High Tumoral STMN1 Expression Is Associated with Malignant Potential and Poor Prognosis in Patients with Neuroblastoma

Abstract

Background: Stathmin 1 (STMN1), a marker for immature neurons and tumors, controls microtubule dynamics by destabilizing tubulin. It plays an essential role in cancer progression and indicates poor prognosis in several cancers. This potential protein has not been clarified in clinical patients with neuroblastoma. Therefore, this study aimed to assess the clinical significance and STMN1 function in neuroblastoma with and without MYCN amplification.

Methods: Using immunohistochemical staining, STMN1 expression was examined in 81 neuroblastoma samples. Functional analysis revealed the association among STMN1 suppression, cellular viability, and endogenous or exogenous MYCN expression in neuroblastoma cell lines.

Result: High levels of STMN1 expression were associated with malignant potential, proliferation potency, and poor prognosis in neuroblastoma. STMN1 expression was an independent prognostic factor in patients with neuroblastoma. Furthermore, STMN1 knockdown inhibited neuroblastoma cell growth regardless of endogenous and exogenous MYCN overexpression.

Conclusion: Our data suggest that assessing STMN1 expression in neuroblastoma could be a powerful indicator of prognosis and that STMN1 might be a promising therapeutic candidate against refractory neuroblastoma with and without MYCN amplification.

Keywords: STMN1; neuroblastoma; oncoprotein 18; prognostic markers.

Figure 1

Immunohistochemical staining of STMN1 protein in ganglioneuroma, ganglioneuroblastoma, and neuroblastoma samples. Representative sections of (A) ganglioneuroma tissue without STMN1 expression, (B) ganglioneuroblastoma tissue with low STMN1 expression, (C) neuroblastoma tissue with low STMN1 expression, and (D) neuroblastoma tissue with high STMN1 expression. Scale bar = 50 μm.

Figure 2

Kaplan–Meier survival curves of patients with neuroblastoma (NB) based on STMN1 expression. (A) Kaplan–Meier survival analysis based on SMNT1 expression, overall survival in our cohort of all NB patients (n = 81, p < 0.001, left panel), non-MYCN-amplified (n = 69, p = 0.0049, middle panel), and MYCN-amplified (n = 8, p = 0.765, right panel) NB cases. MYCN status in four cases was not evaluated. * p < 0.05. (B) Kaplan–Meier survival analysis based on SMNT1 expression in the R2 database of all NB (n = 782, p = 0.019, left panel), non-MYCN-amplified (n = 629, p < 0.001, middle panel), and MYCN-amplified (n = 153, p = 0.044, right panel) NB patients. We used an online R2 Genomics Analysis and Visualization Platform to verify the relevance of STMN1 mRNA expression to the overall survival of patients with NB: R2 internal identifier, ps_avgpres_dgc2102a786_dgc2102.

Figure 3

STMN1 knockdown inhibited cellular viability in non-MYCN-amplified and MYCN-amplified neuroblastoma cell lines. Cell lines were transfected with STMN1-specific siRNAs and control siRNA. β-actin expression was used as gel loading control. Cellular viability was verified using the Cell Counting Kit-8. (A) Left panel: Western blot showing a decrease in STMN1 expression levels in lysates from non-MYCN-amplified SK-N-AS and NB69 neuroblastoma cells. Right panel: cellular viability. (B) Left panel: Western blot showing a decrease in STMN1 and endogenous MYCN expression levels in lysates from MYCN-amplified LAN-5 neuroblastoma cells. Right panel: Cellular viability. The control siRNA group was defined as the control for Dunnett’s multiple comparison tests. * p < 0.05. All WB images are included in File S1.

Figure 4

STMN1 suppression inhibited the MYCN expression and MYCN-induced proliferation in neuroblastoma cell lines overexpressing exogenous MYCN. Non-MYCN-amplified SK-N-AS and NB69 neuroblastoma cell lines were transduced for overexpressing exogenous MYCN (MYCN OE). (A) Western blot showing a decrease in STMN1 and exogenous MYCN expression levels in lysates from cells transduced with STMN1-specific siRNAs compared with the negative control siRNA transfectants. β-actin expression was used as gel loading control. These experiments were performed in triplicate. (B) MYCN-induced proliferation in cells with MYCN OE or MYCN OE + STMN1 siRNAs. Cell viability in control siRNA, MYCN OE, and MYCN OE + STMN1 siRNA groups was verified using the Cell Counting Kit-8. The MYCN OE control siRNA group was defined as the control for Dunnett’s multiple comparison tests. * p < 0.05. These experiments were performed in triplicate. All WB images are included in File S1.