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SORBS3-β suppresses lymph node metastasis in cervical cancer by promoting the ubiquitination of β-catenin

Journal of Translational Medicine volume 23, Article number: 406 (2025) Cite this article

Abstract

Background

Cervical cancer (CC) is a prevalent gynecological malignancy, with lymph node metastasis (LNM) serving as a critical factor influencing patient prognosis. SORBS3, an adaptor protein with two known isoforms (α and β), has been implicated in tumor suppression, but the specific roles of its isoforms in CC metastasis remains unexplored. This study aimed to identify the functional isoform of SORBS3 driving LNM suppression and elucidate its mechanisms.

Methods

Proteomic analysis of clinical CC tissues and metastatic lymph nodes revealed progressive downregulation of SORBS3. The mRNA and protein levels of SORBS3-α and SORBS3-β were subsequently examined in normal cervical epithelial and CC cell lines. Functional studies, including siRNA-mediated knockdown of SORBS3-α, lentiviral-mediated overexpression and knockdown of SORBS3-β, Transwell migration, lymphangiogenesis assays, and in vivo footpad xenograft models, were conducted to evaluate the role of SORBS3 isoforms in LNM. SORBS3 DNA methylation mechanisms were analyzed by MSP and Targeted Bisulfite sequencing. Mechanistic insights were derived from Co-IP, ubiquitination assays, RNA-seq, and LC–MS/MS.

Results

Knockdown of SORBS3-α had no effect on CC cell migration, invasion, or lymphangiogenesis. In contrast, SORBS3-β overexpression markedly suppressed CC cell invasion, lymphangiogenesis, and adhesion to lymphatic endothelial cells, whereas its knockdown significantly promoted these phenotypes. Promoter hypermethylation driven by DNMT-1 inhibited SORBS3 expression in CC. SORBS3- β directly binds to β-catenin and recruits UBA1 to enhance its ubiquitination and degradation, thereby inhibiting Wnt/β-catenin signaling. This inhibition reduced accumulation of β-catenin and downregulated the pro-lymphangiogenic gene VEGFC, ultimately suppressing lymphangiogenesis and LNM. In vivo, SORBS3-β overexpression attenuated lymphatic metastasis in nude mice, whereas its knockdown promoted metastasis.

Conclusion

SORBS3-β is the major isoform of SORBS3 that inhibits lymphatic metastasis of cervical cancer by degrading β-catenin through UBA1-mediated ubiquitination, blocking Wnt/β-catenin signaling and downstream lymphangiogenesis pathways, thereby inhibiting lymphatic metastasis. Our findings elucidate key molecular mechanisms underlying cervical cancer lymph node metastasis, offering potential therapeutic targets.metastasis.

Introduction

Cervical cancer (CC) is one of the most common malignancies affecting the female reproductive system and continues to pose a major public health challenge, especially in countries such as China, where the incidence of CC remains relatively high [1]. While the widespread adoption of cervical cancer screening has led to a marked reduction in advanced-stage cases, a significant proportion of patients still develop metastasis during treatment. Lymph node metastasis is the predominant mode of spread and a key determinant of poor prognosis in patients with cervical cancer [2]. Lymph node metastasis has been identified as an independent prognostic factor that is strongly linked to higher recurrence rates and poorer overall survival (OS) outcomes in patients [3, 4]. Investigating the molecular mechanisms underlying cervical cancer lymphatic metastasis is crucial for understanding its pathogenesis, predicting disease progression, developing targeted therapies, and improving patient outcomes.

Lymph node metastasis involves lymphangiogenesis, tumor cell invasion into lymphatic vessels, transport to regional lymph nodes, and subsequent colonization and growth, all of which are regulated by various factors [5]. Local lymphangiogenesis and epithelial‒mesenchymal transition (EMT) are critical steps in tumor lymph node metastasis. Research indicates that intratumoral lymphangiogenesis significantly affects survival in patients without metastatic disease [6]. Lymphangiogenesis requires the coordinated actions of lymphatic endothelial cells, including proliferation, sprouting, migration, and tube formation, similar to angiogenesis [7, 8]. Tumor-derived cytokines, such as VEGF-C, VEGF-D, IGF1, FGF2, HGF, and IL-7, drive this process, contributing to lymph node metastasis [5, 9]. Simultaneously, tumor cells undergo EMT, lose polarity and gain invasive abilities, which facilitate lymphatic invasion. Key signalling pathways, including the TGF, Wnt/β-catenin, FGF, HGF, and Notch pathways, regulate EMT and lymph node metastasis [10]. However, the specific molecular mechanism of cervical cancer lymphatic metastasis has not been fully elucidated.

Sorbin and SH3 Domain Containing 3(SORBS3) is a member of the SORBS protein family, a novel class of adaptor proteins involved in regulating various pathophysiological processes, including cell adhesion, cytoskeletal organization, and growth factor signalling [11]. Early studies linked SORBS3 to diseases such as Alzheimer's disease and atherosclerosis by modulating smooth muscle contractility and cardiac conduction [12, 13]. Recent research has shown frequent loss of the chromosome 8p region, where SORBS3 is located, in hepatocellular carcinoma. SORBS3 has been found to bind and inhibit proteins such as STAT3 and STAT1, suppressing tumor growth by downregulating the IL-6/STAT3 signalling pathway [14]. Additionally, prognostic studies in uveal melanoma have revealed that ZNF704 restores tumor activity by inhibiting SORBS3, promoting cancer cell proliferation [15]. In this study, our proteomic analysis revealed a progressive decrease in SORBS3 expression from normal cervical tissue adjacent to tumors to primary cervical cancer and further to lymph node metastases. These findings suggest that the loss of SORBS3 expression is associated with the malignant progression of cervical cancer and may play a role in promoting lymph node metastasis.

Materials and methods

Clinical tissue specimens

The cervical cancer and adjacent tissue samples used in this study were obtained from the Department of Obstetrics and Gynaecology of the First Affiliated Hospital of Soochow University. The study was conducted with the consent of the patients and their families.The cervical pathological diagnosis was normal in the controls.

Cell culture

HeLa, SiHa, CaSki, and C33A cervical cancer cell lines were purchased from the Cell Bank of Type Culture Collection of The Chinese Academy of Sciences, as was Hcer-Epic, a normal cervical epithelial cell line. SiHa, CaSki, and C33A cells were grown in RPMI-1640 (HyClone, USA) supplemented with 10% foetal bovine serum (Biosera, France) and 1% penicillin‒streptomycin (Beyotime, China). HeLa and Hcer-Epic cells were cultured in DMEM with the same supplements. All the cells were maintained at 37 °C in a humidified incubator with 5% CO2.

Lentiviral infection

Lentiviral vectors carrying SORBS3-β short hairpin RNA (shRNA) were purchased from GenePharma (Suzhou, China), while overexpression vectors were obtained from Shanghai Genechem Co., Ltd. (Shanghai, China). An empty vector served as the control. SiHa and HeLa cells were infected with lentiviral particles at an MOI of 10. After 72 h, the cells were selected with puromycin (2 μg/mL for HeLa cells or 1 μg/mL for SiHa cells) for 7 days. The transduction efficiency was assessed via GFP fluorescence, and the infection efficacy was confirmed via RT‒qPCR and Western blotting.

Cell counting kit-8 (CCK-8)

HeLa or SiHa cells transfected with lentivirus were collected at 24-h intervals up to 120 h posttransfection and incubated with 10 μl of Cell Counting Kit-8 (New Cell and Molecular Biotech Co., Ltd., Suzhou, China) for 1 h. Finally, a Multiskan FC microplate photometer (Thermo Scientific, Massachusetts, USA) was used to measure the absorbance at 450 nm.

Total RNA extraction and RT–qPCR

Total RNA was extracted via the FreeZol Reagent Kit (#R711-01, Vazyme, Nanjing, China) following the manufacturer’s protocol. One microgram of total RNA from each sample was used for cDNA synthesis with the HiScript III RT SuperMix Kit (#R323, Vazyme, Nanjing, China). PCR was conducted on a CFX96 Touch Real-Time PCR System (Bio-Rad, CA, USA) under the following conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The data were analysed via the 2−ΔΔCt method. Use GAPDH as the internal reference gene. ΔCt = target gene CT value—internal reference gene CT value. ΔCt = experimental groupΔCt value—control group ΔCt value.

Transwell assay

A suspension of 8 × 104 cells in serum-free medium was prepared and introduced into the chambers, which were positioned in a 24-well plate filled with complete medium. To evaluate cell invasion, the upper chambers were coated with Matrigel (BD Biosciences), while the uncoated chambers were used for migration assays. After 24 h, the cells that had passed through the chamber were stained with 0.5% crystal violet (Biyuntian Biotech, Shanghai, China) and subsequently examined via a microscope (Leica Microsystems, Germany).

Western blot

The cells were lysed in RIPA buffer (#P0013B, Beyotime) and sonicated on ice at 20% amplitude for 1 min. The total protein concentration was determined via an enhanced BCA protein assay kit (#P0009, Beyotime). Proteins were separated by SDS‒PAGE (#PG110, #PG112, #PG113; Epizyme Biotech) and transferred to PVDF membranes (#IPVH00010; Merck Millipore, Germany). The membranes were blocked with 5% nonfat milk at room temperature for 1 h, followed by overnight incubation at 4 °C with primary antibodies. HRP-conjugated secondary antibodies were applied for 1 h at room temperature. After washing, the bands were detected with enhanced chemiluminescence (ECL) reagents (#10,100, NCM Biotech) and visualized via a ChemiDoc™ MP imaging system (Bio-Rad, CA, USA).

Tubule formation experiment

In total, 2 × 106 cervical cancer cells were seeded into 6-well plates. Once the cells adhered, the medium was replaced with culture medium containing 1% FBS. After 24 h, the aspirated medium was centrifuged at 800 r/min for 10 min, and the resulting supernatant was collected as the conditioned medium. Human lymphatic endothelial cells (HLECs) were then resuspended in the conditioned medium to a concentration of 6 × 105 cells/mL. Subsequently, 100 μL of the suspension was added to each well of a 96-well plate containing 50 μL of solidified Matrigel. After 6 h, the tubular networks were imaged via an inverted microscope and analysed with ImageJ.

Co-IP

To investigate protein‒protein interactions, SiHa cells were transfected with SORBS3-β-Flag and β-catenin-His plasmids as required for the experimental conditions and lysed with IP buffer. Flag gel and His magnetic beads were added to the corresponding lysate and incubated with rotation at 4 °C overnight. The beads were washed five times with washing buffer using a magnetic stand. The bound proteins were eluted with SDS‒PAGE sample buffer at 95 °C for 5 min and analysed by Western blotting.

In vivo assay

Nude mice, aged 3 to 5 weeks, were obtained from Nanjing GemPharmatech Biotechnology Co., Ltd. All the animal procedures were approved by the Institutional Animal Care and Use Committee of Soochow University (No. 202307A0134). SiHa cells were transfected with either control or SORBS3-β-overexpressing lentivirus, and HeLa cells transfected with control or SORBS3-β-knockdown lentivirus were dissociated with trypsin and rinsed twice in sterile PBS. A suspension of 1 × 105 cells in 0.1 mL of Matrigel was injected into the footpads of the mice. Four weeks postinjection, the mice were euthanized, and primary tumors from the footpads along with popliteal lymph nodes were harvested. Tumor volumes and popliteal lymph node sizes were measured after euthanasia. Tumor and lymph node tissues were subjected to immunohistochemistry (IHC) on the basis of established protocols.

DNA extraction and methylation-specific PCR (MSP)

DNA from cells and tissues was extracted according to the protocol of the DNA extraction kit (#D1700, Beijing Solabio Technology Co., Ltd.). Methylated DNA standards&Non-Methylated DNA standards(#D5014-1,#D5014-2, Zymo Research, CA, USA) were used as positive and negative controls.The genomic DNA was sulfite-converted via EZ DNA Methylation-DirectTM (Zymo Research, CA, USA). MSP (#EM101, Beijing Tiangen Biotechnology Co., Ltd.) was performed using sulfite-converted DNA as a template at 95 °C for 5 min; 35 cycles of 94 °C for 20 s, 60 °C for 30 s, and 72 °C for 20 s; followed by 72 °C for 5 min.

RNA-Seq assay

Total RNA extracted from cells via TRIzol reagent (Invitrogen) was sent to Shanghai Jinwei Biotechnology Co., Ltd., for RNA sequencing.

Liquid chromatography‒mass spectrometry (LC‒MS/MS)

With informed consent from patients, paired specimens of primary cervical cancer tissues and metastatic lesions were collected from 5 patients. To identify downstream molecules of SORBS3-β, SiHa cells transfected with control or SORBS3-β-overexpressing lentivirus were collected. To identify proteins that interact with β-catenin, immunoprecipitation assays were performed to pull down the proteins. All the above samples were tested via PTM BIO (Hangzhou, China).

Statistical analysis

All the data were analysed via GraphPad Prism 8 (GraphPad Software, San Diego, CA), and the results are presented as the means ± standard deviations (SDs) unless otherwise indicated. Comparisons between groups were performed via Student's t test or one-way/two-way analysis of variance (ANOVA), and a p value of < 0.05 was considered statistically significant. Different asterisks in the figure show the corresponding differences (*P < 0.05; **P < 0.01; ***P < 0.001).

Results

SORBS3 is downregulated in cervical cancer

We performed proteomic analysis on clinical samples from five cervical cancer patients with lymph node metastasis. Each case included four paired samples: primary cervical cancer, cervical lymph node metastasis, adjacent normal cervical tissue, and normal pelvic lymph nodes (Fig. 1A). The analysis showed consistency across sample sets (Fig. 1B). Among the 6209 quantifiable proteins identified, 490 were downregulated in primary cervical cancer compared with normal tissue, whereas 169 were downregulated in lymph node metastasis compared with primary cancer (Fig. 1C). Cross-analysis of the proteomic data revealed that 27 proteins, including SORBS3, were significantly downregulated in cervical cancer tissue, with even lower expression in lymph node metastases than in primary tumors (Fig. 1D). GEPIA analysis further revealed that SORBS3 expression is reduced or lost in various cancers (Fig. 1E), and its expression in cervical cancer tissues is significantly lower than that in normal cervical tissues (Fig. 1G). K-M plot analysis revealed that low SORBS3 expression was associated with poor prognosis in cervical cancer patients (Fig. 1F). Immunohistochemistry of five paired cervical epithelial, primary tumor, and lymph node metastatic tissues confirmed a gradual decrease in SORBS3 expression from normal cervical tissue to primary cancer and metastatic cancer in lymph nodes (Fig. 1H, I). These findings suggest that the abnormal downregulation of SORBS3 may play a critical role in the malignant progression of cervical cancer.

Fig. 1
figure 1

SORBS3 is downregulated in cervical cancer cells. A Flow chart for proteomic analysis of paired cervical cancer samples. B Similarity analysis between paired cervical cancer samples. C Quantity of differentially expressed proteins from proteomic analysis. D The cross-analysis of differentially expressed proteins revealed that 27 proteins were potential metastasis-associated proteins associated with cervical cancer (N: normal cervical tissue adjacent to cancer; T: primary cervical cancer; LT: cervical cancer lymph node metastasis; LN: normal lymph node tissue. E The GEPIA database revealed low expression of SORBS3 in many tumors across tumor samples and paired normal tissues. The height of bar represents the median expression of certain tumor type or normal tissue. F K‒M plot analysis revealed that cervical cancer patients with higher SORBS3 expression levels had better prognoses than those with lower SORBS3 expression levels. G The expression level of SORBS3 in the GEPIA database was significantly lower than that in normal cervical tissues. The Y axis values represent log2(TPM + 1). H Paired samples from patients with cervical cancer were subjected to immunohistochemical analysis to verify the expression of SORBS3 in normal cervical tissues adjacent to cervical cancer, primary cervical lesions and lymph node metastases from patients with cervical cancer. I Positive rate of SORBS3 expression in paired cervical cancer samples. Data are presented as mean ± SD. ns: no significance, **p < 0.01, ***p < 0.001 by Student’s t test (I)

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SORBS3-β is the predominant isoform SORBS3 in cervical cancer

SORBS3 has two splice variants, α and β. The α isoform is highly expressed in skeletal muscle with limited expression in other tissues, whereas the β isoform is ubiquitously expressed across various tissues. SORBS3-β plays a role in the organization of the actin cytoskeleton, cell spreading, and the regulation of signal transduction[11]. SORBS3-β is localized in the extracellular matrix and at cell–cell junctions [16], while its expression can also be detected in the cytoplasm and nucleus [17, 18].We assessed the RNA and protein expression levels of both isoforms in human cervical epithelial cells (HcerEpic) and cervical cancer cell lines via qPCR and Western blotting. The results demonstrated that SORBS3-β is the predominant isoform expressed in all the cell lines. Furthermore, there was a significant reduction in SORBS3-β expression in cervical cancer cell lines compared with HcerEpic (Fig. 2A, B). To elucidate the function of reduced SORBS3-β expression in cervical cancer progression, we generated SiHa cells stably expressing SORBS3-β cDNA (SiHaOE−con, SiHaOE−SORBS3−β) and HeLa cells with stable knockdown of SORBS3-β (Helash−con, Helash−SORBS3−β) (Fig. 2C–F). As a next step, we examined the effects of SORBS3-β on the proliferation of cervical cancer cells. Colony formation and CCK8 assays revealed that SORBS3-β had no notable effect on the proliferation of cervical cancer cells (Fig. 2G–L).

Fig. 2
figure 2

SORBS3-β has no effect on cervical cancer proliferation. A, B RNA and protein expression levels of different SORBS3 subtypes in normal cervical cells and cervical cancer cell lines. C–F SORBS3-β was overexpressed or knocked down by lentivirus infection in SiHa and HeLa cells, and the results were verified at the RNA and protein levels. G–L SORBS3-β had no significant effect on the proliferation of cervical cancer cells. Data are presented as mean ± SD. ns: no significance, **p < 0.01, ****p < 0.0001 by Student’s t test (E, F, H, J)

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SORBS3-β inhibits cervical cancer cell invasion and lymphangiogenesis in vitro

In subsequent scratch and transwell assays, SORBS3-β overexpression in cervical cancer cells led to a significant reduction in invasive potential compared with that in vector control cells, whereas SORBS3-β silencing increased invasion (Fig. 3A–J). Tube formation assays demonstrated that, compared with HcerEpic cells, human lymphatic endothelial cells (HLECs) induced more tubule formation when cultured with conditioned medium obtained from cervical cancer cell lines (Fig. 3K, F). Notably, SORBS3-β silencing markedly enhanced, whereas SORBS3-β overexpression suppressed the ability of cervical cancer cells to promote HLEC tubule formation (Fig. 3M–P). At the same time, To exclude the possibility that SORBS3-α may have a function in our model, we designed small interfering RNA targeting only SORBS3-α (Supplementary Fig. 1A), and performed transwell assays on Hela cells transfected with si-SORBS3-α. The results showed that knocking down SORBS3-α did not significantly affect the migration and invasion ability of cervical cancer cells (Supplementary Figs. 1B, 1C). The culture supernatant of Hela cells transfected with si-SORBS3α also had no significant effect on the ability of HLEC to form tubules (Supplementary Fig. 1D).Additionally, cell adhesion assays revealed that SORBS3-β overexpression reduced the adhesion of cervical cancer cells to lymphatic endothelial cells, whereas SORBS3-β knockout increased adhesion (Fig. 3Q–R). These findings strongly suggest that SORBS3-β plays critical anti-invasion and anti-lymphangiogenesis roles in cervical cancer, which may contribute to the inhibition of cancer cell migration during metastatic progression. VEGFC is an important regulatory molecule in lymphatic metastasis [6]. Therefore, we detected the VEGFC content in the supernatant of SiHaOE−con/SiHaOE−SORBS3−β and Helash−con/Helash−SORBS3−β via ELISA, and the results revealed that, after SORBS3-β was overexpressed, VEGFC expression in the supernatant decreased, and after SORBS3-β was knocked down, VEGFC expression in the supernatant increased (Fig. 3S, T).

Fig. 3
figure 3

SORBS3-β regulates lymphatic metastasis in cervical cancer. A, B, E, F, G Scratch and Transwell assays revealed that SORBS3-β overexpression inhibited the migration and invasion of cervical cancer cells. C, D, H, I, J Scratch tests and Transwell assays revealed that SORBS3-β knockdown enhanced cervical cancer migration and invasion. K, L The ability of HLECs cultured with the four cervical cancer cell supernatants to form tubules was greater than that of HLECs cultured with the normal cervical cell supernatant. M–P When the ability of human lymphatic endothelial cells (HLECs) to form tubules was tested, the ability of HLECs cultured with SiHaOE−SORBS3−β cells to form tubules was significantly lower than that of HLECs cultured with SiHaOE−con cells. The ability of HLECs cultured with Helash−SORBS3−β cell culture medium was significantly greater than that of HLECs cultured with Helash−con cell culture medium. Q, R After SORBS3-β was overexpressed in SiHa cells, cell adhesion ability was significantly weakened. The adhesion ability of HeLa cells was significantly enhanced after SORBS3-β knockdown. S, T The expression levels of VEGFC in the cell supernatant were significantly decreased after SORBS3-β overexpression. After SORBS3-β knockdown, the VEGFC expression level in the cell supernatant significantly increased. Data are presented as mean ± SD. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t test (B, D, F, G, I, J, O, P, Q, R, S, T) or by one-way ANOVA (L).

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SORBS3-β suppresses lymphatic metastasis of cervical cancer in vivo

The impact of SORBS3-β on xenograft tumor growth in patients with cervical cancer was further examined in vivo via a footpad inoculation model. SiHa cells overexpressing SORBS3-β, HeLa cells with SORBS3-β knockdown, and vector control cells were injected into the footpads of nude mice (n = 6 per group). These findings indicated that neither the overexpression nor the silencing of SORBS3-β resulted in a significant difference in the size of primary tumors in the footpads of nude mice compared with those in the control group. Notably, SORBS3-β overexpression led to a reduction in popliteal lymph node size, whereas SORBS3-β knockdown led to an increase in popliteal lymph node size compared with that in the control group (Fig. 4A–H). Notably, the tumors formed by SiHaOE-SORBS3-βcells presented lower D2-40 levels than did the control tumors (Fig. 4I, J). Conversely, D2-40 expression was markedly increased in the tumors formed by the HeLash-SORBS3-β cells (Fig. 4M–N). Moreover, after the lymph node metastases were stained with a cytokeratin marker (Pan-CK), the results revealed that the overexpression of SORBS3-β resulted in decreased lymph node metastasis, and the knockdown of SORBS3-β resulted in increased lymph node metastasis (Fig. 4K–P). We also performed VEGFC staining on lymph nodes, and the results showed that SORBS3-β overexpression led to a decrease in VEGFC expression in lymph nodes, while SORBS3-β knockdown led to an increase in VEGFC expression in lymph nodes (Supplementary Fig. 2A–D), which is consistent with the above in vitro experimental results. Taken together, these findings indicate that SORBS3-β inhibits lymphatic metastasis of CC in vivo.

Fig. 4
figure 4

SORBS3-β suppresses the lymphatic metastasis of cervical cancer in vivo. A After SORBS3-β was overexpressed, lymph node metastasis was significantly reduced in nude mice. B–D Size of primary tumors in the lymph nodes and foot pads of nude mice after overexpression of SORBS3-β. E Lymph node metastases in nude mice were significantly increased after SORBS3-β knockdown. F–H The size of primary tumor tissue in the lymph nodes and foot pads of nude mice after SORBS3-β deletion. I, J, M, N D2-40 expression was detected by immunohistochemistry in these foot pad tumors. After SORBS3-β was overexpressed, D2-40 expression decreased, and after SORBS3-β was knocked down, D2-40 expression increased. K, L, O, P. PAN-CK expression was detected by immunohistochemistry in the popliteal lymph nodes. After SORBS3-β overexpression, PAN-CK expression decreased, and after SORBS3-β knockdown, PAN-CK expression increased. Data are presented as mean ± SD. ns: no significance, **p < 0.01, ***p < 0.001 by Student’s t test (C, D, G, H, J, N, L, P)

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DNA methylation contributes to loss of SORBS3 expression in cervical cancer

To investigate the mechanism of loss of SORBS3 expression in cervical cancer, we collected tumor tissues and adjacent tissues from three clinical cervical cancer patients. Firstly, the mRNA expression levels SORBS3 were measured by QPCR, and the results showed that SORBS3 mRNA expression in cancer tissues was lower compared to paracancerous tissues (Fig. 5A). Previous study have shown that DNA methylation contributes to SORBS3 downregulation in obesity patients [19]. Methylation-specific (MS)-PCR revealed that SORBS3 promoter was more methylated in tumor tissues than adjacent tissues (Fig. 5B). In addition, targeted bisulfite sequencing (TBS) of these tissues revealed that SORBS3 was more methylated in tumor tissues than in adjacent tissues (Fig. 5C). These results suggest that promoter methylation may be an important regulatory mechanism for reducing SORBS3 expression. We subsequently examined the expression levels of DNMT-1, DNMT-3A, and DNMT-3B in three paired clinical samples and SiHa cell lines. The results showed that DNMT-1 exhibited the highest expression in both the clinical samples and SiHa cells, with significantly higher DNMT-1 mRNA levels observed in tumor tissues compared to paired adjacent normal tissues in three clinical samples. In contrast, no significant differences were observed in the expression levels of DNMT-3A and DNMT-3B between tumor and adjacent normal tissues (Fig. 5D, E). To investigate the roles of DNMT-1, DNMT-3A, and DNMT-3B in regulating SORBS3 methylation, we used small interfering RNAs (siRNAs) to individually knock down their expression (Fig. 5F–I). The results demonstrated that knockdown of either DNMT-1 or DNMT-3B significantly reduced the methylation level of the SORBS3 promoter region, with DNMT-1 knockdown exhibiting the most pronounced effect. In contrast, knockdown of DNMT-3A did not result in any noticeable change (Fig. 5J). Furthermore, we assessed the mRNA and protein expression levels of SORBS3 following knockdown of the three methyltransferases. The results showed that silencing DNMT-1 or DNMT-3B effectively restored SORBS3 expression, with DNMT-1 knockdown resulting in the most substantial increase, whereas DNMT-3A knockdown had no significant impact on SORBS3 expression (Fig. 5K, L).Collectively, these findings suggest that DNA methylation plays a critical role in the downregulation of SORBS3 expression in cervical cancer. DNMT-1 appears to be the most crucial regulator of SORBS3 methylation and expression in cervical cancer cells.

Fig. 5
figure 5

Decreased expression of SORBS3 in cervical cancer is associated with promoter methylation status. A Quantitative PCR (qPCR) analysis of SORBS3 mRNA expression levels in the aforementioned clinical tissue samples. B Methylation-specific PCR (MSP) analysis showing the methylation status of SORBS3 in paired clinical cervical cancer tissue samples from three patients.MS: Methylated DNA standards, non-MS: Non-Methylated DNA standards. C Targeted bisulfite sequencing revealing the methylation status of the SORBS3 promoter in the same clinical tissue samples. D QPCR analysis of the expression levels of three DNA methyltransferases (DNMT-1, DNMT-3A, and DNMT-3B) in clinical tissue samples. E QPCR analysis of the expression levels of three DNA methyltransferases (DNMT-1, DNMT-3A, and DNMT-3B) in SiHa cells. F QPCR analysis of DNMT-1, DNMT-3A, and DNMT-3B knockdown efficiency in SiHa cells. G–I Western blot (WB) analysis confirming the knockdown efficiency of DNMT-1, DNMT-3A, and DNMT-3B in SiHa cells. J MSP analysis of the SORBS3 promoter methylation status following the knockdown of DNMT-1, DNMT-3A, and DNMT-3B in SiHa cells. K QPCR analysis of SORBS3 mRNA expression levels following the knockdown of DNMT-1, DNMT-3A, and DNMT-3B in SiHa cells. L Expression of SORBS3 protein following the knockdown of DNMT-1, DNMT-3A, and DNMT-3B in SiHa cells examined by Western blot (WB). Data are presented as mean ± SD. ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001 by Student’s t test (A, F) or by one-way ANOVA (K) or by two-way ANOVA (D)

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SORBS3-β inhibits the Wnt/β-catenin pathway by reducing nucleolar β-catenin levels

To further explore the mechanism by which SORBS3-β inhibits lymphatic metastasis in cervical cancer, we performed proteomic analysis on SiHa cells overexpressing SORBS3-β (Fig. 6A‒C). The analysis revealed a reduction in β-catenin expression levels. Through its role in regulating cytoskeletal reorganization, angiogenesis, apoptosis, and cell proliferation, Wnt/β-catenin signalling is a key player in tumor metastasis [20]. β-catenin is a key indicator of Wnt pathway activation and translocates to the nucleus to initiate the transcription of downstream target genes upon activation [21]. Co-IP assays (Fig. 6D, E) revealed that β-catenin interacts with SORBS3-β, while Western blot analysis revealed that SORBS3-β overexpression significantly reduced β-catenin expression (Fig. 6F). In contrast, β-catenin levels increased after SORBS3-β knockdown (Fig. 6G). Immunofluorescence and nuclear‒cytoplasmic fractionation assays further revealed that nuclear β-catenin levels decreased upon SORBS3-β overexpression and increased following SORBS3-β knockdown (Fig. 6H‒K). These findings suggest that SORBS3-β inhibits the Wnt/β-catenin pathway by reducing nucleolar β-catenin levels, thus potentially suppressing lymphatic metastasis in patients with cervical cancer.

Fig. 6
figure 6

SORBS3-β inactivates the Wnt/β-catenin pathway by regulating β-catenin. A After lentiviral infection of SiHa cells overexpressing SORBS3-β and obtaining stable cell lines, proteomic analysis was performed to identify the regulatory protein of SORBS3-β. B Proteomic analysis revealed that, after SORBS3-β overexpression, 70% of the proteins whose expression changed were downregulated, and 30% were upregulated, suggesting that SORBS3 might negatively regulate the expression of a series of proteins, including important proteins such as β-catenin. C Proteomic analysis revealed that 37% of the proteins that SORBS3-β could regulate were nuclear proteins, 23% were cytoplasmic proteins, and secreted proteins. D, E Flag gel and proteinA/G agarose beads were used for IP experiments, and the interaction between SORBS3-β and β-catenin was detected. F Western blot results confirmed that the protein expression level of β-catenin decreased significantly when SORBS3-β was overexpressed in SiHa cells. G Western blot results confirmed that the protein expression level of β-catenin was significantly increased when SORBS3-β was knocked down in HeLa cells. H, J Immunofluorescence experiments revealed that the expression level of β-catenin decreased after SORBS3-β was overexpressed and increased after SORBS3-β was knocked down. I, K Nucleoplasmic separation experiments revealed that the protein expression level of β-catenin decreased after SORBS3-β was overexpressed and increased after SORBS3-β was knocked down

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SORBS3-β promotes UBA1-mediated ubiquitination of β-catenin

Ubiquitination-mediated degradation is a pathway for β-catenin degradation. To investigate the impact of SORBS3-β on β-catenin protein stability and its ubiquitination-mediated degradation, a CHX chase assay revealed that SORBS3-β reduced the half-life of the β-catenin protein. The results demonstrated that β-catenin protein stability decreased following SORBS3-β overexpression (Fig. 7A–D). Additionally, a ubiquitination IP experiment in SiHa cells overexpressing SORBS3-β revealed that β-catenin ubiquitination was increased upon SORBS3-β overexpression (Fig. 7E). Given that SORBS3-β promotes β-catenin ubiquitination and degradation, we employed mass spectrometry to identify ubiquitin ligases associated with β-catenin. His-β-catenin was transfected into control and SORBS3-β-overexpressing SiHa cells, followed by MG132 treatment to ensure consistent levels of His-β-catenin. Immunoprecipitation via His-Beads and subsequent LC‒MS/MS analysis identified proteins whose binding increased due to SORBS3-β overexpression (Fig. 7F). Among these, we screened for ubiquitin ligases and identified potential enzymes that could promote β-catenin ubiquitination via SORBS3-β (Fig. 7G). UBA1, the protein with the highest binding abundance, was validated in subsequent experiments. A co-IP experiment in Flag-SORBS3-β-transfected SiHa cells confirmed that UBA1 binds to SORBS3-β (Fig. 7H). Furthermore, co-IP experiments in SiHa cells transfected with His-β-catenin revealed that the interaction of UBA1 with β-catenin increased upon SORBS3-β overexpression (Fig. 7I). Finally, UBA1 was silenced in SiHa cells overexpressing SORBS3-β, and the results showed that silencing UBA1 antagonized the SORBS3-β-induced ubiquitination of β-catenin (Fig. 7J). These results suggest that SORBS3-β mediates the ubiquitination and degradation of β-catenin via UBA1.

Fig. 7
figure 7

SORBS3-β mediates the ubiquitination and degradation of β-catenin via UBA1. A–D CHX and MG132 were used to verify the effect of SORBS3-β on the stability of the β-catenin protein. E The results of the ubiquitination IP experiment revealed that the ubiquitination and degradation of β-catenin increased after SORBS3-β overexpression. F Venn diagram showing the proteins that interact with β-catenin in the control and SORBS3-β overexpression groups. G List of β-catenin interacting proteins. H After SiHa cells were transfected with Flag-SORBS3-β, immunoprecipitation was performed via an anti-Flag antibody, with IgG used as a negative control. Western blot analysis revealed that UBA1 bound to Flag-SORBS3-β. I In SiHa cells transfected with His-β-catenin, co-IP using His magnetic beads was conducted, with IgG as a negative control, and UBA1 was detected via Western blotting. J SiHa cells were transfected with UBA1 siRNA, His-β-catenin, Flag-SORBS3, and HA-ub, and the ubiquitination level of β-catenin was assessed. Data are presented as mean ± SD. ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA (B, D)

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SORBS3-β suppresses lymphangiogenesis in cervical cancer by inhibiting VEGFC transcription

SORBS3-β can inhibit the Wnt/β-catenin pathway, while the activated Wnt/β-catenin pathway can initiate the transcription of a series of genes related to tumor metastasis and angiogenesis. Therefore, we further explored whether SORBS3-β affects the transcription of some of these genes at the transcriptomic level (Fig. 8A). The RNA-seq results suggest that, after SORBS3 is overexpressed, the mRNA levels of VEGFC, PROK2, IGFBP5 and other genes are significantly reduced (Fig. 8B). This result is consistent with the decreased expression of VEGFC in the supernatant of cells overexpressing SORBS3-β (Fig. 3S). QPCR experiments also confirmed the results of RNA-Seq: after SORBS3 knockdown, the mRNA expression levels of VEGFC, PROK2 and IGFBP5 increased, whereas after SORBS3 overexpression, the mRNA expression levels of VEGFC, PROK2 and IGFBP5 decreased (Fig. 8C, D). Moreover, WB results revealed that, after SORBS3 overexpression, VEGFC, PROK2 and IGFBP5 protein expression levels decreased, whereas VEGFC, PROK2 and IGFBP5 protein expression levels increased after SORBS3 knockdown (Fig. 8E–J). These results suggest that SORBS3 can inhibit the expression of VEGFC, IGFBP5, PROK2 and other genes at the transcriptional level. Additionally, the transfection of a β-catenin overexpression plasmid into SORBS3-β-overexpressing cells restored VEGFC expression (Fig. 8K). Therefore, SORBS3-β affects downstream VEGFC expression through the Wnt/β-catenin pathway, thereby regulating lymphatic metastasis in patients with cervical cancer (Fig. 9).

Fig. 8
figure 8

SORBS3-β inactivates the Wnt/β-catenin pathway by regulating β-catenin. A After SiHa cells were infected with lentivirus and overexpressing SORBS3-β and stable cell lines were obtained, transcriptomic analysis via RNA-seq was performed to identify the regulatory protein of SORBS3-β. B RNA-seq analysis revealed that the mRNA expression levels of VEGFC, IGFBP5 and PROK2 were significantly decreased after SORBS3-β overexpression. C QPCR results revealed that the mRNA expression levels of VEGFC, IGFBP5 and PROK2 decreased significantly after SORBS3-β gene overexpression. D QPCR results showing that the mRNA expression levels of VEGFC, IGFBP5 and PROK2 were significantly increased after SORBS3-β gene knockout. E, F, G WB results revealed that the expression levels of VEGFC, IGFBP5 and PROK2 were significantly decreased after SORBS3-β overexpression. H, I, J WB results revealed that the expression levels of VEGFC, IGFBP5 and PROK2 were significantly increased after SORBS3-β knockdown. K Western blot analysis of VEGFC expression after the overexpression of β-catenin. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s t test (C, D)

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Fig. 9
figure 9

A schematic diagram of the mechanism by which SORBS3-β degrades β-catenin through UBA1 ubiquitination and inhibits VEGFC transcription and suppresses lymph node metastasis in cervical cancer

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Discussion

Cervical cancer remains one of the most common gynaecological malignancies, with lymph node metastasis being a major factor contributing to poor patient prognosis [22, 23]. Despite its clinical importance, the molecular mechanisms underlying lymph node metastasis in cervical cancer are not fully understood. In this study, we observed a progressive decrease in SORBS3 expression from normal cervical tissues to metastatic lymph nodes. Furthermore, SORBS3 expression correlated positively with clinical outcomes in cervical cancer patients. Research has indicated that SORBS3 plays a role in regulating cell motility and invasion [24] [25]. Notably, compared with those in the normal cervical epithelial cell line HCerEpic, both isoforms of SORBS3 were downregulated in cervical cancer cells, with SORBS3-β being more expressed. SORBS3-β, initially identified as a focal adhesion binding partner for cytoskeletal proteins, is a shorter isoform of SORBS3 with three SH3 domains. It acts as a scaffolding protein, recruiting various signalling and cytoskeletal factors to regulate cell signalling and cytoskeletal rearrangement [16, 26]. However, the role of SORBS3-β in cervical cancer metastasis has not been thoroughly investigated. Our findings show that, SORBS3-α has no obvious function in lymph node metastasis of cervical cancer. While SORBS3-β overexpression does not affect cervical cancer cell proliferation, it significantly inhibits cell migration, invasion, and lymphangiogenesis, indicating that SORBS3-β is the main isoform in cervical cancer and that SORBS3-β expression may serve as a prognostic marker in cervical cancer and warrants further exploration.

DNA methylation is a common form of epigenetic modification that can lead to heritable regulation of gene expression without altering the DNA sequence itself [27] Increasing evidence has demonstrated that aberrant DNA methylation is closely associated with the progression of various human cancers [28, 29]. Day et al. reported that promoter methylation could be a key regulatory mechanism for SORBS3 gene expression [19]. Consistent with these findings, our study revealed that promoter hypermethylation may serve as a critical regulatory mechanism contributing to the downregulation of SORBS3 expression in cervical cancer. By comparing SORBS3 expression and promoter methylation levels between cervical cancer tissues and adjacent normal tissues, we observed that SORBS3 promoters exhibited low or unmethylated status in adjacent normal tissues, while they were significantly hypermethylated in cervical cancer tissues.Furthermore, through targeted bisulfite sequencing (TBS), we confirmed that the methylation level of SORBS3 promoter in cervical cancer tissues was significantly higher than that in adjacent normal tissues, further supporting the hypothesis that promoter hypermethylation may contribute to the downregulation of SORBS3 expression in cervical cancer. It is well-established that DNA methyltransferases (DNMTs) catalyze the addition of a methyl group to the cytosine residues within CpG dinucleotides, resulting in chromatin remodeling and gene silencing [30]. In mammals, DNMTs, including DNMT1, DNMT3A, and DNMT3B, are responsible for establishing and maintaining DNA methylation patterns during embryonic development and somatic tissue differentiation [31]. In our study, DNMT1 exhibited the highest expression level, and its expression was significantly elevated in cancer tissues compared to adjacent normal tissues. SORBS3 promoter methylation was partially reversed, particularly after DNMT1 knockdown, suggesting that DNMT1 may play a dominant role in regulating SORBS3 promoter methylation in cervical cancer.

The Wnt/β-catenin signalling pathway regulates tumor metastasis through mechanisms such as cytoskeletal remodelling, cell proliferation, apoptosis modulation, and angiogenesis [32, 33]. Aberrant activation of this pathway can drive tumorigenesis in various cancers [20, 34, 35]. β-catenin is a crucial signalling molecule for detecting Wnt activation. When the pathway is activated, β-catenin accumulates in the nucleus and initiates the transcription of downstream target genes [36, 37]. In our investigation of the mechanisms underlying lymph node metastasis (LNM) in cervical cancer (CC), we found that SORBS3-β significantly reduces β-catenin expression. Interestingly, we found that overexpression of SORBS3-β significantly inhibited nuclear β-catenin accumulation, suggesting that SORBS3-β may negatively regulate β-catenin levels in the nucleus. Further investigation revealed that SORBS3-β could promote β-catenin ubiquitination and subsequent proteasomal degradation, suggesting that SORBS3-β may suppress the activation of the Wnt/β-catenin signaling pathway by facilitating the ubiquitin–proteasome degradation of β-catenin. UBA1, a key enzyme in the ubiquitin‒proteasome system, is crucial for ubiquitin activation and participates in regulating cell signalling, the cell cycle, and stress responses, and its dysfunction is associated with cancer and neurodegenerative diseases [38]. Our proteomic and immunoprecipitation analyses revealed that SORBS3-β interacts with UBA1 and promotes its association with β-catenin, indicating that UBA1 is a key enzyme in the SORBS3-β-mediated degradation of β-catenin.

Furthermore, we found that SORBS3-β overexpression reduced the expression levels of several genes, including VEGFC, PROK2, and IGFBP5. PROK2 is a secretory protein of the prokineticin family that promotes tumor progression by regulating angiogenesis and myeloid cell infiltration [39–41]. IGFBP5, another secretory protein, is significantly associated with lymph node metastasis in patients with breast cancer [42]. VEGFC is a well-known lymphangiogenic factor that induces proliferation, migration, and lymphatic vessel formation and plays a crucial role in lymphatic system development and function [6, 43]. Studies have demonstrated that activation of the Wnt/β-catenin signaling pathway can enhance the secretion of VEGF-C, thereby facilitating lymph node metastasis in various tumors [44, 45].Our results also indicate that SORBS3-β reduces VEGFC levels in the supernatant of cervical cancer cells. Notably, we observed that overexpression of β-catenin in SORBS3-β-overexpressing cells restored the expression of VEGFC, suggesting that SORBS3-β may regulate the downstream effector VEGFC through the Wnt/β-catenin signaling pathway. Therefore, our proposed mechanism indicates that SORBS3-β overexpression promotes β-catenin ubiquitination and degradation through UBA1, thereby inhibiting the Wnt/β-catenin signaling pathway and subsequently reducing the transcription and secretion of VEGFC.

These findings not only deepen our understanding of CC progression but also suggest that demethylating agents could be leveraged to reactivate SORBS3-β anti-metastatic functions, a strategy meriting further preclinical exploration. 5-Azacytidine (5-AZA) is one of the most commonly used DNA methyltransferase (DNMT) inhibitors, which has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of myelodysplastic syndromes (MDS). It functions by reactivating abnormally silenced genes caused by DNMT-mediated DNA hypermethylation, thereby restoring their normal biological functions [46, 47]. In the future, DNMT inhibitors could be utilized to reverse promoter hypermethylation and reactivate SORBS3-β expression in cervical cancer. Alternatively, gene therapy approaches, including adenoviral or lentiviral vectors encoding SORBS3-β, could directly restore its expression in metastatic lesions. Additionally, exploring SORBS3-β methylation status as a predictive biomarker for patient stratification holds promising potential for personalized cancer therapy.

Conclusion

In conclusion, our study elucidates the multifaceted role of SORBS3-β in the regulation of cervical cancer progression and metastasis. The stepwise reduction in SORBS3 due to promoter hypermethylation, coupled with its impact on β-catenin degradation and Wnt/β-catenin pathway inhibition (Fig. 9), underscores its potential as a therapeutic target. These findings are particularly significant in the context of recent advances in understanding the mechanisms of lymphatic metastasis in cervical cancer. Future research should focus on the development of strategies to restore SORBS3-β expression or function, which may offer promising avenues for the treatment of cervical cancer.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

Not applicable

Funding

The National Natural Science Foundation of China to Jinhua Zhou (No. 82172609, 81772773), the Jiangsu Social Development Project (No. BE2022729, BE2021647), the National Human Genetic Resources Sharing Project (No. YCZYPT[2020]06–2), Gusu Medical Youth Talent to Jinhua Zhou (No. GSWS2019034), Changzhou City's “14th Five Year Plan" High level Health Talent Training Project-Top Talents (2022CZBJ087), the Suzhou Basic Research Pilot Project to Fang Wang (NO.SSD2024066), and the Suzhou Basic Research Pilot Project to Jinhua Zhou (NO.SSD2024027).

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Author notes

  1. Huating Sun and Yinghui Zhang are equal contributors and co-first authors.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China

    Huating Sun, Yinghui Zhang, Fang Wang, Zizhao Wang, Youguo Chen & Jinhua Zhou

  2. Department of Obstetrics and Gynecology, the Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Suzhou Municipal Hospital, Nanjing Medical University, No. 26, Daoqian Street, Suzhou, 215002, Jiangsu, China

    Yuhong Zhang

  3. Changzhou Maternal and Child Health Care Hospital, Changzhou, China

    Li Wang

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Contributions

Huating Sun: Writing – original draft, visualization, data curation, conceptualization. Yinghui Zhang: Methodology, Formal analysis. Fang Wang: Investigation. Zizhao Wang: Visualization, Data curation. Yuhong Zhang: Validation. Youguo Chen: Writing – review & editing, Supervision. Li Wang: Project administration, Funding acquisition. Jinhua Zhou: Writing – review & editing, Resources, Funding acquisition.

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Correspondence to Youguo Chen, Li Wang or Jinhua Zhou.

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Ethics approval and consent to participate

This study involving human participants and human tissue was approved by the Ethics Committee of The First Affiliated Hospital of Soochow University ((2024) Research Approval No. 270). The study was conducted with the consent of the patients and their families.

The animal experiments were approved by the Ethics Committee of Soochow University (No. 202307A0134) and were conducted in accordance with the institutional guidelines for animal care and use.

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Supplementary material 1

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Supplementary material 2: Supplementary figure 1. SORBS3-α has no significant effect on lymph node metastasis of cervical cancer.western blot was used to detect the knockdown of SORBS3-α protein in HeLa cells transfected with SORBS3-α small interfering RNA.Transwell assays revealed that the migration and invasion abilities of HeLa cells transfected with SORBS3-α small interfering RNA.The supernatant of Hela cells transfected with SORBS3-α small interfering RNA was collected to detect its effect on the tubule formation ability of HLECS. Data are presented as mean ± SD. ns: no significance by one-way ANOVA

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Supplementary material 3: Supplementary figure 2. SORBS3-β inhibited VEGFC expression in lymph nodes.. VEGFC expression was detected by immunohistochemistry in the popliteal lymph nodes. After SORBS3-β overexpression,VEGFC expression decreased.. VEGFC expression was detected by immunohistochemistry in the popliteal lymph nodes, and after SORBS3-β knockdown, VEGFC expression increased. Data are presented as mean ± SD. ***P<0.001, ****P<0.0001 by Student’s t test

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Sun, H., Zhang, Y., Wang, F. et al. SORBS3-β suppresses lymph node metastasis in cervical cancer by promoting the ubiquitination of β-catenin. J Transl Med 23, 406 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06409-2

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06409-2

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Journal of Translational Medicine

ISSN: 1479-5876

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