P21Waf1 expression is regulated by nuclear intermediate filament vimentin in neuroblastoma

Xénia Mergui,Marie-Line Puiffe,Jean Bénard,Mounira Amor-Guéret,Marc Lipinski,Dominique Valteau-Couanet

doi:10.1186/1471-2407-10-473

Abstract

BackgroundHuman neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21Waf1 cyclin-dependent kinase inhibitor. We here investigated whether this correlation extend to the mRNA level in NB cell lines as well as in patients' tumors. We also further explored the relationship between expression of vimentin and p21, by asking whether vimentin could regulate p21 expression.MethodsVimentin and p21 mRNA levels in NB cell lines as well as in patients' tumors (n = 77) were quantified using Q-PCR. Q-PCR data obtained from tumors of high risk NB patients (n = 40) were analyzed in relation with the overall survival using the Log-rank Kaplan-Meier estimation. siRNA-mediated depletion or overexpression of vimentin in highly or low expressing vimentin cell lines, respectively, followed by protein expression and promoter activation assays were used to assess the role of vimentin in modulating p21 expression.ResultsWe extend the significant correlation between vimentin and p21 expression to the mRNA level in NB cell lines as well as in patients' tumors. Overall survival analysis from Q-PCR data obtained from tumors of high risk patients suggests that lower levels of p21 expression could be associated with a poorer outcome. Our data additionally indicate that the correlation observed between p21 and vimentin expression levels results from p21 transcriptional activity being regulated by vimentin. Indeed, downregulating vimentin resulted in a significant decrease in p21 mRNA and protein expression as well as in p21 promoter activity. Conversely, overexpressing vimentin triggered an increase in p21 promoter activity in cells with a nuclear expression of vimentin.ConclusionOur results suggest that p21 mRNA tumor expression level could represent a refined prognostic factor for high risk NB patients. Our data also show that vimentin regulates p21 transcription; this is the first demonstration of a gene regulating function for this type III-intermediate filament.

Highlights

Human neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes
We report that vimentin partly localizes to the nucleus of NB cells and regulates p21 transcription
Expression levels of vimentin and p21 correlate in NB cell lines and tumor samples with low p21 expression being associated with worse prognosis in high risk patients We initially observed that levels of vimentin and p21 were correlated at the protein level in S-type-like NB cell lines [4]. To test whether this was true at the mRNA level, ten NB cell lines were tested by real-time quantitative PCR (Q-PCR)

Summary

Introduction

Human neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21Waf cyclin-dependent kinase inhibitor. Neuroblastoma (NB), the most common extracranial solid tumor in children, derives from the sympathetic nervous system It may follow various courses, from spontaneous regression (stage 4S) to aggressive treatment-resistant diseases, which is often metastatic (stage 4) when diagnosed in children aged 12 months or older. NB tumors display various morphologies [1] and NBderived cell lines are classified into two subtypes on the basis of their appearance and expression profiles, the “N” (Neuronal) and “S” (Substrate-adherent) types [3]. In the course of this work, we noticed that the expression of the type-III intermediate filament vimentin, a marker of Stype cells, correlated with that of the p21Waf cyclindependent kinase (CDK) inhibitor [4]. Vimentin, which is known to interact with DNA directly via its N-terminal non-helical domain [6], binds mobile and repetitive sequence elements as well as nuclear matrix attachment regions (MARs) and displays a strong affinity for four-way junctions [7,8]

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Conclusion