Abstract
To overcome the lack of effective pharmacological treatments for high-risk neuroblastoma (HR-NB), the development of novel in vitro and in vivo models that better recapitulate the disease is required. Here, we used an in vitro multiclonal cell model encompassing NB cell differentiation stages, to identify potential novel pharmacological targets. This model allowed us to identify, by low-density RT-PCR arrays, two gene sets, one over-expressed during NB cell differentiation, and the other up-regulated in more malignant cells. Challenging two HR-NB gene expression datasets, we found that these two gene sets are related to high and low survival, respectively. Using mouse NB cisplatin-treated xenografts, we identified two genes within the list associated to the malignant stage (MCM2 and carbonic anhydrase 9), whose expression is positively correlated with tumor growth. Thus, we tested their pharmacological targeting as potential therapeutic strategy. We measured mice survival and tumor growth rate after xenografts of human NB treated with cisplatin in the presence of MCM2/carbonic anhydrase 9 inhibitors (ciprofloxacin and acetazolamide). MCM2 or carbonic anhydrase 9 inhibition significantly increased cisplatin activity, supporting their possible testing for NB therapy.
Highlights
It leads to reduced expression of multidrug resistance proteins (MDRs) in malignant NB cells, increasing susceptibility to cytotoxic drugs commonly used in NB therapy [1,9]
In a subcutaneous xenotransplant NB model the growth index of NB tumor nodules, the rate of GD2 positive malignant cells and the cell proliferation index are highly correlated with the expression level of two genes of our malignant set, MCM2 and carbonic anhydrase 9 (CA9), validating these genes as possible novel targets for NB therapy
In order to assess the progressive acquisition of neuronal traits during NB differentiation from Mock to S1.1 cells, we first measured in the four clones by the means of IF intensity signal the expression of four markers of neural differentiation: nestin (NES), Tubulin β3 Class III (TUBβ3), Doublecortin (DCX), and Microtubule Associated Protein 2 (MAP2)
Summary
In order to investigate neuroblastoma (NB) cell differentiation, we recently developed a novel in vitro model based on the controlled expression of NDM29 non-coding (nc)RNA in SKNBE-2 cell line [1].Biomedicines 2020, 8, 471; doi:10.3390/biomedicines8110471 www.mdpi.com/journal/biomedicinesNDM29 RNA is part of a set of ncRNAs transcribed by RNA Polymerase III, recently isolated and studied as pivotal regulators of different key processes in nervous system cells [2,3,4,5,6].In particular, NDM29 is located in the first intron of the ASCL3 (Achaete Scute-like homologue 3) gene; its over-expression promotes differentiation of NB cells into cells with neuron-like phenotype and causes inhibition of NB development and malignancy [1,7,8]. In order to investigate neuroblastoma (NB) cell differentiation, we recently developed a novel in vitro model based on the controlled expression of NDM29 non-coding (nc)RNA in SKNBE-2 cell line [1]. It leads to reduced expression of multidrug resistance proteins (MDRs) in malignant NB cells, increasing susceptibility to cytotoxic drugs commonly used in NB therapy [1,9]. This NB model is composed by four SKNBE-2 clones genetically engineered to integrate extra copies of NDM29 ncRNA transcriptional unit. We demonstrated that the induction of differentiation commitment in the context of anticancer therapy is possible by the pharmacological induction of NDM29 ncRNA expression which, in turn, increase the efficacy of the anticancer treatments
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