Protein arginine methyltransferase 1 coordinates the epithelial-mesenchymal transition/proliferation dichotomy in gastric cancer cells

doi:10.1016/j.yexcr.2017.10.035

Experimental Cell Research

Volume 362, Issue 1, 1 January 2018, Pages 43-50

https://doi.org/10.1016/j.yexcr.2017.10.035 Get rights and content

Abstract

Protein arginine methyltransferase 1 (PRMT1) is up-regulated and promotes migration, invasion and proliferation in wide range of cancers. However, we for the first time identify that PRMT1 promotes migration and invasion and inhibits proliferation in gastric cancer cells, a phenomenon called “migration-proliferation dichotomy”. First, we find that PRMT1 overexpression promotes migration and invasion and inhibits proliferation, whereas PRMT1 knockdown reverses the above abilities. Next, PRMT1 reduces the expression of epithelial marker E-cadherin and increases the expression of mesenchymal markers including N-cadherin, Vimentin, snail and β-catenin in gastric cancer cells. Furthermore, our studies show that PRMT1 silencing promotes the phosphorylation of LATS1, and then induces YAP phosphorylation, while overexpression of PRMT1 down-regulates the phosphorylation of LATS1 and YAP, indicating that PRMT1 inhibits EMT probably via Hippo signaling. Collectively, the present study reveals important roles of PRMT1 in progression of gastric cancer. Given the dual functions of PRMT1, it is as a potential drug target of gastric cancer with extreme caution.

Introduction

Gastric cancer is the most common lethal malignancy and the third leading cause of cancer-related mortality worldwide with a 5-year survival rate of 20% [1]. This poor prognosis and ultimate cause of death are mainly due to the high metastasis rate and low chemotherapy sensitivity among gastric cancer patients after surgical resection despite improvements in surgery and other therapies [2]. This explains why most current therapies fail to significantly extend gastric cancer patients’ life span. Therefore, there is an urgent need to develop an in-depth understanding of the underlying molecular mechanisms of invasion and metastasis of gastric cancer, and suppression of metastasis is the key to improve the survival rate of gastric cancer [3].

Family members of the protein arginine methyltransferases (PRMTs), been able to methylate a variety of protein substrates [4], play a crucial role in several physiological and pathological processes, such as gene transcription regulation, RNA processing, DNA damage repair, signal transduction, protein translocation and tumorigenesis [5]. PRMT1 is one of the most important types of protein in PRMT family members, and it is reported to methylate histone H4 arginine 3 side chain, which is used to form asymmetric dimethylation and monomethylation and plays a critical role in modification for active chromatin and regulating protein function [6]. Recently, a large number of documents have been reported that aberrant expression of PRMT1 is related to a variety of cancers, including breast cancer, lung cancer, colon cancer, bladder cancer, acute myeloid leukemia and mixed lineage leukemia [7]. Meanwhile, over expression of PRMT1 is correlated with a poor prognosis of lung cancer and colon cancer development [8], [9]. However，we found that PRMT1 orchestrated two cellular processes: promoted migration and invasion and inhibited proliferation in gastric cancer cells.

Epithelial-mesenchymal transition (EMT) has a major role in tumor invasion and metastasis, in which epithelial cells lose their cell-cell adhesion and polarized organization, and acquire invasive properties and stem cell-like features with enhanced cell migration and invasion in various cancers [10]. Although EMT is first described in embryogenesis and development [11], cancer progression has similarities to the process of EMT found during embryonic development. And during EMT process cells down-regulate E-cadherin and up-regulate Vimentin and N-cadherin expression, resulting in cell motility and a change in cell morphology [12]. EMT is increasingly accepted as a crucial step for tumor infiltration and metastasis [13]. Recent study investigating in vivo the relevance of EMT to tumor metastasis revealed that the EMT-like features were enriched in clinical gastric cancer samples [14], providing convincing support for the actual role of EMT in gastric cancer metastasis. However, the precise mechanism for initiation of EMT in gastric cancer has been not elucidated fully. Hence, better understanding towards the regulation of EMT will shed light on our cancer therapeutic strategies.

In this study, we identified PRMT1 as a novel regulator of epithelial-mesenchymal transition to enhance the capabilities of migration and invasion via Hippo signaling. Interestingly, we also found that PRMT1 depressed the proliferation in gastric cancer cells, which played different roles in other cancers. The present data suggest that PRMT1 is a potential oncogene of gastric cancer and the dual effects of PRMT1 should be considered to provide clues for the development of new epigenetic intervention targeting PRMT1 for advanced gastric cancers.

Section snippets

Cell culture

Human gastric cancer cell lines (AGS, MGC803, BGC823 and SGC7901) were obtained from Shanghai Cancer Institute. All cell lines were maintained in DMEM medium (Hyclone, Beijing, China) supplemented with 10% fetal bovine serum(Gibco, Carlsbad, CA, USA), 100 units/ml penicillin and 100 mg/ml streptomycin, and cultivated at 37 °C in a humidified atmosphere containing 5% CO₂.

Cell transfection and viral infection

BGC823 and SGC7901 cells (5 × 10⁵ per well) were grown in a 6-well plate overnight followed by transfection with pcmv6-PRMT1 or

PRMT1 expression varies in gastric cancer cells

To determine the role of PRMT1 in gastric cancer progression, we first investigated PRMT1 expression levels in gastric cancer cell lines (AGS, MGC803, BGC823 and SGC7901) using western blotting. The data showed that the PRMT1 protein levels remained highly abundant in AGS and MGC803 cells, while had weak expression in BGC823 and SGC7901 cells (Fig. 1A and B). Subsequently, we constructed sh-PRMT1 and pcmv6-PRMT1 plasmids to investigate the role of PRMT1 in gastric cancer, and sh-EGFP or Vector

Discussion

In this study, we find that PRMT1 functions as oncogene to promote EMT via Hippo signaling. Importantly, it is the first time to show that PRMT1 inhibits proliferation in gastric cancer cells, which exhibits the contrary function in other cancers [17], [18].

With the above data, we confirm that PRMT1 presents two cellular processes in gastric cancer cells: promotes migration and invasion and inhibits proliferation. This observation is commonly referred to as the migration/proliferation dichotomy

Conclusions

Our research clarifies that PRMT1 could promote the EMT through the Hippo pathway in gastric cancer cells, whereas inhibit the proliferation, which remains to be further elaborated. The dual functions of PRMT1 should be taken into consideration carefully as a potential drug in the therapy of gastric cancer.

Acknowledgements

We thank the teachers of Institutional Animal Committee of Jiangsu University for the care of animals in experiments.

Conflict of interest

The authors declare no conflict of interest.

Funding

This study was supported by the National Natural Science Foundation of China (81472333, 81372718, 81672402), Provincial Natural Science Foundation of Jiangsu, China (BK20171305), Research Programs of Jiangsu Provincial Commission of Health and Family Planning, China (H201434), “Six One Project” Research Projects of High-level Medical Personnel of Jiangsu Province (LGY2016054), the Natural Science Foundation of Jiangsu Province (BK2013247), the Grants

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Protein Arginine Methyltransferases (PRMTs) are a family of enzymes regulating protein arginine methylation, which is a post-translational modification crucial for various cellular processes. Recent studies have highlighted the mechanistic role of PRMTs in cancer pathogenesis, immunotherapy, and drug resistance. PRMTs are involved in diverse oncogenic processes, including cell proliferation, apoptosis, and metastasis. They exert their effects by methylation of histones, transcription factors, and other regulatory proteins, resulting in altered gene expression patterns. PRMT-mediated histone methylation can lead to aberrant chromatin remodeling and epigenetic changes that drive oncogenesis. Additionally, PRMTs can directly interact with key signaling pathways involved in cancer progression, such as the PI3K/Akt and MAPK pathways, thereby modulating cell survival and proliferation. In the context of cancer immunotherapy, PRMTs have emerged as critical regulators of immune responses. They modulate immune checkpoint molecules, including programmed cell death protein 1 (PD-1), through arginine methylation. Drug resistance is a significant challenge in cancer treatment, and PRMTs have been implicated in this phenomenon. PRMTs can contribute to drug resistance through multiple mechanisms, including the epigenetic regulation of drug efflux pumps, altered DNA damage repair, and modulation of cell survival pathways. In conclusion, PRMTs play critical roles in cancer pathogenesis, immunotherapy, and drug resistance. In this overview, we have endeavored to illuminate the mechanistic intricacies of PRMT-mediated processes. Shedding light on these aspects will offer valuable insights into the fundamental biology of cancer and establish PRMTs as promising therapeutic targets.
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Protein arginine methyltransferase 1 (PRMT1), a type I PRMT, is overexpressed in gastric cancer (GC) cells. To elucidate the function of PRMT1 in GC, PRMT1 expression in HGC-27 and MKN-45 cells was knocked down by short hairpin RNA (shRNA) or inhibited by PRMT1 inhibitors (AMI-1 or DCLX069), which resulted in inhibition of GC cell proliferation, migration, invasion, and tumorigenesis in vitro and in vivo. MLX-interacting protein (MLXIP) and Kinectin 1 (KTN1) were identified as PRMT1-binding proteins. PRMT1 recruited MLXIP to the promoter of β-catenin, which induced β-catenin transcription and activated the β-catenin signaling pathway, promoting GC cell migration and metastasis. Furthermore, KTN1 inhibited the K48-linked ubiquitination of PRMT1 by decreasing the interaction between TRIM48 and PRMT1. Collectively, our findings reveal a mechanism by which PRMT1 promotes cell proliferation and metastasis mediated by the β-catenin signaling pathway.
Expression and localisation of methylthioadenosine phosphorylase (MTAP) in oral squamous cell carcinoma and their significance in epithelial-to-mesenchymal transition
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We previously reported that the nuclear localisation of PRMT5 is involved in EMT in OSCC.23 In gastric and breast cancer, PRMT1 was also found to be a regulator of EMT.29,32,33 These findings raise the interesting possibility that MTAP and PRMTs might be regulated in a coordinated manner in aggressive cancer cells that undergo EMT.
Methylthioadenosine phosphorylase (MTAP) is a rate-limiting enzyme in the methionine salvage pathway, which recycles one carbon unit that is lost during polyamine synthesis back into the methionine cycle. Although MTAP deficiency has been reported in various tumours, MTAP is overexpressed and might promote oncogenesis in other cancers, including prostate and colon cancer. Currently, little is known about the MTAP status of oral squamous cell carcinoma (OSCC). In this study, we immunohistochemically examined the expression of MTAP in surgically resected oral epithelial dysplasia (OED, n=7), carcinoma in situ (CIS) (n=16), and OSCC (n=118). In the normal epithelium, MTAP was only weakly expressed in the cytoplasm of the basal layer cells. In OED, CIS, and OSCC, MTAP was uniformly expressed in the cytoplasm of the dysplastic and cancer cells. In addition to cytoplasmic MTAP expression, 45 of 118 cases (38.1%) exhibited increased nuclear expression of MTAP in the cancer cells at the invasive front. Statistical analysis showed that the concomitant nuclear and cytoplasmic expression of MTAP was associated with a high budding score (p=0.0023); poor differentiation (p=0.0044); aggressive invasion patterns (p=0.0001); and features of epithelial-to-mesenchymal transition (EMT), such as loss of E-cadherin expression (p=0.0003) and upregulated expression of vimentin (p=0.0002), slug (p=0.0002), and laminin 5 (p<0.0001). High expression of protein arginine methyltransferase 1 or 5, the functions of which are reported to be inhibited in MTAP-deficient cancer, was associated with the concomitant nuclear and cytoplasmic expression of MTAP (p<0.0001). Concomitant nuclear and cytoplasmic expression of MTAP was marginally significantly associated with worse 5-year relapse-free survival (p=0.045). These findings suggest that MTAP not only plays a role in the oncogenesis of OSCC, but that it might also make it more aggressive by inducing EMT through its activity in the methionine salvage pathway.
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We previously reported that in mammary epithelial cells, PRMT1 regulates EMT through the TGF-β/Smad pathway via Smad7 methylation (Katsuno et al., 2018). Others also documented PRMT1 functions in cancer cell EMT through various mechanisms (Avasarala et al., 2015; Chen et al., 2019; Federico et al., 2019; Gao et al., 2016; Wei et al., 2019; Zhang et al., 2018b). Here, in epicardial cells, PRMT1 regulates epicardial EMT through a distinct pathway without affecting TGF-β-induced Smad3 signaling (Figures S6C–S6F).
Epicardial cells are cardiac progenitors that give rise to the majority of cardiac fibroblasts, coronary smooth muscle cells, and pericytes during development. An integral phase of epicardial fate transition is epithelial-to-mesenchymal transition (EMT) that confers motility. We uncover an essential role for the protein arginine methyltransferase 1 (PRMT1) in epicardial invasion and differentiation. Using scRNA-seq, we show that epicardial-specific deletion of Prmt1 reduced matrix and ribosomal gene expression in epicardial-derived cell lineages. PRMT1 regulates splicing of Mdm4, which is a key controller of p53 stability. Loss of PRMT1 leads to accumulation of p53 that enhances Slug degradation and blocks EMT. During heart development, the PRMT1-p53 pathway is required for epicardial invasion and formation of epicardial-derived lineages: cardiac fibroblasts, coronary smooth muscle cells, and pericytes. Consequently, this pathway modulates ventricular morphogenesis and coronary vessel formation. Altogether, our study reveals molecular mechanisms involving the PRMT1-p53 pathway and establish its roles in heart development.
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Specifically, PRMT1 activates ER stress and induces apoptosis of mesangial cells [16]. In multiple cancer models, PRMT1 presented activities in modulating EMT and promoting cancer malignancy [18–20]. It is not known, however, whether activating ER stress and/or inducing EMT are integral for the disease-promoting activities of PRMT1 during DN development.
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The level of PRMT1 was compared between the serum from DN patients and healthy individuals by ELISA, and between renal tissues of DN mice and normal mice using RT-qPCR and immunohistochemistry. Using high-glucose-treated PTEC cell line, HK2 cells as the model system, the significance of PRMT1 in ER stress and EMT was assessed by shRNA targeting PRMT1 (sh-PRMT1) and/or by overexpressing PRMT1. Mechanistic studies focused on three major pathways controlling ER stress: protein kinase R-like ER kinase (PERK), inositol requiring-1α (IRE1α), and activating transcription factor 6 (ATF6).
PRMT1 was up-regulated in the serum of DN patients and renal tissues of DN mice. High glucose administration induced elevation of PRMT1 expression in HK2 cells in vitro, accompanied with ER stress and EMT activation. PRMT1 knockdown attenuated high glucose-induced ER stress and apoptosis by inactivating PERK and ATF6, but not IRE1α. PRMT1 activated ATF6 by recruiting H4R3me2as to the promoter. Furthermore, PRMT1-induced ER stress was concomitant with the activation of an EMT-like state. Specifically, inhibition of ATF6, but not PERK blocked PRMT1-induced EMT in high-glucose-treatment HK2 cells.
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¹: These authors have contributed equally to this work.

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Protein arginine methyltransferase 1 coordinates the epithelial-mesenchymal transition/proliferation dichotomy in gastric cancer cells

Abstract

Introduction

Section snippets

Cell culture

Cell transfection and viral infection

PRMT1 expression varies in gastric cancer cells

Discussion

Conclusions

Acknowledgements

Conflict of interest

Funding

Cell

J. Biol. Chem.

J. Biol. Chem.

Global cancer statistics. 2012

CA Cancer J. Clin.

Prevention strategies for gastric cancer: a global perspective

Clin. Endosc.

The role of microRNAs in cancers of the upper gastrointestinal tract

Nat. Rev. Gastroenterol. Hepatol.

Immunoaffinity enrichment and mass spectrometry analysis of protein methylation

Mol. Cell Proteom.

Protein arginine methyltransferases and cancer

Nat. Rev. Cancer

Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor

Science

Role of PRMTs in cancer: could minor isoforms be leaving a mark?

World J. Biol. Chem.

CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features

Biochimie

The PRMT1 gene expression pattern in colon cancer

Br. J. Cancer

Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits

Nat. Rev. Cancer

An overview of epithelio-mesenchymal transformation

Acta Anat.

The protein tyrosine phosphatase Pez regulates TGF beta, epithelial-mesenchymal transition, and organ development

J. Cell Biol.

The basics of epithelial-mesenchymal transition

J. Clin. Investig.