Abstract

1. 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 also 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. Standard treatment consists of surgical resection and radiotherapy in combination with temozolomide (TMZ) chemotherapy [10,11]. GBM has poor prognosis with only 28.4% of patients surviving one year and 3.4% surviving to year five [12,13,14]. Brain cancer cells are implicated in manipulating not only the tumour microenvironment but also systemic immunity to their advantage [15,16].

Extracellular vesicles (EVs) are recognised players in mediating such changes, being lipid bilayer-enclosed structures, 30–1000 nm in diameter, released from cells and acting as key-mediators for intra/inter-tumour communication through horizontal transfer of functional proteins and nucleic acids (mRNA, miRNA, lncRNA, sncRNA) [17,18,19,20]. EVs have great potential as diagnostic and prognostic biomarkers in a range of pathologies, including GBM, and therefore increased understanding of EV-mediated functions in GBM biology is urgently needed [21,22]. In GBM, EV protein-cargo has for example been associated with the phenotypic signature of GBM cells [23], while TMZ treatment has been shown to increase EV release in GBM and to promote more pro-oncogenic EV signatures [24]. Similarly, as in other cancers, GBM cells use EVs for inter-cellular communication in the tumour and to influence the surrounding microenvironment to promote tumour growth, angiogenesis, metabolism and invasion [20,25,26,27,28,29,30]. The regulation of EV biogenesis has therefore received increasing attention in recent years as an interceptive strategy in cancer, both to sensitize cancer cells to chemotherapy and to limit tumour growth in vivo [8,9,31,32,33,34,35,36,37,38]. The peptidylarginine deiminase (PAD)-mediated pathway of EV biogenesis has been highlighted as a novel and significant contributor to EV release in a range of cancer cells [8,9,33,36], including most recently in GBM by our group [9]. We previously showed that pan-PAD inhibition, using Cl-amidine, significantly modulated EV signatures, both with respect to EV numbers released, as well as by changing EV-related microRNA cargo to a more anti-oncogenic signature [9]. As Cl-amdine showed different effects on LN18 and LN229 GBM cells and due to the fact that it is a pan-PAD inhibitor with inhibitory effects on PAD2, PAD3 and PAD4 isozymes [39], further studies on assessing PAD2, PAD3 and PAD4 isozyme-specific inhibitors in these two GBM cell lines are warranted. Novel interventions for targeted EV regulation and inhibition may be of particular importance in GBM treatment, considering recent reports on TMZ-mediated increase in EV release and modulated EV cargos dedicated to cell adhesion processes therefore possibly increasing pro-tumoral communication by standard TMZ treatment [24].

Besides effects on EV release, PADs are well known for their contribution to a range of pathologies, including cancers, by converting arginines into citrulline, causing post-translational deimination and changes in target protein tertiary structure and function [1,2,3,8,40]. PADs are gaining increased attention in the context of GBM research. Previous studies have established that grade IV GBM patient samples show an increase in cytoplasmic and nuclear deiminated proteins, albeit not identifying specific protein candidates [7]. A recent study by our group established that the pan-PAD inhibitor Cl-amidine reduced deimination of the mitochondrial house-keeping protein prohibitin (PHB) and deimination of histone H3, as well as identifying a range of protein candidates that are deiminated in LN18 and LN229 cells under normal growth conditions [9]. This included prohibitin (PHB), a multifaceted protein with key roles in mitochondrial housekeeping and tumorigenesis [41,42,43,44]; Stromal interaction molecule 1 (STIM-1) which is a membrane ER-resident protein with important roles in calcium-homeostasis and cancer invasion [45,46,47]; and moesin, a critical factor for cell migration, filopodia formation [48] and associated with more aggressive forms of GBM [49,50]. The assessment of proteins involved in mitochondrial function, cancer progression and invasion is of considerable relevance both with respect to PAD-inhibitor mediated changes in total protein levels and with respect to their post-translational deimination, as this may affect protein structure, function and protein–protein interactions [1,40].

As five isozyme-specific PADs are known in mammals [1], which display tissue specific expression and different preferences for target proteins, the difference in prominence of the main three isozymes related to cancer and the CNS (PAD2,3 and 4) is of additional interest for isozyme-specific targeting relating to cancer types and cancer sub-types, including heterogeneous cancers like GBM. Indeed, in LN18 and LN229 GBM cell lines these three PAD isozymes have been found to be differently expressed [9] (Supplementary Figure S1A). An increase in PAD4 staining has been reported in undescribed astrocytomas [51], while PAD2 and PAD3 upregulation via cAMP-PKA signalling has been shown in U251MG astrocytoma cells [52]. This indicates some differences in PAD isozyme expression in different glioma and astrocytoma cells. In addition, PAD-upregulation was shown in a study assessing response to hypoxia in malignant gliomas [53].

Hypoxia related pathways may be of considerable relevance due to the hypoxic core of a tumour mass, which also contains the therapy resistant glioma stem-like cells, a well-recognized problem in the standard treatment of GBM tumours [10,11]. PAD activation has indeed been linked to hypoxia in the CNS [53,54,55] and deiminated KEGG (Kyoto Encyclopedia of Genes and Genomes) protein pathways for HIF-1 regulation have been identified to be enriched in animal models of hypoxia- and cancer-resistance [56,57]. PADs also modulate neuronal stem cell growth and death [58], which is of importance considering that GBM tumour invasion, progression and chemo-resistance of recurrent GBM are partly linked to stem-ness [59,60]. Therefore, effects of PAD inhibitors on PAD mediated pathways including EV-mediated export of a range of cancer and hypoxia related microRNAs are also of considerable interest. As we have previously shown pan-PAD inhibition to be effective for regulating EV release and two key microRNAs (miR21 and miR126) in GBM, we furthermore 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]. Effects of the PAD-isozyme specific inhibitors on the invasion ability of GBM cells was also assessed, revealing isozyme-specific differences in the two different GBM cell lines, which also related to the effects observed on EV release inhibition. Furthermore, the PAD2, 3 and 4 isozyme-specific inhibitors differentially reduced total protein levels of PHB, moesin and STIM-1, showing overall an anti-oncogenic regulation of these 3 proteins following PAD-inhibitor treatment. In summary, our findings indicate PAD isozyme-specific regulation in the pro-oncogenic communication in GBM and highlight the potential of using PAD-isozyme specific inhibition for tailored treatment of GBM subtypes.

2. Results

2.1. PAD Isozyme-Specific Inhibitors Differently Modulate EV Release in LN18 and LN229 GBM Cells Following 1 h Treatment

Both LN18 and LN229 cells showed significant changes in EV release following 1 h PAD isozyme-specific inhibitor treatment, and this varied between the two cell lines (Figure 1). Both PAD2 and PAD4 inhibitors showed stronger effects on reducing EV release in LN18 cells (50%), although not reaching statistical significance (Figure 1A). In the LN229 cells on the contrary, both PAD2 and PAD4 inhibitor treatment resulted in some increased EV release, although not statistically significant (Figure 1B). After 1 h treatment with the PAD3 inhibitor, a significant reduction in numbers of EVs released was observed in the LN229 cells only (5-fold, p = 0.0334), while no significant change was observed in the LN18 cells.

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Figure 1

Peptidylarginine deiminase (PAD)2, PAD3 and PAD4 isozyme-specific inhibitor treatment shows glioblastoma multiforme (GBM) cancer cell line specific regulation of extracellular vesicle (EV) release. (A) Effects of PAD2 and PAD4 inhibitors on EV release in LN18 cells. (B) Effects of PAD2 and PAD4 inhibitors on EV release in LN229 cells. (C) Effects of PAD3 inhibitor on EV release in LN18 cells. (D) Effects of PAD3 inhibitor on EV release in LN229. (D). For each set of histograms, respectively, the PAD isozyme-specific inhibitor-treated and control-treated cells were run under the same experimental conditions. Exact p-values are indicated (* indicates significant differences with p < 0.05; n = 3 biological replicates for all).

Figure 2 furthermore shows representative nanoparticle tracking analysis (NTA) profiles for EV size distribution of LN18 and LN229 control and PAD isozyme-specific treated GBM cells (Figure 2A–H), alongside characterisation of EVs by western blotting using the EV-specific markers CD63 and Flot-1; the absence of β-actin in EVs was assessed to rule out cell-contamination (Figure 2I). Typical morphology of EVs was verified by TEM (Figure 2J).

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Figure 2

NTA size distribution profiles of EVs released from LN18 and LN229 cells following PAD isozyme-specific inhibitor treatment for 1 h and EV characterisation. Representative NTA profiles of LN18 cells following 1 h PAD inhibitor treatment (A–D): (A) Control DMSO treated cells; (B) PAD2 inhibitor treated cells; (C) PAD3 inhibitor treated cells; (D) PAD4 inhibitor treated cells. Representative NTA profiles of LN229 cells following 1 h PAD inhibitor treatment (E–H): (E) control DMSO treated cells; (F) PAD2 inhibitor treated cells; (G) PAD3 inhibitor treated cells; (H) PAD4 inhibitor treated cells. (I) Western blotting analysis (WB) showing that EVs isolated from LN18 and LN229 cells are positive for the EV specific markers CD63 and Flot-1; β-actin is absent from the EVs but present in the cells. (J) Transmission electron microscopy (TEM) images showing characteristic EV morphology for EVs isolated from both cell lines; the scale bar indicates 50 µm. In the NTA curves the black line represents the mean of the 5 repetitive readings per individual sample and the red line represents standard error (+/−) between those same 5 readings per sample. Each treatment group was measured in 3 biological replicates.

EV modal size was overall not affected by any of the PAD inhibitors following 1 h treatment (Figure 3A,B), except for some increase observed in EV modal size (from 125 nm to 175 nm) following 1 h treatment with the PAD2 inhibitor in LN18 cells (p = 0.0022) (Figure 3A).

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Figure 3

Effects of PAD2, PAD3 and PAD4 isozyme-specific inhibitor treatment on EV modal size in GBM cells, following 1 h treatment. (A) Modal size of EVs released from LN18 cells and LN229 cells, respectively, following 1 h PAD2 and PAD4 inhibitor treatment. (B) Modal size of EVs released from LN18 cells and LN229 cells, respectively, following 1 h PAD3 inhibitor treatment. Exact p-values are indicated, error bars show SD (* indicates significant differences with p < 0.05; ns = non-significant change; n = 3 biological replicates for all).

2.2. MicroRNA EV-cargo is Differently Modulated in Response to 1 h PAD Isozyme-Specific Inhibitor Treatment in LN18 and LN229 GBM Cells

When assessing EV cargo for pro-cancerous, GBM and hypoxia related microRNAs (miR21, miR126, miR210), respectively, some significant expression changes were observed, specific to the two cell lines and in response to the different PAD inhibitors (Figure 4). In LN18 cells, PAD3 inhibitor had no significant effects while both PAD2 and PAD4 inhibitors significantly changed EV miR cargo as follows: pro-cancerous miR21 was significantly reduced by PAD2 and PAD4 inhibitors in LN18 cells by 1055-fold and 131-fold, respectively (Figure 4A); the GBM protective microRNA marker miR126 was significantly increased by 3.8-fold and 3.9–fold following PAD2 and PAD4 inhibitor treatment, respectively (Figure 4B); and the hypoxia related miR210 was significantly reduced by 9.8-fold and 10.6-fold in LN18 cell-derived EVs following PAD2 and PAD4 inhibitor treatment, respectively (Figure 4C). Overall, PAD3 inhibitor was more effective in the LN229 cells and significantly reduced miR21 by 535-fold (Figure 4D); significantly increased miR126 by 2.4-fold (Figure 4E); and significantly reduced miR210 by 11.4-fold in LN229 cell-derived EVs (Figure 4F). In LN229 cells, both PAD2 and PAD4 inhibitor also had some significant anti-oncogenic effect by reducing miR21 by 4.6 and 3.4-fold, respectively (Figure 4D), but did neither up-regulate miR126 (protective in GBM) nor have significant effects on miR210 in cell-derived EVs (Figure 4F).

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Figure 4

PAD isozyme-specific inhibitor mediated effects on EV microRNA cargo is GBM cell line specific. LN18 cell-derived EVs following 1 h PAD inhibitor treatment are shown in A–C: (A) PAD2, 3, and 4 isozyme-specific inhibitor-mediated effects on the pro-oncogenic miR21 in EVs derived from LN18 cells. (B) PAD2, 3, and 4 isozyme-specific inhibitor mediated effects on the anti-oncogenic miR126 in EVs derived from LN18 cells. (C) PAD2, 3, and 4 isozyme-specific inhibitor mediated effects on the hypoxia-related and pro-oncogenic miR210 in LN18 cells. LN229 cell-derived EVs following 1 h PAD inhibitor treatment are shown in D–F: (D) PAD2, 3, and 4 isozyme-specific inhibitor-mediated effects on the pro-oncogenic miR21 in EVs derived from LN229 cells. (E) PAD2, 3, and 4 isozyme-specific inhibitor mediated effects on the anti-oncogenic miR126 in EVs derived from LN229 cells. (F) PAD2, 3, and 4 isozyme-specific inhibitor mediated effects on the hypoxia-related and pro-oncogenic miR210 in LN229 cells. Results are represented as relative miR expression compared to the internal control miRs (2^Λ(−DDCT)) and normalised to expression in control-treated cells; exact p-values are indicated, error bars show SD (*indicates significant differences with p < 0.05; n = 3 biological and 3 technical replicates for all).

2.3. PAD Isozyme-Specific Inhibitors Affect PHB, STIM-1 and Moesin Protein Expression Differently in LN18 and LN229 GBM Cells Following 1 h Treatment

Following 1 h treatment with PAD2, PAD3, and PAD4 isozyme-specific inhibitors, respectively, the protein levels of PHB, STIM-1 and moesin were assessed by western blotting (Figure 5). The levels of PHB were somewhat reduced in LN18 cells both by PAD2 inhibitor (7% to 66%) and PAD4 inhibitor (1% to 49%), although not reaching statistical significance (Figure 5A); a similar pattern was seen in LN229 cells, with some reduction for PAD2 (7% to 21%) or PAD4 (19% to 80%) inhibitor (Figure 5A), but not reaching statistical significance. For effects on STIM-1 protein levels, both cell lines showed some reduction in this invasion protein following PAD2 and PAD4 inhibitor treatment. In LN18 cells, a 15% to 90% reduction (albeit non-significant) in STIM-1 was observed following PAD2 inhibition, while following PAD4 inhibition a significant (p = 0.0374) 33% to 90% reduction of STIM-1 protein levels was observed (Figure 5B). For LN229 cells, a significant (p = 0.0254) 29% to 34% reduction was seen in STIM-1 protein levels following PAD2 inhibition, while PAD4 inhibitor treatment resulted in 17% to 39% reduction in STIM-1 protein levels, almost reaching significance (p = 0.0571). For changes in moesin protein levels, neither PAD2 nor PAD4 inhibitor resulted in significant changes in moesin protein levels in LN18 cells, while in LN229 cells, moesin protein levels were significantly (p = 0.0163) reduced by 39% to 49% following PAD2 inhibitor treatment and by 13% to 27% (but non-significant) following PAD4 inhibitor treatment (Figure 5C).

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Figure 5

PAD2 and PAD4 isozyme-specific inhibitor 1 h treatment affects prohibitin (PHB), stromal interaction molecule 1 (STIM-1) and moesin protein levels in LN18 and LN229 GBM cell lines. (A) PHB protein levels in LN18 and LN229 cells, respectively, following 1 h treatment with the PAD2 and PAD4 inhibitors, compared to control-treated cells. (B) STIM-1 protein levels in LN18 and LN229 cells respectively, following 1 h treatment with the PAD2 and PAD4 inhibitors, compared to control-treated cells. (C) Moesin protein levels in LN18 and LN229 cells, respectively, following 1 h treatment with the PAD2 and PAD4 inhibitors, compared to control-treated cells. Representative blots are shown; the density ratios of the various proteins analysed and actin are presented as normalised quantified data (mean ±S.D.) for treatment with the PAD2 and PAD4 specific inhibitors, compared to DMSO treated controls. The corresponding molecular weight size standard is indicated in kilodaltons (kDa) on each blot. Exact p-values are indicated, error bars show SD (* indicates significant differences with p < 0.05; n = 3 biological replicates for all).

Following PAD3 inhibitor treatment, PHB protein levels were significantly reduced in LN18 cells by 27% to 38% (p = 0.0231), and up to 45% in LN229 cells, albeit non-significantly, following 1 h treatment (Figure 6A). PAD3 inhibitor did not significantly change STIM-1 protein levels in LN18 cells but resulted in significantly reduced STIM-1 protein levels in LN229 cells (26% to 53%; p = 0.0110) (Figure 6B). Moesin protein levels were not significantly affected by PAD3 inhibitor either in LN18 or LN229 cells, although a 1% to 68% (but non-significant) reduction was observed in LN229 cells (Figure 6C).

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Figure 6

PAD3 isozyme-specific inhibitor 1 h treatment affects PHB, STIM-1 and moesin protein levels in two GBM cell lines. (A) PHB protein levels in LN18 and LN229 cells, respectively, following 1 h treatment with the PAD3 inhibitor, compared to control-treated cells. (B) STIM-1 protein levels in LN18 and LN229 cells, respectively, following 1 h treatment with the PAD3 inhibitor, compared to control-treated cells. (C) Moesin protein levels in LN18 and LN229 cells, respectively, following 1 h treatment with the PAD3 inhibitor, compared to control-treated cells. Representative blots are shown; the density ratios of the various proteins analysed and actin are presented as normalised quantified data (mean ±S.D.) for treatment with the PAD3 specific inhibitor, compared to DMSO treated controls. The corresponding molecular weight size standard is indicated in kilodaltons (kDa) on each blot. Exact p-values are indicated, error bars show SD (* indicates significant differences with p < 0.05; n = 3 biological replicates for all).

Besides assessing a ranging effect of the three isozyme-specific inhibitors on changes of total protein levels of PHB, moesin and STIM-1, all three proteins were verified to be post-translationally deiminated as assessed by blotting the F95 protein eluates from both cell lines with the PHB, moesin and STIM-1 antibodies (Supplementary Figure S1B). Furthermore, FoldIndex© analysis revealed a number of disordered regions in moesin and STIM-1 (Supplementary Table S1).

2.4. PAD Isozyme-Specific Inhibitors Differently Affect Invasion in LN18 and LN229 GBM Cells

The invasiveness of LN18 and LN229 cells was studied in Boyden chambers with Matrigel. LN18 cells demonstrated noticeable invasion over 16 h (Figure 7A, A.1), while invasiveness of LN229 cells was far lower compared to that observed for LN18 cells. Incubation for 16 h with PAD2 and PAD4 inhibitors resulted in a significant suppression of invasiveness in LN18 cells by 39.3% (p ≤ 0.0001) and 23.2% (p = 0.0020) respectively, while less effect was observed following treatment with the PAD3 inhibitor (9.1%, p = 0.0215) (Figure 7A, A.1; n = 3). Cellular invasion of LN229 cells was overall lower than for LN18 cells (Figure 7B, control panel), and was significantly suppressed only by the PAD3 inhibitor (16.5%, p = 0.0019) (Figure 7B, B.1; n = 3), while neither PAD2 nor PAD4 inhibitors showed any significant effect on invasion in LN229 cells (0.4% and 1.25%, respectively; ns; n = 3) (Figure 7B, B.1). There was no significant change in cell proliferation over the 16 h incubation time with the PAD inhibitors for either cell line, compared to control treated cells (Figure 8A,B; n = 3).

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Figure 7

Cell invasion is differently affected by PAD isozyme-specific inhibitors in LN18 and LN229 GBM cells. Representative images of LN18 (A) and LN229 (B) cells following 16 h treatment with the three PAD-isozyme specific inhibitors, compared to control (DMSO)-treated cells following crystal violet treatment (imaged using a 10× objective). A.1. The corresponding histogram for the MTT assay for LN18 cell invasion following 16 h treatment with all three PAD isozyme-specific inhibitors. B.1. The corresponding histogram for the MTT assay for LN229 cell invasion following 16 h treatment with all three PAD isozyme-specific inhibitors. Exact p-values are indicated, and error bars show SD (n = 3 biological and 3 technical replicates for all).

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Figure 8

Cell proliferation assay for PAD isozyme-specific inhibitors in LN18 and LN229 GBM cells. (A). Representative images of LN18 and LN229 cells following 16 h treatment with the three PAD isozyme-specific inhibitors, compared to control (DMSO)-treated cells. (B) (imaged using a 10× objective). Chresyl violet assay revealed no significant effects (ns) of any of the three inhibitors on cell proliferation, following 16 h incubation, compared to control (DMSO)-treated cells (n = 3 biological and 3 technical replicates for all).

2.5. Deiminated Protein Targets and KEGG Networks Enriched in Deiminated Proteins Differ in LN18 and LN229 GBM Cells under Standard Culture Conditions

STRING analysis revealed some common deiminated KEGG pathways (glycolysis/gluconeogenesis, ribosome, splicosome, protein processing in ER, carbon metabolism, oestrogen signalling pathway, biosynthesis of amino acids, arrhythmogenic right ventricular cardiomyopathy, antigen processing and presentation, Huntington’s disease, pathogenic Escherichia. coli (E. coli) infection) in both GBM cell lines, while several pathways were enriched for deiminated proteins in LN18 cells only (gap junction, aminoacyl-tRNA biosynthesis, pentose phosphate pathway; phagosome, necroptosis, Epstein–Barr virus infection, legionellosis, salmonella infection, mRNA surveillance pathway, longevity regulating pathway, central carbon metabolism in cancer, HIF-1 signalling pathway, thyroid hormone synthesis) or in LN229 cells only (pyruvate metabolism, IL-17 signalling pathway). Overall, more protein hits were identified as deiminated in LN18 cells (417 protein hits; Supplementary Table S2) compared to in LN229 cells (300 hits; Supplementary Table S3). KEGG pathways for deiminated proteins identified in both cell lines are presented in Supplementary Figures S3 and S4 for LN18 and LN229 cells, respectively (see also Supplementary Tables S2 and S3 for full LC-MS/MS data analysis of all protein hits). The Venn diagram in Figure 9 summarises common and distinct KEGG pathways enriched in deiminated proteins in LN18 and LN229 cells under normal culture conditions (see Supplementary Figures S3 and S4 for protein–protein interaction networks showing all identified KEGG pathways enriched in deiminated proteins).

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Figure 9

Deiminated Kyoto Encyclopedia of Genes and Genomes (KEGG) protein networks differ between LN18 and LN229 GBM cells. A Venn-diagram representing shared and different KEGG pathways identified to be enriched in deiminated proteins in the two GBM cell lines under study (LN18 and LN229), under standard culture conditions. For detailed network analysis highlighting the different KEGG pathways in both cell lines see Supplementary Figures S3 and S4.

3. Discussion

Regulation of EV release is critical for cellular communication and modulation of the microenvironment in cancer progression and metastasis and recognised to be of increasing importance in GBM [21,22]. GBM-derived EVs have furthermore been shown to induce tumour-promoting transformation of subventricular zone resident neural stem cells, possibly contributing to GBM recurrence [64]. Differences in EV release profiles between GBM cell lines, including EV sub-populations and during normal growth conditions, have previously been reported by us and others [9,38,65]. Several studies have assessed various types of EV cargo in GBM, including protein and genetic material [9,20,38,66,67,68]. Due to a range of pro-oncogenic cargo components transported by GBM cells for intra-tumour communication and the penumbra, including evidence for increased pro-oncogenic EV-release in response to standard chemotherapeutic treatment with TMZ [24], adjunct therapies that can modulate EV release and result in anti-oncogenic signatures, irrespective of or in conjunction with changes in EV numbers released, are of considerable interest. In the current study, both significant reduction in EV release, as well as anti-oncogenic modulation of microRNA expression following PAD isozyme-specific inhibitor treatment was observed in the two GBM cell lines, representative of a chemo-resistant (LN18) and chemo-sensitive (LN229) type [69]. A slight but significant increase in EV modal size was furthermore observed in LN18 cells following PAD2 inhibitor treatment and may have some relevance in cellular communication which will need further exploration, although aggregation of EVs contributing to this shift in modal size identified by NTA analysis cannot be excluded at this stage. Effects of the PAD isozyme-specific inhibitors on the modulation of microRNA EV-cargo highlight approaches for targeted modulation of EV cargo to change GBM intra- and inter-tumour communication.

Both microRNAs and short non-coding RNAs have been identified in GBM EVs, with miR21 being one of the highest expressed microRNAs [20]. Changes in miR21 have been shown to affect viability, senescence and invasion in GBM [70,71,72], with miR21 silencing leading to decreased tumour size and improved survival in GBM animal models [73]. Furthermore, exosomes engineered to suppress miR21 were recently shown to reduce tumour volume in vivo in a rat GBM model [74]. Inhibition of miR21 has also been shown to enhance chemo-sensitivity of TMZ-resistant GBM cells in vitro [75]. We recently identified that miR21 was significantly reduced in LN18 and LN229 cells following pan-PAD inhibitor treatment and identified in the current study that the PAD2 and PAD4 inhibitors were most potent at reducing relative expression of miR21 in EV cargo of LN18 cells, while PAD3 inhibitor showed strongest relative reduction in miR21 expression in LN229 cell-derived EVs, out of the three PAD inhibitors tested. This also correlated with that PAD2 and PAD4 inhibitor most significantly reduced cell invasion in LN18 cells, as assessed by Matrigel™ Invasion Chamber assay, while only PAD3 inhibitor significantly affected cell invasion in LN229 cells.

In GBM-derived patient samples, miR126 has been found to be decreased and related to high histopathological grades; while higher intra-tumoural miR126 levels are indicative of significantly improved survival duration, compared to patients with lower miR126 levels [76]. Over-expression of miR126 has been shown to suppress glioma cell proliferation and invasion in vitro [77]. Previously we found that miR126 was significantly elevated in GBM cells and cell-derived EVs following pan-PAD inhibitor treatment and have in the current study identified that in LN18 cell-derived EVs PAD2 and PAD4 inhibitors were significantly effective in raising relative miR126 expression, while PAD3 specific inhibitor was not effective in increasing relative miR126 expression in LN18 cell-derived EVs. In LN229 cell-derived EVs, only PAD3 inhibitor significantly increased relative levels of miR126 while PAD2 and PAD4 inhibitors slightly (but significantly) decreased miR126; therefore, only PAD3 inhibitor achieved the “protective” anti-oncogenic response by elevating relative levels of miR126 in LN229 cell-derived EVs. Interestingly, cannabidiol, which is effective in GBM treatment [78,79] and was also recently identified as an potent EV modulatory agent [37,38], has also been found to elevate miR126 and reduce miR21 EV-cargos from LN18 and LN229 cells, possibly explaining some of its protective functions in GBM [37].

miR210 has previously been identified as a major miR induced under hypoxia and has important roles in mitochondrial metabolism, DNA damage response, cell proliferation and apoptosis [80]. MiR210 is furthermore implicated in the regulation of cell glycolytic activity, is linked to inflammation [81] and is also involved in angiogenesis and vascular remodelling [82], including via EV-mediated transport [83]. miR210 has been identified as a regulator of the hypoxia pathway [84,85] and belongs to a group of hypoxia-mediated microRNAs, which are upregulated in glioma compared to normal brain tissue [86] and upregulated in patient-derived glioblastoma spheroids upon hypoxia exposure [87]. Elevated plasma expression of miR210 has been found in GBM compared to low-grade astrocytoma patients [88], as well as being elevated in serum and associated with tumour grade and poor patient outcome [62]. miR210 is also elevated and associated with worse prognosis in higher grade GBM tumours, compared to lower grade gliomas [61] and promotes hypoxic survival and chemoresistance in GBM cells [89]. Furthermore, miR210 has recently been found to belong to miRs that encourage radioresistance of GBM [63]. In the current study PAD2 and PAD4 inhibitors significantly reduced relative miR210 levels in LN18 cell-derived EVs, while in LN229 cell-derived EVs only the PAD3 inhibitor significantly reduced relative miR210 levels.

The observed decrease in pro-oncogenic miR21, hypoxia related and pro-oncogenic miR210 and the increase in anti-oncogenic miR126 levels in GBM cell-derived EVs, caused by the different PAD isozyme-specific inhibitors in the current study, indicates GBM cell type selective anti-GBM functions of the different PAD isozyme-specific inhibitors. These observations highlight heterogeneity of PAD isozyme expression in different GBM cell lines.

Modulation of proteins involved in cancer invasion and progression is of pivotal importance for targeting cancer, including GBM. Targeted modulation of the invading GBM tumour edge, which can act as a seed for recurrence [90], is therefore of considerable importance. New strategies for regulation of invasion-proteins including via inhibition of post-translational deimination, which may facilitate, for example, actin assembly and proteins involved in filopodia formation, as well as targeting proteins linked to stem-ness in the tumour periphery, promoting tumour invasion and rapid tumour progression, may therefore be of interest.

Previously we established that pan-PAD inhibitor Cl-amidine modulated cancer promoting proteins, including in GBM [9,33]. Prohibitin is a multifaceted protein involved in cell survival and apoptosis and a key mitochondrial house-keeping protein [41,44]. We had previously identified that pan-PAD inhibitor Cl-amidine reduced PHB expression somewhat following 1 h treatment, in addition to affecting post-translational deimination, in the two GBM cell lines under study [9], although such effects were more pronounced in the LN18 cell line [9]. In the current study, PAD isozyme-specific inhibition highlighted differences between the two GBM cell lines and PAD isozyme-specificity on regulation of PHB protein levels, following 1 h treatment. In LN18 cells, PAD3 inhibitor led to a significant reduction in PHB levels, correlating with previous findings that Cl-amidine (pan-PAD inhibitor with most specificity for PAD3) affected PHB levels in this GBM cell line [9]. Both PAD2 and PAD4 inhibitor less effectively modified PHB levels in LN18 cells, with PAD2 inhibitor somewhat more effective in lowering PHB levels (albeit not significantly) than PAD4 inhibitor, following 1 h treatment. In LN229 cells PHB reduction was more pronounced following 1 h treatment with PAD2 and PAD3 inhibitors, although not significant and overall less than for LN18 cells. This correlates with previous findings showing non-significant changes in PHB levels in LN229 cells using pan-PAD inhibitor Cl-amidine for the same time duration [9].

STIM-1 was previously identified to be a deiminated protein candidate in GBM cells by our group [9], and folding dynamics of STIM-1 have been discussed with respect to adaption to differential roles in Ca²⁺ homeostasis and signalling [45]. Roles for STIM-1 in Ca²⁺ entry mechanisms have been highlighted in a range of pathophysiological processes including infection and cancer [91]. As STIM-1 plays roles in Ca²⁺ regulation, it also has effects on EV release, which is in part a Ca²⁺ mediated mechanism and furthermore may impact mitochondrial function. Reduction in STIM-1 protein expression observed in the current study, following PAD isozyme-specific inhibitor treatment, correlates with a significant reduction observed in EV release. STIM-1 activity has indeed been shown to be essential for GBM invasion [46]. In studies on GBM human U251 and rat C6 cells, STIM-1 knockdown has been shown to reduce Ca²⁺ influx and to inhibit tumour cell proliferation and induce apoptosis [92]. Higher STIM-1 expression is correlated with migration, tumour size and clinical outcome in cervical cancer [93] and such correlation with tumour size is also seen in breast cancer [94]. Furthermore, STIM-1 enhances cell migration and promotes metastasis of cancer cells [93,95]. The findings reported in the current study, showing reduction of STIM-1 protein following PAD isozyme-specific inhibitor treatment, align with these previous studies as the PAD inhibitors lead to reduced STIM-1 protein and also to reduced cell migration, as assessed by reduced invasion observed for GBM cells following PAD inhibitor treatment. In LN18 cells, PAD4 inhibitor was most effective at lowering STIM-1 levels, while in LN229 cells PAD3 inhibitor was significantly and most effective, with PAD2 and PAD4 inhibitor leading to some (but not significant) reduction of STIM-1.

Moesin is an ezrin-radixin-moesin (ERM) family member and involved in the regulation of cell adhesion, polarity and migration [96]. Moesin connects the actin cytoskeleton to transmembrane receptors, and its upregulation is correlated with increased cell invasion and migration in GBM [49]. Moesin has been associated with filopodia formation, which are dynamic actin-rich membrane protrusions important for cell adhesion, membrane trafficking (including EV internalisation) [48] and therefore also of importance in cancer cell adhesion and invasion [97,98,99]. The correlation of moesin overexpression with higher grade GBM has also been related to its ability to increase stem cell neurosphere formation [50,100], which may furthermore promote stem-ness (which correlates with aggressiveness and chemo-resistance) in GBM. Increased moesin expression is related to invasion and metastasis and is also correlated to a progressive pathological state of pancreatic [101] and breast cancer [102]. In the current study PAD isozyme-specific inhibition resulted in some reduced moesin levels, which differed between the two GBM cell lines and PAD isozyme-specific inhibitor treatment. Moesin was not reduced by any PAD-isozyme inhibitor treatment in the LN18 cells, which overall have been reported to also be more chemo-resistant [69], while in LN229 cells PAD2 and PAD3 inhibitor lowered moesin levels following 1 h treatment.

It must be noted that the current study assessed changes in total protein levels only, following 1 h PAD inhibitor treatment of the three proteins under study, while PAD inhibition may also affect changes in post-translational deimination of the proteins, affecting protein–protein interactions, and this will need further detailed evaluation, including at longer time-points. The results of the invasion cell assay were assessed after a longer time-point, following 16 h, and do correlate with the anti-oncogenic signatures observed following 1 h PAD isozyme-specific treatment. Overall, more target proteins were found to be deiminated in LN18 cells compared to LN229 cells, and this also correlated with more significant effects of PAD inhibition on invasion in LN18 cells. We verified that all three proteins under study are post-translationally deiminated in both cell lines (Supplementary Figure S1B) while FoldIndex© analysis [103] furthermore shows that both STIM-1 and moesin are highly unfolded proteins (Supplementary Table S1), which makes them susceptible for post-translational deimination [40,104,105]. While PHB does not show any unfolded regions, a high number of arginines are present, and these pose as sites for citrullination/deimination (Supplementary Table S1). Furthermore, we previously showed that PHB deimination was reduced in GBM cells following 1 h pan-PAD inhibition [9]. Further in-depth investigation into roles for the most critical arg/cit conversions in the context of PAD-inhibitor treatment and PAD isozyme-specific preferences for deimination of target proteins could for example be explored using site-directed mutagenesis in selected proteins.

Our findings indicate differences in function of PAD isozymes 2, 3 and 4 in the two GBM cell lines, also differently affecting pro-oncogenic protein expression. PAD isozyme-specific inhibitor treatment in the two GBM cell lines reflects to some extent the finding that PAD2 and PAD4 are higher expressed in LN18 cells compared to PAD3, while PAD3 is higher expressed in LN229 cells compared to PAD2 and PAD4 [9] (Supplementary Figure S1A). This is in accordance with the PAD3 inhibitor being most effective at reducing EV release from LN229 cells and most effectively reducing STIM-1 protein levels in LN229 cells. Furthermore, PAD3 inhibitor most effectively modulated the three microRNAs under study to an anti-oncogenic signature in the LN229 cell-derived EVs. A similar trend was observed for LN18 cells, where PAD2 and PAD4 inhibitors most effectively reduced EV release (while PAD3 inhibitor had no significant effect) and also significantly modulated the three microRNAs to an anti-oncogenic signature in the cell-derived EVs. The PAD-inhibitor mediated effects on reducing protein levels of STIM-1, moesin and PHB, all pro-oncogenic proteins, was less prominently pronounced, although overall a trend for PAD2 and PAD4 inhibitors was observed in LN18 and for the PAD3 inhibitor in LN229 cells. The PAD isozyme-specific inhibitors affected cell invasiveness differently in the two GBM cell lines and were overall more effective in reducing invasiveness in LN18 cells, with PAD2 inhibitor being most effective but overall all three PAD inhibitors showing significantly reduced invasion of LN18 cells, while in LN229 cells only PAD3 inhibitor significantly reduced invasion following 16 h treatment. Furthermore, relative expression of miR21, involved in cell invasion, was most significantly reduced by PAD2 and PAD4 inhibitors in EV cargo from LN18 cells and by the PAD3 inhibitor in EV cargo from LN229 cells. Overall, this indicates isozyme-specific selection of target proteins and miRs involved in invasion, also as PAD isozyme expression significantly differed between the two cell lines (Supplementary Figure S1A; [9]). In both cell lines, the PAD inhibitors seemed to promote invasiveness independently of proliferation. This furthermore highlights current challenges in the treatment of GBM, which are known to be a highly heterogeneous group of tumours.

In both GBM cell lines under study, enrichment of deiminated proteins in a number of KEGG pathways was identified under normal culture conditions. Some pathways were common to both cell lines (including glycolysis/gluconeogenesis, ribosome, splicosome, oestrogen signalling pathway, carbon metabolism, bacterial infection, antigen processing and presentation, Huntington’s disease), while some KEGG pathways differed between the two cell lines. Interestingly, deiminated KEGG pathways for HIF-1 were identified in LN18 cells only, but not in LN229 cells, under normal conditions, and this may be indicative of some differences in hypoxia regulation via post-translational deimination. HIF-1 expression plays a major role in GBM development and progression, participating in glucose uptake, cancer proliferation, cell mobility, cancer stem cell metabolic reprogramming and chemo-resistance [106,107,108,109]. Inhibition of HIF-1 has been shown to induce cell death of GBM [110], and HIF-1 over-expression and silencing studies have furthermore shown regulation of specific miRNAs, including miR210, to be HIF-1 dependent [89]. To what extent deimination of these pathways plays roles in the regulation of hypoxia resistance and chemo-resistance remains to be further investigated. Interestingly, deimination of HIF-1 related KEGG pathways has recently been described in animal models of cancer resistance and hypoxia tolerance [56,57]. Furthermore, KEGG pathways for thyroid hormone synthesis were identified to be enriched for deiminated proteins in LN18 cells only. Thyroid hormone synthesis has been suggested as one of the pathogenic factors of GBM, with implications in formation and course of GBM pathogenesis [111,112]. LN18 cells also showed enrichment for deiminated proteins in KEGG pathways for Epstein–Barr virus infection (EBV). The relationship between EBV and GBM still remains to be fully understood [113] and has been recognized to be associated with worse patient prognosis [114]. In LN18 cells, deiminated proteins were enriched for the necroptosis pathway, which has been highlighted as a novel therapeutic target for GBM, as necrosis seems to be related to GBM proliferation, angiogenesis and invasion [115]. As necroptosis is a pathological and radiological hallmark of GBM [116], it has been proposed that modulating necroptosis in GBM could circumvent apoptosis resistance, which is common in GBM [115,117,118]. Therefore, regulation via post-translational deimination of these pathways, which could be targeted via selective PAD-inhibitors, may be of some interest. KEGG pathways for phagosome were also found to be enriched in protein deimination in LN18 cells. Phagosome KEGG pathways have been identified to be associated with TMZ resistance in GBM at the proteomic level [119] and related to the progression of glioma into glioblastoma by gene expression profiling [120]. Therefore, regulation via deimination may also be of considerable interest, particularly as this pathway was detected in LN18 cells, which are reported to be the more chemo-resistant of the two GBM cell lines [69]. While KEGG pathways for glycolysis, which is of high relevance for GBM [121], were identified to be deiminated in both GBM cell lines, the pentose phosphate pathway (PPP) was identified as a deimination KEGG pathway in LN18 cells only and the KEGG pathway for pyruvate metabolism in LN229 only. Both are related to glycolysis, with the PPP associated with cell migration and glial tumour aggressiveness [122,123]. Pyruvate plays a key role in GBM metabolic reprogramming [124] and modulation of the pyruvate pathway induces alterations of metabolic and stress-related pathways in GBM [121] also with putative roles in hypoxia-dependent resistance of GBM [110]. While carbon metabolism KEGG pathway was identified for deiminated proteins in both GBM cell lines, KEGG pathway for central carbon metabolism in cancer was identified in LN18 cells only. Carbon metabolism is related to metabolic adaption and growth in GBM [125,126], including in TMZ resistance [127] and in the control of glioma cell transformation to a higher aggressive stage [109]. Roles for post-translational deimination in such metabolic control have not been investigated and may be of relevance as this pathway was prominent for deimination in LN18 cells, which present a more aggressive and chemo-resistant form than LN229 [69]. Interestingly, enrichment of deiminated proteins in carbon metabolism KEGG pathway has also been recently recognized in whales [57], which are hypoxia tolerant and long-lived mammals displaying cancer resistance [128]. Only LN229 cells showed enrichment of deiminated proteins in IL-17 KEGG pathway, and this was not found in LN18 cells. Regulation of IL-17 via post-translational deimination may be of interest for recent IL-17 inhibitor treatment approaches, which were shown to decrease GBM tumour hypoxia, angiogenesis and tumour growth in animal models [129]. Furthermore, KEGG pathways for bacterial infection were identified to be deiminated in both GBM cell lines (pathogenic E. coli) but more prominently for LN18 cells (E. coli, salmonella and legionellosis). This may be of relevance for success of using bacterial carriers for glioblastoma therapy [130] as well as for management of bacterial infection following GBM surgery and treatment [131,132,133,134]. Indeed, critical roles for PADs, including pan-PAD and PAD-isozyme specific regulation of bacterial membrane vesicle release, critical for host-pathogen interactions, and in bacterial drug resistance, have recently been described [135]. Furthermore, the modulation of the host’s immune proteins via post-translational deimination by bacterial arginine deiminases is also a recognized mechanism for immune evasion [136].

The differences identified in the current study in deiminated KEGG pathways between the two GBM cell lines may indicate selective differences in the preference of target proteins for deimination by the different PAD-isozymes, particularly as in LN18 cells PAD2 and PAD4 isozymes are more prominently expressed while PAD3 is found at higher levels compared to PAD2 and PAD4 in LN229 cells [9].

In summary, our findings indicate PAD isozyme-specific regulation in the pro-oncogenic communication in GBM and highlight the potential of using PAD-isozyme specific inhibition for tailored treatment in GBM subtypes.

4. Materials and Methods

5. Conclusions

In summary, the findings of the current study highlight effects of PAD isozyme-specific regulation of EV release, modulation to anti-oncogenic microRNA signatures of EVs and effects on GBM cell invasion, including via effects on proteins involved in mitochondrial metabolism and invasion. Furthermore, our findings suggest differences in the regulation of KEGG pathways and protein–protein interaction networks, underlying pathogenesis of GBM tumours, via post-translational deimination. The findings presented here highlight recently identified roles for PADs in GBM, the need to assess PAD isozyme-specific processes in the heterogeneity of GBM and the potential for tailored treatment of GBM subtypes, using targeted PAD isozyme-specific inhibitors.

Acknowledgments

Abbreviations

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/21/4/1495/s1. Supplementary Figure S1. PAD isozyme detection and deimination of PHB, STIM-1 and moesin in LN18 and LN229 GBM cells. A. PAD2 and PAD4 protein detection is relatively higher in LN18, compared to LN229 cells (by 63% and 90%, respectively), while PAD3 protein detection is relatively higher in LN229, compared to LN18 cells (by 36%). B. PHB, STIM-1 and moesin are post-translationally deiminated in both LN18 and LN229 cells as assessed by blotting the F95-enriched proteins (IP:F95) from both cell lines against the three antibodies. Relative densitometry compared to internal actin-control is represented by “R”. Supplementary Figure S2. GBM cell viability following 1 h PAD2, PAD3 and PAD4 isozyme-specific inhibitor treatment. LN18 and LN229 cell viability was assessed by PrestoBlue assay following 1 h incubation with the PAD2 (AMF30a; 5 µM), PAD3 (Cl4-amidine; 5, 10, 50 and 100 µM) and PAD4 (GSK199; 10 µM) isozyme-specific inhibitors. Supplementary Figure S3. Protein–protein interaction network analysis of deiminated proteins in LN18 cells. KEGG pathways identified for F95 enriched proteins in LN18 cells are highlighted for: (A) immune responses, (B) metabolic pathways, (C) cancer-related pathways. Supplementary Figure S4. Protein–protein network analysis of deiminated proteins in LN229 cells. KEGG pathways identified for F95 enriched proteins in LN229 cells are highlighted for: (A) immune responses, (B) metabolic pathways, (C) cancer-related pathways. Supplementary Table S1. FoldIndex© analysis of prohibitin, moesin and STIM-1. All three proteins were verified to be deiminated in LN18 and LN229 GBM cells under normal culture conditions. The number of disordered regions, residue length of the longest disordered region, total number of disordered residues, as well as number of arginines present in the total number of residues for the individual proteins is shown. Supplementary Table S2. LC-MS/MS analysis of F95 enriched proteins in LN18 cells. The full list of protein hits identified following protein immunoprecipitation using the pan-deimination F95 antibody in LN18 GBM cells is presented. Supplementary Table S3. LC-MS/MS analysis of F95 enriched proteins in LN229 cells. The full list of protein hits identified following protein immunoprecipitation using the pan-deimination F95 antibody in LN229 GBM cells is presented.

Author Contributions

Conceptualization, S.L.; methodology, P.U.-O., A.M., I.K. and S.L.; validation, P.U.-O., J.M.I. and S.L.; formal analysis, P.U.-O., A.M., R.M. and S.L.; investigation, P.U.-O., A.M., R.M., and S.L.; resources, S.L., P.U.-O., P.R.T., I.K., J.M.I.; data curation, S.L.; writing—original draft preparation, S.L.; writing—review and editing, P.U.-O., J.M.I., A.M. and S.L.; visualization, P.U.-O., I.K., A.M. and S.L.; supervision, S.L.; project administration, S.L.; funding acquisition, S.L. and P.U.-O. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported in parts by a University of Westminster Start-up Grants to S.L. and P.U.-O.

Conflicts of Interest

The authors declare no conflict of interest.

References