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. 2025 Jul 2;25:249. doi: 10.1186/s12935-025-03871-2

P4HA2 promotes the progression of papillary thyroid cancer by enhancing degradation of IκBα to activate NF-κB signaling pathway

Ruowen Li 1,#, Mingjian Zhao 1,#, Min Sun 1,#, Yongkang Wu 2, Chengxu Miao 1, Liu Fangyu 1, Mengting Wu 2, Xiaojia Shi 2, Jinghui Lu 1,✉,#, Xuetian Yue 2,✉,#
PMCID: PMC12224786  PMID: 40604807

Abstract

Papillary thyroid cancer (PTC) is the most common malignancy of the endocrine system. Collagen prolyl 4-hydroxylase alpha subunit 2 (P4HA2) is a key enzyme involved in collagen metabolism. However, the expression and function of P4HA2 in PTC progression have not been well studied. Our previous proteomic data showed that the differential proteins in human PTC were significantly enriched in metabolic signaling pathways, with P4HA2 being the most up-regulated protein. Here, we found that P4HA2 promotes the proliferation and migration of PTC. The expression of P4HA2 is elevated and its elevation is associated with poor prognosis in human PTC specimens. Functionally, P4HA2 promotes the proliferative and migratory abilities of BHP10-3 and TPC-1 cells. Further studies showed that overexpression of P4HA2 significantly activates the NF-κB signaling pathway. Mechanistically, P4HA2 promotes the ubiquitination and degradation of inhibitor kappa B-alpha (IκBα) by directly binding, leading to activation of NF-κB signaling pathway. Furthermore, BAY 11-7082, an inhibitor of the NF-κB signalling pathway, reversed the promotion of PTC proliferation and metastasis by P4HA2 both in vivo and in vitro. In conclusion, P4HA2 activates the NF-κB signaling pathway by promoting proteasome-dependent degradation of IκBα, which in turn contributes to the proliferation and migration of PTC. Therefore, P4HA2 could be used as a potential therapeutic target for the treatment of PTC patients.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-025-03871-2.

Keywords: P4HA2, Papillary thyroid carcinoma, NF-κB signaling pathway, Proteasome degradation, Ubiquitination

Background

Thyroid neoplasm, originating from follicular epithelial cells or parafollicular cells (C cells), is the most prevalent endocrine system malignancy [1]. Based on their origin and degree of differentiation, thyroid carcinomas can be categorized into poorly differentiated thyroid carcinoma (PDTC), and anaplastic thyroid cancer (ATC), papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC) and medullary thyroid carcinoma (MTC). Among the different subtypes, papillary thyroid cancer is the most frequently encountered [2–4]. In recent years, there has been a substantial increase in the prevalence of thyroid cancer. According to statistics reported in the literature, the global incidence of thyroid cancer in 2020 was reported as 10.1 cases per 100,000 women and 3.1 cases per 100,000 men [5]. Patients with early-stage thyroid cancer can be cured through surgery, while local vascular invasion and distant metastasis significantly reduce survival rates [6]. Therefore, it is of great significance to study the specific molecular pathogenesis of thyroid cancer and to find new drug targets to combat thyroid cancer metastasis.

The gene P4HA2 (Prolyl 4-hydroxylase subunit alpha 2) encodes the P4HA2 protein, which is a component of the P4HA complex playing a crucial catalytic role in collagen modification post-synthesis [7, 8]. Numerous studies have demonstrated that P4HA2 has diverse functions in normal physiological processes and disease progression, including cholestasis [9]vascular intimal formation [10]high myopia [11]and malignant biological activities in various tumors [12–15]. P4HA2 can promote tumor progression by promoting epithelial-mesenchymal transition (EMT) [16–18]affecting collagen deposition [16]or the hydroxylation of other proteins [19, 20]. However, the expression and biological function of P4HA2 in thyroid cancer remain to be elucidated.

The nuclear factor kappa B (NF-κB) pathway is critical in the regulation of cellular immunity, inflammation, apoptosis and various physiological processes [21–23]. NF-κB is extensively activated in prevalent human malignancies and holds significant implications for tumorigenesis and progression [24]. For example, the oncogenic factor CD13 is capable of activating the NF-κB signaling pathway, thereby eliciting drug resistance in hepatocellular carcinoma [25]. NF-κB signalling pathway can be activated by S100A11 and significantly enhance glioblastoma cell invasion by inducing Epithelial-Mesenchymal Transition (EMT) [26]. In its inactive state, the NF-κB binds to the inhibitor kappa B-alpha (IκBα) protein in the cytoplasm. Upon stimulation, phosphorylation and degradation of IκBα proteins lead to the release of NF-κB and its translocation into the nucleus [27]. Previous studies have shown that the NF-κB pathway is involved in the proliferation and anti-apoptotic processes of thyroid tumor cells [28]. However, the precise mechanism underlying the activation of NF-κB signaling in PTC remains incompletely elucidated.

In this study, we have found that P4HA2 is upregulated in PTC and its elevation is associated with an unfavorable prognosis. Subsequent experimental studies have shown that P4HA2 passages can contribute to the activation of the NF-κB pathway by enhancing the level of ubiquitination of IκBα and protein degradation, consequently enhancing proliferation and migration of PTC cells. Our findings indicate that targeting P4HA2 might hold potential therapeutic implications for management of papillary thyroid cancer.

Materials and methods

Patients and clinical samples

Forty-nine papilary thyroid tumor tissues and thirty-four adjacent non-tumor tissues from Qilu Hospital were collected. Written consent was obtained from the patients. The inclusion criteria for the tissue samples were: (1) the patient had no history of thyroid surgery, hypertension, diabetes mellitus, coronary heart disease, hyperlipidemia, non-thyroidal tumors, allergic rhinitis, nephrotic syndrome, ulcerative colitis, and other metabolism-related disorders; (2) intraoperative specimens are collected in a size that meets the requirements for post-procedure testing; (3) postoperative histopathology results were PTC. The detail information of the clinical samples had been displayed in our previous paper [29]. The study was approved by the Ethics Committee of Qilu Hospital of Shandong University (KYLL-2021(ZM)-028).

The RNA expression data related to thyroid cancer in fragments per kilobase of transcript per million mapped reads (FPKM) and clinical information were obtained from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). Kaplan-Meier analysis was used to assess the survival differences between the high and low expression groups of P4HA2. The log-rank test was employed to evaluate the Progression-free survival differences between the two groups.

Cell lines and reagents

The papillary thyroid cancer cell lines BHP10-3(RRID: CVCL_6278) and TPC-1 (RRID: CVCL_6298) were obtained from American Type Culture Collection (ATCC). These cells were cultured in RPMI 1640 (Gibco, Cat No. 11875119) medium supplemented with 10% fetal bovine serum (Gibco, Cat No. A5670701) in a 37℃ incubator containing 5% CO2. The plasmids used in the rest of the experiments were obtained from MiaoLing Plasmid Platform (Wuhan, China). The lentivirus expressing P4HA2 or shP4HA2 was purchased from GeneChem. These plasmids were transfected into PTC cells and HEK293T cells using Liposome™2000 Transfection Reagent.

RNA sequencing analysis

For RNA sequencing analysis (RNA-seq), we collected P4HA2-overexpressing BHP10-3 cells and control BHP10-3 cells for transcriptome analysis. Three replicates were utilized for each group. Then, the Next-Generation Sequencing were carried out on an Illumina platform (Shanghai Personalbio Technology). With log2FC > 1 and p < 0.05 as screening parameters, We analysed differential genes (DEGs) between the two groups using R software (R version 4.2.0). To explore the critical biological pathways regulated by P4HA2, we performed GSEA analysis using the KEGG gene set derived from the Molecular Signature Database (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp).

Immunohistochemistry

The tissue chips were purchased from Shanghai Outdo Biotechnology Company (Cat No. HThyP120CS02). Every chips was incubated with anti-prolyl 4-hydroxylase subunit α2 (P4HA2) antibody at 4℃ overnight. After incubation with secondary antibody and staining by diaminobenzidine (DAB). The results were analysed carefully and in detail by at least three senior pathologists. Staining intensity was scored on a scale in four levels (negative staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3). Percent staining was assessed on a scale in five levels (no staining = 0, 1–25%=1, 26–50%=2, 51–75%=3, and 76–100%=4). The intensity score multiplied by the percentage score was used as the immunohistochemistry score [45]. Immunohistochemical score>6 was considered high expression and score ≤ 6 was considered low expression.

Quantitative reverse transcription PCR

Total RNA was isolated from cells and tissues using TRIzol reagent (Thermo Fisher Scientific, Cat No. A33250). cDNA was reverse transcribed from RNA by Revert Aid First Strand cDNA Synthesis kit (Vazyme Biotech, Cat No. R201). Quantitative reverse transcription PCR (RT-qPCR) was performed using the BioRad C1000 Termal Cycler CFX96 Real-Time System. Primers used in this paper are shown in the Supplementary Table 1. β-actin was used as a loading control. Three independent replicate groups were analyzed.

Western blot

Cells were lysed using NP-40 cell lysis buffer (Beyotime, Cat No. P0013). Protein extracts were analyzed using Detergent Compatible Bradford Protein Assay Kit (Beyotime, Cat No. P0006C). Equal mass proteins were separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane (Merck, Cat No. IPVH00010) and incubated overnight at 4 ℃ with the following primary antibodies: mouse anti-beta actin (Proteintech, 66009, 1:20000), rabbit anti-P4HA2 (Proteintech, 13759-1-AP, 1:2000), rabbit anti-Vimentin (Proteintech, 10366-1-AP, 1:2000), rabbit anti-E-cadherin (Proteintech, 20874-1-AP,1:20000), rabbit anti-N-cadherin (Proteintech, 22018-1-AP, 1:2000), rabbit anti-IκB Alpha(Proteintech, 10268-1-AP, 1:1000), rabbit anti-NF-κB p65 (Proteintech, 10745-1-AP, 1:1000), rabbit anti-Phospho-NF-κB p65 (Cell Signaling, 3033, 1:1000), HRP DYKDDDDK Tag (Proteintech, HRP-66008, 1:10000), anti HA-Tag (ABclonal, AE025, 1:1000), MYC tag (Proteintech, 60003-2-lg, 1:2000). The membranes were then incubated with HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00001-2, 1:10000) or HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00001-1, 1:10000) treatment. Signals were detected using TANON™ECL chemiluminescent substrate and an automated chemiluminescence/fluorescence image analysis system (Tanon, Shanghai).

Co-immunoprecipitation (Co-IP)

Cells transfected with plasmids were incubated at 37 ℃ in an incubator with 5% CO2 and saturated humidity for 48 h and then lysed using BC100 lysis buffer. After centrifugation, the supernatant was taken off and then added with antibody containing conjugated Protein A + G beads at 4 ℃ for overnight incubation. After washing three times, the samples were added with SDS-PAGE loading buffer and incubated with metal bath at 100 ℃ for 10 min. The samples were finally tested by WB. IgG was used as a negative control for the experiment.

Cell proliferation assays

Cell proliferation was determined using Cell Counting Kit 8 (CCK-8). In the CCK-8 assay, 1 × 103 cells were cultured in 96-well plates with five replicate wells per group. CCK-8 reagent (Beyotime, Cat No. C0042) was added to each well. After 2 h of routine incubation, the absorbance of each well was measured at 450 nm. Cell cycle progression was determined by staining of 5-ethyl-2′-deoxyuridine (EdU). In EdU experiments, 5 × 103 cells were cultured in 24-well plates. After incubation with 50 µM EdU for 2 h (RiboBio, Cat No. C10310-1). Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, followed by incubation with apollo 567 solution for 30 min for cell nuclei staining. DAPI was added to stain the cell nuclei for 10 min. The ratio of EdU-positive nuclei was calculated.

Migration and wound healing assays

In transwell assays, chambers (Corning, Cat No. 3422) were placed in 24-well plates and the chambers were soaked with serum-free medium for at least two hours. The treated cells were resuspended in 200 µL of FBS-free medium and 2 × 104 cells were inoculated in the upper chamber. Complete medium containing 10% fetal bovine serum (FBS) was then added to the lower chamber and incubated for 24 h. The cells were fixed with 4% para-formaldehyde for 15 min, stained with 1% crystal violet for 5 min. After removing the non-migrated cells on the upper surface of the chamber, the migrated cells were photographed with a light microscope. In wound healing assay, scratch inserts (ibidi, Cat No. 80206) were placed in a 24-well plate. In the scratching experiment, 1 × 104 cells were inoculated in wells and the inserts were removed after cell apposition. Then, the cells were cultured by serum-free medium for 24 h. Photographs were taken under the microscope (Olympus).

In vivo experiment

All in vivo studies were approved by the Laboratory Animal Ethical and Welfare Committee of Shandong University Cheeloo College of Medicine (Permit No: 23032). BALB/c nude mice (six-weeks old) were purchased from Vital River were randomly divided into four groups. To assess the proliferative capacity of the tumors, P4HA2-overexpressed or control BHP10-3 cells (5 × 106) were suspended in 100 µL PBS and injected into the subcutaneous layer of the right flank of the mice. One week later, two groups of mice were injected intraperitoneally with BAY11-7082 (MedChemExpress, Cat No. HY-13453) at a dose of 5 mg/kg every other day. Tumor volumes were measured every 7 days. After 35 days, the nude mice were executed and the tumor tissues were weighed. And the proliferation marker Ki67 of isolated tumors were verified by IHC analysis. To assess the migration ability of the tumors, P4HA2-overexpressed or control cells (1 × 106) were injected into mice via tail vein. After two weeks, two groups of mice were injected intraperitoneally with BAY 11-7082 at a dose of 5 mg/kg every other day. On the day 60, mice were executed and whole lungs were collected for hematoxylin and eosin (H&E) assay. Tumors were photographed under a microscope (Olympus, Japan) and counted in terms of volume.

Statistical analysis

We used GraphPad Prism 8.0 software and SPSS 27.0 software for statistical analysis. Categorical data were statistically analysed using the chi-square test. Statistical analyses of continuous data were performed using T-test. Survival analyses were performed using the Kaplan-Meier method. P < 0.05, which was considered as statistically significant difference. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Results

P4HA2 expression is increased in PTC and is associated with poor prognosis

Our previous study analyzed differentially expressed proteins between PTC tissues and adjacent non-tumor tissues by proteomics and reported a large number of proteins upregulated in PTC. KEGG (kyoto encyclopedia of genes and genomes) enrichment analysis showed that most of them were significantly enriched in metabolic signaling pathways (Fig. 1A) [29]. The most differentiated protein in the metabolic signaling pathway was P4HA2, which was significantly upregulated in PTC tissues (Fig. 1B). To confirm the proteomic results, we collected 34 pairs of PTC tissues and adjacent non-tumour tissues and examined P4HA2 mRNA levels by RT-qPCR, which showed that P4HA2 was significantly elevated in PTCs (Fig. 1C). To further confirm the expression of P4HA2, we performed western blot (WB) and immunohistochemical (IHC) analyses of P4HA2 content in PTC tissues and para-cancer tissues. The results of WB and IHC showed that P4HA2 was highly expressed in PTC as well (Fig. 1D, E). Besides, P4HA2 mainly located in the cytoplasm of cells from PTC specimens (Fig. 1E). Interestingly, the high expression of P4HA2 was significantly correlated with T and N classifications of PTC according to IHC score (Table 1). Additionally, based on the transcriptional levels of P4HA2 in thyroid cancer patients from the TCGA database, patients were categorized into a high-expression group (n = 103) and a low-expression group (n = 102). The disease-free progression period of thyroid cancer patients with high P4HA2 expression was significantly shorter than that of patients with low P4HA2 expression (Fig. 1F). Above all, these results indicate that P4HA2 involves in the progression of PTC.

Fig. 1.

Fig. 1

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P4HA2 expression is elevated in PTC and which is associated with poor prognosis. (A) KEGG enrichment analysis of differential proteins detected by proteomic analysis in PTC tissues and non-tumor tissues. (B) The volcano plot showed that P4HA2 was the most significantly increased protein among the differential proteins enriched into metabolic pathways. (C) Relative mRNA levels of P4HA2 in PTC and non-tumor tissues were detected by RT-qPCR. n = 34. (D) Protein levels of P4HA2 were determined in PTC and non-tumor tissues by western blot. (E) Relative expression of P4HA2 in PTC and para-cancerous tissues were examined by IHC. Scale bar:100 μm. (F) Progression-free survival curve of thyroid cancer patients with differentially expressed P4HA2 based on TCGA database. *p < 0.05; **p < 0.01

Table 1.

The relationship between P4HA2 expression and PTC clinicopathologic features

Clinicopathologic feature P4HA2 expression p-value
low high
All cases 26 32
Gender
 Male 11 12 0.7093
 Female 15 20
Age
 < 55 21 20 0.1285
 ≥ 55 5 12
T classification
 T1 19 13 0.0135
 T2 ∼ 4 7 19
N classification
 N0 20 11 0.0012
 N1 6 21
AJCC stage
 I 24 24 0.0827
 II 2 8
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P4HA2 overexpression promotes the proliferation and migration of PTC cells

To determine the effect of P4HA2 on PTC progression, we constructed stable P4HA2-overexpressing, P4HA2-silencing, and their matching control BHP10-3 and TPC1 cell lines. RT-qPCR and WB confirmed the overexpressing and silencing efficiency of P4HA2 (Fig. S1A, S1B). We used EdU and CCK-8 methods to compare the growth of PTC cells overexpressing P4HA2 with control cells. The growth of BHP10-3 and TPC-1 cell lines were significantly augmented after P4HA2 overexpression compared to control PTC cells (Fig. 2A). The EdU analysis revealed a significant acceleration in cell cycle progression in PTC cell lines after P4HA2 overexpression (Fig. 2B). Furthermore, we found that overexpression of P4HA2 expression enhanced the migration of BHP10-3 and TPC1 cells by transwell and wound healing assays (Fig. 2C, D). Conversely, the proliferation and migration ability of P4HA2-silencing PTC cells were notably decreased compared to those of shNC cells (Fig. 3A-D). Moreover, RT-qPCR and WB confirmed that P4HA2 overexpression markedly increased N-cadherin and Vimentin levels, while predominantly decreased E-cadherin levels in PTC cell lines (Fig. 2E, F). Reciprocally, the opposite effects of P4HA2 on the expression of EMT markers were found in P4HA2-silencing groups (Fig. 3E, F). The findings suggest that P4HA2 enhances the proliferation and migration of PTC cells.

Fig. 2.

Fig. 2

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Overexpression of P4HA2 promotes the proliferation and migration of PTC cells. (A) CCK-8 assays showed that the cell proliferation is enhanced after the overexpression of P4HA2 in BHP10-3 andTPC-1 cell lines. n = 5. (B) Percentage of EdU stained cells was increased after P4HA2 overexpression in the BHP10-3 and TPC-1 cell lines. The quantitative data was displayed on the right panel. n = 3. Scale bar:20 μm. C-D. The transwell (C) and wound healing (D) assays showed that the migration ability of BHP10-3 and TPC-1 cell lines was enhanced after P4HA2 overexpression. The representative images were showed on the top panel. The quantitative data were presented on the bottom panel. C, n = 8. Scale bar:100 μm. D, n = 3. Scale bar:100 μm. E-F. The mRNA levels (E) and protein levels (F) of N-cadherin, E-cadherin and vimentin were detected after P4HA2 overexpression in the BHP10-3 and TPC-1 cell lines by RT-qPCR and western blot, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ns no significant

Fig. 3.

Fig. 3

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Knockdown of P4HA2 decreases the proliferation and migration of PTC cells. (A) CCK-8 assays showed that the cell proliferation is decreased after P4HA2 knockdown in BHP10-3 and TPC-1 cell lines. n = 5. (B) Percentage of EdU stained cells was decreased after P4HA2 knockdown in the BHP10-3 and TPC-1 cell lines. The quantitative data were showed on the right panel. n = 3. Scale bar:20 μm. C-D. The transwell (C) and wound healing (D) assays showed that the migration ability of BHP10-3 and TPC-1 cell lines was inhibited after P4HA2 knockdown. The quantitative data were presented on the bottom panel. C, n = 8. Scale bar:100 μm. D, n = 3. Scale bar:100 μm. E-F. The mRNA levels (E) and protein levels (F) of N-cadherin, E-cadherin and vimentin were detected after P4HA2 knockdown in the BHP10-3 and TPC-1 cell lines by using RT-qPCR and western blot, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ns no significant

P4HA2 activates the NF-κB signaling pathway to promote PTC

To explore the molecular mechanism by which P4HA2 promotes PTC proliferation and migration, we used RNA-seq to detect differentially expressed genes (DEGs) between P4HA2-overexpressed BHP10-3 cells and control BHP10-3 cells. The results indicated that there are 65 upregulated genes and 85 downregulated genes based on the set parameters of log2 FC > 1 and p < 0.05 (Fig. S1C). The GSEA enrichment analysis revealed a significant enrichment of DEGs in NF-κB signaling pathway (Fig. 4A and Fig. S1C), suggesting the potential involvement of P4HA2 in modulating the NF-κB pathway. Therefore, we further explored the effect of P4HA2 on NF-κB signaling activation by western blot analysis. Overexpression of P4HA2 increased phosphorylation of p65 at Ser536, while reduced the protein level of IκBα (Fig. 4B). However, the expression trend of these key proteins were opposite with P4HA2 overexpression after P4HA2-silencing (Fig. 4C). P4HA2 overexpression significantly increased the mRNA levels of NF-κB target genes such as VEGF, ANGPT2, and CCND3 (Fig. S1D). Besides, after separating the cytoplasmic and nuclear proteins, we found that overexpression of P4HA2 promoted nuclear translocation of p65 in BHP10-3 cells (Fig. 4D). Moreover, the expression of P4HA2 is negatively associated with IκBα levels and positively associated with phosphorylated p65 in PTC and adjacent non-tumor tissues (Fig. 4E). Together, the above experiments demonstrate that P4HA2 activates the NF-κB signaling pathway in PTC.

Fig. 4.

Fig. 4

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P4HA2 promotes tumor malignancy via NF-κB signaling pathway. (A) GSEA enrichment analysis of RNA-seq data identified the top changed pathways in thyroid cancer cells. (B) GSEA analysis showed that most of DEGs were enriched in the NF-κB signaling pathway in P4HA2 transfected BHP10-3 cells. C-D. The expression of IκBα, p65, and phosphorylated p65 was detected by western blot after P4HA2 overexpression (C) or knockdown (D) in BHP10-3 and TPC-1 cell lines, respectively. E. The cytoplasm and nucleus proteins were isolated in BHP10-3 cells with P4HA2 overexpression. The protein level of p65 in the nucleus and cytoplasm was determined by western blot. GAPDH was used as the control of cytoplasm, while histone H3 was used as the control of nucleus. F. Expression of P4HA2 and IκBα in four pairs of thyroid cancer tissues and para-cancerous tissues were tested by western blot. *p < 0.05, **p < 0.01, ***p < 0.001, ns no significant

P4HA2 activates NF-κB pathway by impairing IκBα protein stability

Since P4HA2 was negatively correlated with IκBα at protein levels (Fig. 4E), we speculated whether P4HA2 acts by impairing the stability of IκBα proteins to activate NF-κB signaling pathway. Overexpression of P4HA2 did not notably affect the mRNA levels of IκBα (Fig. S1E). However, P4HA2 overexpression was found to decrease the levels of IκBα protein. And this effect could be reversed by treatment of the proteasome inhibitor (MG132), but not affected by the lysosome inhibitor Chloroquine (CQ) (Fig. 5A). In addition, the half-life of IκBα protein was detected using the cycloheximide (CHX) tracking method. The results showed that the degradation rate of IκBα was accelerated after transfection of P4HA2 overexpressed plasmids in 293T cells (Fig. 5B, C). Next, we performed Co-IP experiments and found that P4HA2 physically interacted with IκBα (Fig. 5D, E). Moreover, we found that the ubiquitination levels of enriched IκBα was significantly elevated after transfection of P4HA2-overexpressed plasmids in 293T cells (Fig. 5F). In contrast, the ubiquitination levels of enriched IκBα were decreased in BHP10-3 cells after transfected with shP4HA2 plasmids (Fig. 5G). Besides, through endogenous Co-IP experiments, we found that overexpression of P4HA2 accelerates the ubiquitination and degradation of IκBα in BHP10-3 cells (Fig. S1F). Therefore, our data demonstrate that P4HA2 activates NF-κB pathway via IκBα, more precisely, by facilitating the proteasome-dependent degradation of IκBα.

Fig. 5.

Fig. 5

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P4HA2 activates NF-κB signaling by promoting the degradation of IκBα. (A) HEK293T cells were transfected with P4HA2, then treated with CQ (25µM) or MG132 (10µM) for 24 h, respectively. The total protein were collected to assess IκBα and P4HA2 levels by western blot. (B-C) IκBα and P4HA2 protein levels were determined in CHX (25 µg/mL) treated HEK293T cells with or without P4HA2 overexpression. The representative blots were showed on (B) The statistic data was display on (C) n = 3. (D-E) HEK293T cell were transfected with Flag-P4HA2 and HA-IκBα, simultaneously or individually. The cell lysis was immunoprecipitated with anti-Flag beads (D) or anti-HA beads (E), following western blot for HA and Flag. (F) HA-IκBα plasmids were co-transfected with P4HA2 in HEK293T cells, respectively. Anti-HA beads were used to immunoprecipitate IκBα, then the Ub levels were detected by MYC antibody. (G) HA-IκBα plasmids were transfected with control or P4HA2-silencing BHP10-3 cells, respectively. Anti-HA beads were used to immunoprecipitate IκBα, then the Ub levels were detected by MYC antibody

Inhibiting the NF-κB signaling pathway reverses P4HA2-induced malignant phenotypes in vitro

To further determine whether activation of the NF-κB pathway plays a essential role in the promotion of PTC progression by P4HA2, we treated PTC cells with BAY 11-7082, an inhibitor of the NF-κB signaling pathway. The cytotoxicity of the BAY 11-7082 was detected by CCK8 assay (Fig. S1G). BAY 11-7082 (1 μm) was used to treat PTC cells. As shown in Fig. 6A and B, CCK8 and EdU assays consistently displayed that the proliferation ability of P4HA2-overexpressed PTC cells was significantly enhanced compared with the control group. After the incubation with BAY 11-7082, the proliferation ability of control and P4HA2-overexpressed PTC cells was significantly inhibited. Notably, BAY 11-7082 treatment eliminated the effect of P4HA2 on promoting proliferation of PTC cells. Meanwhile, transwell and wound healing assays consistently showed that BAY 11-7082 reversed the enhanced migratory ability of P4HA2-overexpressed PTC cells (Fig. 6C, D). Furthermore, the WB experiment showed that treatment with BAY 11-7082 markedly abolished increase of N-cadherin, Vimentin, and phosphorylated p65 and decrease of E-cadherin and IκBα, which were induced by overexpressing P4HA2 in BHP10-3/TPC-1 cells (Fig. 6E). These experiments demonstrate that NF-κB inhibitor reverses the malignant phenotype induced by P4HA2 overexpression in vitro.

Fig. 6.

Fig. 6

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Blocking the NF-κB signaling pathway reverses P4HA2-induced phenotype in vitro. BHP10-3 and TPC-1 cells were transfected with P4HA2, then were treated with BAY 11-7082 (1µM). (A) Cell proliferation were detected by using CCK-8 assay kit. n = 5. (B) Cell cycle progression were examined by using EdU assays. n = 3. (C) The treated cells were resuspended in FBS-free medium and then seeded on the top of the chamber. The lower chamber was added with 10% FBS culture medium. The representative migrated images were presented. Scale bar: 100 μm. (D) The quantitative data for cell migration was presented. n = 8. (E) Wound healing assays were used to determine the ability of cell migration. The statistic data were displayed. n = 3. (F) The protein levels of E-cadherin, N-cadherin, Vimentin, p65, phosphorylated p65, and IκBα were detected in above treated cells by using western blot. Vector: The control group with the same volume of DMSO added. BAY: BAY 11-7082. *p < 0.05, **p < 0.01,***p < 0.001, ns not significant

Inhibiting the NF-κB signaling pathway reverses the P4HA2-induced malignant phenotype in vivo

To further elucidate the role of P4HA2 in the progression of PTC, we established a subcutaneous tumorigenesis model and a lung metastasis model in nude mice. The subcutaneous tumors in mice injected with P4HA2-overexpressed BHP10-3 cells were larger in size and relatively heavier in weight. And, the subcutaneous tumors in the mice treated with daily intraperitoneal injection of BAY 11-7082 (5 mg/kg) were smaller. Interestingly, BAY 11-7082 clearly eliminated the tumorigenesis ability of P4HA2-overexpressed BHP10-3 cells (Fig. 7A-C). WB analysis verified the overexpression of P4HA2 in the subcutaneous tumors and its negative correlation with IκBα and positive corelation with phosphorylated p65 (Fig. S2D). The IHC results showed that P4HA2 overexpression increased the number of ki-67-positive cells and the abundance of Vimentin, but which were reversed by treatment with BAY11-7082 (Fig. 7D). Moreover, the lung metastasis model showed larger lung metastases in the P4HA2 overexpression group than the vector control, while BAY11-7082 treatment abolished the increasing of BHP10-3 cells lung colonization induced by P4HA2 overexpression (Fig. 7E, F). Altogether, P4HA2 promotes PTC progression through activation of NF-κB signaling pathways.

Fig. 7.

Fig. 7

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P4HA2 promotes PTC progression in vivo. Subcutaneous and tail vein injections of stably overexpressing P4HA2 or control BHP10-3 cells were used to establish subcutaneous tumorigenesis and lung metastasis models in nude mice, which were then treated by intraperitoneal injection of BAY 11-7082 (5 mg/kg), respectively. (A-C) The tumor substances (A), volumes (B) and tumor weight (C) were displayed. n = 5. (D) Representative IHC staining of Ki67 and Vimentin in tumor tissues were presented. Scale bar: 20 μm. (E) Representative H&E image of mice lung metastatic tumors from different groups were displayed. Scale bar: 500 μm. (F) Quantification of the percentage of metastatic area in the whole lung nodules was performed. n = 5. BAY: BAY 11-7082. Statistical differences were determined by two-sided Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant

Discussion

Papillary thyroid carcinoma is the subtype with the highest incidence among thyroid tumors [30]. Although surgery and radioiodine therapies can cure the most papillary thyroid cancers, some subtypes of PTC with high aggressiveness and recurrence rates greatly decrease the survival rate of patients [31]. Therefore, it is still urgent to find alternative targets for the treatment of PTC. Here, we found that P4HA2 is elevated in PTC tissues and its elevation is associated with poor prognosis of PTC. Functional assays demonstrated that P4HA2 promotes proliferation and migration of PTC. Further mechanism study revealed that P4HA2 induces elevated levels of IκBα ubiquitination and degradation at the protein level to activate NF-κB signaling pathway during PTC progression. These results also supported by the clinically samples. Importantly, blocking NF-κB signaling pathway using BAY 11-7082, clearly reversed the effects of P4HA2 in promoting PTC proliferation and migration. Therefore, these new findings provide potential therapeutic strategies for PTC.

As a key enzyme in the collagen metabolic pathway, P4HA2 has been clearly demonstrated in many previous studies to play a critical role in a variety of malignancies. Specifically, P4HA2 promotes tumor metastasis by facilitating epithelial-mesenchymal transition (EMT) in breast cancer, glioma, and ovarian cancer [16–18]. Knockdown of P4HA2 reduces tumor growth and metastasis by blocked collagen deposition in breast cance [16]. Moreover, the activation of P4HA2 plays a critical role in the malignant phenotype of IDH1 wild-type gliomas and contributes to their resistance to Temozolomide [32]. Additionally, P4HA2-mediated stabilization of HIF-1α confers resistance to erdafitinib in bladder cancer cells [33]. In this study, our analysis of public databases and tissue samples revealed that P4HA2 exhibits high expression levels in PTC tissues, which positively correlates with poor clinical prognosis. Using cell lines and mice models, we observed that overexpression of P4HA2 enhanced tumor proliferation and migration ability of PTC cells. Conversely, knockdown of P4HA2 resulted in decreasing of PTC progression. Collectively, these results provide compelling evidence supporting the possible utility of P4HA2 as a potential treatment candidate.

The NF-κB signaling pathway plays diverse roles in tumors, including promoting tumor cell survival, resisting apoptotic mechanisms, regulating inflammation and immune evasion, facilitating cell migration and invasion, as well as promoting tumor angiogenesis [34]. Aberrant activation of NF-κB has been observed in numerous tumors, such as brain tumors, gastrointestinal cancers, gynecological cancers [35]. Notably, the involvement of NF-κB in maintaining the malignant phenotype of thyroid cancer has long been documented [36]. In mouse models of thyroid cancer, PTEN or PPARγ gene inactivation leads to excessive activation of NF-κB and subsequently results in more aggressive forms of cancer [37, 38]. The small molecule inhibitor, triptolide, effectively impedes the growth and invasion of thyroid cancer cells by disrupting the interaction between NF-κB p65 subunit and CBP/P300 [39]. Recent studies have demonstrated that knockdown of AHNAK2 gene reduces thyroid cancer progression by suppressing NF-κB pathway activity [40]. In this study, RNA-seq analysis revealed that genes altered after P4HA2 overexpression in BHP10-3 cells were enriched in the NF-κB pathway. Further experiments showed that overexpression of P4HA2 increased phosphorylated p65 levels while decreasing IκBα levels. Moreover, overexpression of P4HA2 significantly augmented the nuclear translocation of p65. Additionally, the NF-κB pathway inhibitor BAY 11-7082 markedly inhibited proliferation and migration capacity in PTC cells induced by overexpressing P4HA2. These findings collectively indicate that activation of the NF-κB pathway is an integral component of P4HA2 in PTC.

Illustrating the molecular mechanism of P4HA2 in regulating NF-κB signaling pathway is an important finding in this study. In the NF-κB signaling pathway, IκBα serves as a crucial negative regulator [41]. Here, our data demonstrated that P4HA2 mediated ubiquitination and degradation of IκBα. Previous studies have demonstrated that IκBα protein degradation occurs through the ubiquitin-proteasome pathway [42]. Importantly, the NF-κB signaling inhibitor BAY 11-7802, blocking IκBα degradation [43]reversed P4HA2 effects in PTC. P4HA2 primarily interacts with target proteins via binding and hydroxylation mechanisms. P4HA2 may influence target proteins through its effect of hydroxylation modification [44]. It was reported that P4HA2 could bind to and stimulate the hydroxylation and degradation of carabin, a negative regulator of B-cell lymphoma proliferation [19]. P4HA2 interacts with YAP1 and facilitates its proline hydroxylation, thereby exerting inhibitory effects on the migration and invasion abilities of prostate cancer cells [20]. Our study revealed that overexpression of P4HA2 accelerates the rate of IκBα degradation. Further experiments demonstrated that P4HA2 interacts with IκBα to facilitate its ubiquitination process. However, it is worth further exploring whether the effect of P4HA2 on IκBα degradation is influenced by hydroxylation.

Overall, our results suggest that P4HA2 is overexpressed in papillary thyroid cancer and promotes PTC progression. P4HA2 can activate the NF-κB signaling pathway by accelerating IκBα degradation to promote the proliferation and migration of PTC cells. Therefore, targeting P4HA2 is a potential therapeutic strategy for the management of PTC.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (479.7KB, docx)

Abbreviations

P4HA2

Collagen prolyl 4-hydroxylase alpha subunit 2

PTC

Papillary thyroid cancer

Co-IP

Co-immunoprecipitation

RT-qPCR

Quantitative real-time PCR

qPCR

Quantitative PCR

IHC

Immunohistochemical

IκBα

Inhibitor kappa B alpha

EMT

Epithelial mesenchymal transition

Author contributions

Conceptualization, RL, XY, and JL; methodology, RL, XY, and MS; formal analysis, RL, and MZ; investigation, RL, and MS; resources, XY, and JL; writing-original draft preparation, RL, MZ, and MS; writing-review and editing, all authors; funding acquisition, XY, and JL. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81972819), Natural Science Foundation of Shandong Province (No. ZR2020YQ57 and No. ZR2021LZL003) and the Wu Jieping Medical Foundation (No.6010125122).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was approved by the Ethics Committee of Qilu Hospital of Shandong University (KYLL-2021(ZM)-028) and the Laboratory Animal Ethical and Welfare Committee of Shandong University Cheeloo College of Medicine (Permit No: 23032).

Consent for publication

All authors agree with the content for publication.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ruowen Li, Mingjian Zhao, Min Sun, Jinghui Lu and Xuetian Yue contributed equally to this work.

Contributor Information

Jinghui Lu, 201262005933@email.sdu.edu.cn.

Xuetian Yue, Email: yuexu@sdu.edu.cn.

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Supplementary Materials

Supplementary Material 1 (479.7KB, docx)

Data Availability Statement

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