Abstract
BackgroundNeuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied.MethodsRNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells.ResultsIn response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts.ConclusionsHuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.
Highlights
Neuronal-origin Hu Antigen D (HuD) (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers
HuD is overexpressed in neuroblastoma and is required for maintaining neuroblastoma viability HuD protein levels are significantly upregulated in patient neuroblastoma (NB) tissue array samples when compared with normal peripheral nerve tissue (Fig. 1A)
At the same time that HuD is suppressing mTOR Complex 1 (mTORC1) activity and enhancing autophagy, we showed that HuD promotes antiapoptosis via ARL6IP1
Summary
Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. The neuron-specific RNA binding protein Hu Antigen D (HuD) ( known as Bishayee et al J Exp Clin Cancer Res (2022) 41:18. The role of HuD in developing neurons, adult neurons and a few other non-cancer types including pancreatic β cells has been fully documented; its role in neuroblastoma remains to be defined [6,7,8]. The above findings in regards to the RNA substrates for HuD and their significance have only focused on neurons and other non-transformed cells
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