RNF113A Promotes Colorectal Cancer Metastasis by Regulating TGF-β Pathway and EMT via Ubiquitin-Mediated Degradation of SNIP1

Abstract

Background

RNF113A is a RING finger protein that is upregulated in colorectal cancer (CRC). Smad nuclear-interacting protein 1 (SNIP1) negatively regulates the transforming growth factor-beta (TGF-β) pathway in CRC and plays a significant role in disease progression; however, its precise regulatory mechanism remains unclear.

Aims

To investigate whether RNF113A is associated with SNIP1 protein stability and TGF-β pathway modulation in CRC and to explore the potential underlying molecular mechanisms.

Study Design

In vitro experimental study.

Methods

Immunohistochemistry and correlation analyses were performed on clinical CRC specimens to assess the expression relationships among RNF113A, SNIP1, and TGF-β1. CRC cell clonogenic capacity and migration were evaluated using colony formation, wound healing, and transwell assays. Co-immunoprecipitation was used to assess protein–protein interactions. Protein stability was examined via RNF113A overexpression and knockdown, with or without proteasome inhibition (MG132). TGF-β pathway-related protein expression and epithelial–mesenchymal transition markers were assessed by qPCR and Western blotting, using TGF-β1 (pathway activator) and SB525334 (ALK5 inhibitor) for pharmacological modulation.

Results

RNF113A expression was inversely correlated with SNIP1 and positively correlated with TGF-β1 in clinical specimens. Functionally, RNF113A promoted CRC cell clonogenic capacity and migration. Mechanistically, RNF113A interacted with SNIP1. RNF113A overexpression decreased SNIP1 protein levels, whereas RNF113A knockdown increased them. This effect was reversed by MG132, suggesting ubiquitin–proteasome-mediated degradation.

Conclusion

This study suggests that RNF113A is associated with SNIP1 ubiquitination and degradation, potentially modulating TGF-β signaling and CRC cell migration. These findings reveal a potential RNF113A/SNIP1/TGF-β regulatory axis and highlight RNF113A as a potential therapeutic target in CRC, warranting further mechanistic validation.

INTRODUCTION

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with metastasis being the primary contributor to poor prognosis.^{1, 2} The transforming growth factor-beta (TGF-β) pathway plays a dual role in cancer, acting as a tumor suppressor in the early stages but promoting invasion and epithelial–mesenchymal transition (EMT) in advanced stages.³ EMT facilitates CRC dissemination by enhancing cell motility and conferring stem-like properties.⁴ However, the regulatory mechanisms linking specific molecules to TGF-β-driven EMT in CRC remain incompletely understood.

RING finger protein 113A (RNF113A) is a member of the RING finger protein family, characterized by a conserved RING domain that is suggestive of ubiquitin ligase activity. Although RNF113A has been implicated in RNA splicing and DNA damage repair through interactions with the BRCA1/BARD1 complex, its role in cancer remains poorly understood.^{5, 6, 7, 8} Emerging evidence suggests that dysregulation of E3 ubiquitin ligases may contribute to tumorigenesis by destabilizing tumor suppressors or stabilizing oncoproteins.^{9, 10} RNF113A has been associated with unfavorable prognosis in cervical cancer and exerts its effects through the miR-197/Prp19/p38 MAPK signaling pathway.¹¹ The pro-proliferative role of RNF113A in cervical cancer is mediated by its suppression of autophagy.¹²

In cancer, aberrant ubiquitination signaling frequently drives key processes such as EMT, metastasis, and therapeutic resistance.^{13, 14, 15, 16} RNF113A, with its RING domain, represents a potentially interesting therapeutic target in cancer, and investigating its specific mechanisms of action could inform novel treatment strategies. Preliminary bioinformatic analyses have revealed elevated RNF113A expression in aggressive cancers, correlating with poor patient survival.¹⁷ However, direct mechanistic studies validating its oncogenic or tumor-suppressive roles are lacking.

Smad nuclear-interacting protein 1 (SNIP1) is a protein primarily localized in the nucleus. Its basic structure consists of an N-terminal nuclear localization signal and a C-terminal forkhead-associated domain.^{18, 19} By binding to Smad4, SNIP1 blocks the recruitment of the coactivator CBP/p300 by Smad4, thereby inhibiting the activation of downstream TGF-β target genes and exerting a negative regulatory effect on this pathway.²⁰ Additionally, SNIP1 competitively inhibits the binding of the NF-κB subunit RelA/p65 to p300, leading to suppression of NF-κB pathway activity.^{21, 22}

Recent studies have indicated that RNF113A is highly expressed in CRC, where it promotes unfavorable patient outcomes and is associated with immune infiltration. However, the mechanistic basis of RNF113A action in CRC remains elusive. Based on the inverse correlation between RNF113A and SNIP1 observed in our preliminary analyses, and given the known role of SNIP1 as an inhibitor of the TGF-β pathway—a key driver of EMT, invasion, and metastasis—we hypothesized that RNF113A may promote CRC progression through modulation of SNIP1 stability and subsequent TGF-β signaling. This study explores the potential association between RNF113A and SNIP1 in CRC and investigates whether this interaction may contribute to CRC progression via TGF-β pathway modulation, providing a foundation for further research into the RNF113A–SNIP1–TGF-β axis as a potential therapeutic target in CRC.

MATERIALS AND METHODS

Antibodies and reagents

The antibodies used in this study were obtained from the following providers: anti-RNF113A antibody (Proteintech, 27018-1-AP, 1:1000), anti-SNIP1 antibody (Proteintech, 14950-1-AP, 1:1000), anti-ubiquitin antibody (Proteintech, 10201-2-AP, 1:3000), anti-TGF-β1 antibody (R22797, Zenbio, 1:1000), anti-phospho-SMAD2 antibody (HA722443, Zenbio, 1:1000), anti-phospho-SMAD3 antibody (R380775, Zenbio, 1:1000), anti-E-cadherin antibody (20874-1AP, Proteintech, 1:4000), anti-N-cadherin antibody (GB121135, Servicebio, 1:1000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (GB15004, Servicebio, 1:5000). HRP-conjugated secondary antibodies were obtained from ZSGB-BIO (Beijing, China, 1:20000).

In addition, the following reagents were used: MG132 (HY-13259, MedChemExpress, Shanghai, China); TGF-β1 (Cat. No. CA59, Affinity Biosciences, Jiangsu, China); SB525334 (HY-10431, MedChemExpress, Shanghai, China), a TGF-β type I receptor/ALK5 inhibitor; and protein A/G-agarose (Beyotime, China).

Patients and tissue samples

This retrospective cohort study included 64 consecutive patients with CRC who underwent surgical resection at the hospital between March 2018 and December 2019. The enrolled patients, aged 35 to 81 years, were staged according to the American Joint Committee on Cancer/Union for International Cancer Control TNM (T-tumor, N-nodes, M-metastasis) staging system for colorectal carcinoma (8^th edition). All tissue specimens were collected with written informed consent from the patients. The study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Guangxi Medical University [approval no: 2026-KLY(012); date: 26/01/2026]. CRC tissue specimens were collected from 64 consecutive patients who underwent surgical resection. Of these 64 patients, 33 paired tumor-adjacent normal tissue samples were obtained; after immunohistochemistry (IHC) staining, 32 tumor tissues and 28 normal tissues yielded valid composite scores (the remaining specimens were excluded because of section detachment or tissue folding). These 32 tumor tissues were used for correlation analyses among RNF113A, SNIP1, and TGF-β1. Additionally, the full set of 64 tumor tissues was used for clinicopathological association analyses (Table 1).

TABLE 1. Correlation Between RNF113A Expression and Clinicopathological Characteristics of Patients with CRC.

RNF113A expression
Clinicopathological characteristic	High	Low	p value
Gender	-	-	0.902
Male	24	15	-
Female	15	10	-
Age	-	-	0.800
< 65	19	11	-
≥ 65	20	14	-
Infiltration depth	-	-	0.557
Mucosa and superficial muscular layer	7	6	-
Deep muscular layer and below	32	19	-
Lymphatic metastasis	-	-	0.035
-	16	17	-
+	23	8	-
Distant metastasis	-	-	0.146
-	36	20	-
+	3	5	-
TNM stage	-	-	0.035

Immunohistochemistry detection

CRC tissue specimens were processed by formalin fixation and paraffin embedding, followed by sectioning for IHC. The IHC slides were independently evaluated by two pathologists who were blinded to all clinical and follow-up data. The percentage of positive tumor cells was graded as follows: 0 (< 5%), 1 (5-25%), 2 (26-50%), and 3 (> 50%). Staining intensity was graded as follows: 0 (achromatic), 1 (light yellow), 2 (yellow), and 3 (brown). An IHC composite score was calculated as the sum of the intensity and proportion scores (range, 0-6). The final score for each core was calculated as the average of five fields. Interobserver agreement was assessed using Cohen’s κ = 0.86 and an intraclass correlation coefficient of 0.91. Discrepancies between the two pathologists were resolved by consensus discussion with a third senior pathologist. The median composite IHC score was selected as the cut-off value for stratifying patients into high- and low-RNF113A-expression groups, as prespecified before outcome analysis. In the present cohort, the median composite score was 3; specimens with composite scores ≥ 3 were defined as having high expression, whereas those with scores < 3 were defined as having low expression.

Cell culture

HT29 and SW480 human CRC cells, obtained from the American Type Culture Collection, were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂, passaged using 0.25% trypsin-EDTA, and routinely screened for mycoplasma contamination.

Lentiviral transduction

For gene overexpression or knockdown, lentiviral particles encoding the gene of interest or short hairpin RNA (shRNA) were produced in 293T cells using a packaging system from Sangon Biotech (Shanghai, China). Target cells were seeded in 6-well plates and transduced with lentivirus in the presence of 8 μg/mL polybrene. After 48 hours, cells were selected with puromycin to generate stable cell lines. The shRNA sequences (Supplementary Table 1) and details of the lentiviral construct (Supplementary Figures 1 and 2).

Wound-healing assay

Confluent cells in 6-well plates were scratched with a sterile 200-μL pipette tip, rinsed with PBS, and then cultured in serum-free medium. Wound images were captured at 0 and 48 hours using an inverted microscope, and the scratch area was quantified using ImageJ. Experiments were performed as three independent biological replicates (n = 3).

Transwell migration assays

Migration was assessed using Transwell inserts with 8-μm pores. The upper chambers were seeded with 5 × 10⁴ cells in serum-free medium, and the lower chambers contained medium supplemented with 10% FBS as a chemoattractant. After 48 hours, cells on the lower surface of the membrane were fixed with 4% PFA, stained with 0.1% crystal violet, and counted microscopically. Three fields per well were averaged and treated as a single biological replicate (n = 3 independent experiments).

Colony formation assay

Cells (500/well) were seeded in 6-well plates and cultured for 14-21 days. After methanol fixation and 0.1% crystal violet staining, colonies were counted. A colony was defined as a cluster of > 50 cells. Experiments were performed as three independent biological replicates (n = 3).

Quantitative real-time PCR (qRT-PCR)

RNA was extracted using TRIzol and reverse-transcribed into complementary DNA using a reverse transcription kit. qRT-PCR was performed using SYBR Green Master Mix in a real-time PCR system, with GAPDH as the internal reference for normalization. Statistical analyses of qRT-PCR data were performed using ΔCt values, which better approximate a normal distribution. Fold changes (2^-ΔΔCt) are presented in the figures for interpretability. Primer sequences are provided in the (Supplementary Table 2).

Western blot analysis

Cells were homogenized in RIPA buffer containing protease/phosphatase inhibitors. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS) and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at room temperature. Target proteins were detected, and densitometric analysis was performed, using an infrared imager and ImageJ. All experiments were performed as three independent biological replicates (n = 3).

Co-immunoprecipitation (Co-IP)

Cells were lysed in IP buffer and precleared with protein A/G beads. Lysates were incubated with the indicated antibody or control IgG overnight at 4 °C, followed by incubation with protein A/G beads for 2 hours. The beads were washed extensively, and bound proteins were eluted by boiling in SDS sample buffer for Western blot analysis.

Ubiquitination assay

To inhibit proteasomal degradation, cells were treated with 10 μM MG132 for 8 hours before lysis. Cells were then lysed in RIPA buffer, and the protein of interest was immunoprecipitated using a specific antibody. The precipitated protein complexes were analyzed by Western blotting to detect ubiquitinated forms using an anti-ubiquitin antibody.

Statistical analysis

All cell-based experiments were performed in triplicate and repeated at least three times. For biological versus technical replicates, independent cell culture experiments (n = 3 for all functional assays) were considered biological replicates. Technical replicates (multiple wells or fields) were averaged and were not treated as independent observations. For IHC data, comparisons were analyzed using non-parametric tests, with the Mann-Whitney U test used for unpaired comparisons. Spearman rank correlation analysis was used to assess correlations. For clinicopathological correlations, Pearson’s chi-square test was used when all expected counts were ≥ 5, whereas Fisher’s exact test was used when any expected count was < 5. p values were nominal and uncorrected for multiple comparisons; Bonferroni-corrected thresholds (α = 0.0083 for six variables) are reported in the text. All clinical correlation analyses were exploratory. A p value < 0.05 was considered statistically significant for nominal comparisons. For cell-based experiments, the unpaired Student’s t test was used for two-group comparisons. For multiple comparisons, one-way ANOVA was applied, followed by Tukey’s post-hoc test for all pairwise comparisons or Dunnett’s post-hoc test for comparisons with the control only. Equal variance was not assumed, and Welch’s correction was applied for t-tests. Data are presented as individual biological replicates (n = 3) with mean ± standard deviation.

RESULTS

RNF113A expression is upregulated in CRC tissues

Analysis of clinical samples revealed that RNF113A was significantly overexpressed in CRC tissues compared with adjacent normal tissues (Figure 1). A statistically significant association was observed between RNF113A expression and lymph node metastasis and advanced TNM stage (nominal p = 0.0351 for both). However, after Bonferroni correction for six comparisons (adjusted α = 0.0083), these associations did not retain statistical significance. No association was found with patient sex, age, infiltration depth, or distant metastasis (Table 1). These associations are considered exploratory.

Figure 1.

Expression of RNF113A in colorectal cancer (CRC) and adjacent normal colon tissues. (a) Representative IHC images of RNF113A expressions in CRC and adjacent normal colon tissues. Scale bar, 20 µm. (b) Chi‑square analysis was performed to evaluate the high‑ and low‑expression status of RNF113A in (CRC, n = 64) and paired adjacent normal tissues (N, n = 28). The data were converted into percentages and visualized in bar charts.***p < 0.001.

RNF113A expression correlates with SNIP1 and TGF-ß1 in CRC

To investigate the role of RNF113A in CRC progression and explore its potential mechanism, we evaluated the expression of RNF113A, TGF-β1, and SNIP1 by IHC staining in 32 CRC tissues and 28 adjacent normal tissues with valid IHC scores. As shown in Figure 2a and b, RNF113A and TGF-β1 were significantly upregulated in CRC tissues compared with adjacent normal tissues (p < 0.001 and p < 0.01, respectively; Mann-Whitney U test), whereas SNIP1 was significantly downregulated (p < 0.001; Mann-Whitney U test).

Figure 2.

RNF113A expressions correlated with SNIP1 and TGF-β1 in CRC tissues. (a) Representative IHC images of RNF113A, SNIP1, and TGF‑β1 expression in CRC and adjacent normal colon tissues. Scale bar, 20 µm. (b) Quantitative analysis of IHC composite scores for RNF113A, SNIP1, and TGF‑β1 in CRC (n = 32) and adjacent normal (n = 28) tissues. Data are presented as median with IQR; ****p < 0.001, ***p < 0.001 (Mann-Whitney U test). (c, d) Spearman’s rank correlation analysis of RNF113A with TGF‑β1 (c) and SNIP1 (d) in 32 CRC tissues.

CRC, colorectal cancer; SNIP1, smad nuclear-interacting protein 1; TGF-β1, transforming growth factor-beta 1; IHC, immunohistochemistry; IQR, interquartile range.

Spearman rank correlation analysis was performed on 32 CRC tissues. RNF113A expression showed a weak positive correlation with TGF-β1 [Spearman’s ρ = 0.3812, 95% confidence interval (CI), 0.015-0.665; nominal p = 0.0314]. A weak negative correlation was observed between RNF113A and SNIP1 (Spearman’s ρ = -0.3696, 95% CI, -0.657 to -0.018; nominal p = 0.0374) (Figure 2c and d). After Bonferroni correction for the two correlations (adjusted α = 0.025), neither correlation retained statistical significance, indicating that these associations are exploratory and require validation in larger cohorts.

Collectively, these findings suggest that RNF113A, SNIP1, and the TGF-β pathway may be involved in CRC progression, although the modest correlation strengths and limited sample size warrant cautious interpretation. Further functional studies are needed to establish causal relationships.

RNF113A promotes CRC cell migration and clonogenic capacity in vitro

To evaluate the functional impact of RNF113A on CRC cells, we modulated its expression through both overexpression and knockdown approaches (Figure 3). Functional assays revealed that overexpression of RNF113A in SW480 cells significantly promoted clonogenic capacity (Figure 4a). Conversely, genetic knockdown of RNF113A in HT29 cells markedly suppressed clonogenic capacity (Figure 4a). Wound-healing and Transwell assays consistently demonstrated that overexpression of RNF113A enhanced cell migration (Figure 4b and c). In contrast, knockdown of RNF113A expression had the opposite effects on CRC cells (Figure 4b and c). All experiments were performed as three independent biological replicates (n = 3). We note that wound healing measures wound closure capacity, which may reflect both migration and proliferation; however, Transwell migration assays, which are less sensitive to proliferation, independently support the effect on cell motility.

Figure 3.

Establishment of HT29 and SW480 cell subline with stable RNF113A overexpression or knockdown. (a, b) Western blot analysis of RNF113A protein expression in HT29 and SW480 cells with RNF113A overexpression (a) or knockdown (b). (c, d) Quantification of western blot bands from three independent biological experiments (n = 3). (e, f) Quantitative reverse transcription‑PCR analysis of RNF113A mRNA levels in HT29 (e) and SW480 (f) cells with RNF113A overexpression or knockdown. Data are shown as individual biological replicates (n = 3) ; *p < 0.001,**p < 0.01 vs. control group (unpaired Student’s t‑test with Welch’s correction).

PCR, polymerase chain reaction.

Figure 4.

RNF113A promotes clonogenic capacity and migration of colorectal cancer cells. (a) Colony formation assay showing clonogenic capacity of HT29 and SW480 cells with RNF113A overexpression or knockdown. (b) Wound healing assay showing wound closure capacity. (c) Transwell assay showing cell migration ability. All experiments were performed as three independent biological replicates (n = 3). *p < 0.001,**p < 0.01 vs. 0.001,****p < 0.001, vs. control group (statistical comparisons between two groups were performed using the Mann‑Whitney U test (non‑parametric test).

Collectively, these results indicate that RNF113A promotes CRC cell migration and clonogenic capacity in HT29 and SW480 cells.

RNF113A interacts with SNIP1 and promotes SNIP1 ubiquitination and degradation

The negative correlation between RNF113A and SNIP1 identified by our earlier IHC analysis prompted further mechanistic investigation. Next, Western blot analysis confirmed that overexpression of RNF113A resulted in decreased SNIP1 protein levels, whereas RNF113A knockdown increased SNIP1 protein levels (Figure 5a). A direct interaction was then verified by reciprocal Co-IP assays (Figure 5c). Given the established identity of RNF113A as an E3 ubiquitin ligase, we asked whether RNF113A promotes SNIP1 degradation via the ubiquitin-proteasome pathway. To test this hypothesis, we employed the proteasome inhibitor MG132. Treatment with MG132 effectively rescued the degradation of SNIP1 induced by RNF113A, demonstrating that RNF113A-mediated SNIP1 degradation depends on the 26S proteasome pathway (Figure 5b). Ubiquitination analysis demonstrated that RNF113A overexpression increased the ubiquitination levels of SNIP1 (Figure 5d and e). These findings indicate that RNF113A promotes SNIP1 ubiquitination and subsequent proteasomal degradation. However, we acknowledge that our data demonstrate an association between RNF113A and SNIP1 ubiquitination/degradation but do not establish direct E3 ligase activity. This limitation is explicitly noted in the Discussion.

Figure 5.

RNF113A drives the degradation of SNIP1 through ubiquitination. (a) Western blot analysis showing SNIP1 protein expression in CRC cells with RNF113A overexpression or knockdown. (b) Proteasome‑dependent degradation of SNIP1: SW480 cells overexpressing RNF113A were treated with 10 µM MG132 for 8 h, and SNIP1 expression was assessed. (c) Co‑immunoprecipitation assay showing the interaction between RNF113A and SNIP1 in HT29 and SW480 cells. (d) Ubiquitination assay showing RNF113A promotes SNIP1 ubiquitination. (e) Western blot analysis of SNIP1 ubiquitination after 8 h treatment with 10 µM MG132. All experiments were performed as three independent biological replicates (n = 3). *p < 0.05 **p < 0.01 vs. control group (unpaired Student’s t‑test with Welch’s correction).

CRC, colorectal cancer; SNIP1, smad nuclear-interacting protein 1.

RNF113A modulates TGF-ß pathway-related protein expression and EMT markers

Given that SNIP1 inhibits the TGF-β pathway in CRC, we found that RNF113A was negatively correlated with SNIP1 and positively correlated with TGF-β1. Based on this association, we investigated whether RNF113A influences TGF-β pathway-related protein expression. Western blot and qPCR analyses confirmed that RNF113A overexpression enhanced, whereas RNF113A knockdown reduced, the expression of TGF-β pathway-related proteins at both the protein and mRNA levels in CRC cells (Figure 6a and b). We note that increased total TGF-β1, SMAD2, or SMAD3 expression does not necessarily indicate pathway activation. Phospho-SMAD2 and phospho-SMAD3 levels were also altered, suggesting potential pathway modulation; however, total SMAD2/3 protein blots were not available for normalization, and SMAD-responsive reporter assays were not performed. Therefore, our conclusions regarding TGF-β pathway modulation are preliminary.

Figure 6.

RNF113A Modulates the TGF-β Pathway and EMT. (a) qPCR analysis of TGF‑β1, Smad2, and Smad3 mRNA levels in CRC cells with RNF113A overexpression or knockdown. (b) Western blot analysis of TGF‑β pathway‑related protein expression (TGF‑β1, p‑SMAD2, p‑SMAD3). (c) Western blot analysis of epithelial (E‑cadherin) and mesenchymal (N‑cadherin) markers following RNF113A knockdown and overexpression. All experiments were performed as three independent biological replicates (n = 3). *p < 0.05 ,**p < 0.01, ***p < 0.001,****p < 0.0001 vs. control group (unpaired Student’s t‑test with Welch’s correction for two groups.

CRC, colorectal cancer; TGF-β1, transforming growth factor-beta 1; EMT, epithelial-mesenchymal transition; qPCR, quantitative polymerase chain reaction.

Because the TGF-β pathway induces EMT, we concurrently assessed EMT markers. RNF113A overexpression decreased E-cadherin and increased N-cadherin protein levels, whereas RNF113A knockdown reciprocally altered their expression (Figure 6c). Collectively, these results indicate that RNF113A modulates TGF-β pathway-related protein expression and EMT markers in CRC cells.

RNF113A promotes migration and EMT in CRC cells in a TGF-ß signaling

We next investigated whether the pro-metastatic effects of RNF113A are mediated through the TGF-β pathway. Pharmacological inhibition of the TGF-β type I receptor/ALK5 with SB525334 markedly reduced the migratory ability of RNF113A-overexpressing SW480 cells. After SB525334 treatment, the initially high migratory capacity of these cells was significantly reduced compared with that of untreated cells. Treatment with TGF-β1 (10 ng/mL, 48 hours) partially restored the EMT and migration capacity of RNF113A-knockdown HT29 cells, whereas treatment with SB525334 (1 μM, 24 hours) reversed the EMT and migration capacity of RNF113A-positive SW480 cells (Figure 7a and b). These findings suggest that the migration-promoting function of RNF113A may depend on TGF-β signaling pathway activity.

Figure 7.

Rescue experiment result. (a) Western blot analysis of TGF‑β pathway and EMT‑related proteins in RNF113A‑overexpressing SW480 cells treated with the ALK5 inhibitor SB525334, and in RNF113A‑knockdown HT29 cells treated with TGF‑β1. (b) Wound healing assay showing migratory capacity of RNF113A‑overexpressing SW480 cells treated with SB525334 and RNF113A‑knockdown HT29 cells treated with TGF‑β1. All experiments were performed as three independent biological replicates (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.001; (one‑way ANOVA with Tukey’s post‑hoc test).

TGF-β1, transforming growth factor-beta 1; EMT, epithelial-mesenchymal transition.

We acknowledge that a formal factorial design with interaction testing would be required to definitively establish whether the effect of RNF113A is modified by TGF-β pathway manipulation. Therefore, these rescue experiments are presented as preliminary and observational, and this limitation is explicitly acknowledged in the Discussion.

DISCUSSION

CRC represents a major global health challenge, ranking as the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths worldwide.²³ The high mortality rate is primarily attributed to the invasive and metastatic characteristics of advanced CRC, for which current therapeutic regimens remain inadequate.^{24, 25} The TGF-β signaling pathway has emerged as a crucial regulator of CRC pathogenesis, demonstrating a paradoxical dual nature during different stages of tumor development. In normal epithelial cells and early-stage adenomas, TGF-β functions as a potent tumor suppressor by inducing cell cycle arrest and promoting apoptosis. However, during malignant progression, cancer cells develop resistance to these growth-inhibitory effects, and the pathway undergoes a functional switch to become a powerful driver of tumor progression and metastasis.^{26, 27, 28, 29, 30} Previous studies have found that RNF113A promotes CRC aggressiveness, with its overexpression predicting poor survival.¹⁷ Functional analyses confirm its critical role in stimulating clonogenic capacity, migration, and EMT.

Targeting the TGF-β pathway may represent a potential strategy for treating advanced CRC. Several types of drugs using this approach are currently in clinical development, including small-molecule inhibitors that block TGF-β receptors, neutralizing antibodies, and fusion proteins that simultaneously block TGF-β signaling and immune checkpoints.^{31, 32, 33, 34} These agents aim to inhibit TGF-β-mediated immunosuppression, EMT, and metastasis. Progress has been made with galunisertib, a TGF-β receptor I inhibitor, which has demonstrated acceptable safety and some efficacy in clinical trials. Another example is bintrafusp alfa (M7824), a bifunctional agent that traps TGF-β and blocks PD-L1 simultaneously. It has demonstrated promising antitumor activity in early studies and targets both the immunosuppressive and pro-metastatic functions of the TGF-β pathway.^{35, 36, 37} Consequently, elucidating the underlying molecular mechanisms and identifying new biomarkers are crucial for advancing cancer drug development and optimizing treatment strategies.

Our findings indicate that RNF113A promotes EMT and migration in CRC, potentially through modulation of the TGF-β pathway. RNF113A is a RING-domain-containing protein that has been implicated as an E3 ubiquitin ligase whose biological functions and pathological significance are increasingly being recognized. RNF113A is involved in the regulation of various cancers, such as cervical cancer, esophageal squamous cell carcinoma, and small cell lung cancer.^{10, 11, 12, 38} Current evidence indicates that RNF113A participates in multiple fundamental cellular processes, including DNA damage repair, RNA processing, and cell cycle regulation, although its complete repertoire of physiological substrates remains to be fully characterized.^{39, 40, 41} The clinical importance of RNF113A is highlighted by its dysregulation in various cancers, where it appears to function as a context-dependent oncogene, potentially through the degradation of tumor suppressor proteins.

The ubiquitin-proteasome system, comprising E1 activating enzymes, E2 conjugating enzymes, E3 ligases, and deubiquitinases, controls the degradation and function of numerous proteins, and its dysregulation is a hallmark of cancer.^{42, 43, 44, 45} Therapeutic strategies targeting this system show considerable promise. Particularly noteworthy are proteolysis-targeting chimeras (PROTACs), which are bifunctional molecules that recruit E3 ligases to target proteins for ubiquitination and degradation.^{46, 47, 48} PROTACs can target proteins that have historically been difficult to drug, act through a catalytic mechanism, and potentially produce sustained effects. They may also help overcome drug resistance. Several candidates are currently being evaluated in clinical trials for cancers such as prostate and breast cancer.^{49, 50}

In this study, administration of the proteasome inhibitor MG132 to RNF113A-overexpressing cells restored SNIP1 protein levels, suggesting that RNF113A may promote SNIP1 degradation via the ubiquitin–proteasome pathway. This finding suggests that RNF113A may contribute to CRC progression through its potential role in regulating protein stability. However, although our findings support an association between RNF113A and SNIP1 ubiquitination and degradation, they do not establish direct E3 ubiquitin ligase activity toward SNIP1. These findings position RNF113A as a compelling subject for further research, particularly to identify its specific substrates, elucidate its regulatory mechanisms in different pathological contexts, and explore its potential as a therapeutic target.

Several limitations of this study should be acknowledged. First, the sample size for clinical correlation analyses was limited. Of the total 64 patients, only 32 tumor tissues and 28 normal tissues yielded valid IHC scores across all three proteins for correlation analysis. Additionally, this was a retrospective, single-center study. Second, STR authentication and mycoplasma testing were not performed for the cell lines used. Third, knockdown and overexpression experiments were conducted in different cell lines (HT29 and SW480, respectively), which may introduce cell line-specific effects. Fourth, the direct E3 ligase activity of RNF113A toward SNIP1 has not been formally demonstrated; ligase-deficient mutants, denaturing IP, and in vitro ubiquitination assays are required to establish direct enzymatic activity. The specific ubiquitin linkage type also remains to be determined. Fifth, total SMAD2/3 protein blots were not available for normalization of phospho-SMAD data, and SMAD-responsive reporter or nuclear localization assays were not performed; therefore, our conclusions regarding TGF-β pathway modulation are preliminary. Sixth, a full factorial design with interaction testing was not performed to definitively establish whether the effect of RNF113A is mediated through SNIP1 or the TGF-β pathway. Genetic rescue experiments (SNIP1 re-expression/deletion and degradation-resistant SNIP1 constructs) are required to establish causality. Seventh, in vivo metastasis models and survival analyses were not performed, so our findings are restricted to in vitro observations. Therefore, our findings should be considered preliminary and hypothesis-generating, and independent validation in additional CRC cell lines with multiple shRNAs, rescue constructs, and large-cohort studies is required to confirm our conclusions.

In summary, our study suggests that RNF113A is associated with SNIP1 ubiquitination and degradation, potentially modulating TGF-β pathway-related protein expression and CRC cell migration. These findings suggest a potential RNF113A/SNIP1/TGF-β regulatory axis in CRC. However, the precise mechanistic details—including whether RNF113A acts as a direct E3 ligase for SNIP1, the specific ubiquitin linkage types involved, and the causal role of this axis in CRC progression—warrant further investigation. Our data provide a foundation for future studies exploring RNF113A as a potential therapeutic target in CRC.

Supplementary Materials

Supplementary Figures

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Supplementary Tables

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