Glioblastoma with PRMT5 gene upregulation is a key target for tumor cell regression

Introduction

Post-translational modification (PTM) is essential to expand the cellular proteome, which can affect protein interaction, stability, and activity with other proteins on multiple sites. This PTM is mediated by a variety of enzymatic processes, including phosphorylation, hydroxylation, acetylation, and methylation [1]. Protein arginine methylation was characterized in 1964 by Hurwitz et al. [2]. They discovered that the enzyme arginine methyltransferase was responsible for adding a methyl group to the amino acid arginine in proteins. This modification plays an important role in the regulation of cellular processes, gene expression, homeostasis, and protein-protein interaction. The main type of protein arginine methyltransferases (PRMT) was PRMT1, and it was followed by nine other members [3, 4]. PRMT family enzymes catalyze three distinct types of methylation. Type I PRMTs, including PRMT1, 2, 3, 4, 6 and 8, catalyze mono-methylarginines (MMA) and asymmetric dimethylarginines (ADMA) synthesis [5]. The type II PRMTs which include PRMT5 and 9, catalyze MMA and symmetric dimethylarginines (SDMA) synthesis. PRMT7, a type III PRMT, catalyzes only MMA [6, 7]. All PRMT family members, which have been found to be highly expressed in several solid tumors, are considered dominant regulators of arginine methylation [8]. Although PRMT mutations are infrequent in these tumors, increased PRMT gene expression has been identified as a predictor of poor prognosis [8].

A new type of PRM, known as PRMT5, was recently discovered in mammals [3]. The protein has two domains: an N-terminal domain, which adopts a triosephosphate isomerase structure and binds to methylosome protein 50 (MEP50) for full methyltransferase activity, and a C-terminal domain, which contains all methyltransferase motifs and is essential for plasma membrane association as a catalytic domain [9]. PRMT5 is an epigenetic modifier that methylates histones to control gene expression. Specifically, it can methylate histone H2A at Arg3 to form H2AR3me2s and histone H3 at both Arg2 and Arg8 to form H3R2me2s and H3R8me2, respectively [10]. As H4R3me2s, PRMT5 can also methylate histone H4 on Arg3 [11]. PRMT5 functionally activates or suppresses gene expression by changing these residues in histone tails [4]. Studies over the last five decades demonstrated that PRMT5 is an oncoprotein that regulates a variety of cellular processes involved in cancer development through several signaling pathways [12]. Epigenetically, upregulated PRMT5 expression has been linked to a negative prognosis in numerous types of cancer [13, 14]. Expression levels in liver, lung, and breast cancers have been associated with large tumor size and advanced tumor grade [15], [16], [17].

The association between PRMT5 and diffuse gliomas has been explored in scattered studies [18], [19], [20], [21]. PRMT5 expression varies in different grades of astrocytomas, either undetectable or very low in low-grade gliomas, whereas high-grade gliomas have the highest levels of expression [19]. A significant increase in PRMT5 expression was seen in Grade 4 astrocytomas compared to normal brain tissue controls [20]. This expression negatively correlates with patient survival [21]. There is evidence to suggest that the growth of gliomas may be reliant on PRMT5 expression, making it a potential new target for glioblastoma therapy. Otani et al. demonstrated that blocking PRMT5 activity led to apoptosis in both differentiated and stem-like glioma tumor cells [22]. Moreover, Yan et al. demonstrated that PRMT5 mediates the methylation of arginine in P53 to control its activity [21]. Their findings implied that the significance of P53 arginine methylation may vary between tumor types and that PRMT5 could be an equally promising target in glioblastoma tumors where P53 is often lost or mutated and linked to a negative prognosis. This is because the findings indicate that cell death triggered by PRMT5 inhibition is unrelated to P53 mutational status [21].

The exact mechanism of PRMT5 upregulation in Grade 4 astrocytoma is not yet fully investigated. Upregulation of some transcriptional factors such as c-Myc or the downregulation of miRNAs may contribute to increased PRMT5 expression in glioblastoma [23, 24]. This increased expression can cause various effects on cellular processes, including alterations in gene expression, epigenetic modifications, and protein functions. Additionally, there was no sufficient evidence to observe the relationship between PRMT5 and isocitrate dehydrogenase (IDH) mutation in gliomas. One study found that IDH-mutant Grade 4 astrocytoma has lower levels of PRMT5 expression compared to IDH-wildtype glioblastoma, suggesting that PRMT5 may be a target of IDH mutation [25]. Our research objective is to investigate the correlation between the expression of the PRMT5 gene and the presence of IDH mutation in Grade 4 astrocytoma. Additionally, we aim to assess the influence of this association on tumor growth and recurrence.

Materials and methods

Protein expression measurement using IHC for anti-PRMT5 and IDH1 antibodies

Anti-PRMT5 antibody (rabbit monoclonal, Cat# EPR5772, Abcam, Cambridge, UK) was used on the 34 sections in IHC. The assay was processed through a GX-automated stainer from Ventana (Tucson, AZ, USA) Using an Ultra-View detection Kit from Ventana. The protocol involved deparaffinization using EZ preparation solution at 75 °C, followed by heat pre-treatment in a cellular medium for 60 min. This was further followed by an optimum incubation of 20 min at 75 °C after adjusting the antibody using a dilution of 1:200.

Anti-PRMT5 was used to identify tumor cells (TC) in the tumor microenvironment. Each section was examined using a light microscope (DM500 Leica, Germany) examined at ×10 high-power field (HPF) and a focal non-necrotic area with anti-PRMT5 expression was re-examined ×25 HPF. Cells with anti-PRMT5 expression were considered as PRMT5 positive while the total cells were defined as cells with expressed PRMT5 and non-stained cells. The labelling index (LI) of PRMT5 expression at each examined area was evaluated using the following equation:

Labelling Index  ( % ) = PRMT 5 + TC Total   cells × 100

IDH1^R132H (monoclonal mouse antibody, clone H09, Abcam, UK) was retrospectively assessed during tumor diagnosis and grading, using an automated stainer from Ventana (Tucson, USA). Sections in which >10 % of tumor cells were positively stained were defined as IDH1-mutant.

Three expression patterns were defined: no expression: 0 %; minimal expression: <20 %; moderate expression: 20–60 %; diffuse expression: >60 %. No tumors were found to have a minimal PRMT5 expression. Therefore, tumors with moderate expression were categorized as having low expression, while those with diffuse expression were categorized as having high expression (Figure 1). IDH1 mutation has been previously assessed using the IHC technique.

Figure 1:

Anti-PRMT5 stains tumor cells in the microenvironment using IHC. (a) Low expression (b) high expression. Magnification (×25)=100 µm on scale bar.

Results

Patients age range between 24 and 80 years (mean 50.2). IDH-wildtype was detected in 13 (38.2 %) tumors. PRMT5 protein was highly expressed in 30 (88.2 %) tumors and low expressed in 4 (11.8 %) tumors. PRMT5 gene expression was upregulated in 33 (97.1 %) tumors and downregulated in single tumor cases (2.9 %). The mean RFI is 15.5 months (standard deviation: 10.2). There was no major difference in diagnostic consistency between the two testing methods used for PRMT5 expression. Testing PRMT5 expression with RT-PCR was 87 % sensitive compared to IHC protein expression. The overall accuracy between the two testing methods was 85.3 %.

There was a significant statistical difference in RFI among tumors with different PRMT5 gene regulations and IDH mutations (p-value<0.001). IDH-wildtype tumors with upregulated PRMT5 gene or protein expression showed earlier tumor recurrence compared to IDH-mutant tumor with upregulated PRMT5 expression (Figure 2).

Figure 2:

The impact of PRMT5 expression on RFI. (a) The impact of PRMT5 protein expression and IDH mutation on RFI; (b) the impact of PRMT5 gene expression and IDH mutation on RFI.

Discussion

PRMT5 regulates the expression of genes involved in cell proliferation, cell cycle regulation, and DNA damage response. It also promotes the growth of cancer cells, which are thought to be responsible for tumor recurrence and resistance to treatment. PRMT5 has also been identified as a potential therapeutic target in many cancers [4]. Targeting PRMT5 with specific inhibitors can lead to the suppression of glioma cell growth and the restoration of the immune response. This approach showed promising results in some preclinical models and is currently being tested in clinical trials as a potential treatment for Grade 4 astrocytoma [18].

The exact mechanisms by which PRMT5 is overexpressed in Grade 4 astrocytoma are not fully understood. One possible mechanism is the upregulation of transcription factors that directly or indirectly activate PRMT5 expression [23]. Jin et al. found that the transcription factor E2F1, a key regulator of the G1 to S phase transition in the cell cycle involved in DNA replication and cell proliferation, can stimulate PRMT5 expression by binding to its promoter [24]. Another possible mechanism is the dysregulation of microRNAs (miRNAs) that target PRMT5 mRNA. In glioblastoma, the expression of these miRNAs is often downregulated, leading to increased PRMT5 expression [28].

Insufficient evidence exists to establish a direct correlation between PRMT5 and IDH mutation in gliomas. It is also unclear if IDH mutation or its products would affect PRMT5 gene activity. Our results revealed that most of the glioblastomas associated with upregulated PRMT5 had earlier tumor recurrence. It means that IDH is more directly linked to tumor recurrence than PRMT5 (Figure 2). Suvà et al. found that IDH-mutant Grade 4 astrocytoma has lower levels of PRMT5 expression compared to IDH-wildtype glioblastoma, suggesting that PRMT5 may be a downstream target of IDH mutation [25]. Additionally, IDH-mutant tumors have a distinct DNA methylation signature, which can lead to the dysregulation of genes involved in cell differentiation and proliferation.

PRMT5 has been shown to play a role in the regulation of DNA methylation, and it is possible that its dysregulation in IDH wild-type gliomas contributes to the altered methylation patterns observed in these tumors [29]. We believe that IDH mutation in Grade 4 astrocytomas with PRMT5 upregulation may lead to decreased cell proliferation and increased differentiation, suggesting that PRMT5 may be involved in maintaining the stem-like state of glioma cells. On the other hand, absent IDH mutation may lead to tumor re-growth and progression.

The relationship between hydroxyglutarate (2-HG), a product of IDH, and PRMT5 in glioma is complex. 2-HG is a structural analog of alpha-ketoglutarate (αKG), which is a cofactor for several enzymes involved in epigenetic regulation, including PRMT5. 2-HG can compete with αKG for binding to PRMT5 and other enzymes, effectively inhibiting their activity [30]. In addition to these direct effects on enzyme activity, 2-HG can also alter the availability of substrates for epigenetic regulation, such as histones, by affecting metabolic pathways in the cell. For example, 2-HG has been shown to inhibit the activity of demethylases, enzymes that remove methylation marks from histones, leading to alterations in the epigenetic landscape that contribute to gliomagenesis [10, 30].

It seems that the interaction between 2-HG and PRMT5 is bidirectional, with PRMT5 activity modulating 2-HG levels in gliomas. 2-HG usually accumulates in cells with IDH mutations, which can inhibit the activity of several enzymes involved in epigenetic regulation, including PRMT5. However, epigenetic dysregulation may occur when 2-HG is not released, and this can cause PRMT5 overexpression [10]. In the end, the exact mechanisms underlying the relationship between PRMT5 and IDH mutation in Grade 4 astrocytoma require further research.

PRMT5 inhibitors are a class of compounds that specifically target and inhibit the activity of the PRMT5 enzyme. In IDH-mutant WHO Grade-4 astrocytoma or glioblastoma, PRMT5 inhibitors have gained attention as potential therapeutic agents due to their ability to disrupt the aberrant PRMT5-mediated processes that contribute to tumor growth and progression [31, 32]. Preclinical studies have shown promising results regarding the use of PRMT5 inhibitors in glioblastoma. These inhibitors have been found to suppress the growth of glioblastoma cells, and induce cell cycle arrest, and apoptosis. Additionally, PRMT5 inhibitors have demonstrated the ability to inhibit the migration and invasion of glioblastoma cells, which are crucial processes involved in tumor metastasis. Based on our findings, we believe that PRMT5 inhibitor would make beneficial anti-tumor activity on IDH-wildtype glioblastoma with PRMT5 upregulation than downregulated tumors. PRMT5 inhibitors have also been shown to sensitize glioblastoma cells to other treatment modalities, such as radiation therapy and chemotherapy [32]. This suggests that combining PRMT5 inhibitors with existing therapies may enhance their effectiveness and improve patient outcomes. Further research and clinical trials are needed to evaluate the safety, efficacy, and potential side effects of these inhibitors in glioblastoma patients.

One acknowledged limitation of our study is the relatively low number of samples analyzed. However, it is important to note that our study aimed to examine the relationship between IDH and PRMT5 in Grade 4 astrocytoma.

Conclusions

IDH mutation directly affects the mechanism of PRMT5 expression in Grade 4 astrocytomas. IDH-wildtype glioblastoma with PRMT5 upregulation can accelerate tumor re-growth, implying that IDH and PRMT5 have a bidirectional mechanism.