Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines.

Pinar Uysal-Onganer,Rayan Mahmoud,Amy Maclatchy,Igor Kraev,Paul R Thompson,Sigrun Lange,Jameel M Inal

doi:10.3390/ijms21041495

Abstract

Glioblastoma multiforme (GBM) is an aggressive adult brain tumour with poor prognosis. Roles for peptidylarginine deiminases (PADs) in GBM have recently been highlighted. Here, two GBM cell lines were treated with PAD2, PAD3 and PAD4 isozyme-specific inhibitors. Effects were assessed on extracellular vesicle (EV) signatures, including EV-microRNA cargo (miR21, miR126 and miR210), and on changes in cellular protein expression relevant for mitochondrial housekeeping (prohibitin (PHB)) and cancer progression (stromal interaction molecule 1 (STIM-1) and moesin), as well as assessing cell invasion. Overall, GBM cell-line specific differences for the three PAD isozyme-specific inhibitors were observed on modulation of EV-signatures, PHB, STIM-1 and moesin protein levels, as well as on cell invasion. The PAD3 inhibitor was most effective in modulating EVs to anti-oncogenic signatures (reduced miR21 and miR210, and elevated miR126), to reduce cell invasion and to modulate protein expression of pro-GBM proteins in LN229 cells, while the PAD2 and PAD4 inhibitors were more effective in LN18 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins relating to cancer, metabolism and inflammation differed between the two GBM cell lines. Our findings highlight roles for the different PAD isozymes in the heterogeneity of GBM tumours and the potential for tailored PAD-isozyme specific treatment.

Highlights

Peptidylarginine deiminases (PADs) are calcium-dependent enzymes which cause structural changes in target proteins via post-translational deimination, which affects protein function, protein–protein interactions, gene regulation and causes generation of neo-epitopes [1,2,3]
As we have previously shown pan-PAD inhibition to be effective for regulating extracellular vesicle (EV) release and two key microRNAs in Glioblastoma multiforme (GBM), we set out to assess the effects of PAD isozyme-specific inhibitors on these two miRs, as well as on the hypoxia-related miR210, which is related to more aggressive forms and poor prognosis in GBM [61,62,63]
In the LN229 cells on the contrary, both PAD2 and PAD4 inhibitor treatment resulted in some increased EV release, not statistically significant (Figure 1B)

Summary

Introduction

Peptidylarginine deiminases (PADs) are calcium-dependent enzymes which cause structural changes in target proteins via post-translational deimination, which affects protein function, protein–protein interactions, gene regulation and causes generation of neo-epitopes [1,2,3]. Protein deimination can facilitate protein moonlighting, allowing proteins to exhibit a range of physiological and pathophysiological functions within one polypeptide chain [4,5]. PADs play important roles in cancer pathogenesis, including in the central nervous system [6,7,8,9]. Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumour in adults. Brain cancer cells are implicated in manipulating the tumour microenvironment and systemic immunity to their advantage [15,16]

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Conclusion