PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

Abstract Protein arginine methyltransferase 5 (PRMT5) has been implicated in the development and progression of human cancers. However, few studies reveal its role in epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells. In this study, we find that PRMT5 is up‐regulated in pancreatic cancer, and promotes proliferation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. Silencing PRMT5 induces epithelial marker E‐cadherin expression and down‐regulates expression of mesenchymal markers including Vimentin, collagen I and β‐catenin in PaTu8988 and SW1990 cells, whereas ectopic PRMT5 re‐expression partially reverses these changes, indicating that PRMT5 promotes EMT in pancreatic cancer. More importantly, we find that PRMT5 knockdown decreases the phosphorylation level of EGFR at Y1068 and Y1172 and its downstream p‐AKT and p‐GSK3β, and then results in down‐regulation of β‐catenin. Expectedly, ectopic PRMT5 re‐expression also reverses the above changes. It is suggested that PRMT5 promotes EMT probably via EGFR/AKT/β‐catenin pathway. Taken together, our study demonstrates that PRMT5 plays oncogenic roles in the growth of pancreatic cancer cell and provides a potential candidate for pancreatic cancer treatment.


| INTRODUC TI ON
Pancreatic cancer is the most common malignancy of the pancreas, with a dismal 5-year survival rate of less than 5% and a median survival of <11 months. This dismal outcome can be attributed to the lack of early diagnoses and effective interventions. Additionally, conventional treatment approaches such as surgery, chemotherapy and radiation have generally had little impact on the course of this aggressive cancer despite efforts over the past several years. 1 Therefore, the detection and diagnosis of pancreatic cancer in the early stage are extremely urgent.
Emerging evidence has demonstrated that epithelial-mesenchymal transition (EMT) plays an essential role in the progression of pancreatic cancer. 2 It is a biologic process in which epithelial cells transform into special cells with mesenchymal phenotypes, resulting in enhanced invasion and metastasis. Concomitantly, epithelial markers such as E-cadherin are down-regulated, whereas mesenchymal markers including Vimentin, collagen I and β-catenin are up-regulated. 3 In 2019, a study provided evidence for the positive effects of SLC34A2 on EMT phenotype in glioma cell lines via the EGFR/PI3K/AKT signalling. 4 However, the molecular mechanisms that act upstream of these factors in various physiological and pathologic contexts in pancreatic cancer are not well characterized. Therefore, it is necessary to discover the specific mechanism in pancreatic cancer to provide novel prognostic and treatment targets.
Protein arginine methyltransferases (PRMTs) plays critical roles in a variety of cellular processes including transcriptional regulation, chromatin regulation, signal transduction, RNA processing and DNA damage repair. 5 PRMT5, the type II protein arginine transferase, catalyses the symmetrical dimethylation of arginine residues on histone and non-histone substrates and plays multiple roles in cellular processes, including differentiation, proliferation, apoptosis and ribosome biogenesis. 6 Furthermore, several studies have shown that PRMT5 plays an important role in the development and progression of human cancers including glioblastoma, 7 colorectal cancer, 8 breast cancer, 9 lymphoma, 10 prostate cancer 11 and lung cancer. 12 Recently, Menin and PRMT5 were found to suppress Glucagon-like-peptide-1 receptor (GLP1R) transcription to inhibit the proliferation of β-cell in pancreatic diseases. 13 In addition, PRMT5 was proved to inhibit the tumour suppressor FBW7, resulting in increasing c-Myc levels to promote the proliferation of and aerobic glycolysis in pancreatic cancer cells. 14 By far, there are not enough studies uncovering the roles of PRMT5 about EMT in pancreatic cancer.
In this study, we examined the roles of PRMT5 in pancreatic cancer and elucidated the underlying mechanism. Our data showed that PRMT5 promoted cell proliferation, migration and invasion in pancreatic cancer cells, and promoted tumorigenesis.

| Cell lines and culture conditions
The pancreatic cancer cell lines PaTu8988 and SW1990 were obtained from ATCC (USA) and Cancer Cell Repository (Shanghai, China). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% foetal bovine serum (FBS) at standard cell culture conditions (37°C, 5% CO 2 in humidified incubator). DMEM, FBS and trypsin were purchased from Gibco (Invitrogen).

| Transient transfection
The plasmids pHA-venus and pHA-PRMT5 were kind gifts from Professor Mo. Cells were plated in six-well plates at a density of 4 × 10 5 cells/well. After 24 hours of culture, the medium was replaced by Opti-MEM (Invitrogen) and cultured. In total, 2 μg plasmid was transfected using 6 μL Lipofectamine ® 2000 Transfection Reagent. After incubation for another 48 hours, the treated cells were determined using Western blot analysis, transwell or cell counting Kit-8 assay.

| Cell counting kit-8 assay
The measurement of viable cell mass was performed with Cell Counting Kit-8 (Promega) according to manufacturer's instructions.
Briefly, 3000 cells/well were seeded in a 96-well plate, grown in an incubator (5% CO 2 , 37°C). Respectively in first day, second day, third day and fourth day, 10 μL CCK-8 was added to each well, cells were incubated at 37°C for 2 hours, and the absorbance was finally determined at 490 nm.

| Colony formation assay
Cells were seeded in six-well plates at a density of 1000 cells per well and cultured in incubator (5% CO 2 , 37°C) for two weeks.
At the end of the incubation, the cells were fixed with 4% para-  Each test was repeated in triplicate.

| Cell invasion assay
Cell invasion was determined using a Boyden chamber assay. 5 × 10 4 PaTu8988 cells and 1 × 10 5 SW1990 cells were seeded in serum-free DMEM in the upper wells, which have already been covered with a layer of BD Matrigel Basement Membrane. The cells were later processed similar to that of cell migration assay, photographed (×20) in five independent fields for each well, and counted. Each test was repeated in triplicate.

TA B L E 1 DNA and RNA nucleotide sequences
The primers for qRT-PCR were in Table 1. Samples were cycled once at 95°C for 2 minutes and then subjected to 35 cycles of 95°C, 56°C and 72°C for 30 seconds each. The relative mRNA content was calculated using the 2 −ΔΔCt method with GAPDH as an endogenous control.

| Statistical analysis
All statistical analyses were carried out using the SPSS statistics software package. All data are presented as mean ± SD from at least three independent experiments. Comparisons between groups were analysed using the Student's t test (two groups) or an one-way ANOVA (multiple groups). Kaplan-Meier survival was analysed using log-rank analysis. P < .05 was considered statistically significant.

| PRMT5 is profiled in pancreatic cancers and different pancreatic cancer cells
To confirm the clinical relevance of PRMT5 expression, we first analysed the PRMT5 protein expression in clinical specimens from the human protein atlas (www.prote inatl as.org). We found that PRMT5 had the positive strong expression in pancreatic cancer and negative weak expression in normal pancreas ( Figure 1A). Furthermore, we analysed the PRMT5 mRNA level in clinical specimens from oncomine (www.oncom ine.org). We found that PRMT5 mRNA level was higher in pancreatic cancer tissues than that in normal pancre-  Figure 1E). Moreover, further analysis showed that PRMT5 expression in stage I was lower than that in stage II + III + IV groups ( Figure 1F).

| PRMT5 promotes cell proliferation in pancreatic cancer cells and tumorigenesis
To investigate the effects of PRMT5 on cell growth in pancreatic cancer cells, we first used the CCK8 assay to determine the growth curves and then evaluated their ability of colony formation. As showed in Figure 2A

| PRMT5 promotes EMT via activating EGFR/ AKT/β-catenin signalling in pancreatic cancer cells
To probe the molecular basis for PRMT5-enhanced cell motility, we next examined some EMT biomarkers such as E-cadherin, collagen I, knockdown on invasion-related signalling ( Figure 5A-B). We found that PRMT5 knockdown decreased the phosphorylation level of AKT, as well as its downstream p-GSK-3β, and then resulted in β-catenin down-regulation. Expectedly, ectopic PRMT5 re-expression reversed these changes. Previous study proved that EGFR is methylated by an arginine methyltransferase PRMT5. 17 Considering EGFR as the upstream signalling of AKT pathway, we speculate that EGFR signalling also regulates PRMT5-induced EMT in pancreatic cancer cells. So, we utilized the Western blot to detect the level of EGFR, p-EGFR (Y1068) and p-EGFR (Y1172). As observed in Figure 5A

| D ISCUSS I ON
In this study, we confirm that PRMT5 is overexpressed in human pancreatic cancer at both mRNA and protein levels, and acts as an independent prognostic factor for patient outcome. Furthermore, we find that PRMT5 promotes EMT via stimulating EGFR/AKT/βcatenin pathway for the first time. All these findings suggest that PRMT5 may function as an oncogene and be a candidate for diagnosis and prognosis in pancreatic cancer.
Previous studies have determined that PRMT5 may function as an oncogene to promote cancer cell growth. 18 More recently, PRMT5 was demonstrated that lead to FBW7 expression to promote tumorigenesis in pancreatic cancer. 14 In this study, we find that PRMT5 promotes cell proliferation, colony formation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. EMT has been associated with various tumour functions, including tumour initiation, malignant progression and tumour cell migration. More importantly, we confirm that PRMT5 promotes EMT through EGFR/AKT/β-catenin pathway in pancreatic cancer cells. Collectively, these data indicate that PRMT5 may function as an oncogene and is a key mediator in carcinogenesis and progression of pancreatic cancer.
It is well-established that β-catenin is dependent on Wnt signalling to promote cancer progression in various tumours. Upon canonical Wnt-signal, Wnt receptors inhibit the β-catenin phosphorylation, and facilitate β-catenin stabilization and β-catenin translocation into the nuclei. 22 Recently, Stephanie Grainger et al 23 found that EGFR-mediated phosphorylation of Fzd9b via β-catenin-dependent Wnt signalling to regulate haematopoietic stem and progenitor cell emergence, indicating that EGFR activation is required as a cofactor for β-catenin-dependent Wnt signalling. Also, it is found that β-catenin activation is independent of canonical Wnt signalling. For example, calreticulin increased β-catenin protein expression to promote EMT via Integrin/EGFR-ERK/MAPK signalling, 24 and EGF-induced nuclear translocation of SHCBP1 directly increased acetylation of β-catenin to enhanced NSCLC cellular stemness, 25 which is suggested that EGFR signalling can drive β-catenin activation via various routes. In this study, we provide a novel F I G U R E 2 PRMT5 promotes cell proliferation in pancreatic cancer cells and tumorigenesis. A-B, CCK-8 assay showed that PRMT5 knockdown inhibited PaTu8988 and SW1990 cell growth rate (Student's t test:*P < .05). C-D, Clone formation assays in PaTu8988 and SW1990 cells. PRMT5 knockdown inhibited cell clone formation (Student's t test:*P < .05). The number of clones with at least 50 cells per colony and strong, high dense staining was counted. The rates of colony formation were 48.33% and 17.67% in sh-EGFP and sh-PRMT5 PaTu8988 cells, and 35.67% and 10.67% in sh-EGFP and sh-PRMT5 SW1990 cells, respectively. E-F, PaTu8988 cells with PRMT5 downregulation were injected (2.0 × 10 6 cells/site) subcutaneously into a mice, and the tumour volume was measured weekly (n = 5 mice). *P < .05. G-H, CCK-8 assay showed that ectopic PRMT5 re-expression in PaTu8988 and SW1990 sh-PRMT5 stable infected cells promoted cell proliferation rate (Student's t test: *P < .05). I-J, Clone formation assays in PaTu8988 and SW1990 sh-PRMT5 stable infected cells. Ectopic PRMT5 re-expression in PaTu8988 and SW1990 sh-PRMT5 stable infected cells promoted cell clone formation (Student's t test: ***P < .001).

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.