ARHGAP4/MYH9/β-catenin/c-Jun positive feedback loop promotes colorectal cancer stemness

Liu, Shaolin; Chen, Yizhen; Tan, Song; Chen, Yifan; Yang, Changshun; Mi, Yulong; Lin, Shengtao; Li, Weihua

doi:10.1038/s41698-025-01022-4

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Published: 15 August 2025

ARHGAP4/MYH9/β-catenin/c-Jun positive feedback loop promotes colorectal cancer stemness

Shaolin Liu^1,2^na1,
Yizhen Chen^1,2^na1,
Song Tan^1,2^na1,
Yifan Chen^1,2,
Changshun Yang²,
Yulong Mi²,
Shengtao Lin² &
…
Weihua Li²

npj Precision Oncology volume 9, Article number: 285 (2025) Cite this article

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Abstract

Rho GTPase-activating protein 4 (ARHGAP4) is implicated in poor prognosis across multiple malignancies. However, its role in driving stemness in colorectal cancer (CRC) remains unclear. In this study, we demonstrate that ARHGAP4 drives CRC stemness through a positive feedback loop involving MYH9/β-catenin/c-Jun, as validated by integrated bioinformatics analysis, in vitro and in vivo tumor stemness assays, co-immunoprecipitation, chromatin immunoprecipitation, and fluorescence recovery after photobleaching. These findings identify ARHGAP4 as a promising therapeutic target in CRC, particularly for addressing cancer stem cells (CSCs).

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Introduction

Colorectal cancer (CRC) remains a leading global health challenge, ranking as the third most prevalent malignancy and a major cause of cancer-related mortality¹. Despite therapeutic advances, tumor relapse and metastasis, driven by cancer stemness, remain key obstacles to improving patient outcomes². A critical unmet need is the identification of molecular drivers that sustain stemness properties, which are tightly linked to tumor progression³. Thus, elucidating the mechanisms governing stemness regulation may provide novel strategies for CSC-targeted therapies.

The Rho GTPase-activating protein (RhoGAPs) family has emerged as a key regulator of oncogenic signaling, modulating cytoskeletal dynamics and cell motility⁴. However, compared to RhoGEFs, RhoGAPs remain poorly characterized, particularly in the context of CRC stemness⁵. Among these, ARHGAP4 as a member implicated in hematopoietic malignancies, has been proposed as a potential CRC biomarker⁶, yet its functional role and mechanisms of interplay with stemness regulators are unknown. Therefore, a study is urgently needed to elucidate the key molecules and the mechanism behind them.

Non-muscle myosin heavy chain 9 (MYH9) has emerged as a multifunctional player in mechanotransduction and cancer progression⁷. In hepatocellular carcinoma and gastric cancer, MYH9 has shown to enhances stemness^8,9,10,11,12 and act as a protein scaffold, facilitating signal transduction^13,14. Intriguingly, MYH9’s coiled-coil domain harbors intrinsically disordered regions (IDRs), suggesting a potential for liquid-liquid phase separation (LLPS)—a process increasingly recognized for organizing oncogenic condensates¹⁵. However, whether MYH9 undergoes LLPS, and how this is regulated by upstream partners remains unexplored.

Our study elucidates a novel mechanistic link between ARHGAP4 and CRC stemness through MYH9-mediated LLPS. By integrating multi-omics and functional analyses, we demonstrate that ARHGAP4 promotes CRC stemness by facilitating MYH9-dependent LLPS, which orchestrates GSK3β degradation complex assembly to activate β-catenin signaling. Furthermore, we reveal that this oncogenic axis is sustained through a c-Jun-mediated positive feedback loop that reinforces ARHGAP4/MYH9 expression. These findings not only establish ARHGAP4 as a promising therapeutic target but also provide the first evidence linking LLPS to stemness regulation in CRC, offering new mechanistic insights for precision oncology interventions.

Results

ARHGAP4 overexpression drives CRC progression

We performed data mining of CRC using the human cancer genome atlas (TCGA) database, from which we identified several differentially expressed genes (Fig. 1A). Among these differentially expressed genes, we intersected them with members of the RhoGAPs gene family (Fig. 1B). However, except for ARHGAP4, the other seven highly expressed genes had no significant effect on the overall survival (OS) of CRC patients (Fig. S1A). In contrast, CRC patients with high ARHGAP4 expression exhibited poorer OS, disease-specific survival, progression-free interval and relapse-free survival compared to those with low ARHGAP4 expression (Fig. 1C–F). Lasso regression analysis of these genes further confirmed that ARHGAP4 was the only gene within the selection criteria (Fig. 1G). To verify the authenticity of the results from the public database, we conducted a prognostic analysis on 78 CRC patients from the Affiliated Provincial Hospital of Fujian University. The results indicated that patients with relatively high ARHGAP4 expression had worse OS compared to those with low expression (Fig. 1H). Additionally, univariate and multivariate COX regression analyses on CRC samples from the TCGA database confirmed that ARHGAP4 is an independent risk factor for CRC (Supplementary Table 5, Fig. S1B).

**Fig. 1: ARHGAP4 is highly expressed in CRC and is associated with poor prognosis.**

Furthermore, paired sample analysis of CRC data from TCGA and GSE44076 revealed a significant upregulation of ARHGAP4 expression in CRC (Fig. 1I, J). Pan-cancer analysis demonstrated conserved ARHGAP4 overexpression across multiple malignancies (Fig. 1K). Immunohistochemical (IHC) testing of cancerous and adjacent non-tumor tissues from primary CRC patients further confirmed this high expression status (Fig. 1L). Clinical data from the Fujian Provincial Hospital affiliated with Fuzhou University suggested that elevated ARHGAP4 expression is associated with higher T-stage, N-stage and clinical stage (Fig. 1M–O). These findings collectively suggest that ARHGAP4 is highly expressed in CRC and plays a role in its onset and progression.

ARHGAP4 promotes the malignant biological behavior of CRC cells

After determining the correlation between ARHGAP4 and CRC, we further systematically investigated its functional role through in vitro and in vivo experiments. Western blot analysis was performed on HCT116, SW48, RKO, SW620, and LoVo cell lines to assess the baseline protein expression levels of ARHGAP4 (Fig. S2A). HCT116 and RKO cell lines were selected for stable transfection to construct overexpression and knockdown models (Fig. S2B). Functional characterization demonstrated that ARHGAP4 overexpression significantly enhanced cellular proliferation in CCK-8 assays, while knockdown exerted opposite effect (Fig. 2A). This proliferative advantage was further confirmed by increased EdU incorporation rates in ARHGAP4-high cells (Fig. 2B, Fig. S2C) and augmented colony formation capacity (Fig. 2C, Fig. S2D). Consistent with these findings, the subcutaneous tumorigenesis experiment showed that ARHGAP4-overexpressing HCT116 cells significantly increased the tumor growth rate, size and weight compared to the control group (Fig. 2D). Furthermore, the Transwell invasion assay validated that ARHGAP4 promotes the in vitro invasive and metastatic capabilities of CRC cells (Fig. 2E, F). These experiments collectively demonstrate that ARHGAP4 enhances the malignant biological behaviors of CRC.

ARHGAP4 promotes stemness in CRC

Given the established link between cancer stemness and tumor aggressiveness¹⁶.We investigate ARHGAP4 role in CRC stemness regulation. TCGA data analysis revealed significant positive correlations between ARHGAP4 expression and established stemness markers (Fig. S3). We further conducted spheroid culture experiments and observed that overexpression of ARHGAP4 enhanced the stemness of CRC cells, while knockdown of ARHGAP4 resulted in the opposite effect (Fig. 3A, B). Side population (SP) cell analysis also demonstrated that the ARHGAP4 overexpression group contained more cells with verapamil-sensitive signals, whereas the knockdown group showed the opposite pattern (Fig. 3C, D). Subsequently, we performed WB to assess the expression of tumor stemness markers, metastasis-related proteins, and cell cycle/proliferation proteins in the four stable transfectant groups. The results indicated that high ARHGAP4 expression enhanced the levels of CRC stemness markers, such as CD44¹⁷, metastasis-related proteins like N-cadherin and Vimentin¹⁸ as well as proliferation and oncogenic proteins, including c-Myc and Cyclin D1¹⁹ (Fig. 3E). Additionally, protein immunoblotting from xenograft tumors in previous subcutaneous tumor models of nude mice also confirmed that in vivo tumorigenic models showed an increase in stemness markers as the expression of ARHGAP4 increased (Fig. 3F). Further, Limiting dilution assays using HCT116 cells in nude mice demonstrated that ARHGAP4 overexpression significantly enhanced tumor initiation capacity, as evidenced by increased tumor incidence, size, and weight at equivalent cell doses (Fig. 3G–I). Corresponding stemness assessments confirmed greater stem-like properties in ARHGAP4-high tumors (Fig. 3J). These comprehensive analyses establish ARHGAP4 as a critical regulator of CRC stemness that drives tumor malignancy.

**Fig. 3: ARHGAP4 promotes the stemness characteristics of CRC cells.**

ARHGAP4 promotes CRC stemness through MYH9

To elucidate the molecular mechanism underlying ARHGAP4-mediated CRC progression and stemness, we performed immunoprecipitation (IP) of the ARHGAP4 protein followed by mass spectrometry analysis. The results revealed MYH9 as the top candidate interacting protein, with the highest numbers of binding peptides, characteristic peptides, and unique peptides in both cell lines (Fig. 4A). This interaction was confirmed endogenously through Co-IP (Fig. 4B) and demonstrated strong intracellular colocalization by immunofluorescence (Fig. 4C). To verify that the increased stemness in CRC cells induced by ARHGAP4 is mediated through MYH9, we performed WB analysis, which demonstrated that knockdown of MYH9 weakened the upregulation of proteins, such as CD44, Cyclin D1, and Vimentin caused by ARHGAP4 overexpression (Fig. 4D). Consistent with these findings, MYH9 depletion significantly impaired ARHGAP4-driven enhancements in colony formation, invasion capacity, and sphere formation (Fig. 4E–G, Fig. S4A–C). Additionally, in vivo subcutaneous tumorigenesis using a limiting dilution assay showed that MYH9 knockdown significantly reduced the tumor formation rate induced by ARHGAP4 overexpression (Fig. 4H, Fig. S4D). Through structural analysis, we identified key interaction sites between ARHGAP4 and MYH9. Site-directed mutagenesis of these residues (Fig. 4I, J) abolished the ARHGAP4-MYH9 interaction (Fig. 4K) and consequently impaired ARHGAP4’s ability to promote malignant phenotypes in vitro (Fig. 4L–N, Fig. S4E–G). These results collectively establish the ARHGAP4-MYH9 interaction as a critical molecular determinant of CRC stemness and progression.

**Fig. 4: ARHGAP4 promotes CRC progression and stemness by interacting with MYH9.**

The biological changes induced by ARHGAP4 are regulated by MYH9/GSK3β/β-catenin signal transduction

To delineate the signaling mechanisms underlying ARHGAP4-mediated CRC progression, which revealed significant enrichment of Wnt/β-catenin signaling pathways (Fig. 5A), a finding corroborated in the GSE44076 dataset (Fig. 5B). Given the established role of Wnt/β-catenin activation in promoting tumor stemness and EMT²⁰. And prior reports linking GSK3β phosphorylation at Ser9 to β-catenin pathway activation in various cancers^10,11,21,22, we investigated whether ARHGAP4 similarly modulates this pathway in CRC. WB results indicated that overexpression of ARHGAP4 led to increased levels of β-catenin and p-GSK3β (Ser9)/GSK3β, while knockdown of ARHGAP4 produced the opposite effect (Fig. 5C). Additionally, immunofluorescence analysis of β-catenin nuclear translocation and WB further demonstrated that the increased expression of ARHGAP4 promotes the accumulation of β-catenin in the nucleus (Fig. 5D, E). Further WB analysis revealed that overexpression of ARHGAP4 in the RKO cell line accelerated GSK3β protein degradation, whereas knockdown of ARHGAP4 in the HCT116 cell line slowed GSK3β degradation (Fig. 5F). Proteasome inhibition with MG132 rescued GSK3β levels in ARHGAP4-overexpressing cells but not in knockdown cells (Fig. 5G). Co-IP experiments further demonstrated that in ARHGAP4 high-expressing cells, GSK3β underwent increased ubiquitination (Fig. 5H).

**Fig. 5: ARHGAP4 regulates the biological behavior of CRC through MYH9/GSK3β/β-catenin signal transduction.**

To determine the key role of MYH9 in this axis, we performed MYH9 knockdown experiments based on ARHGAP4 overexpression to study the upstream and downstream responses. WB results confirmed that MYH9 knockdown effectively reduced the activation of the β-catenin pathway induced by ARHGAP4 overexpression (Fig. 5I). Furthermore, nuclear translocation WB results also confirmed that MYH9 knockdown significantly restored the levels of β-catenin in both the nucleus and the cytoplasm (Fig. 5J). Consistently, MYH9 knockdown also effectively reversed the reduction in GSK3β protein degradation caused by ARHGAP4 overexpression in the HCT116 and RKO cell lines (Fig. 5F, G). Similarly, MYH9 knockdown significantly reversed the increase in ubiquitination of GSK3β associated with ARHGAP4 overexpression (Fig. 5H). Subsequently, we treated control cell lines and ARHGAP4-OE + MYH9-Sh cell lines with Laduviglusib (WNT/β-catenin pathway activator) and treated ARHGAP4-overexpressing cell lines with KYA1797K (WNT/β-catenin pathway inhibitor) (Fig. S5A). We then performed colony formation assays, Transwell invasion assays, and spheroid formation assays. The results indicated that after treatment with pathway modulators, the proliferation, migration, and stemness characteristics of both the control and recovery groups in the HCT116 and RKO cell lines were significantly increased. Meanwhile, the biological changes induced by ARHGAP4 overexpression were reversed by KYA1797K (Figs. S5B–G). In conclusion, our results suggest that ARHGAP4 promotes the progression and stemness of CRC cells through the MYH9/GSK3β axis, which activates the β-catenin pathway.

ARHGAP4-mediated increased MYH9 phase separation promotes GSK3β ubiquitination complex formation

After confirming the signal axis, we were curious about how ARHGAP4 regulates the ubiquitination of GSK3β through MYH9. We observed that under conditions where the expression of ARHGAP4 changed, the E3 ubiquitin ligase for GSK3β, tumor necrosis factor receptor-associated factor 6 (TRAF6), did not show significant changes (Fig. 6A)^23,24. However, when the corresponding ubiquitin ligase did not increase, the enhanced ubiquitination could potentially be attributed to an increase in the assembly of the overall degradation complex^25,26. Therefore, we hypothesized that ARHGAP4 may form a complex with GSK3β, TRAF6 and MYH9 to promote GSK3β ubiquitination. Co-IP experiments revealed that MYH9, GSK3β and TRAF6 form a complex in CRC cells, though ARHGAP4 showed no direct interaction with GSK3β or TRAF6 (Figs. 6B, S6A). We further speculated that ARHGAP4 might influence the interactions among the other three proteins. The Co-IP results indicated that overexpression of ARHGAP4 leads to a relative enhancement of their binding, while downregulation of ARHGAP4 results in the opposite effect (Fig. 6C–E). Although MYH9 is known to play a role as a scaffold protein in multiple signaling pathways, it is not clear whether its activity involves phase separation²⁷. Structural analysis shows that the Coiled-coil domain of MYH9 contains IDRs, which is a sign of phase separation drive (Fig. S6B)²⁸. The prediction analysis of multiple databases (POUNDR, catGRANULE, PSPHunter) consistently shows that the domain (Fig. 6F, G, Supplementary Table 8) has a strong phase separation tendency. The formation of cytoplasmic droplets of MYH9-GFP was verified by experiments, and the dissolution of condensate after FRAP (Fig. 6H, I) and 1,6-hexanediol treatment confirmed its dynamic liquid-like properties (Fig. 6J). Furthermore, we constructed a truncated form of MYH9 to determine whether the CC domain drives the phase separation of MYH9 (Fig. 6K). Coagulation experiments in vitro and in vivo showed that MYH9-GFP-CCΔ (coiled-coil deletion) lost phase separation ability, while MYH9-GFP-NTΔ (N-terminal deletion) retained phase separation ability (Fig. 6L).

**Fig. 6: ARHGAP4 promotes the formation of GSK3β ubiquitination complex.**

However, we also observed that the addition of ARHGAP4 further promotes the formation of the GSK3β complex. Based on this, we hypothesize that the spontaneous phase separation ability of MYH9 may be enhanced through interaction with ARHGAP4. Molecular dynamics simulations confirmed that the interaction between ARHGAP4 and MYH9 is mediated by ionic interactions and presents a relatively stable interaction state (Fig. 7A). Furthermore, phase separation mediated by the Coiled-coil domain is further enhanced by interactions, such as charge and ionic interactions. To verify this, we co-transfected ARHGAP4-mCherry and MYH9-GFP plasmids into 293 T cells and observed that the presence of ARHGAP4 induced the formation of droplets with MYH9, with more aggregates observed in cells expressing ARHGAP4 (Fig. 7B). FRAP analysis confirmed that these droplets were formed by phase separation (Fig. 7C). The 1,6-hexanediol experiment also demonstrated the fluidity of these droplets (Fig. 7D). Subsequently, purified the two proteins in vitro and performed the corresponding experiments, observing that the phase separation ability of MYH9 was enhanced with increasing ARHGAP4 protein concentration, and these droplets retained fluidity (Fig. 7E, F). Further experiments with the previously constructed ARHGAP4-WT and ARHGAP4-MUT plasmids revealed that only ARHGAP4 capable of interacting with MYH9 enhanced the formation of aggregates (Fig. 7G). Co-IP experiments showed that only the supplementation of ARHGAP4-WT in both cell types promoted the formation of the GSK3β complex (Fig. 7H, Fig. S6C, D). Recruitment imaging of the aggregates also demonstrated that GSK3β was recruited into the aggregates only when MYH9 phase separation ability was enhanced, while MYH9 lacking phase separation did not form aggregates nor recruit GSK3β (Fig. 7I). Consistent with these findings, ARHGAP4-enhanced MYH9 phase separation was identified as a key factor in promoting the ubiquitination of GSK3β and the subsequent activation of the β-catenin pathway (Fig. S6E, Fig. 7J, K). These molecular biology results align with the analysis from GEPIA, which similarly indicates that groups with higher expression of both ARHGAP4 and MYH9 exhibit poorer OS (Fig. S6F). These data collectively represent the first characterization of MYH9 phase separation ability and its enhancement through interaction with ARHGAP4. This interaction serves as the direct cause for the increased formation of the GSK3β complex, facilitated by ARHGAP4 in MYH9 scaffolding role.

**Fig. 7: ARHGAP4 enhances MYH9 phase separation and mediates the recruitment and assembly of GSK3β.**

ARHGAP4 upregulates c-Jun in CRC to form a positive feedback loop

Previous studies have shown that the up-regulation of Wnt/β-catenin pathway activates the increase of downstream target gene c-Jun, and MYH9 is one of the transcriptional regulatory targets of c-Jun²⁹. Our data also show that ARHGAP4-mediated MYH9 phase separation activates the β-catenin pathway, but whether it will increase the expression level of MYH9 based on this signal axis needs further verification. we performed a correlation analysis between ARHGAP4 and MYH9 using CRC data from TCGA. The results indicated a strong positive correlation between ARHGAP4 and MYH9 (Fig. S7A). Previous studies have shown that c-Jun acts as an upstream positive regulator of MYH9 expression, serving as a transcription factor for MYH9 regulation³⁰. To investigate this, we also analyzed the correlation between c-Jun and MYH9 using CRC data from TCGA. The results indicated a positive correlation between the two (Fig. S7B). Furthermore, to verify the accuracy and authenticity of the bioinformatics analysis, we performed RT-qPCR on cell lines, which also confirmed this positive correlation (Fig. 8A, B). Notably, the upregulation of the WNT/β-catenin pathway has been shown to promote the increase of c-Jun expression and our subsequent protein immunoblotting experiments confirmed this finding (Fig. 8C, D). Additionally, after treating the corresponding cells with pathway regulators, we further confirmed that the overexpression of c-Jun and the subsequent changes in MYH9 are mediated through ARHGAP4 regulation of the β-catenin pathway (Fig. 8E, F).

**Fig. 8: In CRC, ARHGAP4 promotes the increase of c-Jun to form a positive feedback loop.**

Next, we explored the upstream transcription factors of ARHGAP4. Using the Knock TF and GTRD databases, we analyzed the transcription factors upstream of ARHGAP4. Surprisingly, we discovered that c-Jun is also a transcription factor for ARHGAP4 (Fig. 8G). External validation of this data was conducted using the JASPAR database (Fig. 8H). Furthermore, the correlation analysis using TCGA data showed a significant positive correlation between c-Jun and ARHGAP4 expression (Fig. 8I). Subsequently, ChIP assays on the c-Jun protein confirmed that c-Jun binds to the promoter region of ARHGAP4 DNA (Fig. 8J). Additionally, promoter activity reporter assays further confirmed the transcriptional activity of c-Jun on the ARHGAP4 promoter region (Fig. 8K). Moreover, after inhibiting or overexpressing c-Jun, we measured the protein and RNA levels of ARHGAP4 and MYH9. The results indicated a significant positive correlation between c-Jun and both at the protein and RNA levels (Fig. 8L, M). In conclusion, our data suggest that ARHGAP4 not only upregulates the β-catenin pathway by indirectly mediating the assembly of the GSK3β ubiquitination complex but also promotes the upregulation of MYH9 and the transcription factor c-Jun in the context of pathway activation. This signaling ultimately forms a positive feedback loop.

Discussions

The emergence of CSCs is one of the key factors contributing to CRC progression and poor treatment outcomes³¹. Increasing evidence suggests that targeting CSCs could be a promising therapeutic strategy for CRC in the future³². However, the high heterogeneity of CSCs presents significant challenges to such targeted therapies³³. Therefore, identifying and targeting the critical signaling pathways that drive CSCs transformation in CRC represents a relatively feasible approach for targeted treatment³⁴. In CRC, ARHGAP4 emerges as a key regulator of stemness, distinct from other RhoGAP family members, such as ARHGAP29, which governs signaling in melanoma³⁵, unlike its counterparts, ARHGAP4 predominantly exerts its oncogenic effects in CRC through non-canonical pathways. Our findings confirm the significance of ARHGAP4 as a therapeutic target for CRC and further support recent reports on members of the RhoGAPs family, which play a diverse key role in a wide range of signal transduction.

Protein-protein interactions, especially the formation of complexes, play a crucial role in signal transduction pathways^36,37. For instance, mutations and aberrant formation of subunits in the SWI/SNF complex can significantly affect chromatin remodeling, leading to diseases, particularly cancer³⁸. Additionally, in the past five years, the discovery of PANoptosis bodies, which are also formed by protein-protein interactions, has garnered attention³⁹. Abnormal assembly of these complexes induces an inflammatory form of cell death, characterized by apoptosis, necrosis, and pyroptosis, which contributes to disease development⁴⁰. Studying how to target these abnormally increased complexes or exploring the mechanisms behind their aberrant formation holds great potential for the treatment of diseases, particularly cancer^41,42. Reports have shown that MYH9 plays a role in promoting cancer progression across various tumor types^43,44,45. Recent studies indicate that multiple upstream proteins facilitate tumor progression through their interaction with MYH9^14,46,47. MYH9 acts as a scaffolding protein that regulates the ubiquitination of GSK3β and activates the β-catenin pathway, thereby promoting malignant tumor progression¹⁰. Current research has confirmed that the N-terminal of MYH9 interacts with GSK3β, which could be a functional site responsible for altering the ubiquitination levels of GSK3β¹¹. Furthermore, the involvement of chaperone proteins that interact with MYH9 might promote the formation of this protein complex, thereby disrupting GSK3β, a key negative regulatory molecule in the β-catenin pathway. Our study is the first to confirm the presence of this protein complex in cCRC and provides evidence that ARHGAP4 acts as an important regulatory molecule in this disruptive complex. Moreover, we demonstrate that reducing the interaction between ARHGAP4 and MYH9 significantly diminishes the abnormal pro-cancer signaling cascade.

Recently, the introduction of LLPS has provided researchers with a more comprehensive understanding of the molecular mechanisms underlying diseases, such as cancer⁴⁸. However, there remains a significant gap in our understanding of the fundamental forces driving phase separation. Recent studies have confirmed that Coiled-coil regions exhibit phase separation capabilities, further solidifying the foundation of research in this field⁴⁹. In our study, we found that MYH9 also possesses phase separation ability. An increasing number of studies suggest that proteins with phase separation capabilities promote the formation of condensates and downstream signaling pathways with the assistance of chaperone proteins. As an uncharacterized phase-separating molecule, we verified the phase separation ability of MYH9 and its dependence on interacting partners for enhanced phase separation. Moreover, combining this with our finding that MYH9, upon losing its interactions, failed to promote the formation of the GSK3β ubiquitination complex, we were able to confirm the central role of enhanced phase separation in driving abnormal signaling. This further highlights the significance of targeting aberrant phase separation as a strategy to delay or even treat tumors. Overall, we have effectively broadened the scope of phase separation research and provided a clearer explanation of the mechanisms behind MYH9-mediated complex formation.

It is important to note that targeting positive feedback loops in tumor stem cells is a critical direction in cancer therapy⁵⁰. As mentioned earlier, the disruption of GSK3β is achieved by increasing the destruction of complex assembly. This disruption in complex formation promotes the activation of the β-catenin pathway, which in turn upregulates the expression of c-Jun, an upstream transcription factor of MYH9²⁹. Furthermore, we have identified that c-Jun is also a transcription factor for ARHGAP4. Its upregulation leads to increased expression of both ARHGAP4 and MYH9, thereby facilitating the formation of the positive feedback loop. Notably, the elevation of ARHGAP4, especially its interaction with MYH9, plays a crucial role in the assembly of downstream complexes, which serves as the key initiator in the formation of this feedback loop. Future targeted therapies aimed at ARHGAP4 or its interaction sites with MYH9 could effectively weaken the positive feedback activation that regulates the malignant progression of CRC.

However, this study has several limitations. First, the selection of ARHGAP4 in this study was based on high-expression genes within the RhoGAPs family. Future research should focus on investigating the roles of low-expression RhoGAPs family genes in CRC. Second, our pathway exploration primarily concentrated on the WNT/β-catenin pathway. We did not further analyze whether ARHGAP4 could mediate biological regulation through molecular mechanisms beyond stemness. This aspect also warrants further investigation in future studies. Third, whether the enhanced phase separation of MYH9 by ARHGAP4 regulates complex assembly beyond GSK3β remains to be further elucidated. Lastly, the lack of additional preclinical models to assess the potential of targeting the ARHGAP4-MYH9 axis in clinical translational therapies remains a limitation.

Overall, this study reveals that the stable interaction between ARHGAP4 and MYH9 enhances the phase separation ability of MYH9. The increased phase separation of MYH9 subsequently recruits and mediates the formation of the ubiquitination complex of GSK3β, which directly activates the Wnt/β-catenin pathway, thereby promoting stemness and malignant biological behavior in CRC. Furthermore, the activation of this pathway induces the upregulation of the downstream target gene c-Jun, which in turn transcriptionally activates ARHGAP4 and MYH9, establishing a positive feedback loop (Fig. 9). This complex mechanism crosstalk underscores the potential therapeutic significance of targeting the ARHGAP4-MYH9 interaction.

**Fig. 9: The mechanism map of ARHGAP4 promoting CRC stemness and progression.**

Methods

Bioinformatics analysis

The data used for analysis were obtained from two datasets: TCGA and GSE44076 (from the GEO database), focusing on CRC. The data were processed, analyzed, and visualized using R Studio (R Version 4.4.0). The specific R packages used include “tidyverse”, “ggplot2”, “dplyr” and “TCGAbiolinks.”

Cell lines and clinical samples

Cell lines HCT116, RKO, SW48, SW620, Lovo and HEK293T were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum (cat # AUS-01S-02, Cell-Box, Australia) (Shanghai BasaMedia Biological Technology, China). Clinical specimens were collected from 78 patients after surgery and ten pairs of paraffin sections of colorectal tumor biopsy in Fujian Provincial Hospital. The specific information of clinical data is shown in Supplementary Table 1, and the baseline information of clinical patients is shown in Supplementary Table 2.

Lentiviral and plasmid transfection vectors

ARHGAP4 overexpression and no-load and nonsense sequence and knockdown sequence lentivirus design and coating, MYH9 nonsense sequence and knockdown sequence lentivirus, MYH9-GFP, GFP, ARHGAP4-mcherry, MYH9-GFP-NTΔ, YH9-GFP-CCΔ and GSK3β-mcherry were provided by Wuhan Puyun Biotechnology Co., Ltd.Technical support. The PCDNA, wild-type and mutant plasmids of ARHGAP4 were provided by Shanghai Hanheng Biotechnology Co., Ltd. See Supplementary Table 3 for the specific sequence.

Western-blotting (WB)

Cells were washed with PBS after removing the culture medium. Protein extraction was performed using NP40 lysis buffer (Herui Biotechnology, China) with phosphatase (100x, MCE, China) and protease inhibitors (100x, MCE, China) at 4 °C, followed by centrifugation. The supernatant was mixed with 5X loading buffer (Herui Biotechnology, China) and boiled at 100 °C for 10 min. SDS-PAGE gels (Biyuntian Biotechnology, China, P0012A) with 12% concentration were used for Western blotting. Protein transfer was conducted on 0.45 μm PVDF membranes (Millipore, USA, IPFL00010). The membranes were blocked with BSA solution (Merck Biotech, Germany, V900933) and incubated with primary antibodies overnight. After washing with TBST (3 × 15 min), secondary antibody incubation was performed for 1 h. Membranes were then washed and developed using ECL detection reagent (GLPBIO, USA, GK10008) on a BIO-RAD system. Antibody details are listed in Supplementary Table 4.

IP Assay (Co-IP)

The Co-IP kit was purchased from Thermo Fisher, China. Antibody details are in Supplementary Table 3. Briefly, cells were washed with PBS and lysed on ice for 15 min in 1 mL of pre-chilled lysis/wash buffer supplemented with phosphatase and protease inhibitors (100×; MCE, China). Lysates were centrifuged at 12,500 g for 15 min at 4 °C. For input samples, the supernatant was mixed with 5× loading buffer and denatured at 100 °C for 10 min. For IP, 100 µL of supernatant was incubated with 2 µL of IgG or IgG1 antibody (Wuhan Sanjing Biotech, China) overnight at 4 °C. The following day, protein complexes were processed as per the kit instructions. Co-IP samples were then subjected to electrophoresis and transferred for WB using standard protocols.

Immunohistochemical staining (IHC)

Tissue sections from Fujian Provincial Hospital were deparaffinized, antigen retrieved, and incubated with ARHGAP4 antibody (1:200) at 4 °C for 8 h. DAB chromogen staining was applied, followed by hematoxylin counterstaining. Slides were dehydrated, mounted with neutral gum, and air-dried before observation.

EdU cell proliferation assay

Adherent cells were treated with EdU solution for 2 h, fixed with paraformaldehyde, and permeabilized with Triton-X100. EdU staining was applied for 30 min, followed by DAPI nuclear staining. The cells were washed and analyzed by microscopy.

Immunofluorescence Staining (IF)

Adherent cells were washed with PBS, fixed with paraformaldehyde, and permeabilized with Triton-X100. After blocking with goat serum, cells were incubated with primary antibodies (ARHGAP4, MYH9, β-catenin) overnight. After washing, fluorescent secondary antibody was applied, followed by DAPI staining. Images were captured using a Leica DMI8 microscope.

CCK-8 proliferation assay and clone assay

For the CCK-8 assay, cells were seeded in 96-well plates and incubated with CCK-8 solution for 2 h, with absorbance measured. Data were analyzed using GraphPad Prism. For the clonogenic assay, cells were seeded in 6-well plates, stained with crystal violet after two weeks, and quantified using ImageJ, followed by statistical analysis with GraphPad Prism.

Suspension spheroid formation assay

After cell digestion, cells were resuspended and counted. Cell suspension containing 400 cells per well was prepared using spheroid culture medium (Sicogee Biotechnology Co., Ltd., China). 1 mL of the cell suspension was added to each well, and cells were gently mixed. After 1–2 weeks, spheroid formation was analyzed by counting the spheroids in each well and taking images. Data were processed using GraphPad Prism (GraphPad Prism 10.1.2), with three independent replicates per group for statistical analysis.

SP cell sorting

Reagents used for SP cell sorting included Hoechst 33342 (Sicogee Biotechnology Co., Ltd., China) and verapamil (Sicogee Biotechnology Co., Ltd., China). The procedure followed that described by Wei Rao et al. ⁵¹. The final result measured was the proportion of cells with Hoechst 33342 efflux. The final concentration of verapamil used in the experiment was unified at 40 uM/ml.

Transwell invasion assay

Matrigel was diluted in serum-free DMEM/F12 medium and applied to the Transwell chamber membrane to polymerize at 37 °C. Cells were prepared, resuspended in serum-free medium, and seeded into the upper chamber. After incubation, cells were fixed, stained with crystal violet, and images captured. The number of invasive cells was counted and data analyzed using GraphPad Prism.

Subcutaneous tumor formation and tumor experiment in nude mice

The experimental animals used were male BALB/C nude mice devoid of a thymus. Tumor formation experiments were conducted by subcutaneously injecting cells at three different concentrations: 1 × 10⁵, 5 × 10⁵, and 5 × 10⁴ cells per mouse. In the subcutaneous tumor formation experiment, 2 × 10⁶ cells per mouse were used for injection. Mice were euthanized when the tumor size was less than 1500 mm³, and the tumors were subjected to imaging, weighing, and growth curve plotting for xenograft evaluation. Tumor sizes were recorded every two days from day 7 post-inoculation until euthanasia, and tumor growth curves were drawn during subsequent analysis. All statistical analysis and graphical representations were performed using GraphPad Prism version 10.1.2.

Protein mass spectrometry analysis

Protein mass spectrometry was performed using an LC-MS/MS method for enzyme digestion analysis. Following the purification of ARHGAP4 from RKO and HCT116 cells via magnetic bead pull-down, the purified proteins underwent subsequent analysis. After the cleaning steps, the magnetic beads were processed using a C18 membrane-packed column for peptide desalting, followed by mass spectrometry analysis. This entire process was managed by Luming Biotechnology Co., Ltd., Shanghai, China.

Detection of protein degradation and recovery

Specifically, this experiment is to detect proteasome pathway-mediated protein determination⁵². HCT116 and RKO stable cell lines were treated with 100 µM of cycloheximide for 1, 2, and 4 h. Protein extraction was followed by Western blotting to detect changes in GSK3β protein levels. Further analysis was performed by treating HCT116 and RKO stable cell lines with 20 µM of the proteasome inhibitor MG132 for 12 h, followed by protein extraction and Western blotting to examine changes in GSK3β protein levels.

Phase separation prediction

The IDRs of MYH9 were analyzed using the PONDR database (https://www.pondr.com/). The phase separation potential of MYH9 was further analyzed using the PSPHunter database (http://psphunter.stemcellding.org/) and the catGRAUNLE database (https://tools.tartaglialab.com/catgranule). The amino acid sequence of MYH9 was obtained from the Uniprot database (https://www.uniprot.org/).

Preparation of relevant systems for phase separation experiments

In vivo phase separation experiments were performed by transfecting the appropriate plasmids into HEK293T cells. For in vitro studies, proteins were initially obtained through protein purification. All proteins were expressed in Escherichia coli BL21 (DE3) cells (Herrui Biotechnology Co., Ltd., China) with the addition of 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by incubation at 25 °C for 16 h. The cell pellet from 500 mL of cultured cells was resuspended in a binding buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 10 mM imidazole) containing a protease inhibitor mixture (1 mM PMSF) and subjected to sonication. The resulting supernatant was purified using Ni-NTA agarose (Invitrogen, R901-15), followed by elution with an elution buffer (50 mM Tris-HCl, pH 7.4, 1 M KCl, 5% glycerol, and 200 mM imidazole). The protein was further concentrated using a Millipore filter and dialyzed against a storage buffer (50 mM Tris-HCl, 300 mM KCl, 1 mM DTT, and 5% glycerol). The purified protein was stored at -80 °C. For the experiment, the protein was resuspended in LLPS buffer (25 mM Tris-HCl, 150 mM KCl, 0.5 mM DTT, 8% PEG8000, and 2.5% glycerol) and adjusted to the required concentrations (The concentration of MYH9 protein in vitro was fixed at 20 uM).

Live cell imaging technology

Cells were seeded in confocal culture dishes (NEST, 801002) and transfected with 2 μg of plasmid for 48 h. Live cell imaging was conducted using a Leica TCS SP8 STED microscope (Leica Microsystems) at 37 °C and 5% CO2. All procedures adhered to the manufacturer’s guidelines, and images were captured in confocal mode with a 100× oil immersion objective.

Fluorescence recovery after photobleaching (FRAP) experiment

FRAP analysis of in vitro or cellular biomolecular condensates was carried out at room temperature using the FRAP module of a Leica TCS SP8 STED confocal microscope system (Leica Microsystems). The droplet to be bleached was located using the 100× oil immersion objective, and a circular region was photobleached with full laser power. Time-lapse images were then captured. Fluorescence recovery was measured against the initial fluorescence intensity using ImageJ, and the data were plotted using GraphPad Prism.

Uptake experiment after treatment with 1,6-Hexanediol

Following the same protocol as live cell imaging, 48 h after transfection, 2% 1,6-hexanediol was introduced into the culture medium. Time-lapse imaging was then performed using the Leica TCS SP8 STED confocal microscope system with a 100× oil immersion objective.

Prediction and correlation analysis of upstream transcription factors

The prediction of upstream transcription factors was performed using the Knock TF database and the GTRD database (https://gtrd18-06.biouml.org/). The binding sites of transcription factors were predicted using the JASPAR database (https://jaspar.elixir.no/), and the promoter sequence of ARHGAP4 was obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/). Correlation analysis between genes was conducted using Pearson correlation analysis on CRC data from the TCGA database.

Chromatin immunoprecipitation (ChIP) assay

Chromatin immunoprecipitation (ChIP) assays were conducted using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003) from CST Biotechnology. The procedure was carried out according to the instructions provided in the kit’s manual. The antibody for c-Jun (JUN) was purchased from CST.

Promoter Activity Assay

The promoter region of ARHGAP4 was retrieved from the NCBI website (https://www.ncbi.nlm.nih.gov/). A ‘promoter-reporter gene-terminator’ vector construction method was used, with the luciferase (LUC) gene as the reporter. The reporter gene vector and c-Jun plasmid were co-transfected into HEK293T cells. Luciferase activity was measured with a microplate reader to evaluate the transcriptional regulatory function.

Statistical methods

Bioinformatics analysis was performed using RStudio, with the necessary R packages installed for the analysis. Statistical analysis of the experimental data was done using SPSS 27.0 and GraphPad Prism 10.1.2. All experiments were repeated at least three times. Survival analysis was conducted with the Kaplan-Meier method, followed by the Log-rank test. Data are presented as mean ± standard deviation (mean ± SD). The independent sample t-test was used for comparing two groups, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. A p value of <0.05 was considered statistically significant. nsP represents > 0.05, *P represents < 0.05, **P represents < 0.01, ***P represents < 0.001.

Data availability

The data sets used in this study have all been shown in the method. The original data of WB has been stored in the supplementary materials. The original data can be provided according to reasonable requirements.

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Acknowledgements

Acknowledgments: This work was supported by financial grants from the Natural Science Foundation of Fujian Province (grant number: 2022J011024) to Weihua Li.

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These authors contributed equally: Shaolin Liu, Yizhen Chen, Song Tan.

Authors and Affiliations

Shengli Clinical Medical College of Fujian Medical University, Fuzhou, China
Shaolin Liu, Yizhen Chen, Song Tan & Yifan Chen
Department of Gastrointestinal Surgery, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou, Fujian, China
Shaolin Liu, Yizhen Chen, Song Tan, Yifan Chen, Changshun Yang, Yulong Mi, Shengtao Lin & Weihua Li

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Shaolin Liu
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Yizhen Chen
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Song Tan
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Yifan Chen
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Changshun Yang
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Yulong Mi
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All authors agree to be responsible for all aspects of the work, and ultimately approve the version to be published. L.-S.L., M.-Y.L., and C.-Y.Z.: material preparation, retrieval, and data collection. T.-S., L.-S.L., and C.-Y.F.: Visualization. L.-S.L. and C.-Y.Z.: Write the original. L.-S.T., Y.-C.S., and L.-W.H.: Supervision and conceptualization. L.-S.T. and L.-W.H.: Revised draft. L.-W.H.: Funding supports.

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Liu, S., Chen, Y., Tan, S. et al. ARHGAP4/MYH9/β-catenin/c-Jun positive feedback loop promotes colorectal cancer stemness. npj Precis. Onc. 9, 285 (2025). https://doi.org/10.1038/s41698-025-01022-4

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Received: 20 March 2025
Accepted: 21 June 2025
Published: 15 August 2025
DOI: https://doi.org/10.1038/s41698-025-01022-4