ODF3B Promotes the Progression of Clear Cell Renal Cell Carcinoma via the JAK/STAT Signaling Pathway

Introduction

Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma (RCC), accounting for approximately 80% of cases [1,2]. Despite advances in surgical techniques and targeted therapies, the prognosis for advanced ccRCC remains poor, with high rates of metastasis and therapeutic resistance [3,4]. Understanding the molecular mechanisms underlying ccRCC progression is critical for identifying novel therapeutic targets.

Among the various signaling cascades implicated in ccRCC, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has emerged as a critical mediator of tumorigenesis. Activation of the JAK/STAT pathway promotes proliferation, survival, angiogenesis, and immune evasion in various cancers, including renal cell carcinoma [5,6]. Aberrant STAT3 activation, in particular, has been associated with poor prognosis and aggressive tumor behavior in ccRCC [7,8]. Despite these insights, the upstream regulatory factors and molecular players that modulate JAK/STAT signaling in ccRCC remain incompletely characterized. In renal cell carcinoma specifically, pharmacologic blockade of STAT3 with the small-molecule inhibitor WP1066 has been shown to suppress tumor angiogenesis, reduce STAT3 activation, and inhibit renal cell carcinoma xenograft growth in vivo, providing preclinical proof of concept that JAK/STAT targeting may be therapeutically actionable in this disease [9].

Outer dense fiber of sperm tails 3B (ODF3B), initially characterized as a structural component of sperm flagella, has garnered attention for its potential extragonadal roles [10,11]. Consistent with this concept, outer dense fiber proteins as a family have been recognized as testis-enriched, sperm-tail–localized structural proteins whose aberrant expression has been detected in several malignancies, suggesting that ODF members are a subset of cancer/testis-like antigens with potential biomarker and therapeutic value [12]. Although the biological functions of ODF3B outside the reproductive system remain largely unexplored, emerging transcriptomic data hint at its aberrant expression in various malignancies. Given its limited investigation, ODF3B remains a promising, albeit understudied, candidate for elucidating novel oncogenic pathways in ccRCC.

Beyond its structural role in sperm flagella, ODF3B belongs to a testis-enriched protein family in which several members have been re-identified as cancer/testis antigens with oncogenic functions in somatic tumors. Reactivation of germ-cell–restricted genes is a recognized mechanism contributing to tumorigenesis through cytoskeletal remodeling, metabolic reprogramming, and altered signaling output. Notably, a recent study in glioma demonstrated that ODF3B can activate the JAK/STAT pathway, suggesting that this protein may exert non-canonical regulatory functions beyond the reproductive system. Given that current upstream regulators of JAK/STAT in ccRCC are largely limited to cytokine-driven or receptor-mediated mechanisms, investigating a structurally distinct, cancer/testis-like protein such as ODF3B provides an opportunity to uncover previously unrecognized modulators of this pathway. These features highlight ODF3B as a biologically plausible and unexplored target in ccRCC.

Importantly, ccRCC is typified by frequent inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, leading to constitutive activation of hypoxia-inducible factors (HIFs) and subsequent transcription of genes promoting angiogenesis and metabolic reprogramming [13,14]. While the VHL-HIF axis is well established as a central driver of ccRCC, it does not fully account for the complex landscape of tumor progression and resistance. Crosstalk between the HIF pathway and other signaling networks, including JAK/STAT, suggests a more intricate regulatory environment [15–17]. Thus, investigating factors such as ODF3B, which may intersect with multiple oncogenic pathways, could illuminate additional layers of ccRCC pathobiology.

Currently, therapeutic strategies targeting the JAK/STAT pathway are under active investigation in multiple malignancies. Small molecule inhibitors of JAK kinases, such as ruxolitinib and tofacitinib, and STAT3 inhibitors have demonstrated efficacy in preclinical models and early-phase clinical trials [18–20]. However, resistance mechanisms and compensatory pathway activation remain significant challenges. Understanding the upstream regulators of JAK/STAT signaling, such as ODF3B, may aid in the development of more effective combination therapies or novel targeted approaches. Recent multi-omics and transcriptomic studies have demonstrated the value of pathway-level enrichment analyses in elucidating regulatory networks in renal cancer, supporting the analytical strategies used in our study [21,22].

In this study, we aimed to clarify ODF3B’s role in ccRCC progression and its link to JAK/STAT activation, hypothesizing that it is an oncogenic driver that promotes tumor growth and metastasis. Using bioinformatics, functional assays, and mechanistic studies, we defined ODF3B’s clinical significance, advancing ccRCC understanding and suggesting it as a biomarker and therapeutic target.

Material and Methods

CELL CULTURE:

Normal renal epithelial cell lines (HK2 and HEK-293) and renal carcinoma cell lines (786-O, ACHN, Caki-1, and OSRC-2) were obtained from the Department of Urology, Peking University First Hospital. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 50 U/mL penicillin–streptomycin. Cultures were maintained at 37°C in a humidified incubator containing 5% CO₂ and were subcultured regularly to ensure optimal cell viability and growth stability.

WESTERN BLOTTING:

Total protein was extracted by lysing cells on ice for 20 min in RIPA buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors. Protein concentrations were determined using the bicinchoninic acid assay (Beyotime). Equal amounts of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). After blocking at room temperature for 20 minutes, membranes were incubated overnight at 4 °C with primary antibodies. Following 3 washes with tris-buffered saline with Tween, membranes were incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies. Bands were visualized using an enhanced chemiluminescence detection kit (Bio-Rad), imaged with the ChemiDoc system, and analyzed using Image Lab software.

CELL TRANSFECTION:

Small interfering RNA (siRNA) targeting ODF3B (sequence: 5′-CCCACACgUgUUUgCUUAAAgTT-3′) and a scrambled negative control were synthesized by Tianyi Huiyuan Life Science & Technology Co, Ltd. Cells in the logarithmic growth phase (786-O, ACHN, Caki-1, OSRC-2) were seeded into 6-well plates (5×10⁴ cells/well) 24 hours prior to transfection. Upon reaching 60% to 70% confluence, transfection was conducted using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 20 nM siRNA and 5 μL Lipofectamine 3000 were separately diluted in 250 μL serum-free DMEM, incubated for 5 minutes at room temperature, mixed, and incubated for another 5 minutes. The complexes were added dropwise to each well, and cells were incubated for 6 hours under standard conditions before replacing the medium with complete serum-containing DMEM.

CELL COUNTING KIT 8 PROLIFERATION EXPERIMENT:

After trypsinization, 786-O cells were seeded at 500 cells/well and OSRC-2 cells at 1000 cells/well into 96-well plates, with 6 replicates per group. After 24 hours, the medium was replaced with 90 μL fresh DMEM and 10 μL Cell Counting Kit 8 (CCK-8) reagent (Dojindo Laboratories). Plates were incubated for 2 hours in the dark, and absorbance was measured at 450 nm using a microplate reader. Measurements were taken at 24, 48, 72, and 96 hours after seeding. Data were analyzed and visualized with GraphPad Prism.

CELL COLONY FORMATION EXPERIMENT:

Cells were digested, counted, and seeded at 500 cells/well in 6-well plates, followed by incubation at 37°C with 5% CO₂ for 10 days. Colonies were washed twice with ice-cold phosphate-buffered saline (PBS), fixed in 1 mL methanol for 20 minutes, and stained with 0.1% crystal violet for 20 minutes. Excess stain was removed with PBS washes, and plates were air-dried. Images of the colonies were captured, and colony numbers were quantified using ImageJ software with size-based thresholding.

WOUND HEALING ASSAY:

Cells were seeded into 6-well plates at 3×10⁵ cells/well and grown to full confluence. Linear scratches were made using a sterile 200 μL pipette tip, and wells were washed 3 times with ice-cold PBS to remove debris. Images were acquired at 0, 12, and 24 hours after scratch using a phase-contrast microscope (Motic AE31, China) with identical imaging settings for all replicates.

TRANSWELL ASSAY OF CELL MIGRATION AND INVASION:

We evaluated cell migration and invasion using Transwell chambers (8 μm pore size; Corning). For migration assays, 5×10⁴ cells were seeded in the upper chambers without Matrigel coating, and the lower chambers contained medium with 10% fetal bovine serum as a chemoattractant. After 24 hours of incubation, non-migrated cells were removed, and migrated cells were fixed and stained with 0.1% crystal violet. For invasion assays, the upper chamber was pre-coated with diluted Matrigel (BD Biosciences) and incubated for 2 hours before seeding cells under the same conditions. Five random microscopic fields per insert were counted, and experiments were performed in triplicate.

FLOW CYTOMETRIC ANALYSES OF CELL APOPTOSIS:

Apoptosis was measured using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Pharmingen, USA) according to the manufacturer’s protocol. At 48 hours after siRNA transfection, 1×10⁵ cells were collected, washed with ice-cold PBS, and resuspended in 100 μL binding buffer. Cells were stained with 5 μL Annexin V-FITC and 5 μL propidium iodide, incubated for 15 minutes at room temperature in the dark, and diluted with 400 μL binding buffer. Samples were analyzed by flow cytometry, and early and late apoptotic cells were quantified using FlowJo software. All assays were conducted in triplicate.

COLIVELIN TFA RESCUE TREATMENT:

To evaluate whether the JAK/STAT pathway mediates the biological effects of ODF3B, a STAT3-selective agonist, Colivelin TFA (MedChemExpress), was applied to ODF3B-silenced 786-O and OSRC-2 cells. Colivelin TFA was used at a final working concentration of 100 nM, which is a widely adopted and experimentally established dose in previous studies examining STAT3 activation. This concentration reliably induces STAT3 phosphorylation without causing detectable cytotoxicity under standard culture conditions. Cells were treated with Colivelin TFA for 24 hours following siRNA transfection, and subsequent assays for proliferation, apoptosis, and protein expression were performed as described above.

PUBLIC DATA ACQUISITION:

RNA-seq expression profiles (HTSeq-FPKM) and corresponding clinicopathological data for patients with kidney renal clear cell carcinoma (KIRC) were downloaded from The Cancer Genome Atlas (TCGA) database via the UCSC Xena browser on March 15, 2025.

STATISTICAL ANALYSIS:

Data were analyzed using SPSS 22.0 and GraphPad Prism 9.0. Survival analysis (Kaplan-Meier curves and log-rank tests) was performed using the GEPIA2 online platform (http://gepia2.cancer-pku.cn/) on March 15, 2025, based on TCGA-KIRC data.

Before applying parametric tests, data distribution was assessed using the Shapiro-Wilk test for normality and the Levene test for homogeneity of variance. Comparisons between 2 groups were performed using the t test, while one-way ANOVA followed by Bonferroni post hoc testing was applied for multiple-group comparisons. A P value <0.05 was considered statistically significant.

For high-throughput enrichment analyses (Kyoto Encyclopedia of Genes and Genomes [KEGG] and gene set enrichment analysis [GSEA]), P values were adjusted using the Benjamini-Hochberg false discovery rate method. As this study involved exploratory in vitro experiments, no formal sample size calculation (power analysis) was performed. CCK-8 assays included 6 technical replicates per group, whereas all other functional assays were conducted in at least 3 independent biological replicates, consistent with standard practice for cellular functional studies in cancer research.

Results

INCREASED ODF3B LEVELS IN CCRCC TISSUES AND CELL LINES:

As shown in Figure 1A, a comparative analysis across multiple cancer types revealed that ODF3B expression was significantly elevated in 6 malignancies, including bladder urothelial carcinoma, esophageal carcinoma, glioblastoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, and liver hepatocellular carcinoma. Analysis of the TCGA-KIRC dataset consisting of 533 tumor samples and 72 normal kidney tissues further demonstrated markedly higher ODF3B expression in tumor tissues, compared with normal counterparts (Figure 1B). Consistently, immunoblotting of normal renal epithelial cell lines (HEK-293, HK2) and renal carcinoma cell lines (786-O, ACHN, OSRC-2, CAKI-1) confirmed pronounced upregulation of ODF3B protein in all 4 ccRCC cell lines (Figure 1C, 1D). Among these tumor cell lines, 786-O exhibited the highest endogenous ODF3B level, whereas OSRC-2 displayed the lowest but still detectable expression. These 2 lines were therefore selected as complementary models representing distinct ODF3B expression backgrounds to comprehensively evaluate the effects of ODF3B knockdown in ccRCC.

ASSOCIATION OF ODF3B EXPRESSION WITH AGGRESSIVE CLINICOPATHOLOGICAL FEATURES AND POOR PROGNOSIS IN CCRCC:

Clinical and pathological data from 529 KIRC cases in the TCGA database were analyzed to assess the relationship between ODF3B expression and patient characteristics. Consistent with these findings, logistic regression analysis further confirmed that elevated ODF3B expression was significantly associated with advanced T stage, M stage, and higher clinical stage (Table 1). As depicted in Figure 1E–1J, across most clinicopathological characteristics, including age and lymph node status, ODF3B expression did not differ significantly between the high- and low-expression groups. In contrast, clear trends emerged when stratifying by tumor burden and disease stage. Tumors classified as T3–T4 showed markedly higher ODF3B expression compared with T1–T2 lesions, and patients with distant metastasis (M1) exhibited significantly elevated levels relative to those without metastasis (M0). Similarly, clinical stage III–IV tumors displayed higher ODF3B expression than stage I–II tumors. Survival analysis indicated that patients with high ODF3B expression had significantly shorter disease-free survival (Figure 1K), with a similar pattern observed for overall survival (Figure 1L). These findings suggest that elevated ODF3B in renal cancer is closely linked to unfavorable clinical outcomes and may serve as a prognostic biomarker in renal cell carcinoma.

ODF3B SILENCING SUPPRESSES CCRCC CELL GROWTH AND MOTILITY WHILE PROMOTING APOPTOSIS:

To explore the functional role of ODF3B in ccRCC, siRNA-mediated knockdown was performed in 786-O and OSRC-2 cells, which display high endogenous ODF3B expression. 786-O showed the highest level among ccRCC cell lines, whereas OSRC-2 showed the lowest among tumor cell lines but remained clearly higher than that in normal renal epithelial cells. Si2-ODF3B was identified as the most efficient sequence for gene silencing (Figure 2A, 2B). CCK-8 and colony formation assays showed that ODF3B knockdown significantly reduced cell proliferation (Figure 2C–2F). Flow cytometric analysis demonstrated increased proportions of early and late apoptotic cells following ODF3B depletion (Figure 2G, 2H). Consistent with these observations, Western blotting showed reduced expression of the anti-apoptotic protein B-cell lymphoma 2 (BCL2) and increased cleaved caspase 3 levels in ODF3B-silenced cells (Figure 2I).Transwell and wound-healing assays revealed that silencing ODF3B markedly impaired cellular motility (Figure 3A–3C). Collectively, these data indicate that ODF3B promotes ccRCC progression by stimulating proliferation and motility while suppressing apoptosis, highlighting its potential as a therapeutic target.

ODF3B ACTIVATES THE JAK-STAT PATHWAY TO DRIVE CCRCC PROGRESSION:

To investigate the molecular mechanisms underlying ODF3B’s oncogenic activity in ccRCC, KEGG pathway enrichment analysis was performed using TCGA-derived ODF3B expression profiles. ODF3B is enriched in pathways such as endoplasmic reticulum processing, endocytosis, apoptosis, and vascular endothelial growth factor signaling (Figure 4A). GSEA further demonstrated a strong positive correlation between ODF3B expression and the JAK/STAT signaling pathway (Figure 4B). Given the established role of this pathway in regulating proliferation, apoptosis, and immune responses, its involvement in ODF3B-mediated tumor progression was further examined. Western blotting showed that ODF3B knockdown substantially reduced phosphorylation of JAK1, JAK2, JAK3, and STAT3 (Figure 4C–4H), indicating that ODF3B positively regulates JAK/STAT pathway activation in ccRCC cells.

JAK/STAT SIGNALING MEDIATES THE EFFECTS OF ODF3B ON CCRCC CELL GROWTH AND APOPTOSIS:

To determine whether the JAK/STAT pathway mediates ODF3B’s effects on ccRCC cells, ODF3B-silenced 786-O and OSRC-2 cells were treated with Colivelin TFA, a selective STAT3 agonist. STAT3 activation reversed the growth inhibition caused by ODF3B depletion, restoring proliferation rates to levels comparable to those of controls (Figure 5A, 5B). The increase in apoptosis induced by ODF3B knockdown was also partially attenuated following STAT3 activation (Figure 5C–5F). At the molecular level, Colivelin TFA treatment reinstated p-STAT3 expression, increased BCL2 levels, and decreased cleaved caspase 3 expression (Figure 5G–5I). These results demonstrate that ODF3B facilitates ccRCC progression by enhancing proliferation and limiting apoptosis through JAK/STAT pathway activation, underscoring its potential as a therapeutic target in renal cell carcinoma.

Discussion

In this study, we identified ODF3B as a novel promoter of ccRCC progression, exerting its effects predominantly through activation of the JAK/STAT signaling pathway. Our findings highlight ODF3B as a potential diagnostic biomarker and therapeutic target in ccRCC. This work extends prior knowledge about the roles of cytoskeletal-associated proteins in cancer biology and underscores the intricate regulatory networks involved in renal carcinogenesis.

ODF3B, known for its role in sperm tail structure, is underexplored in somatic tissues and cancers. Although research on ODF3B in malignancies remains limited, a recent study in glioma reported that ODF3B is upregulated and promotes tumor cell proliferation and apoptosis through JAK/STAT pathway activation, suggesting that its oncogenic potential may extend beyond reproductive tissues [23]. Our results reveal that ODF3B is significantly upregulated in ccRCC tissues, compared with in adjacent normal renal tissues, a finding corroborated by bioinformatic analyses of TCGA datasets. Importantly, elevated ODF3B expression was associated with advanced tumor stage, higher histologic grade, and poorer patient survival, indicating its clinical relevance. However, the survival analyses in this study were based on Kaplan-Meier curves generated from the GEPIA2 platform, which performs univariate log-rank testing but does not provide access to complete individual-level TCGA covariates needed for fully adjusted multivariate Cox regression. Therefore, multivariable Cox analysis could not be performed, and residual confounding cannot be fully excluded. While prior research had not linked ODF3B with carcinogenesis, the aberrant reactivation of germ-cell–specific genes in cancer – a phenomenon termed “cancer/testis antigen” expression – is a well-documented mechanism that can drive tumorigenesis [24–27]. Thus, the aberrant expression of ODF3B in ccRCC may represent another example of this phenomenon.

Mechanistically, we demonstrated that ODF3B promotes ccRCC cell proliferation, migration, and invasion. These oncogenic effects were shown to be mediated by activation of the JAK/STAT pathway, a canonical signaling cascade that regulates numerous aspects of cancer cell biology, including survival, proliferation, immune evasion, and metastasis [28–30]. Our data indicate that ODF3B enhances phosphorylation of JAK1, 2, 3 and STAT3, leading to transcriptional activation of downstream targets, such as BCL2 and cleaved caspase 3, which are known contributors to tumor progression. Notably, inhibition of the JAK/STAT pathway attenuated the tumor-promoting effects of ODF3B, further confirming the pathway’s pivotal role in mediating ODF3B-driven oncogenesis.

Our findings align with and expand upon previous studies implicating the JAK/STAT pathway in ccRCC pathophysiology. For instance, increased STAT3 activation has been linked to resistance to tyrosine kinase inhibitors, a standard therapeutic approach for advanced ccRCC [31,32]. Moreover, STAT3 has been shown to promote an immunosuppressive tumor microenvironment by upregulating PD-L1 and other immune checkpoint molecules [33–35]. Given these observations, it is plausible that ODF3B may contribute not only to intrinsic tumor cell aggressiveness but also to the modulation of the tumor microenvironment, although this hypothesis warrants further investigation.

Our study also raises several exciting avenues for future research. First, given the emerging importance of immunotherapy in ccRCC treatment, it would be highly informative to investigate whether ODF3B modulates immune responses within the tumor microenvironment, perhaps by regulating cytokine secretion or immune checkpoint expression. Second, exploring ODF3B’s expression and function across different renal cell carcinoma subtypes (eg, papillary renal cell carcinoma, chromophobe renal cell carcinoma) could reveal whether its oncogenic role is specific to ccRCC or more broadly applicable. Third, given the heterogeneity of ccRCC, single-cell transcriptomic analyses could identify specific tumor subpopulations with high ODF3B expression, potentially informing precision medicine approaches.

Despite the strengths of this study, several limitations should be acknowledged. First, all functional experiments were performed in vitro, and in vivo validation using subcutaneous or orthotopic xenograft models will be essential to confirm the biological relevance of ODF3B in ccRCC. Second, although ODF3B was significantly upregulated in ccRCC, its upstream regulatory mechanisms remain unclear. Given the central role of the VHL-HIF axis in ccRCC pathogenesis, future studies should investigate whether ODF3B expression is influenced by hypoxia-inducible transcription factors. Third, while ODF3B knockdown reduced phosphorylation of STAT3, the present study did not establish whether ODF3B physically interacts with components of the JAK/STAT pathway; co-immunoprecipitation or interaction proteomics will be required to clarify this point. Finally, because ccRCC is characterized by a highly complex immune microenvironment, further work is needed to assess whether ODF3B influences immune infiltration or modulates the tumor immune ecosystem.

Conclusions

Overall, this study identifies ODF3B as a previously unrecognized oncogenic driver in ccRCC and provides mechanistic evidence supporting its role in activating the JAK/STAT signaling pathway. The findings highlight the novelty of ODF3B as a germline-associated structural protein aberrantly reactivated in renal cancer and underscore its potential biological significance. Future studies will focus on validating the oncogenic function of ODF3B in vivo, elucidating its upstream regulatory mechanisms – including potential VHL-HIF-dependent regulation – and determining whether ODF3B influences the tumor immune microenvironment. These investigations will further clarify the translational potential of targeting ODF3B in ccRCC therapy.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.