EPZ020411, a PRMT6 inhibitor, suppresses colorectal cancer cell proliferation, migration, and invasion through targeting the TGM2/MAPK axis

Abstract

BACKGROUND

Colorectal cancer (CRC) remains a highly fatal cancer worldwide, while metastasis is the main challenge in clinical treatment. Protein arginine methyltransferase 6 (PRMT6), a type I PRMT family member, catalyzes asymmetric methylation of arginine residues and contributes to tumor cell proliferation, invasion, metastasis and chemoresistance by both epigenetic and post-translational mechanisms. Notably, PRMT6 is overexpressed in CRC tissues and is associated with a poor survival outcome for patients. However, its targeted therapy and related mechanisms remain unclear.

AIM

To investigate the antitumor activity and molecular mechanisms of EPZ020411 in CRC HCT116 and CACO-2 cells.

METHODS

Human CRC cell lines (HCT116 and CACO-2) were treated with EPZ020411, and cell proliferation, migration and invasion were evaluated by CCK-8, EdU, colony formation, wound healing and Transwell assays. RNA sequencing was performed to screen differentially expressed genes, and the results were validated by reverse transcription-quantitative polymerase chain reaction and western blotting. To assess the functional role of transglutaminase 2 (TGM2), we generated a TGM2 over-expression model. The antitumor efficacy in vivo was evaluated using the xenograft mouse model, and tumor growth, histology and immunohistochemistry were performed.

RESULTS

EPZ020411 inhibited CRC cell proliferation, migration, and invasion dose-dependently. IC₅₀ values of EPZ020411 in HCT116 and CACO-2 cells at 48 hours were 126 μmol/L and 106 μmol/L. At 24 μmol/L, colony formation decreased by 32% and 50%, migration by ~62% in both lines, and invasion by 64% and 45%, respectively. Transcriptomic analysis revealed marked downregulation of TGM2, which was confirmed at both mRNA and protein levels. Mechanistically, EPZ020411 reduced phosphorylation of JNK and p38, indicating inactivation of the mitogen-activated protein kinases (MAPK) signaling pathway. Functional rescue experiments demonstrated that TGM2 overexpression partially reversed the inhibitory effects of EPZ020411. In vivo, EPZ020411 effectively suppressed tumor growth (45.1% inhibition at 10 mg/kg) without toxicity and reduced TGM2 expression in tumor tissues.

CONCLUSION

EPZ020411 inhibits CRC in vitro and in vivo via TGM2/MAPK, linking PRMT6-TGM2-MAPK, and suggesting PRMT6 as a potential therapeutic target.

Key Words: Colorectal cancer; EPZ020411; Protein arginine methyltransferase 6; Transglutaminase 2; Mitogen-activated protein kinases

Core Tip: In this study, we have found that EPZ020411 suppresses the growth and metastasis of colorectal cancer (CRC) cells. Mechanistically, EPZ020411 downregulates transglutaminase 2 (TGM2) and inhibits the JNK/p38 mitogen-activated protein kinases (MAPK) signaling pathway. Notably, TGM2 overexpression partially reverses these effects, highlighting its critical role as a downstream mediator. Overall, this study identified a novel protein arginine methyltransferase 6 (PRMT6)-TGM2-MAPK regulatory axis and provided a preliminary experimental basis for the potential of targeting PRMT6 in CRC, and new insights into the potential of targeting PRMT6 as a therapeutic strategy for CRC.

INTRODUCTION

Colorectal cancer (CRC) continues to be one of the principal causes of cancer-related deaths all over the world, largely due to late cancer diagnosis, and high rates of tumor metastases and recurrence following treatment. Although surgery, chemotherapy and targeted approaches have improved the treatment of patients with metastatic CRC, the prognosis is poor, highlighting the need to understand the molecular mechanisms underlying CRC invasion and metastasis, and find new targets for CRC treatment[1]. CRC most commonly metastasizes to the liver and the management of CRC liver metastases has changed; the combination of systemic therapy (including targeted agents), surgical resection, and adjunctive therapies has increased patient survival[2]. However, the molecular mechanisms responsible for the ability of CRC cells to gain the abilities to invade and metastasize are still not fully defined.

The role of epigenetic regulation is now recognized as being an important driver in the progression of cancer, especially CRC metastasis. Epigenetic regulation affects the expression of genes without changing the underlying sequence of DNA, and can be accomplished in a number of ways, including DNA methylation, histone modification and non-coding RNA-mediated regulation, which can modulate tumor cell plasticity, invasion and potential[3-5]. Protein arginine methyltransferases (PRMTs) are of particular interest as epigenetic regulators, and they are well known for their critical role in arginine methylation. Through the modification of either histone or the non-histone proteins, the PRMTs can affect the structure of the chromatin, the transcriptional programs, and signal transduction; all these are important processes in the formation of tumors and metastasis[6,7]. PRMT6, a type I PRMT, asymmetrically dimethylates arginine residues and has been implicated in various cancers, including lung adenocarcinoma[8], glioblastoma[9], and breast cancer[10] with the effects being mediated through epigenetic and post-translational mechanisms by PRMT6 in these cancers.

Interestingly, the marked upregulation of PRMT6 in CRC tissues vs normal mucosa, coupled with its inverse correlation with patient survival, points to a contributory function of this enzyme in the development and progression of CRC[11]. Functional studies reveal that PRMT6 supports the proliferation and invasion of CRC cells, in part through the activation of oncogenic signaling pathways like MYC and the regulation of the expression of cell cycle regulators[12]. PRMT6 is overexpressed in CRC, and its positive expression is closely correlated with shorter disease-free survival in CRC patients[13]. The results here provide a basis for the development of CRC therapeutics that could target PRMT6.

EPZ020411 is a highly potent and selective small molecule PRMT6 inhibitor. EPZ020411 is a cell membrane-penetrating compound which has been shown to specifically lower cellular levels of asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a) with minimal activity against other members of the PRMT family[14]. EPZ020411 has demonstrated potent inhibition of the methylation activity of PRMT6 and functional effects in cultured cells in cancer research. EPZ020411, for example, was validated for suppression of tumor cell metastasis in breast cancer studies through inhibition of methylation (R729) of signal transducers and activators of transcription 3 by EPZ020411[10]. In gliomas, EPZ020411 suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against glioma stem cell brain tumor xenografts[15]. Furthermore, EPZ020411 has been used as a probe tool for the functional studies of PRMT6. In a neuropathic pain model, it has been used for proof of the crucial role of PRMT6 in microglial glycolysis and neuroinflammation[16]. While EPZ020411 has been reported to modulate mismatch repair to influence immune surveillance in CRC, its role in regulating tumor cell migration and invasion remains unclear[17]. This leaves a significant opportunity to investigate the role of EPZ020411 in CRC metastasis.

Transglutaminase 2 (TGM2) is a multifunctional protein involved in protein cross-linking, signal transduction, and cell adhesion, and it plays a well-established tumor-promoting role in various cancers, including CRC[18]. In CRC, TGM2 is frequently overexpressed and is closely associated with tumor progression, epithelial-mesenchymal transition, and poor patient prognosis[19,20]. Moreover, TGM2 has been shown to promote tumor cell survival and proliferation through activation of key signaling cascades, including the mitogen-activated protein kinases (MAPK) pathway[21-24]. Overall, TGM2 represents a compelling target for mechanistic investigation in the context of PRMT6 inhibition.

To answer some of these questions, we first asked what the effect of EPZ020411 was on malignant phenotypes of CRC cells, which were shown to be significantly inhibited by EPZ020411 in CRC cell proliferation, migration and invasion abilities. During the investigation that led to understanding the underlying molecular mechanisms, we conducted transcriptomic sequencing analysis and found that unexpectedly, EPZ020411 was found to significantly downregulate TGM2 expression. Given the critical function of both TGM2 in tumor invasion and metastasis, we wanted to confirm this regulatory connection in CRC cells. We have shown in our experiment that expression of TGM2 was reduced upon treatment with PRMT6 inhibitor, thus altering its downstream MAPK signaling pathway. Our data provide a novel insight into how pharmacological inhibition of PRMT6 may affect CRC cells’ invasive capacity via modulation of TGM2 expression.

MATERIALS AND METHODS

Cell culture

The human CRC cell lines HCT116 and CACO-2 were purchased from iCell Bioscience Inc. (Shanghai, China; Cat. No. iCell-h071 and iCell-h032). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and MEM medium supplemented with 20% FBS, respectively. FBS was purchased from Nanjing Hongchuang Biotechnology Co., Ltd. (Cat. No. FSP500, Nanjing, Jiangsu Province, China). All cells were maintained in a humidified incubator at 37 °C with 50 mL/L CO₂. Cells were authenticated by short tandem repeat profiling and were negative for mycoplasma contamination.

Plasmids construction and lentiviral transfection

To overexpress TGM2, human TGM2 cDNA was cloned and inserted into the lentiviral vector pLV3-CMV-Puro at EcoRI and BamHI sites. The OE-TGM2 lentivirus was produced in HEK293T cells by co-transfection with pLV3-CMV-TGM2-Puro (2 μg), the viral envelope plasmid pMD2.G, and the viral packaging plasmid psPAX2 using Lipofectamine 3000 (Invitrogen, CA, United States) following the manufacturer’s instructions. At 48 hours after infection, the viral supernatants were harvested and used to infect cells or stored at -80 °C. HCT116 and Caco-2 cells in the logarithmic phase of growth were seeded into 6-well plates one day before transduction, with the seeding density optimized to reach 30%-40% confluence at the time of viral infection. After 48 hours of infection, stably expressing cells were selected in complete medium containing puromycin.

Quantitative real-time polymerase chain reaction and western blot analysis

Total RNA was extracted from cell lines using TRIzol™ reagent. Reverse transcription and quantitative polymerase chain reaction were performed using the HiScript III All-in-one RT SuperMix Perfect for qPCR Kit and PerfectStart Green qPCR SuperMix Kit, respectively. The primer sequences were as follows: TGM2 forward, 5’-TCTGGGACTCAGGGGAGTTT-3’ and reverse, 5’-CTCTCTAAGACCAGCTCCTCG-3’; GAPDH forward, 5’-TCATGACCACAGTCCATGCC-3’ and reverse, 5’-TTCTAGACGGCAGGTCAGGT-3’. Relative mRNA expression levels of TGM2 were quantified using the 2^-∆∆Ct method. Cells were lysed in RIPA buffer, denatured in loading buffer, separated by 10% sodium-dodecyl sulfate gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Blots were blocked with skim milk/TBST and incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-TGM2, anti-p-JNK, anti-JNK, anti-p-p38, and anti-p38. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using imaging system.

Cell viability assay

Cell viability was assessed using a CCK-8 kit. Cells were counted with a hemocytometer, and 5000 cells per well were seeded into a 96-well plate. After cell attachment, transfection was performed. At indicated time points (24, 48, and 72 hours), 10 μL of CCK-8 reagent and 100 μL of medium were added to each well, followed by incubation at 37 °C for 1 hour. Absorbance was measured at 450 nm.

Wound healing assay

HCT116 and CACO-2 cells in good growth condition were seeded into 6-well plates. When cells reached 70%-80% confluence, a straight scratch was made using a 100 μL pipette tip with the aid of a ruler. The detached cells were removed by washing with phosphate buffered saline (PBS). Images were captured at 0 hour using an inverted microscope. After drug treatment, images were taken at 24 hours and 48 hours, and the wound healing rate was calculated.

EdU proliferation assay

HCT116 and CACO-2 cells in the logarithmic growth phase were seeded into 6-well plates. Upon reaching appropriate density, the cells were incubated with 10 μmol/L EdU (Cat. No. CX002, Yaji Biotechnology, China) for 2 hours at 37 °C under 50 mL/L CO₂. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. EdU detection was performed according to the manufacturer’s instructions using Alexa Fluor 488-conjugated azide in a click reaction mixture, followed by incubation in the dark for 30 minutes at room temperature. The cells were washed with PBS containing 0.5% Tween-20, and Hoechst was then used to stain the nuclei. Images were taken under a fluorescent microscope, and the proportion of EdU-positive cells was scored from random fields.

Colony formation assay

Cells were seeded at a density of 1000 per well in 6-well plates and cultured for 15 days. The cloned cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 5 g/L crystal violet solution for 15 minutes. After a final wash with PBS, colonies were dried and photographed.

Transwell assay

The transfected cells, suspended in FBS-free RPMI-1640, were seeded into the Matrigel-precoated upper chamber, and the lower chamber was filled with RPMI-1640 supplemented with serum. After 24 hours, the migrated cells in the lower chamber were fixed with 4% paraformaldehyde and then stained with crystal violet. Images were acquired with a light microscope and then analyzed using ImageJ software.

In vivo xenograft mouse model

Animal experiments were approved by the Ethics Committee of Bengbu Medical University. Eighteen male SPF nude mice (6-8 weeks, 20-24 g) were purchased from Jiangsu Qinglong Laboratory Animal Technology Co., Ltd. and randomly divided into three groups (n = 6 per group): Control, low-dose EPZ020411, and high-dose EPZ020411. HCT116 cells (5 × 10⁶) suspended in 50 μL RPMI-1640 and 50 μL Matrigel were subcutaneously injected into the right flank. When tumors reached ~100 mm³, mice received intraperitoneal injections of EPZ020411 at 5 mg/kg and 10 mg/kg, or equal volume of vehicle (PBS with 0.1% DMSO), once daily for 18 days. Tumor dimensions and body weights were measured every three days (by an investigator blinded to the treatment groups); tumor volume was calculated as length × width² × 0.5. At endpoint, mice were euthanized under anesthesia; tumors and major organs (heart, liver, spleen, kidney) were collected for histopathological analysis (all data analyses were performed blinded to the group assignments).

Hematoxylin and eosin staining

Tissue sections were deparaffinized in xylene and rehydrated in a series of graded ethanol solutions. Hematoxylin staining was performed for 5-10 minutes, followed by a rinse under running water, differentiated in 1% acid alcohol, and bluing in running water. Counterstaining was carried out with eosin for 1-3 minutes. After staining, the sections were dehydrated through graded ethanol, cleared in xylene, and mounted with neutral resin. Stained sections were observed and imaged under a light microscope.

Immunohistochemical staining

Immunohistochemical staining was done on paraffin-embedded tissue sections. Sections were first dewaxed, rehydrated, and heated in citrate buffer for antigen retrieval. Endogenous peroxidase activity was blocked with 0.3% H₂O₂ (10 minutes) and then with goat serum (20 minutes). The sections were incubated with anti-TGM2 (1:500, Cat. No. ER1902-28, HUABIO, Hangzhou, Zhejiang Province, China) and anti-Ki67 (1:200, Cat. No. HA721115, HUABIO, Hangzhou, Zhejiang Province, China) overnight at 4 °C and then with secondary antibodies. Subsequently, the sections were developed with DAB and counterstained with hematoxylin. Images were captured under a light microscope.

Statistical analysis

All data are presented as the mean ± SD from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, United States). Comparisons were performed using the Student’s t test (two groups) or one-way analysis of variance (multiple groups). P < 0.05 was considered statistically significant. For tumor growth curves involving repeated measurements over time, two-way analysis of variance (ANOVA) with repeated measures was performed, followed by Bonferroni’s post-hoc test for multiple comparisons. For comparisons of final tumor weights and body weights between two groups, Student’s t-test was used.

RESULTS

EPZ020411 inhibits proliferation, migration, and invasion of CRC cells in a concentration-dependent manner

EPZ020411 treatment suppressed proliferation of HCT116 and CACO-2 cells in a dose-dependent manner, with calculated IC₅₀ values of 126 μmol/L and 106 μmol/L, respectively (Figure 1A and B). This anti-proliferative effect was further corroborated by colony formation assays (Figure 1C and D) and EdU incorporation assays (Figure 1E and F), both of which showed marked reductions upon EPZ020411 treatment. We then determined if EPZ020411 affected the migration and invasion of CRC cells. Transwell assays revealed that EPZ020411 dose-dependently inhibited migration and invasion of both HCT116 and CACO-2 cells (Figure 2A and B). To further test cell migration, wound healing assays were carried out and indicated that EPZ020411 treatment caused decreased wound closure rates (Figure 2C and D). Collectively, these results demonstrate that EPZ020411 effectively suppresses the malignant phenotypes of CRC cells.

Figure 1 Effects of EPZ020411 at varying concentrations on the proliferation of colorectal cancer cells. A and B: IC₅₀ values were calculated by nonlinear regression analysis; C and D: Representative images of colony formation assays of HCT116 (C) and CACO-2 (D) cells treated with increasing concentrations of EPZ020411; E and F: EdU staining was performed to detect the proliferation of HCT116 (E) and CACO-2 (F) cells treated with increasing concentrations of EPZ020411 (scale bar: 50 μm). Error bars represented the mean ± SEM. ^bP < 0.001.

Figure 2 EPZ020411 inhibits the migration and invasion of colorectal cancer cells in a concentration-dependent manner. A and B: Transwell assays showing the effects of increasing concentrations of EPZ020411 on the migration and invasion of HCT116 (A) and CACO-2 (B) cells; C and D: Wound healing assays assessing the migratory ability of HCT116 (C) and CACO-2 (D) cells after EPZ020411 treatment. Data are presented as mean ± SD from three independent experiments (n = 3). Error bars represented the mean ± SEM. ^bP < 0.001.

EPZ020411 downregulates TGM2 expression and inactivates the JNK/p38 signaling pathways

To elucidate the underlying mechanism, RNA sequencing analysis of EPZ020411-treated HCT116 cells revealed significant downregulation of TGM2, a tumor progression-associated gene (Figure 3A and B). Reverse transcription-quantitative polymerase chain reaction and western blotting confirmed that EPZ020411 reduced TGM2 mRNA and protein levels in both HCT116 and CACO-2 cells (Figure 3C-F). TGM2 was reported to act downstream of the MAPK pathway to regulate proliferation in cancer cells, and hence the effect was studied on its effector molecules. We examined JNK and p38 phosphorylation. EPZ020411 treatment markedly decreased p-JNK and p-p38 levels (Figure 3G and H), indicating inactivation of these pro-tumorigenic signaling cascades. Collectively, these results suggest that the antitumor activity of EPZ020411 is at least partially mediated through the TGM2/JNK/p38 axis.

Figure 3 EPZ020411 inhibits transglutaminase 2 gene expression and suppresses the JNK and p38 signaling pathways. A and B: The heatmap of RNA sequencing displays the differentially expressed genes in HCT116 cells treated with EPZ020411; C and D: Reverse transcription-quantitative polymerase chain reaction was used to detect the expression levels of transglutaminase 2 (TGM2) mRNA in EPZ020411-treated HCT116 and CACO-2 cells; E and F: Western blot was used to detect the expression levels of TGM2 protein in EPZ020411-treated HCT116 and CACO-2 cells; G and H: Western blot analysis of JNK, p-JNK, p38 and p-p38 protein levels in HCT116 and CACO-2 cells treated with varying concentrations of EPZ020411. Data are presented as mean ± SD from three independent experiments (n = 3). Error bars represented the mean ± SEM. ^aP < 0.01, ^bP < 0.001. TGM2: Transglutaminase 2.

TGM2 overexpression enhances CRC cell proliferation, migration, and invasion

To gain insight into the functional significance of TGM2 in CRC progression, we first generated HCT116 and CACO-2 cells stably overexpressing TGM2 using a lentiviral vector carrying the human TGM2 gene (Figure 4A). The expression of the TGM2 protein was confirmed by western blot in both cell lines (Figure 4B and C). CRC cells with a high level of TGM2 expression exhibited prominently greater colony-forming ability and increased EdU incorporation (Figure 4D-G), indicating high proliferative ability. In addition, overexpression of TGM2 resulted in enhanced cell migration and invasion through transwell and wound healing assays (Figure 5). The results indicate that TGM2 can be a tumor promoting agent in CRC, and the downregulation of TGM2 by EPZ020411 can be involved in the effect of the compound against CRC.

Figure 4 Transglutaminase 2 overexpression enhances colorectal cancer cell proliferation. A: Circular plasmid map of lentiviral vector pLV3-CMV-TGM2 (human)-5-Puro; B and C: Western blot was used to detect the expression levels of transglutaminase 2 protein levels in HCT116 (B) and CACO-2 (C) cells; D and E: Representative image of colony formation assays of HCT116 (D) and CACO-2 (E) cells; F and G: EdU staining was performed to detect the proliferation of HCT116 (F) and CACO-2 (G) cells (scale bar: 50 μm). Error bars represented the mean ± SEM. ^aP < 0.01, ^bP < 0.001. TGM2: Transglutaminase 2.

Figure 5 Transglutaminase 2 overexpression rescues EPZ020411-inhibited migration and invasion in colorectal cancer cells. A and B: Transwell assays showing the migration and invasion of HCT116 (A) and CACO-2 (B) cells under the indicated treatments (control, EPZ020411, and EPZ020411 + transglutaminase 2 overexpression); C and D: Wound healing assays assessing the migratory ability of HCT116 (C) and CACO-2 (D) cells under the same treatment conditions. Data are presented as mean ± SD from three independent experiments (n = 3). Error bars represented the mean ± SEM. ^aP < 0.01, ^bP < 0.001. TGM2: Transglutaminase 2.

EPZ020411 inhibits subcutaneous tumor growth and modulates TGM2 expression in vivo

In a subcutaneous xenograft model, EPZ020411 treatment significantly suppressed tumor growth, as reflected by reduced tumor volumes and final tumor weights, without affecting body weight or causing major organ toxicity (Figure 6A-E). Significantly, immunohistochemical staining of tumor tissues from mice treated with EPZ020411 revealed decreased TGM2 expression, consistent with our in vitro findings (Figure 6F). These findings demonstrate that EPZ020411 inhibits CRC tumor growth in vivo, potentially through the regulation of TGM2 expression levels.

Figure 6 EPZ020411 inhibits the growth of colorectal cancer subcutaneous tumors and affects transglutaminase 2 expression. A: Mice body weights; B-D: Tumor growth curves (B), representative images of excised tumors (C), and tumor weights (D); E: Representative hematoxylin and eosin staining images of organs. Original magnification (scale bar: 100 μm); F: Representative immunohistochemistry staining of transglutaminase 2 in subcutaneous tumors from EPZ020411-treated mice. Original magnification (scale bar: 100 μm). Error bars represented the mean ± SEM. ^aP < 0.01, ^bP < 0.001. TGM2: Transglutaminase 2.

DISCUSSION

In the present study, the specific PRMT6 inhibitor EPZ020411 was used to focus on the inhibitory effects on the proliferation, migration and invasion of CRC cells HCT116 and CACO-2 and the underlying mechanisms. Mechanistically, EPZ020411 downregulates TGM2 expression and inactivates the JNK/p38 MAPK signaling pathway, with functional rescue experiments confirming TGM2 as a critical downstream mediator. The results of in vivo animal experiments indicated certain efficacy and a favorable safety profile, suggesting that PRMT6 inhibitors may have potential value in the treatment of CRC; however, further investigation is still required to confirm this.

The anti-proliferative effects of EPZ020411 in CRC cells are consistent with previous reports showing that PRMT6 inhibition compromises cell cycle regulator function in glioblastoma stem cells[24], and exerts precise control on proliferation through chromatin remodeling and transcription factor activity[9]. The observed inter-cell-line differences in IC₅₀ values suggest heterogeneous PRMT6 regulation within tumor cell populations, highlighting the need for further investigation into the selectivity of PRMT6 inhibitors. Based on the multi-targeted and molecularly targeted therapy principles, EPZ020411 is considered a candidate for combination therapy, to have improved antitumor activity.

Our study identifies a previously unrecognized mechanism by which EPZ020411 suppresses CRC cell migration and invasion, specifically through downregulation of TGM2 and subsequent inactivation of the MAPK pathway. Previous studies have linked PRMT6 to invasiveness in glioblastoma through EZH2 protein stability and downstream transcription factor targets[9], as well as to cell motility via methylation-mediated changes in extracellular matrix remodeling, cytoskeletal dynamics, and adhesion molecules[10]. The results indicate that the specific effects of PRMT6 inhibition on tumor cell proliferation are not confined to this process, but rather to several essential aspects of tumor metastasis. Therefore, it is possible that EPZ020411 may have anti-metastatic activity through the modulation of the tumor microenvironment and cell migration based signaling pathways.

Transcriptomic analysis revealed that EPZ020411 downregulates TGM2 expression, leading to inactivation of the MAPK signaling pathway, suggesting a critical role for this axis in the antitumor activity of EPZ020411. The transmembrane enzyme TGM2 has commonly been identified as a tumor-promoting factor and is associated with crosslinking of the extracellular matrix and signal transduction[10]. The pathway stimulated by the activation of EPZ020411 treatment indirectly targets the MAPK pathway by downregulating the expression of TGM2, in line with previously reported models involving downmodulation of different MAPK signaling pathways through non-histone protein methylation pathways by the modulation of PRMT6 bioactivity states[9,10]. Additionally, this study is distinct from those that have investigated only individual targets, providing for the first time a molecular network connecting this axis (PRMT6 inhibition/TGM2/MAPK signaling) and suggesting a novel axis of regulation, expanding the theoretical knowledge of PRMT6-involved multi-layered regulation in tumor signaling pathways.

Functional rescue experiments further demonstrated that TGM2 overexpression partially reverses the inhibitory effects of EPZ020411 on CRC cell proliferation and migration, confirming TGM2 as a downstream effector of PRMT6. Consistent with prior observations, TGM2 drives tumor cell migration and invasion and acts as a downstream effector of PRMT6[10]. Furthermore, TGM2 promotes extracellular matrix crosslinking and signal complex assembly, thereby modulating intra- and extracellular signaling to regulate tumor cell motility and survival - a known versatile function[9]. This is the first study to point out the necessity of TGM2 in the antitumor activity of PRMT6 inhibitors, with the functional rescue experiments, and to offer a molecular basis for further designing drugs targeting PRMT6-TGM2 axis in CRC which was missed previously.

In vivo, EPZ020411 suppressed tumor growth without apparent toxicity, supporting its favorable safety profile. Previous studies have shown that PRMT6 inhibitors can reshape the tumor microenvironment in immune-tolerant CRC, suggesting potential synergy with immune checkpoint blockers[15]. However, the optimal concentration and treatment schedule require further investigation to maximize clinical benefit. Additionally, EPZ020411 may exert broad antitumor activities through modulation of multiple PRMT6-associated signaling pathways, potentially enhancing its efficacy as a monotherapy or in combination regimens.

However, there are some drawbacks to this study. The mechanistic link between PRMT6 inhibition and TGM2 downregulation remains correlative first. PRMT6 is a transcription co-regulator that can potentially be enriched at TGM2 promoter region, catalyzing the histone H3R2me2a modification which regulates its transcription. Alternatively, PRMT6 can directly modify the TGM2 protein to either alter protein structure or protein activity, such as by methylation of arginine. Further characterization may be performed by chromatin immunoprecipitation sequencing to examine PRMT6 enrichment at the TGM2 promoter and/or by mass spectrometry to identify arginine methylation sites on TGM2 proteins known to be modified by PRMT6 to gain insight in the transcriptional or post-translational modification level. Second, the study is performed primarily in the human colon cancer cell lines HCT116 and CACO-2 and their xenografts, thus relatively limited sample size. So there’s a possibility that our results might be limited in generalizability. Additional cell lines or patient-derived organoids or patient-derived xenografts of CRC cell lines are required to validate the efficacy of EPZ020411 and clinical translation should be investigated in conjunction with chemotherapy or immunotherapy to provide a more comprehensive experimental basis. Finally, although EPZ020411 showed no overt toxicity in this study, its long-term safety and potential synergistic effects with standard chemotherapeutic agents (e.g., 5-fluorouracil or oxaliplatin) require systematic evaluation. Therefore, while our results are encouraging, they are preclinical in nature and do not warrant immediate clinical translation.

CONCLUSION

Overall, this study identifies the ability of EPZ020411, a PRMT6 inhibitor, to efficiently suppress the proliferation, migration and invasion capacity of CRC cells in vitro and in vivo by reducing the expression of TGM2 and blocking the MAPK pathway. Together, our results offer a novel conceptual basis for PRMT6-directed therapy in CRC and supply a key experimental foundation for moving EPZ020411 toward clinical application.