FOXR2 Stabilizes MYCN Protein and Identifies Non- MYCN-Amplified Neuroblastoma Patients With Unfavorable Outcome

Abstract

Purpose: Clinical outcomes of patients with neuroblastoma range from spontaneous tumor regression to fatality. Hence, understanding the mechanisms that cause tumor progression is crucial for the treatment of patients. In this study, we show that FOXR2 activation identifies a subset of neuroblastoma tumors with unfavorable outcome and we investigate the mechanism how FOXR2 relates to poor outcome in patients.

Materials and methods: We analyzed three independent transcriptional data sets of in total 1030 primary neuroblastomas with full clinical annotation. We performed immunoprecipitation for FOXR2 and MYCN and silenced FOXR2 expression in two neuroblastoma cell lines to examine the effect on cellular processes, transcriptome, and MYCN protein levels. Tumor samples were analyzed for protein levels of FOXR2 and MYCN.

Results: In three combined neuroblastoma data sets, 9% of tumors show expression of FOXR2 but have low levels of MYCN mRNA. FOXR2 expression identifies a group of patients with unfavorable outcome, showing 10-year overall survival rates of 53%-59%, and proves to be an independent prognostic factor compared with established risk factors. Transcriptionally, FOXR2-expressing tumors are very similar to MYCN-amplified tumors, suggesting that they might share a common mechanism of tumor initiation. FOXR2 knockdown in FOXR2-expressing neuroblastoma cell lines resulted in cell cycle arrest, reduced cell growth, cell death, and reduced MYCN protein levels, all indicating that FOXR2 is essential for these tumors. Finally, we show that FOXR2 binds and stabilizes MYCN protein and MYCN protein levels are highly increased in FOXR2-expressing tumors, in several cases comparable with MYCN-amplified samples.

Conclusion: The stabilization of MYCN by FOXR2 represents an alternative mechanism to MYCN amplification to increase MYCN protein levels. As such, FOXR2 expression identifies another subset of neuroblastoma patients with unfavorable clinical outcome.

FIG 1.

FOXR2 is expressed in a distinct subset of neuroblastomas. (A) Cohort overview of the primary neuroblastoma RNA-seq data set (N = 498). (B) Expression of FOXR1, FOXR2, MYC, and MYCN is in nearly all cases mutually exclusive from another. (C) Percentage of FOXR1-, FOXR2-, MYC-, and MYCN-expressing cases within the INSS stages 1, 2, 3, 4, and 4S. The FOXR2 group is significantly enriched in stages 3 and 4 when compared with stages 1, 2, and 4S. (D) Percentage of MYCN-, FOXR2-, FOXR1-, and MYC-expressing cases within the non–high-risk and the high-risk group (N = 1,016; legend of [C] applies). (E) Oncoplot of the RNA-seq data set (N = 498), showing the status of the FOXR1, FOXR2, MYC, MYCN, and TERT expression and the ALT phenotype. ALT, alternative lengthening of telomeres; INSS, International Neuroblastoma Staging System; RPM, Reads per million mapped reads. **P ≤ .005.

FIG 2.

Survival data of distinct molecular groups. (A) OS and EFS from initial diagnosis of patients with neuroblastoma of the RNA-seq data set (N = 498) separated by the groups FOXR1, FOXR2, MYC, MYCN, and others. (B) OS and EFS of exclusively high-risk patients in the RNA-seq data set (n = 173). ^aThe FOXR1 group is excluded because of low number (n = 2). (C) Multivariable Cox regression analyses of the RNA-seq data set (N = 498) for OS taking into account FOXR2 and the established prognostic factors age above 18 months at diagnosis, MYCN amplification, INSS 4, and FOXR2 and risk group in a separate analysis. HRs with 95% CIs and P values are indicated. ^bThe MYCN amplification status is unavailable for five cases in the RNA-seq data set, and therefore, this multivariate analysis was conducted for 493 of 498 cases. EFS, event-free survival; HR, hazard ratio; OS, overall survival.

FIG 3.

FOXR2 silencing reduces proliferation. (A) FOXR2 KD in neuroblastoma cell lines SK-N-AS (left) and SK-N-FI (right) shown by quantitative real-time polymerase chain reaction and western blot. (B) Growth curve of SK-N-AS (left) and SK-N-FI (right) upon FOXR2 KD reveals reduced cell proliferation. (C) Venn diagram indicating the FOXR2 KD signature overlap of SK-N-FI (red) and SK-N-AS (blue). Ctr, control; KD, knockdown. **P ≤ .005.

FIG 4.

FOXR2 and MYCN tumors are transcriptionally similar. (A) Applying the FOXR2 tumor signature on the RNA-seq data set (N = 498), the MYCN group resembles the FOXR2 group transcriptionally. (B) FOXR2 expression plotted against the MYC(N) signature score reveals a positive MYC(N) activity score for the FOXR2 group.

FIG 5.

FOXR2 protein binds to and stabilizes MYCN protein. FOXR2, MYCN, and a combination of both overexpressed in HEK 293T cells as shown by (A) fluorescence immunostainings, (B) on protein level, and (C) on RNA level. (D) Immunoprecipitation analysis revealed that FOXR2 binds to MYCN. (E) CHX assays of FOXR2- and MYC(N)-overexpressing HEK 293T cells show MYC and MYCN stabilization by FOXR2 on western blot (upper panels). Band intensities were quantified and signal intensities are shown for each time point, normalized to β-actin and the initial signal intensity at the time point 0 hour (lower panel). (F) CHX assays of the FOXR2 knockdown cell lines SK-N-AS and SK-N-FI indicate that MYCN and MYC degrade more rapidly upon FOXR2 knockdown (upper panel). Band intensities were quantified, and signal intensities are shown for each time point, normalized to β-actin and the initial signal intensity at the time point 0 hour (lower panel). CHX, cyclohexamide.

FIG 6.

MYCN protein levels are high in FOXR2-expressing neuroblastoma. (A) mRNA levels and (B) western blot of seven FOXR2-expressing neuroblastomas, three MYCN-amplified neuroblastomas, and three low-risk neuroblastomas reveal that MYCN protein levels are highly increased in most FOXR2 expressing neuroblastoma samples despite low MYCN expression.