐off value of 0.05 was used to dichotomize the patients into two groups (high MYCN expression >0.05; low MYCN expression ≤0.05).

Figure 2

WT1 expression is inversely correlated to MYCN amplification and expression in NB cell lines. A. Expression profiles of WT1 and MYCN in neuroblastoma cell lines measured by quantitative PCR. Expression values were normalized to GAPDH housekeeping gene. Error bars correspond to S.E.M. B. Whole cell extracts of NB cells were separated by SDS‐PAGE and probed with anti‐WT1, MYCN, Vimentin, NSE and NF70 antibodies. GAPDH was used as a loading control. MNA: MYCN‐amplified; non MNA: MYCN‐non‐amplified; S: substrate adherent type (F/S); N: neuronal type; I: intermediate type.

Figure 3

Mechanistic analysis of the link between WT1 and MYCN. A. LAN‐1 NB cells were transiently transfected with two human WT1: WT1 (+17AA/−KTS) and WT1 (−17AA/−KTS), and the empty vector as a control (EV). B. Endogenous WT1 expression was targeted by transduction of a shRNA against WT1 (sh) or a scrambled control RNA (scr). C. MYCN was activated in SK‐N‐AS/MYCN‐ER cells by the addition of 4‐OHT for the indicated time. D. MYCN expression was switched off for 7 days by tetracycline treatment (+tet) in SH‐EP21N cells, a subclone of the MYCN single copy SHEP cell line transfected with a tetracycline regulated MYCN expression vector. Subsequently MYCN was re‐expressed by removal of tetracycline (no tet) for 7 days (+7/−7). WT1 and MYCN protein levels were determined by western blot. Loading of equal protein amounts was assessed by the detection of GAPDH.

Figure 4

High WT1 expression is significantly associated with lower patient survival in non‐MYCN‐amplified neuroblastoma. Kaplan–Meier curves of overall survival for low vs high WT1 expression in the entire cohort (67 patients, right panel), in 24 MYCN amplified (middle panel) and 43 MYCN non‐amplified patients (left panel). The cohort of NB patients was dichotomized using the median value of WT1 expression (9.75) into two classes: high (WT1 expression >9.75) and low (WT1 expression ≤9.75). The log‐rank test was used to assess differences in survival of the neuroblastoma subsets indicated. The association of high WT1 expression with poor prognosis is statistically significant (p = 0.0157) only in MYCN non‐amplified NB.