The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro

Abstract

Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

“…Importantly, we found no change in gene expression of Vimentin , a hallmark in cells undergoing EMT (high Vimentin and low E-cadherin expression) [37]. A number of other genes involved in EMT in neuroblastoma, such as ACTN4 , MYH9 , ROCK1 and TCF3 [38, 39], failed to show any significant changes in our studies. Finally, we did not observe obvious cell morphological changes 72 hours after transient overexpression of TFAP4 .…”

Nanoscale Quantifying the Effects of Targeted Drug on Chemotherapy in Lymphoma Treatment Using Atomic Force Microscopy

“…showed that cellular surface changed remarkably after drug stimulations and the change of stimulation conditions (the addition of Rtx, the increase of stimulation concentration, the increase of stimulation time) could result in more significantly morphological alterations on the cells (e.g., distinct particles and pores occurred on the cell surface, cellular shape became irregular, cellular nuclei were discerned). Traditional studies for investigating the anticancer effects of chemotherapy drugs or monoclonal antibodies were commonly based on the ensemble results on a large number of cells by using various biochemical methods [26]- [28], e.g., flow cytometry, western immunoblotting, polymerase chain reaction (PCR), and proliferation/death assays. The flaw of these studies was that they cannot reveal the nanoscale situations taking place on single cells during the process of drug actions.…”

“…Cellular physiological activities are dynamic essentially. However, traditional biochemical methods (such as fluorescence, western immunoblotting, and PCR) destroy the natural structures of cells (e.g., labeling, staining, and lysis) [26]- [28], meaning that we cannot dynamically monitor the changes of cellular physiological parameters. Cellular surface roughness and mechanics detected by AFM are label free, and, thus, we can real-time monitor their dynamic changes on single cells, which can provide novel insights into our understanding of the dynamical cellular activities.…”

Neuroblastoma pathogenesis: deregulation of embryonic neural crest development

“…In NB, general EMT pathways have been associated with the development of drug resistance and correlate with lower overall survival. In an analysis of cisplatin resistant human NB cell lines, CHP-212Cis100, KellyCis83, and SK-N-ASCis24, all resistant lines compared to their cisplatin-sensitive counterparts were found to be enriched in gene pathways of a mesenchymal phenotype, including actin cytoskeleton signaling, integrin-linked kinase signaling, and epithelial adherens junction signaling (Piskareva, et al, 2015). In a similar analysis, SK-N-SH and SK-N-BE doxorubicin resistant cell lines had enriched expression of genes associated with epithelial to mesenchymal transition and had a more invasive phenotype as compared to their parenteral strains (Naiditch, et al, 2015).…”