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

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. Neuroblastoma (NB) is an embryonic pediatric tumor that originates from neural crest progenitors cells 1,2. NB is the most common tumor in young children, about the 90% of cases happen in children less than 5 years old 3. NB is characterized by clinical, biologic and genomic remarkable heterogeneity that affected the survival of NB patients despite intensive therapy 4,5. Cancer stem cells (CSCs) are suggested to be responsible for drug resistance and relapse due to their ability to self-renew, and to differentiate into heterogeneous lineages of cancer cells 6. Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor, and from genetic or epigenetic differences among the cancer cells themselves 7,8. CSCs are the exclusive sources of all tumor cells, survive and persist after cancer therapy and are responsible for tumor relapse and metastasis 9. The concept of CSCs has immediate therapeutic consequences: if cancer growth is sustained by CSCs, then curative therapy will require targeting of this specific sub-population 10. The clinical presentation and the treatment response of advanced NB, which results in relapse and a refractory state after a good response to the initial chemotherapy, suggests the possibility that CSCs exist in NB tumors 11. Moreover, the heterogeneity of NB tumor histology and biology may suggest the existence of selfrenewing multipotent CSCs 12. NB tumors with unfavorable prognosis have been shown to contain a population of undifferentiated stem cells responsible for their malignant state 13. Putative CSCs identification in prima...

Genomic Analysis Made It Possible to Identify Gene-Driver Alterations Covering the Time Window between Diagnosis of Neuroblastoma 4S and the Progression to Stage 4

“…Exosomes for immunofluorescence analysis were resuspended in 100 µL 1× PBS for Cytospin cell preparation and incubated with anti-GD2 mAb (1:500; secreted from hybridoma cell line 14.G2, a generous gift by R.A. Reisfeld, The Scripps Research Institute, La Jolla, CA, USA), followed by a specific secondary antibody conjugated with Alexa 488 (green). Immunofluorescence analysis was performed as previously described [ 55 ]. Results were photographically documented using fluorescence microscope Axio Imager M2 (Carl Zeiss, Oberkoche, Germany).…”

Cancer Stem Cells and Nucleolin as Drivers of Carcinogenesis

“…Interestingly, treatment of neuroblastoma cells with Roniciclib, which induced cell differentiation and impaired neurospheres formation, also inhibited the expression of nucleolar nucleolin and of CSC markers such as CD44v6 and CD114 [ 177 ].…”

“…Importantly, in limiting the dilution functional assay it was shown that sorted nucleolin + triple negative breast cells were more tumorigenic in NOD scid gamma mice than nucleolin low/cells, evidencing that cell surface nucleolin per se enables the selection of efficient tumorigenic cells [14]. Interestingly, treatment of neuroblastoma cells with Roniciclib, which induced cell differentiation and impaired neurospheres formation, also inhibited the expression of nucleolar nucleolin and of CSC markers such as CD44v6 and CD114 [177].…”

Synthetic Heterocyclic Derivatives as Kinase Inhibitors Tested for the Treatment of Neuroblastoma

“…Very recently, it has been reported that roniciclib is active also on NB stem cells, which are a population of undifferentiated cells leading to an unfavorable prognosis. Ognibene et al demonstrated the anticancer effects of the compound on neurospheres derived from NB stem-cell-like and in an orthotopic NB mouse model [ 37 ].…”