EZH2 promotes chemoresistance in colorectal cancer by inhibiting autophagy through NRP1 suppression Open Access

Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality worldwide [1], driven by its aggressive nature and frequent resistance to chemotherapy [2-4]. Enhancer of zeste homolog 2 (EZH2), a critical component of the polycomb repressive complex 2 (PRC2), serves as a pivotal regulator of gene silencing through its histone methyltransferase activity [5,6]. Dysregulation of EZH2 has been implicated in various malignancies, including CRC, highlighting its role in promoting tumor progression [7], metastasis [8], and chemoresistance [9]. Despite these insights, the precise mechanisms by which EZH2 contributes to the proliferation and chemoresistance of CRC remain incompletely understood.

Several recent studies suggest that EZH2 modulates autophagy [10,11], a crucial cellular degradation process that maintains homeostasis and influences cancer cell survival under stress [12]. Additionally, neuropilin-1 (NRP1), a membrane-bound glycoprotein, has emerged as a critical mediator of cancer progression [13,14] and chemoresistance [15,16]. Evidence indicates that NRP1 contributes to tumor growth by modulating autophagy [17,18] and alternatively, interacts with autophagy to promote tumor progression and the acquisition of chemoresistance [19]. Understanding the connection between EZH2 and NRP1 and how their interaction drives tumor growth and therapeutic resistance through autophagy regulation could provide novel insights for therapeutic strategies.

In this study, we investigated the role of EZH2 in CRC by assessing its expression in tumor tissues and cell lines, evaluating its effects on cell proliferation and chemoresistance, and exploring its influence on autophagy. Furthermore, we analyzed the regulatory interaction between EZH2 and NRP1, elucidating how this axis contributes to the development of irinotecan resistance. Our findings highlight EZH2 and NRP1 as potential therapeutic targets in CRC and propose new strategies for overcoming chemotherapy resistance.

EZH2 is a critical epigenetic regulator known to be dysregulated across various cancer types, playing a pivotal role in tumorigenesis. In this study, we observed a striking up-regulation of EZH2 in CRC tissues compared with normal colorectal tissues, as reported in The Cancer Genome Atlas (TCGA). Notably, while elevated EZH2 expression did not correlate with the TNM stage (Figure 1A–1D), it was associated with increased chemotherapy resistance in CRC (Figure 1E). The diagnostic potential of EZH2 was further underscored by an area under the curve (AUC) of 0.968 (Figure 1F). To corroborate the findings from TCGA, we examined EZH2 expression in CRC cell lines (HCT116, SW480, and SW48) compared with normal colonic epithelial cells (NCM460). Consistent with the TCGA data, EZH2 expression was markedly elevated in the HCT116, SW480, and SW48 cell lines at both RNA and protein levels (Figure 1G and H). Collectively, these results suggest that heightened EZH2 expression may contribute to the pathogenesis, diagnosis, and chemotherapeutic resistance of CRC.

To investigate the functional role of EZH2 in CRC cells, cell viability assays were performed using the SW480 and HCT116 cell lines. The results indicated that silencing EZH2 prominently inhibited the proliferation of both SW480 and HCT116 cells, whereas overexpression of EZH2 enhanced their proliferation (Figure 2A–2F).

Subsequently, the impact of elevated EZH2 expression on the efficacy of chemotherapeutic agents was investigated. An analysis of the RNAactDrug database (http://bio-bigdata.hrbmu.edu.cn/RNAactDrug) revealed a negative correlation between EZH2 expression and the sensitivity to drugs such as paclitaxel and irinotecan (Figure 3A). To further investigate the role of EZH2 in modulating irinotecan efficacy, proliferation assays were conducted on HCT116 and SW480 cells with either knockdown or overexpression of EZH2. The results indicated that the IC₅₀ values for irinotecan were lower in wildtype (WT) HCT116 and SW480 cells compared with those in the EZH2 knockdown cells (Figure 3B and C). Conversely, EZH2 overexpression increased IC₅₀ values in both HCT116 and SW480 cells (Figure 3D and E).

To further elucidate the mechanism by which elevated EZH2 expression contributes to chemotherapy resistance, its role in regulating autophagy was examined by assessing LC3bI/II levels. The results illustrated that EZH2 knockdown in HCT116 and SW480 cells led to an increase in LC3b-II expression, indicating enhanced autophagy (Figure 4A). Conversely, EZH2 overexpression resulted in a decrease in LC3bII levels, suggesting reduced autophagy inhibition (Figure 4B).

To confirm that irinotecan promotes autophagy in CRC cells by inhibiting EZH2 expression, we investigated its effects on both mRNA and protein levels of EZH2. The results showed that irinotecan remarkably reduced EZH2 expression in CRC cells (Figure 4C and D). Following irinotecan treatment of HCT116 and SW480 cells, a pronounced increase in LC3b-II expression was observed at 48 hours (Figure 4E). Furthermore, EZH2 overexpression partially reversed the irinotecan-induced increase in LC3b-II expression in these cells (Figure 4F). These findings suggest that irinotecan-mediated suppression of EZH2 is crucial for inducing autophagy in CRC cells.

To investigate whether elevated EZH2 expression in CRC cells contributes to irinotecan resistance by inhibiting autophagy, we examined the impact of EZH2 modulation on cell viability and proliferation following irinotecan treatment. Using either EZH2 knockdown or EZH2 overexpression, we analyzed the effect of irinotecan on HCT116 and SW480 cells. Irinotecan obviously reduced both proliferation and viability in these cells. Moreover, EZH2 knockdown via siRNA enhanced the inhibitory effect of irinotecan on cell proliferation (Figure 5A–5D). In contrast, ectopic overexpression of EZH2 attenuated the ability of irinotecan to suppress proliferation in HCT116 and SW480 cells (Figure 5E–5H). These findings highlight the pivotal role of EZH2 in mediating resistance to irinotecan chemotherapy in CRC.

To elucidate how EZH2 regulates autophagy and contributes to irinotecan resistance in CRC, we analyzed its role in modulating NRP1 expression. Knockdown or depletion of EZH2 in tumor cells significantly enhanced the inhibitory effects of paclitaxel, docetaxel, and cisplatin on tumor cells, leading to a striking reduction in NRP1 levels (Figure 6A). Clinical analysis using the TCGA dataset revealed that NRP1 expression was significantly higher in CRC patients with T4-stage tumors compared with those with T1-stage tumors (Figure 6B). Additionally, CRC patients with elevated NRP1 expression exhibited shorter overall survival (Figure 6C–D). Furthermore, a positive correlation between EZH2 and NRP1 expression was observed (Figure 6E).

To further validate this regulatory relationship, we observed that knockdown EZH2 reduced NRP1 levels, whereas EZH2 overexpression significantly increased NRP1 expression in CRC cells (Figure 6F and G). Mechanistically, we discovered that EZH2 directly binds to the promoter region of NRP1. EZH2 was enriched at the NRP1 promoter region (Figure 6H and I). Collectively, these findings suggested that EZH2 regulated the transcription of NRP1 by binding to its promoter region in CRC cells.

Our findings indicated that NRP1 knockdown significantly suppressed the growth of HCT116 and SW480 cells (Figure 7A). Furthermore, NRP1 knockdown partially reversed the effects of EZH2 overexpression on cell proliferation (Figure 7B). Irinotecan reduced NRP1 expression and increased LC3b-II expression in HCT116 and SW480 cells (Figure 7C and D). In contrast, ectopic overexpression of NRP1 attenuates the irinotecan-induced increase in LC3b-II expression in HCT116 and SW480 cells (Figure 7D). These results suggest that EZH2 promotes CRC cell proliferation and suppresses autophagy by regulating NRP1 expression, thereby contributing to irinotecan resistance.

Our study aimed to explore the mechanism by which EZH2 contributes to chemotherapeutic resistance in CRC by inhibiting autophagy. Specifically, elevated levels of EZH2 in CRC tissues and cells promote tumor cell proliferation and chemoresistance to irinotecan by suppressing autophagy. Mechanistically, EZH2 directly binds to the NRP1 promoter, up-regulating its expression and further enhancing chemoresistance in CRC cells. Depletion of either EZH2 or NRP1 sensitizes CRC cells to irinotecan, suggesting that the EZH2-NRP1 axis plays a critical role in modulating therapeutic resistance.

EZH2, a conserved histone lysine methyltransferase [20], is frequently overexpressed or mutated in a wide range of cancers, where it promotes tumor initiation, progression, and poor clinical outcomes [21-24]. Corroborating with these previous findings, our study demonstrated that EZH2 significantly overexpressed in CRC tissues and cell lines. The high diagnostic accuracy of EZH2, reflected by the AUC of 0.968, underscores its potential as a biomarker for CRC diagnosis and prognosis. EZH2 promoted the proliferation of CRC cells, as evidenced by reduced cell viability and colony formation following EZH2 depletion and enhanced proliferation upon EZH2 overexpression. EZH2 exerts oncogenic effects through various mechanisms, including phosphorylation-mediated modulation of its function [25], its role in the PRC2 complex with EED and SUZ12 to drive histone methylation [26,27], and its promotion of metabolic reprogramming by enhancing tricarboxylic acid cycle activity [28]. Additionally, previous studies have shown that EZH2 activates the PI3K/Akt pathway, contributing to oxaliplatin resistance [29]. Although we observed no strong correlation between EZH2 expression and TNM stage, our data indicated that elevated EZH2 levels were associated with increased resistance to chemotherapy, particularly irinotecan.

In our study, the mechanism through which EZH2 exerts its pro-tumorigenic effects is derived from its chemoresistance, which is imculpated to involve the inhibition of autophagy, which is a primary cellular degradation process responsible for removing damaged macromolecules and organelles [11]. Our results indicated that EZH2 depletion induced autophagy in CRC cells, as indicated by increased LC3bII expression, whereas EZH2 overexpression suppressed this process. Notably, irinotecan induced autophagy by down-regulating EZH2 expression. However, this effect was partially reversed by ectopic EZH2 expression, reinforcing the notion that EZH2 plays a central role in modulating autophagy in response to chemotherapy. These findings suggest that EZH2-mediated autophagy inhibition provides a survival advantage to CRC cells, enabling them to withstand chemotherapeutic stress.

Previous studies have illustrated the regulation of autophagy by EZH2 through an mTOR-dependent pathway [11], which can be suppressed by NRP1 complex [30]. Given that NRP1 is involved in tumor progression across various cancer types [31-36], our study identified synchronous changes and positive correlation between EZH2 and NRP1 in CRC. Additionally, we demonstrated that NRP1 is a downstream target of EZH2, providing evidence that EZH2 directly binds to the NRP1 promoter and induces its expression. This drives tumor progression and resistance to irinotecan, with NRP1 knockdown partially reversing the effects of EZH2 overexpression on both cell proliferation and autophagy suppression.

Collectively, our findings suggest that EZH2 and NRP1 co-operatively contribute to CRC progression and chemoresistance through the modulation of autophagy. Targeting the EZH2-NRP1 axis may represent a promising therapeutic strategy to enhance the efficacy of chemotherapy in CRC patients.

Human normal colon epithelial cells (NCM460) and human CRC cell lines (HCT116, SW48, and SW480), originally obtained from the American Type Culture Collection, were used in this study. NCM460 cells were cultured in M3:10A™ medium, HCT116 cells in McCoy’s 5A medium, and SW48 and SW480 cells in Dulbecco’s modified Eagle medium. All cells were maintained in a humidified atmosphere at 37°C with 5% CO₂. The culture media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Irinotecan hydrochloride (#I1406, Sigma) was prepared as a 10-mM stock solution in DMSO.

Total RNA was isolated and purified using the High Pure RNA Isolation Kit (Roche), according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 µg of RNA per sample using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using Fast SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA). The _ΔΔCt method was used for relative gene expression analysis, with β-actin serving as the internal control for normalization. The sequences of qPCR primers are provided in Supplementary Table S1.

HCT116 and SW480 cells were seeded into 96-well plates at a density of 3000 cells per well. Cells were transfected with the indicated siRNAs, EZH2 pcDNA3.1, or vector controls. After 24 hours, the cells were treated with the specified concentrations of irinotecan for 48 hours. Following the treatment, 10 µl of MTT solution (0.5 mg/ml) was added to each well and incubated for 4 hours. The culture medium was then carefully removed, and 100 µl of DMSO was added to dissolve the formazan crystals. After mixing the plate, absorbance was measured at 570 nm using the Varioskan system (Thermo Fisher).

Cells were lysed in RIPA buffer supplemented with protease inhibitors (Roche) and PhosSTOP™ phosphatase inhibitor tablets (Roche). Lysates were sonicated for 5 seconds and centrifuged at 15,000 rpm for 20 minutes at 4°C. The supernatant was collected, and protein concentrations were quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). For each sample, 40 µg of protein was separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel. Proteins were transferred onto immobilon PVDF membranes (Millipore) following standard protocols (Bio-Rad Laboratories). Imaging was performed using the ECL (Millipore) and visualized with LI-COR Odyssey FC imaging system (Bad Homburg, Germany). The primary antibodies were shown as follows: anti-β-tubulin (2146 cell, signaling), anti-EZH2 (5246, cell signaling), and anti-LC3b-I/II (3868, cell signaling).

Cells were seeded in six-well plates and transfected with the indicated siRNA, EZH2 pcDNA3.1, or vector for 24 hours before treatment with irinotecan for 48 hours. Cells were resuspended and 2000 cells per well were re-seeded into new six-well plates. Colony formation was assessed after two weeks of incubation. Colonies were imaged using a digital camera and quantified with ImageJ software.

HCT116 cells were transfected with EZH2 pcDNA3.1 for 48 hours to activate EZH2. ChIP experiments were performed using the PureBinding® Chromatin Immunoprecipitation Kit. The sequences of qChIP primers are provided in Supplementary Table S2.

Gene expression levels were normalized using housekeeping genes, and the geometric mean of ranks (rank product) was calculated for per-sample gene expression. For differential gene expression analysis of unpaired samples, data from UCSC XENA’s unified processing of TCGA and GTEx RNA-seq data (in TPM format, processed by Toil) were used, comprising 51 normal human colorectal tissue samples and 179 CRC tissue samples. RNA-seq data from the TCGA CRC project (level 3, HTSeq-FPKM format) were utilized for correlation analysis of potentially related molecules. FPKM values were converted to TPM and log₂-transformed to allow for the comparison of expression levels. The Mann–Whitney U test (Wilcoxon rank-sum test) and Spearman’s correlation analysis were conducted using the ggplot2 (version 3.3.3) R package.

Data were presented as mean ± SD from triplicate experiments and analyzed using GraphPad Prism 9 Software (GraphPad, U.S.A.), which was used for all graphical representations. Statistical comparisons between groups were performed using Student’s t-test, while the Wilcoxon signed-rank test was applied to account for unequal variances between groups. A P value of <0.05 was considered the threshold for statistical significance.

1. Elevated enhancer of zeste homolog 2 (EZH2) expression in colorectal cancer (CRC is associated with chemoresistance).

2. EZH2 promotes CRC cell proliferation and chemoresistance by suppressing autophagy.

3. EZH2 directly regulates neuropilin-1 (NRP1) expression, driving tumor growth and resistance to irinotecan.

4. NRP1 knockdown mitigates EZH2-mediated chemoresistance and partially restoring autophagy.

The authors declare no conflict of interest.

This work was supported by the [Science and Technology Department of Sichuan Province] under Grant [2025YFHZ0210] and [2025ZNSFSC1890]; [Sichuan Medical Association] under Grant [Q18010];[Southwest medical university] under Grant [2018-ZRQN-093].

H.D. and L.R.: Methodology. H.D., Q.X., and Q.Z.: Formal analysis and data curation. C.L.: Formal analysis. H.D. and L.R.: Writing—original draft. L.R.: Supervision.

cDNA

complementary DNA

CRC

colorectal cancer

EZH2

enhancer of zeste homolog 2

NRP1

neuropilin-1

TGCA

The Cancer Genome Atlas