Neuroblastoma (NBL) is the most common extracranial solid tumor in children. Whilst, advances in treatment has significantly improved the survival rate of low-risk NBL patients, the overall survival rate of high-risk NBL group, especially N-Myc amplified patients, is poor (1). Hence, there is an urgent need to understand the molecular mechanisms that regulate treatment resistance and develop therapeutic strategies to improve the survival rate of high-risk NBL patients. In this study, p120ctn was identified as one of the key players in regulating treatment resistance and highlighted a novel avenue of targeting cell lineage to treat NBL.
RNA-Seq data analysis and immunohistochemistry was performed on 206 NBL patients. RNAi-based knockdown, CRISPR/Cas9-based knockout, immunofluorescence, xenograft models, quantitative proteomics, co-immunoprecipitation, flow cytometry and phosphoproteomics analysis were performed to uncover the role of p120ctn in NBL pathogenesis.
RNA-Seq and immunohistochemistry analysis highlighted the mesenchymal-like signature of NBL tissue samples. It was revealed that high expression of p120ctn positively correlated with metastasis and poor outcome. Knockdown of p120ctn activated Wnt signaling and reduced the expression of the N-Myc. Depletion of p120ctn induced mesenchymal-to epithelial transition which was highlighted by label-free quantitative proteomics and qPCR analysis of transcriptional factors. Moreover, contrary to the function of p120ctn as a tumour suppressor, p120ctn knockdown resulted in attenuation of angiogenesis, migration, invasion and sensitized the NBL cells to doxorubicin.
The molecules that are regulating this opposing function of p120ctn was further investigated by using confocal microscopy, co-immunoprecipitation and phosphoproteomics analysis. These analyses revealed the differential activation of RhoA in a cell type-dependent manner. Our study exhibits that p120ctn regulates the phosphorylation of Twist (S68) and thereby regulates EMT. Importantly, the regulation pf pTwist by p120ctn is cell type-dependent and correlates with the aggressive phenotype of NBL cells. Inducing a mesenchymal phenotype in epithelial colorectal cells did not change the function of p120ctn however, overexpression of E-cadherin in NBL cells reduced the expression of p120ctn and reversed epithelial-to-mesenchymal transition.
Overall, this study highlights the cell linage dependent roles of p120ctn and alteration of cell type as an alternative to treat high-risk NBL patients.