Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma

Marzia Ognibene,Annalisa Pezzolo

doi:10.1038/s41598-020-69499-6

Abstract

Neuroblastoma, an embryonic tumor arising from neuronal crest progenitor cells, has been shown to contain a population of undifferentiated stem cells responsible for the malignant state and the unfavorable prognosis. Although many previous studies have analyzed neuroblastoma stem cells and their therapeutic targeting, this topic appears still open to novel investigations. Here we found that neurospheres derived from neuroblastoma stem-like cells showed a homogeneous staining for several key nucleolar proteins, such as Nucleolin, Nucleophosmin-1, Glypican-2 and PES-1. We investigated the effects of Roniciclib (BAY 1000394), an anticancer stem cells agent, on neurospheres and on an orthotopic neuroblastoma mouse model, discovering an impressive inhibition of tumor growth and indicating good chances for the use of Roniciclib in vivo. We demonstrated that Roniciclib is not only a Wnt/β-catenin signaling inhibitor, but also a nucleolar stress inducer, revealing a possible novel mechanism underlying Roniciclib-mediated repression of cell proliferation. Furthermore, we found that high expression of Nucleophosmin-1 correlates with patients’ short survival. The co-expression of several stem cell surface antigens such as CD44v6 and CD114, together with the nucleolar markers here described, extends new possibilities to isolate undifferentiated subpopulations from neuroblastoma and identify new targets for the treatment of this childhood malignancy.

Highlights

Neuroblastoma, an embryonic tumor arising from neuronal crest progenitor cells, has been shown to contain a population of undifferentiated stem cells responsible for the malignant state and the unfavorable prognosis
This study proposes the involvement of several key nucleolar proteins, such as NCL25–28, Nucleophosmin-1 (NPM1)[40], Glypican-2 (GPC2)[41], and Pescadillo Ribosomal Biogenesis Factor-1 (PES1)[42] in the nucleolar stress induced by Roniciclib in both stem and non-stem NB cells
Primary neurospheres enzymatically digested after 5–7 days of culture and re-plated as single-cell suspension, generated a second and third passage of spheres, a feature that has been associated to CSCs10

Summary

Introduction

Neuroblastoma, an embryonic tumor arising from neuronal crest progenitor cells, has been shown to contain a population of undifferentiated stem cells responsible for the malignant state and the unfavorable prognosis. The co-expression of several stem cell surface antigens such as CD44v6 and CD114, together with the nucleolar markers here described, extends new possibilities to isolate undifferentiated subpopulations from neuroblastoma and identify new targets for the treatment of this childhood malignancy. Putative CSCs identification in primary tumor sphere derived from NB patients, and from cell lines was reported p reviously[14,15,16,17,18,19,20,21] In this respect, our group showed that the Frizzled receptor 6 (Fzd-6) is a new surface marker of aggressive NB cells with stem celllike features[22]. Fzd-6+ NB cells formed neurospheres with high efficiency, resistant to doxorubicin killing, and expressing high levels of mesenchymal markers such as Twist-1 and Notch-122 These data suggested that targeting CSCs signatures might affect chemo-resistant NB cells. The action mechanisms of Roniciclib are not yet completely understood

Methods

Results

Conclusion