Quantitative proteomic analysis to identify differentiation pathways activated by a potential combination therapy to treat neuroblastoma

Date
2020
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Neuroblastoma is a pediatric sympathetic nervous system cancer with a unique feature of remission in some patients due to spontaneous differentiation of metastatic tumors. A differentiation inducer, 13-cis RA, is currently used in the clinic to eradicate minimal residual disease (MRD). This treatment has improved outcomes but relapse is still seen in many high-risk patients. MRD is comprised of small cells including tumor initiating cells which are poorly differentiated stem-like cells that are unresponsive to chemotherapy and radiation. ☐ Combinatorial therapies have been shown to be more effective in oncotherapy and since cathepsin inhibition has been shown to reduce tumor size I treated SK-N-SH neuroblastoma cells with 13-cis RA or the cathepsin inhibitor (K777) or both and used shotgun proteomics to identify proteins affected by each treatment in order to identify changes in biological processes affected by networks of proteins. Remarkably, bioinformatic analysis showed that cathepsin inhibition was more effective in increasing proteins involved in neuronal differentiation and neurite outgrowth, when compared to 13-cis RA alone and that the combination of both compounds enhanced neuronal differentiation more than either individual treatment. 13-cis RA reduced levels of proteins that were involved in extracellular matrix synthesis and organization and increased levels of proteins that were involved in neurofilament bundle assembly, focal adhesion and granins while K777 increased proteins involved in neuronal differentiation, neurotrophin interactome, the APP interactome, MAPK signaling, homeostasis, transport, vesicles, endosome, lysosome, and autophagocytosis. Combining 13-cis RA and K777 also enhanced all of the processes increased by individual treatment, strengthening neuronal differentiation, focal adhesion, MAPK processes and neurotrophin interactome. Most markedly, the dual treatment expanded the repertoire of increased proteins that contribute to the neurotrophin interactome. Thus the combination of 13-cis RA and K777 leads to amplification of more comprehensive and collaborative signaling pathways that induce differentiation. Major proteins that led to the alterations in the biological processes were increased levels of RET, GDF15, PLAT, SCG2, NEFM, NEFL, APP, integrins, adhesion proteins and CRABP2; and reduced levels of CHGA, FSTL1, PRRX1 and collagens. Others have shown that CHGA, SCG2, PRRX1, and RET may be useful as biomarkers in NB patients. In particular CHGA and PRRX1 are increased in NB TIC. PLAT, a secreted protein was strikingly increased at the transcript, protein and proteomic level in three different cell lines and some studies show that it is positively correlated to neuronal differentiation, thus may be a potential biomarker. Expanding the NB biomarker signature repertoire as a result of this project can lead to more reliable testing. ☐ In this study, I established a comprehensive proteome network that identified pathways that lead to neuronal differentiation. Drug resistance often ensues due to drugs not affecting all of the heterogeneous cells in tumors, so drug combinations that affect comprehensive networks that lead to tumor differentiation or death are more likely to target all cells within the tumor. This proteome map may be used to identify biomarkers of disease prediction and drug efficacy. The key pathways and networks may be targeted to develop novel drugs to treat neuroblastoma.
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Keywords
Cathepsin inhibitor, Differentiation, Mass spectrometry, Neuroblastoma, Retinoic acid
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