Aberrant gene expression is a prominent feature of metastatic cancer. Translational initiation is a vital step in fine-tuning gene expression. Thus, exploring translation initiation regulators may identify therapeutic targets for preventing and treating metastasis. Herein, we identified that DHCR24 was overexpressed in lymph node (LN) metastatic bladder cancer and correlated with poor prognosis of patients. DHCR24 promoted lymphangiogenesis and LN metastasis of bladder cancer in vitro and in vivo. Mechanistically, DHCR24 mediated and recognized the SUMO2 modification at lysine 108 of hnRNPA2B1 to foster TBK1 mRNA circularization and eIF4F initiation complex assembly by enhancing hnRNPA2B1–eIF4G1 interaction. Moreover, DHCR24 directly anchored to TBK1 mRNA 3′-untranslated region to increase its stability, thus forming a feed forward loop to elevate TBK1 expression. TBK1 activated PI3K/Akt signaling to promote VEGFC secretion, resulting in lymphangiogenesis and LN metastasis. DHCR24 silencing significantly impeded bladder cancer lymphangiogenesis and lymphatic metastasis in a patient-derived xenograft model. Collectively, these findings elucidate DHCR24-mediated translation machinery that promotes lymphatic metastasis of bladder cancer and supports the potential application of DHCR24-targeted therapy for LN-metastatic bladder cancer.

Significance:

DHCR24 is a SUMOylation regulator that controls translation initiation complex assembly and orchestrates TBK1 mRNA circularization to activate Akt/VEGFC signaling, which stimulates lymphangiogenesis and promotes lymph node metastasis in bladder cancer.

Lymph node (LN) metastasis is the common metastatic route for most solid tumors, which informs the treatment options and serves as an established prognostic indicator for patients with cancers (1–3). Over the past decade, extensive studies have reported that the uncontrolled synthesis of oncogenic proteins is a pivotal driver that stimulates cancer LN metastasis by sustaining the overactivation of oncogenic signaling pathways (4–7). However, protein synthesis is a highly conserved and orderly process from RNA transcription to translation under physiological conditions (6, 8). Thus, it is of fundamental importance to develop promising therapeutic targets for LN-metastatic cancers to fully understand the molecular mechanism of aberrant oncogenic protein expression.

Translational control is crucial for fine-tuning gene expression, especially in translation initiation complex assembly, dysregulation of which is the rate-limiting step by which translation reprogramming promotes cancer metastasis (4, 9). The functional protein complex-mediated interaction between the 5′ m7G cap and the 3′ poly (A) tail plays a pivotal role in mRNA circularization and translation initiation (10). Physiologically, the direct binding between poly (A)-binding protein cytoplasmic 1 (PABPC1) in the 3′ poly (A) tail and eukaryotic initiation factor 4G1 (eIF4G1) in the 5′ m7G cap is the main mechanism facilitating mRNA looping and driving the assembly of the translation initiation complex (5, 11, 12). However, the precise mediator participating in oncogene translation initiation to accelerate oncoprotein synthesis and stimulate tumor LN metastasis has not yet been reported.

Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is a crucial RNA-binding protein that plays an important role in enhancing or inhibiting the translation of a variety of RNAs (13). The quite different effects of structurally conserved hnRNPA2B1 in targeting the mRNA translation process mainly depend on its incorporated regulatory elements, and the molecular mechanism mediating the accessibility of hnRNPA2B1 to different regulatory elements is under extensive exploration (14–16). The emergence of proteomics technologies has revealed that the majority of hnRNPA2B1 presents distinct posttranslational modifications (PTM) to acquire functional diversity during cancer progression, among which, SUMOylation is a common and essential PTM governing the precise recognition of hnRNPA2B1 with specific elements to induce cancer metastasis (15, 16). However, whether and how SUMOylation is involved in hnRNPA2B1-mediated oncogene translation control to promote cancer LN metastasis remain unknown.

In the present study, we identified an intriguing protein, DHCR24, that promotes lymphangiogenesis and LN metastasis in bladder cancer by driving mRNA translation initiation. Mechanistically, DHCR24 facilitated the preferential conjugation of hnRNPA2B1 with the SUMO2 isoform via UBC9 overactivation to mediate TANK-binding kinase 1 (TBK1) mRNA looping and eIF4F initiation complex assembly by enhancing the hnRNPA2B1–eIF4G1 interaction. Moreover, DHCR24 directly anchored to the 3’-untranslated region (3’-UTR) of TBK1 mRNA and increased TBK1 mRNA stability, thus constituting a feed forward loop to upregulate TBK1 expression. The induced expression of TBK1 via the DHCR24-induced feed forward loop activated the PI3K/Akt signaling pathway to promote the secretion of VEGFC, resulting in the lymphangiogenesis and LN metastasis of bladder cancer. These findings uncover a novel mechanism of DHCR24 in inducing SUMO2 modification of hnRNPA2B1 and clarify its specific role in mediating TBK1 mRNA translation initiation, highlighting that DHCR24 might be a therapeutic target in bladder cancer lymphatic metastasis.

Clinical samples

In the present study, a total of 296 pairs of tumor tissues and normal adjacent tissues (NAT) derived from patients with bladder cancer (containing 218 males and 78 females) and the corresponding clinical information were harvested at Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University (Guangzhou, China) for RNA and protein extraction, IHC, and statistical analysis. The histologic and pathologic types of each clinical sample were identified by three experienced pathologists. Collection of each specimen was performed with the written informed consent of patients and was approved by the Ethics Committees of Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University (Guangzhou, China).

Cell lines

Human bladder cancer cell lines, including T24(ATCC Cat# HTB-4, RRID: CVCL_0554), UM-UC-3 (ATCC, Cat# CRL-1749, RRID: CVCL_1783), 5637 (ATCC Cat# HTB-9, RRID: CVCL_0126), and human normal bladder epithelial cells (SV-HUC-1, ATCC Cat# CRL-9520, RRID: CVCL_3798) were purchased from the ATCC, and human lymphatic endothelial cells (HLEC) were purchased from ScienCell Research Laboratories.

Experimental animals

Four- to six-week-old female nude mice and NOD/SCID/IL2rγ-null (NCG) mice were purchased and kept in the animal center of Sun Yat-sen University, with the approval of the Institutional Animal Care and Use Committee (IACUC), Sun Yat-Sen University, for the duration of the experiment.

Cell culture

T24 and 5637 cells were cultured in RPMI-1640 (Invitrogen), UM-UC-3 cells were cultured in DMEM (Invitrogen), SV-HUC-1 cells were cultured in F-12K medium (HyClone), and HLECs were cultured in endothelial cell medium (ScienCell Research Laboratories). The T24, UM-UC-3, 5637, and SV-HUC-1 media were supplemented with 10% FBS (Gibco), whereas the HLEC medium was supplemented with 5% FBS and 1% matched growth factor (ScienCell Research Laboratories). The cell lines mentioned above were cultured in a humidified incubator at 37°C with 5% CO2. All cell lines were authenticated by STR DNA profiling and tested for Mycoplasma contamination.

High-throughput sequencing

To investigate the downstream target genes of DHCR24, the high-throughput sequencing was conducted in three pair DHCR24-overexpressing UM-UC-3 cells and the control cells. Total RNA was extracted with TRIzol Reagent (Invitrogen). The mRNA libraries were constructed and sequenced on a Hiseq4000 platform by Gene Denovo Biotechnology Co., Ltd.

Popliteal lymphatic metastasis assay

The animal assay was approved by the Sun Yat-sen University IACUC and performed according to the institutional guidelines. Female BALB/c nude mice (4–5-weeks-old, 18–20 g) were purchased from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Then, a 30 μL suspension with a total of 5 × 105 of the indicated bladder cancer cells was implanted into the footpads of mice. Subsequently, the mice were imaged weekly with an in vivo imaging system (IVIS; Xenogen Corporation) to monitor lymphatic metastasis. The primary footpad tumors and popliteal LNs were resected when the tumors reached approximately 200 mm3 and subjected to IHC. All of the operations or scanning of the mice were performed after anesthetizing the mice with pentobarbital.

Polysome profiling

Bladder cancer cells (1 × 107) were treated with chloramphenicol (100 mg/mL) for 15 minutes and then lysed in polymeric lysate supplemented with DTT (MAP9802, MesGen Biotechnology) on ice for 15 minutes. After that, the lysate was centrifuged at 16,000 × g at 4°C to remove the nucleus and mitochondria. Then, a 5%–50% sucrose gradient solution was added to the supernatant, which was centrifuged at 20,000 × g at 4°C for 2 hours. Finally, the absorbance at 260 nm of each group was measured using a BioComp Piston Gradient Fraction, and the total RNA was extracted using TRIzol (Takara). The level of the TBK1 mRNA in each component was measured using qRT‒PCR analysis.

MS2 tethering assay

The MS2 tethering assay is based on the high affinity of the MS2 protein and MS2-binding site (17). A dual luciferase reporter vector was constructed as previously described, and the assays were conducted under the guidance of the manufacturer's instructions (Promega, Cat# E1910). The relative RLuc luciferase activity and relative RLuc mRNA were calculated by the RLuc activity/FLuc activity and RLuc mRNA/FLuc mRNA ratios, respectively. Then, the translation efficiency was measured as the relative RLuc activity/relative RLuc mRNA ratio.

Proximity ligation assay

A proximity ligation assay (PLA) was used to detect and visualize hnRNPA2B1 and DHCR24 or eIF4G1 interactions in bladder cancer cells. Briefly, bladder cancer cells were seeded in a confocal dish a day prior and then fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, the PLA assay was conducted following the instructions from the DUOLINK: PLA kit (Sigma‒Aldrich) by using antibodies against hnRNPA2B1, DHCR24, and eIF4G1. The images were captured by laser scanning confocal microscopy (Zeiss).

Cap-association assay with m7GTP-agarose

To investigate the association of DHCR24-mediated hnRNPA2B1 SUMOylation and cap-binding protein complex assembly, we performed a cap-association assay with m7GTP-agarose. Briefly, the protein lysates of UM-UC-3 cells with different treatments were incubated with m7GTP-agarose (Jena Bioscience, AC-155S) for 2 hours at 4°C. Then, the proteins bound to m7GTP-agarose were washed off and analyzed by Western blot assays.

Construction of the patient-derived xenograft model

The patient-derived xenograft (PDX) model was constructed with the approval of the Sun Yat-sen University IACUC and according to the institutional guidelines. Briefly, fresh bladder cancer tissue was surgically implanted subcutaneously into 4-week-old female NOD/SCID/IL2rγ-null (NCG) mice (first-generation, F1, Yaokang Biotechnology Co., Ltd.). When the tumors grew to 400 mm3, the tumors were excised and implanted into second-generation mice (F2). Subsequently, the tumor tissues from F2 were used to construct third-generation mice (F3). When the tumor volume of F3 reached 200 mm3, the mice were randomly divided into two groups and injected with in vivo–optimized lentivirus downregulating DHCR24 (sh-DHCR24) and the control lentivirus (sh-NC). The tumor volume was monitored every 3 days, and the tumors were excised after 36 days for further analysis. All of the operations or scanning of the mice were performed after anesthetizing the mice with pentobarbital.

Bioinformatic analysis

The expression of DHCR24 in human cancers and its association with the prognosis of patients were investigated through The Cancer Genome Atlas (TCGA) database at GEPIA (http://gepia.cancer-pku.cn/index.html). The precise SUMOylation site on hnRNPA2B1 was predicted at GPS-SUMO (http://sumosp.biocuckoo.org). The 3D structures of hnRNPA2B1, DHCR24, and eIF4G1 were modeled using SWISS-MODEL (https://swissmodel.expasy.org/). The interaction of hnRNPA2B1 and eIF4G1 was predicted on ZDOCK (https://zdock.umassmed.edu/).

Further applied methods

The detailed methods are available in the Supplementary Materials and Methods.

Statistical analyses

All quantitative data are presented as the mean ± standard deviation of three independent experiments. The detailed methods of statistical analysis for each experiment are described in the corresponding figure legends. The threshold for statistical significance was set at a P value of <0.05. All statistical tests were performed using the Statistical Package for the Social Sciences (SPSS) 13 (SPSS Inc.).

Data availability

The sequence data generated in this study are publicly available in GEO at GSE219211. The data analyzed in this study are available from GEO at GSE106534 and from the bladder urothelial carcinoma dataset in TCGA database at https://portal.gdc.cancer.gov/. All raw data generated in the present study are available upon request from the corresponding author.

DHCR24 is positively correlated with LN metastasis of bladder cancer

To identify the crucial oncogene–driving bladder cancer LN metastasis, high-throughput RNA sequencing (RNA-seq) was conducted on five paired bladder cancer tissues and NATs and on another five paired LN-positive and LN-negative bladder cancer tissues (Gene Expression Omnibus ID GSE106534). As shown in Fig. 1A, compared with NATs, bladder cancer tissues exhibited 49 protein-coding genes that were upregulated and 43 that were downregulated by more than 10-fold (fold change > 10, P < 0.01). Compared with LN-negative bladder cancer tissues, LN-positive bladder cancer tissues exhibited 30 protein-coding genes that were upregulated and 23 that were downregulated by more than 10-fold (fold change > 10, P < 0.01). These differentially expressed genes were intersected with the upregulated genes in bladder cancer tissues compared with NATs and in LN-positive compared with LN-negative bladder cancer tissues from TCGA database to reveal that three genes, including DHCR24, SPP1, and PRSS8, were consistently upregulated in both the bladder cancer tissues and LN-positive samples from our high-throughput RNA-sequencing results and the TCGA database (Fig. 1B). Further validation in our large clinical cohort consisting of 296 cases of bladder cancer by qRT‒PCR analysis revealed that DHCR24 was significantly overexpressed in bladder cancer tissues compared with NATs (Fig. 1C). Moreover, the overexpression of DHCR24 in bladder cancer tissues compared with NATs was also confirmed at the protein level by Western blot analysis in 40 paired bladder cancer and NATs (Fig. 1D and E). Notably, Kaplan‒Meier curve analysis demonstrated that DHCR24 overexpression was associated with shorter disease-free survival (DFS) and overall survival (OS) in patients with bladder cancer (Fig. 1F and G). Univariate and multivariate Cox proportional hazards analyses showed that DHCR24 was an independent prognostic factor for the OS and DFS of patients with bladder cancer (Supplementary Tables S1–S3). Consistently, analysis of the TCGA database revealed that DHCR24 was upregulated in multiple human cancers and associated with the poor prognosis of patients, including bladder cancer (Fig. 1H and I; Supplementary Fig. S1A–S1R).

Figure 1.

DHCR24 positively correlates with LN metastasis of bladder cancer. A, Heat map of protein-coding genes differentially expressed in bladder cancer tissues and NATs and in bladder cancer tissues with or without LN metastasis. B, Schematic illustration of the screening process of co-upregulated protein-coding genes in both bladder cancer tissues and LN-positive bladder cancer tissues and the TCGA database. C, qRT‒PCR analysis of DHCR24 expression in bladder cancer tissues and NATs (n = 296). D, Representative Western blot images of DHCR24 expression in bladder cancer tissues and NATs (n = 40). E, Quantification of DHCR24 expression by Western blot analysis in 40 paired bladder cancer and NATs. F and G, Kaplan‒Meier survival analysis of the DFS (F) and OS (G) of patients with bladder cancer with low versus high DHCR24 expression. The cutoff value is the median. H, Analysis of DHCR24 expression in bladder cancer tissues and NATs from the TCGA database. I, Kaplan‒Meier survival analysis of the OS of patients with bladder cancer with low versus high DHCR24 expression from the TCGA database. The cutoff value is the median. J, qRT‒PCR analysis of DHCR24 expression in bladder cancer tissues with or without LN metastasis (n = 296). K, Analysis of DHCR24 expression in bladder cancer tissues with or without LN metastasis from the TCGA database. L, Representative IHC images and quantification of DHCR24 expression in NATs and LN-negative and LN-positive bladder cancer tissues. Scale bars, 50 μm. H&E, hematoxylin and eosin. M and N, Representative IHC images and quantification of DHCR24 expression and LYVE1–indicated lymphatic vessels in peritumoral (M) and intratumoral (N) regions of bladder cancer tissues. Scale bars, 50 μm. Significant differences were assessed through the nonparametric Mann–Whitney U test in C, E, H, J, and K and the χ2 test in L and N. *, P < 0.05; **, P < 0.01. BCa, bladder cancer.

Figure 1.

DHCR24 positively correlates with LN metastasis of bladder cancer. A, Heat map of protein-coding genes differentially expressed in bladder cancer tissues and NATs and in bladder cancer tissues with or without LN metastasis. B, Schematic illustration of the screening process of co-upregulated protein-coding genes in both bladder cancer tissues and LN-positive bladder cancer tissues and the TCGA database. C, qRT‒PCR analysis of DHCR24 expression in bladder cancer tissues and NATs (n = 296). D, Representative Western blot images of DHCR24 expression in bladder cancer tissues and NATs (n = 40). E, Quantification of DHCR24 expression by Western blot analysis in 40 paired bladder cancer and NATs. F and G, Kaplan‒Meier survival analysis of the DFS (F) and OS (G) of patients with bladder cancer with low versus high DHCR24 expression. The cutoff value is the median. H, Analysis of DHCR24 expression in bladder cancer tissues and NATs from the TCGA database. I, Kaplan‒Meier survival analysis of the OS of patients with bladder cancer with low versus high DHCR24 expression from the TCGA database. The cutoff value is the median. J, qRT‒PCR analysis of DHCR24 expression in bladder cancer tissues with or without LN metastasis (n = 296). K, Analysis of DHCR24 expression in bladder cancer tissues with or without LN metastasis from the TCGA database. L, Representative IHC images and quantification of DHCR24 expression in NATs and LN-negative and LN-positive bladder cancer tissues. Scale bars, 50 μm. H&E, hematoxylin and eosin. M and N, Representative IHC images and quantification of DHCR24 expression and LYVE1–indicated lymphatic vessels in peritumoral (M) and intratumoral (N) regions of bladder cancer tissues. Scale bars, 50 μm. Significant differences were assessed through the nonparametric Mann–Whitney U test in C, E, H, J, and K and the χ2 test in L and N. *, P < 0.05; **, P < 0.01. BCa, bladder cancer.

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Furthermore, statistical analysis revealed that LN-positive bladder cancer tissues possessed higher DHCR24 expression than LN-negative bladder cancer tissues in our large clinical cohort (Fig. 1J). Importantly, validation in another bladder cancer cohort from the TCGA database also revealed that DHCR24 was overexpressed in LN-positive bladder cancer tissues compared with LN-negative bladder cancer tissues (Fig. 1K). Consistently, IHC revealed that DHCR24 was significantly overexpressed in bladder cancer with LN metastasis compared with those without LN metastasis (Fig. 1L). DHCR24 overexpression was accompanied by an increased density of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)–marked microlymphatic vessels in both intratumoral and peritumoral regions (Fig. 1M and N), indicating that DHCR24 plays an important role in LN metastasis and lymphangiogenesis in bladder cancer. Collectively, these results suggest that DHCR24 is closely related to LN metastasis of bladder cancer.

DHCR24 promotes lymphangiogenesis of bladder cancer in vitro

Because lymphangiogenesis is the rate-limiting step of tumor LN metastasis (18), we explored the biological function of DHCR24 in the regulation of bladder cancer lymphangiogenesis in vitro. First, the expression of DHCR24 in various bladder cancer cell lines was evaluated to reveal that UM-UC-3, T24, and 5637 cells possessed a higher level of DHCR24 than SV-HUC-1 cells (Supplementary Fig. S2A and S2B). Considering that the bladder cancer cell lines UM-UC-3 and T24 possess highly invasive and metastatic properties, we selected UM-UC-3 and T24 to detect the effect of DHCR24 in bladder cancer in vitro. The expression of DHCR24 in UM-UC-3 and T24 cells was downregulated or upregulated by transfection with siRNA-targeting DHCR24 or an overexpression plasmid in bladder cancer cells, respectively (Supplementary Fig. S2C–S2F). Then, culture media (CM) derived from equal DHCR24-overexpressing or DHCR24-knockdown bladder cancer cells and control bladder cancer cells were harvested to treat HLECs. As shown in Fig. 2A; Supplementary Fig. S2G, the tube formation and migration of HLECs were markedly impaired after incubation with CMs from DHCR24-knockdown UM-UC-3 and T24 cells compared with the controls. Conversely, treatment with CMs from DHCR24-overexpressing UM-UC-3 and T24 cells markedly enhanced the tube formation and migration of HLECs compared with those treated with CMs from UM-UC-3 and T24 cells transfected with empty vector plasmid (Fig. 2B; Supplementary Fig. S2H), suggesting that DHCR24 significantly enhances the lymphangiogenesis of bladder cancer. LN metastasis in tumors is a fine-tuning and multiple process mediated by various factors. In addition to tumor cells inducing the generation of new lymphatic vessels, the alteration of tumor cell invasive properties plays a distinct role in triggering tumor LN metastasis (19). Thus, we performed Transwell assays to evaluate the role of DHCR24 in regulating the invasiveness of bladder cancer cells. Invasion and migration of UM-UC-3 and T24 cells were inhibited after knocking down DHCR24, whereas overexpressing DHCR24 significantly promoted the invasion and migration of bladder cancer cells (Supplementary Fig. S2I–S2T). Together, these results demonstrate that DHCR24 promotes the lymphangiogenesis of bladder cancer in vitro.

Figure 2.

DHCR24 promotes lymphangiogenesis and LN metastasis of bladder cancer in vitro and in vivo. A and B, Representative images and quantification of tube formation and migration of HLECs treated with culture media from DHCR24 knockdown or -overexpressing UM-UC-3 cells. Scale bars, 100 μm. C and D, Schematic diagram of the construction of a nude mouse popliteal LN metastasis model. E and F, Representative images and quantification of bioluminescence of the popliteal metastatic LNs (n = 12). Red arrows, footpad tumor and metastatic popliteal LN. G, Representative images and bioluminescence of the popliteal LNs from mice (n = 12). H, Representative IHC images of anti-GFP analysis in the popliteal LNs from mice (n = 12). Red scale bars, 500 μm; black scale bars, 50 μm. I, The table shows the popliteal LN-metastatic rate in different groups (n = 12). J and K, Representative IHC images and quantification of DHCR24 expression and LYVE1–indicated lymphatic vessel density in the intratumoral (J) and peritumoral (K) regions of primary footpad tumor tissues. Scale bar, 50 μm. Significant differences were assessed through one-way ANOVA, followed by the Dunnett test in A; the χ2 test in I; and the two-tailed Student t test in B, F, J, and K. Error bars, SD. *, P < 0.05; **, P < 0.01. H&E, hematoxylin and eosin.

Figure 2.

DHCR24 promotes lymphangiogenesis and LN metastasis of bladder cancer in vitro and in vivo. A and B, Representative images and quantification of tube formation and migration of HLECs treated with culture media from DHCR24 knockdown or -overexpressing UM-UC-3 cells. Scale bars, 100 μm. C and D, Schematic diagram of the construction of a nude mouse popliteal LN metastasis model. E and F, Representative images and quantification of bioluminescence of the popliteal metastatic LNs (n = 12). Red arrows, footpad tumor and metastatic popliteal LN. G, Representative images and bioluminescence of the popliteal LNs from mice (n = 12). H, Representative IHC images of anti-GFP analysis in the popliteal LNs from mice (n = 12). Red scale bars, 500 μm; black scale bars, 50 μm. I, The table shows the popliteal LN-metastatic rate in different groups (n = 12). J and K, Representative IHC images and quantification of DHCR24 expression and LYVE1–indicated lymphatic vessel density in the intratumoral (J) and peritumoral (K) regions of primary footpad tumor tissues. Scale bar, 50 μm. Significant differences were assessed through one-way ANOVA, followed by the Dunnett test in A; the χ2 test in I; and the two-tailed Student t test in B, F, J, and K. Error bars, SD. *, P < 0.05; **, P < 0.01. H&E, hematoxylin and eosin.

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DHCR24 facilitates LN metastasis of bladder cancer in vivo

To evaluate the effect of DHCR24 on the LN metastasis of bladder cancer in vivo, a footpad–popliteal LN metastasis model was constructed. In the footpad–popliteal LN metastasis model, tumor cells in the mouse footpad metastasize to the popliteal LN and subsequently to the internal and external iliac LNs along their lymphatic drainage pathway, which aptly emulates the biological behavior of lymphatic metastasis in bladder cancer and enhances the sensitivity of in vivo assessments of lymphatic metastasis (20). GFP-labeled DHCR24-overexpressing or DHCR24-silenced UM-UC-3 cells were equally injected into the footpads of nude mice as described previously (7). Then, the LN-metastatic status was measured weekly, and the tissues and popliteal LNs were resected and subjected to IHC (Fig. 2C and D). The IVIS results showed that the fluorescence intensity in popliteal LNs was markedly increased in the DHCR24-overexpressing group compared with the control group but decreased in the DHCR24-silenced group (Fig. 2EG; Supplementary Fig. S3A), indicating that DHCR24 prominently facilitates the metastasis of primary tumor cells to the popliteal LNs. In addition, IHC showed that overexpressing DHCR24 significantly increased the popliteal LN-metastatic rate of mice compared with the control group but rarely affected pulmonary or liver metastasis (Fig. 2H and I; Supplementary Fig. S3B–S3D). Consistently, silencing DHCR24 reduced the LN-metastatic rate compared with that of the control group (Supplementary Fig. S3E). Importantly, the density of LYVE1–indicated lymphatic vessels was increased in the DHCR24-overexpressing group but decreased in the DHCR24-silenced group compared with the control group in both the intratumoral and peritumoral regions (Fig. 2J and K; Supplementary Fig. S3F and S3G), suggesting that DHCR24 induces lymphangiogenesis in bladder cancer. DHCR24 overexpression exhibited little effect on Ki67 expression in primary tumor tissues compared with the control group (Supplementary Fig. S3H and S3I). Considering that hematogenous dissemination could be the potential dissemination path for tumors (21, 22), we constructed a tail vein injection mouse model to detect the effect of DHCR24 on hematogenous dissemination. GFP-labeled DHCR24-overexpressing bladder cancer cells or control cells were equally injected into the tail veins of mice. The IVIS results showed that compared with the control group, DHCR24 overexpression rarely affected the metastasis of bladder cancer cells to the lungs of mice (Supplementary Fig. S3J and S3K). No significant difference in the metastasis ratio or number of metastatic foci was observed between the DHCR24 overexpression and control groups (Supplementary Fig. S3 L and S3M), indicating that DHCR24 exhibits little effect on the distant metastasis of bladder cancer through hematogenous dissemination. Collectively, our results suggest that DHCR24 facilitates the lymphangiogenesis and LN metastasis of bladder cancer in vivo.

DHCR24 induces SUMO2 modification on lysine 108 (K108) of hnRNPA2B1

Given that subcellular location largely determines the biological function of proteins (23), we conducted immunofluorescence analysis and subcellular fractionation assays to investigate the cellular localization of DHCR24 in bladder cancer cells. As shown in Fig. 3A and B, DHCR24 was predominantly localized in the cytoplasm of UM-UC-3 and T24 cells. Previous studies have reported that cytoplasmic proteins usually exert their biological effect via the formation of protein complexes (24). Therefore, coimmunoprecipitation (co-IP) assays were conducted to investigate the crucial proteins that interact with DHCR24. The results of silver staining revealed that DHCR24 enriched an apparent band with a molecular size of 40–55 kDa, which was subjected to mass spectrometry analysis and validated as hnRNPA2B1 (Fig. 3C and D; Supplementary Fig. S4A–S4F). Western blot analysis confirmed the interaction between DHCR24 and hnRNPA2B1 in UM-UC-3 cells (Fig. 3E). Moreover, confocal microscopy analysis revealed that DHCR24 and hnRNPA2B1 were mainly colocalized in the cytoplasm of UM-UC-3 and T24 cells (Fig. 3F). PLA between DHCR24 and hnRNPA2B1 in UM-UC-3 and T24 cells further verified the interaction between DHCR24 and hnRNPA2B1 (Fig. 3G; Supplementary Fig. S5A), indicating that DHCR24 might interact with hnRNPA2B1 to function in bladder cancer.

Figure 3.

DHCR24 induces SUMO2 modification at K108 of hnRNPA2B1. A and B, Detection of the intracellular localization of DHCR24 in bladder cancer cells. Scale bars, 5 μm. C and D, Silver staining and mass spectrometry analysis for the detection of DHCR24-interacting proteins. E, Western blot analysis after co-IP assays with anti-DHCR24 or IgG in UM-UC-3 cells. F, Immunofluorescence assays for the colocalization of DHCR24 and hnRNPA2B1 in bladder cancer cells. Scale bars, 5 μm. G, PLA showing the interaction between hnRNPA2B1 and DHCR24 in UM-UC-3. Scale bars, 5 μm. H, Co-IP assays with DHCR24 to detect the PTM type of hnRNPA2B1 involved in the DHCR24 and hnRNPA2B1 interaction. I, Western blot analysis for the investigation of the SUMOylation type of hnRNPA2B1 in UM-UC-3 cells. J, Western blot analysis for the validation of SUMO2 modification on hnRNPA2B1. K, Western blot analysis to evaluate the effect of DHCR24 on SUMO2 modification on hnRNPA2B1 L. Western blot analysis to confirm the SUMO2 modification site on hnRNPA2B1. M, Western blotting analysis of SUMO2 modification on hnRNPA2B1 in indicated UM-UC-3 cells. N and O, qRT‒PCR of SUMOylation-related enzyme expression in the indicated UM-UC-3 cells. P, Detection of the interaction between DHCR24 and hnRNPA2B1 after hnRNPA2B1K108R mutations or using SENP3 in the indicated cells. Q, Western blot analysis to investigate the interaction between DHCR24 and hnRNPA2B1 after SIM mutation of DHCR24 in the indicated cells. Significant differences were assessed through the two-tailed Student t test in N and one-way ANOVA, followed by the Dunnett test in O. Error bars, SD. *, P < 0.05; **, P < 0.01.

Figure 3.

DHCR24 induces SUMO2 modification at K108 of hnRNPA2B1. A and B, Detection of the intracellular localization of DHCR24 in bladder cancer cells. Scale bars, 5 μm. C and D, Silver staining and mass spectrometry analysis for the detection of DHCR24-interacting proteins. E, Western blot analysis after co-IP assays with anti-DHCR24 or IgG in UM-UC-3 cells. F, Immunofluorescence assays for the colocalization of DHCR24 and hnRNPA2B1 in bladder cancer cells. Scale bars, 5 μm. G, PLA showing the interaction between hnRNPA2B1 and DHCR24 in UM-UC-3. Scale bars, 5 μm. H, Co-IP assays with DHCR24 to detect the PTM type of hnRNPA2B1 involved in the DHCR24 and hnRNPA2B1 interaction. I, Western blot analysis for the investigation of the SUMOylation type of hnRNPA2B1 in UM-UC-3 cells. J, Western blot analysis for the validation of SUMO2 modification on hnRNPA2B1. K, Western blot analysis to evaluate the effect of DHCR24 on SUMO2 modification on hnRNPA2B1 L. Western blot analysis to confirm the SUMO2 modification site on hnRNPA2B1. M, Western blotting analysis of SUMO2 modification on hnRNPA2B1 in indicated UM-UC-3 cells. N and O, qRT‒PCR of SUMOylation-related enzyme expression in the indicated UM-UC-3 cells. P, Detection of the interaction between DHCR24 and hnRNPA2B1 after hnRNPA2B1K108R mutations or using SENP3 in the indicated cells. Q, Western blot analysis to investigate the interaction between DHCR24 and hnRNPA2B1 after SIM mutation of DHCR24 in the indicated cells. Significant differences were assessed through the two-tailed Student t test in N and one-way ANOVA, followed by the Dunnett test in O. Error bars, SD. *, P < 0.05; **, P < 0.01.

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Importantly, we found that DHCR24-immunoprecipitated hnRNPA2B1 exhibited a higher molecular weight (>40 kDa) than the input component (<40 kDa; Fig. 3E), indicating that DHCR24-bound hnRNPA2B1 presented with PTM. Subsequently, a series of inhibitors targeting the different PTMs were used to explore the specific PTM type on DHCR24-bound hnRNPA2B1 in bladder cancer cells. Only treatment with 2-D08, a specific inhibitor of SUMOylation, markedly inhibited the interaction between DHCR24 and hnRNPA2B1 (Fig. 3H), suggesting that DHCR24-bound hnRNPA2B1 is SUMOylated in bladder cancer cells. To investigate the specific SUMOylation type of hnRNPA2B1, co-IP assays with an anti-His antibody were performed in His-tagged SUMO1-, SUMO2-, and SUMO3-overexpressing plasmid-transfected UM-UC-3 cells. Anti-His only enriched hnRNPA2B1 in His-SUMO2–overexpressing UM-UC-3 cells (Fig. 3I). Moreover, Western blot analysis after co-IP assays with anti-hnRNPA2B1 showed an obvious band of SUMO2 (Fig. 3J), confirming that hnRNPA2B1 is SUMOylated with SUMO2 in UM-UC-3 cells. Overexpression of DHCR24 significantly promoted the SUMO2 conjugation of hnRNPA2B1 (Fig. 3K), indicating that SUMOylation of hnRNPA2B1 is mediated by DHCR24. Because the modification residues are crucial for the biological effect of SUMOylation on its target proteins, we then used GPS-SUMO (25), a web tool for predicting the SUMOylation site on proteins, to identify two potential SUMO modification sites on hnRNPA2B1: K108 and lysine 125 (K125; Supplementary Fig. S5B). These potential lysine residues were substituted with arginine (R; hnRNPA2B1K108R, hnRNPA2B1K125R) and subjected to co-IP assays (Supplementary Fig. S5C). As shown in Fig. 3L, hnRNPA2B1K108R, rather than hnRNPA2B1K125R, significantly decreased the SUMO2 modification level of hnRNPA2B1, supporting that hnRNPA2B1 is SUMOylated on the K108 residue. Notably, we demonstrated that DHCR24 overexpression promoted SUMO2 modification on hnRNPA2B1, which was markedly attenuated by hnRNPA2B1K108R mutation (Fig. 3M), indicating that DHCR24 promotes SUMO2 modification at the K108 residue on hnRNPA2B1. Because the conjugation with SUMOs represents the consequence of a cascade of enzymatic reactions (26), we further investigated the essential SUMOylation-associated enzymes contributing to DHCR24-induced hnRNPA2B1 SUMOylation. qRT-PCR and Western blot analysis revealed that among the various SUMOylation-associated enzymes, ubiquitin carrier protein 9 (UBC9), the single E2 SUMO-conjugating enzyme for SUMO modification (27, 28), was most significantly decreased after knocking down DHCR24 in bladder cancer cells, while overexpressing DHCR24 enhanced the expression of UBC9 (Fig. 3N and O; Supplementary Fig. S5D and S5E). We further performed dual-luciferase assays to determine the potential mechanism of DHCR24 in promoting UBC9 expression. The results revealed that DHCR24 overexpression showed little effect on the UBC9 promoter region, whereas a significant increase in luciferase activity was observed on the 3′-UTR of UBC9 mRNA (Supplementary Fig. S5F and S5G), suggesting that DHCR24 may affect UBC9 expression through the 3′-UTR rather than affecting the promoter region of UBC9. It has been well established that the 3′-UTR is important for mRNA stability in cells (29). Actinomycin D assays showed that overexpressing DHCR24 significantly prolonged the half-life of UBC9 mRNA, whereas downregulating DHCR24 shortened the half-life of UBC9 mRNA (Supplementary Fig. S5H and S5I), confirming that DHCR24 promotes UBC9 expression by increasing UBC9 mRNA stability. To explore the underlying mechanism, RNA immunoprecipitation (RIP) assays were conducted and revealed that DHCR24 directly interacted with UBC9 mRNA (Supplementary Fig. S5J). Moreover, the interaction site of DHCR24 on UBC9 mRNA was further predicted to localize in the 992–1081 nt region on the 3′-UTR of UBC9 mRNA (Supplementary Fig. S5K). Mutating these sequences dramatically abolished the binding between DHCR24 and UBC9 mRNA (Supplementary Fig. S5L). Importantly, mutation of DHCR24-binding sequences on the 3′-UTR of UBC9 mRNA significantly decreased the ability of DHCR24 to induce luciferase activity on the 3′-UTR of UBC9 mRNA and to promote the stability of UBC9 mRNA (Supplementary Fig. S5M and S5N), indicating that DHCR24 upregulates UBC9 expression by interacting with the 992–1081 nt region on the 3′-UTR of UBC9 mRNA and enhancing its stability. In addition, we demonstrated that DHCR24 overexpression promoted the SUMOylation of hnRNPA2B1, which was impaired by knocking down UBC9 (Supplementary Fig. S5O), indicating that DHCR24 upregulates UBC9 expression to induce the SUMOylation of hnRNPA2B1.

Intriguingly, we found that mutation of the K108 residue on hnRNPA2B1 or treatment with SENP3 (SUMO-specific peptidase 3), a deSUMOylase mainly targeting SUMO2 conjunction (30), significantly impaired the interaction between DHCR24 and hnRNPA2B1 (Fig. 3P), confirming that SUMO2 binding to hnRNPA2B1 is essential for the interaction between DHCR24 and hnRNPA2B1. Considering that SUMO modification enhances protein interactions through the recognition of SUMO interaction motifs (SIM) on target proteins (31), we further evaluated whether SUMOylated hnRNPA2B1 binds to DHCR24 by recognizing SIMs on DHCR24. Prediction by GPS-SUMO and an automatic protein docking server, ZDOCK Server (32), revealed that DHCR24 possessed a SIM (amino acid sequence: LLPLSLIFD) docked with the SUMO2 modification region of hnRNPA2B1 (Supplementary Fig. S5P). Mutation of the SIM on DHCR24 significantly impaired the interaction between DHCR24 and SUMO2-conjugated hnRNPA2B1 (Fig. 3Q), indicating that DHCR24 recognizes the SUMO2 modification via SIM to bind with SUMOylated hnRNPA2B1. Collectively, these results demonstrate that DHCR24 induces the overexpression of UBC9 to promote SUMO2 modification at the K108 residue on hnRNPA2B1 and binds with SUMOylated hnRNPA2B1 via SIM.

DHCR24 promotes TBK1 mRNA stability and translation initiation by forming a SUMO2/hnRNPA2B1/EIF4G1 feed forward loop

To explore the molecular mechanism of DHCR24-induced hnRNPA2B1 SUMOylation in mediating the lymphangiogenesis and LN metastasis of bladder cancer, high-throughput RNA-seq was carried out in DHCR24-overexpressing bladder cancer and control cells (Gene Expression Omnibus ID GSE219211). The results showed that 44 genes were significantly upregulated and 88 were downregulated (fold change >2) in DHCR24-overexpressing bladder cancer cells compared with the controls (P < 0.01, Supplementary Fig. S5Q). Subsequently, these differentially expressed genes were subjected to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, which verified that the PI3K/Akt signaling pathway was the most significantly enriched pathway (Supplementary Fig. S5R). The expression profile of pivotal genes enriched in PI3K/Akt signaling in our sequencing data was further confirmed by qRT-PCR assays and Western blot analysis. The results revealed that TBK1 was significantly upregulated in DHCR24-overexpressing UM-UC-3 cells and downregulated after DHCR24 was knocked down (Fig. 4A and B; Supplementary Fig. S6A–S6D).

Figure 4.

DHCR24 promotes TBK1 mRNA stability and translation initiation by forming a SUMO2/hnRNPA2B1/EIF4G1 feed forward loop. A and B, qRT‒PCR analysis of the expression of the indicated genes enriched in the PI3K/Akt signaling pathway in DHCR24-knockdown or DHCR24-overexpressing UM-UC-3 cells. C, Luciferase assays showed the effect of DHCR24 overexpression on the 3′-UTR of TBK1 mRNA. D–G, Quantification and representative agarose electrophoresis images of actinomycin D assays for TBK1 mRNA in DHCR24 knockdown or DHCR24-overexpressing UM-UC-3 cells. H, RIP assay to investigate the interaction between DHCR24 and the 3′-UTR of TBK1 mRNA in UM-UC-3 cells. I, Schematic illustration of the predicted binding region for DHCR24 on the 3’-UTR of TBK1 mRNA from catRAPID analysis. J, RIP assays to investigate the interaction between DHCR24 and the 3′-UTR of TBK1 mRNA after S1 and S2 mutations in the TBK1 3′-UTR. K and L, Quantification and representative agarose electrophoresis images of actinomycin D assays for TBK1 mRNA in DHCR24-overexpressing UM-UC-3 cells with the S1 mutation in the TBK1 3′-UTR. M and N, Quantification and representative agarose electrophoresis images of actinomycin D assays for TBK1 mRNA in DHCR24-overexpressing UM-UC-3 cells with hnRNPA2B1K108R mutation or SENP3 treatment. O, Western blot analysis to investigate TBK1 expression after hnRNPA2B1K108R mutation or SENP3 treatment in DHCR24-overexpressing UM-UC-3 cells. P, Polysome profiles in DHCR24-overexpressing UM-UC-3 cells after hnRNPA2B1K108R mutation or SENP3 treatment. Q, Relative distribution of TBK1 mRNA across the polysome fractions in DHCR24-overexpressing UM-UC-3 cells after hnRNPA2B1K108R mutation or SENP3 treatment. R, Dual-luciferase reporter assays for overexpression of MS2 and MS2–hnRNPA2B1 fusion proteins in UM-UC-3 cells with or without downregulation of DHCR24 in regulating TBK1 mRNA translation. S, Western blot analysis after co-IP assays with anti-hnRNPA2B1 or IgG in UM-UC-3 cells. T, PLA showing the interaction between hnRNPA2B1 and eIF4G1. Scale bars, 5 μm. U, Western blot analysis to investigate the interaction between hnRNPA2B1 and eIF4G1 after hnRNPA2B1 or eIF4G1 sequence mutation in bladder cancer cells. V and W, m7G cap pulldown in hnRNPA2B1WT or hnRNPA2B1KO UM-UC-3 cells confirming DHCR24-mediated hnRNPA2B1 SUMOylation to enhance TBK1 mRNA circularization and eIF4F complex assembly. Significant differences were assessed through one-way ANOVA, followed by the Dunnett test in A, D, K, M, Q, and R; and two-tailed Student t test in B, C, F, H, and J. Error bars, SD. *, P < 0.05; **, P < 0.01.

Figure 4.

DHCR24 promotes TBK1 mRNA stability and translation initiation by forming a SUMO2/hnRNPA2B1/EIF4G1 feed forward loop. A and B, qRT‒PCR analysis of the expression of the indicated genes enriched in the PI3K/Akt signaling pathway in DHCR24-knockdown or DHCR24-overexpressing UM-UC-3 cells. C, Luciferase assays showed the effect of DHCR24 overexpression on the 3′-UTR of TBK1 mRNA. D–G, Quantification and representative agarose electrophoresis images of actinomycin D assays for TBK1 mRNA in DHCR24 knockdown or DHCR24-overexpressing UM-UC-3 cells. H, RIP assay to investigate the interaction between DHCR24 and the 3′-UTR of TBK1 mRNA in UM-UC-3 cells. I, Schematic illustration of the predicted binding region for DHCR24 on the 3’-UTR of TBK1 mRNA from catRAPID analysis. J, RIP assays to investigate the interaction between DHCR24 and the 3′-UTR of TBK1 mRNA after S1 and S2 mutations in the TBK1 3′-UTR. K and L, Quantification and representative agarose electrophoresis images of actinomycin D assays for TBK1 mRNA in DHCR24-overexpressing UM-UC-3 cells with the S1 mutation in the TBK1 3′-UTR. M and N, Quantification and representative agarose electrophoresis images of actinomycin D assays for TBK1 mRNA in DHCR24-overexpressing UM-UC-3 cells with hnRNPA2B1K108R mutation or SENP3 treatment. O, Western blot analysis to investigate TBK1 expression after hnRNPA2B1K108R mutation or SENP3 treatment in DHCR24-overexpressing UM-UC-3 cells. P, Polysome profiles in DHCR24-overexpressing UM-UC-3 cells after hnRNPA2B1K108R mutation or SENP3 treatment. Q, Relative distribution of TBK1 mRNA across the polysome fractions in DHCR24-overexpressing UM-UC-3 cells after hnRNPA2B1K108R mutation or SENP3 treatment. R, Dual-luciferase reporter assays for overexpression of MS2 and MS2–hnRNPA2B1 fusion proteins in UM-UC-3 cells with or without downregulation of DHCR24 in regulating TBK1 mRNA translation. S, Western blot analysis after co-IP assays with anti-hnRNPA2B1 or IgG in UM-UC-3 cells. T, PLA showing the interaction between hnRNPA2B1 and eIF4G1. Scale bars, 5 μm. U, Western blot analysis to investigate the interaction between hnRNPA2B1 and eIF4G1 after hnRNPA2B1 or eIF4G1 sequence mutation in bladder cancer cells. V and W, m7G cap pulldown in hnRNPA2B1WT or hnRNPA2B1KO UM-UC-3 cells confirming DHCR24-mediated hnRNPA2B1 SUMOylation to enhance TBK1 mRNA circularization and eIF4F complex assembly. Significant differences were assessed through one-way ANOVA, followed by the Dunnett test in A, D, K, M, Q, and R; and two-tailed Student t test in B, C, F, H, and J. Error bars, SD. *, P < 0.05; **, P < 0.01.

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We next performed dual-luciferase assays to determine the mechanism by which DHCR24 promotes TBK1 expression. DHCR24 overexpression exhibited little effect on the TBK1 promoter region (Supplementary Fig. S6E and S6F), whereas strikingly increased luciferase activity was observed on the TBK1 3′-UTR (Fig. 4C; Supplementary Fig. S6G), supporting that DHCR24 may affect TBK1 expression through the 3′-UTR rather than affecting the promoter region of TBK1. We further applied the actinomycin D assay to explore the effect of DHCR24 on TBK1 mRNA stability. The results revealed that downregulation of DHCR24 remarkably shortened the half-life of TBK1 mRNA (Fig. 4D and E; Supplementary Fig. S6H and S6I), whereas DHCR24 overexpression prolonged the half-life of TBK1 mRNA (Fig. 4F and G; Supplementary Fig. S6J and S6K), suggesting that DHCR24 mediates the 3′-UTR of TBK1 mRNA to stabilize TBK1 mRNA in bladder cancer cells. Given that the direct interaction with a specific sequence of the 3′-UTR that is highly enriched with adenylate and uridylate (AU)-rich elements (ARE) is pivotal for mediating the mRNA stability of target genes (33), we conducted RIP assays to reveal that DHCR24 significantly bound to the 3′-UTR of TBK1 mRNA (Fig. 4H). Moreover, catRAPID, a tool for predicting the interaction site between proteins and RNA (34), was used to predict two potential DHCR24 binding sequences with high ARE abundance on the 3′-UTR of TBK1 mRNA (Fig. 4I). RIP assays showed that mutation of the 2457–2476 nt region, referred to as S1, on the 3′-UTR of TBK1 mRNA significantly impaired the interaction between DHCR24 and the TBK1 3′-UTR, whereas mutation of other predicted sites had no obvious effect (Fig. 4J). Moreover, mutation of the S1 region on the 3′-UTR of TBK1 mRNA abolished the ability of DHCR24 to prolong the TBK1 mRNA half-life (Fig. 4K and L), demonstrating that DHCR24 anchors the 2457–2476 nt region of the 3′-UTR in TBK1 mRNA to increase the stability of TBK1 mRNA.

SUMOylation is reported to be a crucial PTM contributing to the stability of mRNA (28, 35). The observation that DHCR24 directly interacted with hnRNPA2B1 to induce SUMO2 modification at the K108 residue of hnRNPA2B1 prompted us to hypothesize that SUMOylation of hnRNPA2B1 might be involved in DHCR24-promoted TBK1 mRNA stability. Interestingly, we found that upregulation of TBK1 mRNA stability induced by DHCR24 overexpression was rarely impaired by mutation of the K108 residue of hnRNPA2B1 or treatment with SENP3, whereas the TBK1 protein level was markedly decreased after mutation of the K108 residue of hnRNPA2B1 or treatment with SENP3 (Fig. 4MO), indicating that SUMOylated hnRNPA2B1 may affect TBK1 protein expression in a mRNA stability-independent manner. In addition to alterations in mRNA abundance or stability, the expression levels of proteins are also regulated by translation processes (4, 10–12). Thus, we conducted polysome fractionation assays to evaluate the potential effect of DHCR24-mediated SUMOylation of hnRNPA2B1 on TBK1 mRNA translation. The results showed that overexpression of DHCR24 significantly enhanced the enrichment of TBK1 mRNA in heavy polysomal fractions and decreased the association with light and free polysomal fractions, whereas inhibition of the SUMOylation of hnRNPA2B1 via mutation of the K108 residue on hnRNPA2B1 or treatment with SENP3 markedly impaired this effect (Fig. 4P and Q), indicating that DHCR24 mediates SUMO2 modification at the K108 residue of hnRNPA2B1 to promote the translation of TBK1 mRNA. Then, MS2 tethering assays were performed to assess the underlying mechanism of DHCR24-induced SUMOylation of hnRNPA2B1 in regulating the translation of TBK1 mRNA. As shown in Fig. 4R, tethering hnRNPA2B1 to the 3′-UTR of TBK1 mRNA via DHCR24-induced SUMO2 modification or MS2-hnRNPA2B1 significantly increased the ratio of luciferase reporter activity and TBK1 mRNA compared with that in the control group, confirming that DHCR24 induces the SUMOylation of hnRNPA2B1 to secure the attachment of hnRNPA2B1 to the 3′-UTR of TBK1 mRNA and enhance its translation efficiency. The functional protein complex-mediated interaction between the 5′ terminus and 3′-UTR of mRNA to promote mRNA circularization is the rate-limiting step for translation initiation, within which, eIF4G1 serves as the scaffold component of the eIF4F–m7G cap complex and is crucial for this process (4, 10–12). Therefore, we then investigated whether SUMO2-modified hnRNPA2B1 recruited by DHCR24 on the 3′-UTR of TBK1 mRNA initiated TBK1 mRNA translation efficiency by binding with eIF4G1 to cyclize TBK1 mRNA. Notably, co-IP assays showed that hnRNPA2B1 physically interacted with eIF4G1 in bladder cancer cells (Fig. 4S). The PLA further verified the interaction between hnRNPA2B1 and eIF4G1 (Fig. 4T). We then predicted the interaction region between hnRNPA2B1 and eIF4G1 through molecular docking simulations, which were confirmed by serially truncating the predicted fragments (Fig. 4U), demonstrating that hnRNPA2B1 specifically binds with eIF4G1 through the recognition of EDAFYSWES in eIF4G1 by EVRKALSR. Furthermore, m7G pulldown assays, which specifically detected the potential proteins interacting with the 5′ cap of mRNA, were performed and revealed that DHCR24 significantly bound with the m7G cap; this binding was attenuated by inhibiting the SUMOylation of hnRNPA2B1 (Fig. 4V and W), suggesting that DHCR24-induced SUMOylation of hnRNPA2B1 promotes mRNA circularization to mediate the binding of DHCR24 with the 5′ cap of mRNA. Given that mRNA circularization triggers eIF4F complex (eIF4G1/4A/4E) assembly to promote translation initiation (36), we further evaluated the effect of DHCR24-induced SUMOylation of hnRNPA2B1 on the assembly of the eIF4F complex. The results of m7G pulldown assays showed that DHCR24 overexpression markedly facilitated the assembly of the eIF4F complex, while inhibiting the SUMOylation of hnRNPA2B1 by muting its K108 residue suppressed the promotive effect of DHCR24 on eIF4F complex assembly (Fig. 4V and W), supporting the role of DHCR24 in inducing the SUMO2 modification of hnRNPA2B1 to mediate TBK1 mRNA translation initiation by stimulating TBK1 mRNA circularization and the assembly of the eIF4F complex. Interestingly, we revealed that overexpressing or knocking down DHCR24 had a limited effect on the global synthesis of proteins (Supplementary Fig. S6L–S6O), indicating that DHCR24 specifically recognizes TBK1 mRNA to promote its protein synthesis without affecting global protein synthesis. Together, these findings demonstrate that DHCR24 directly anchors to the 3′-UTR of TBK1 mRNA to increase its stability and recruits SUMOylated hnRNPA2B1 to promote TBK1 mRNA circularization and eIF4F assembly, thus forming a feed forward loop to upregulate TBK1 expression.

DHCR24 activates the TBK1/PI3K/Akt signaling pathway to promote VEGFC secretion by bladder cancer

Because TBK1 is a serine/threonine kinase that plays a central role in promoting the progression of tumors by mediating the phosphorylation of Akt (37), Western blot analysis was conducted to explore whether DHCR24 upregulates TBK1 to phosphorylate Akt in bladder cancer. As shown in Fig. 5A and B; Supplementary Fig. S7A and S7B, DHCR24 overexpression markedly increased TBK1 expression and phosphorylation of Akt in bladder cancer cells, while downregulating DHCR24 exhibited the opposite effect, suggesting that DHCR24 upregulates TBK1 expression and promotes the phosphorylation of Akt in bladder cancer.

Figure 5.

DHCR24 activates the TBK1/PI3K/Akt signaling pathway to promote VEGFC secretion by bladder cancer. A and B, Western blot analysis was performed to evaluate the expression of associated genes in the PI3K/Akt signaling pathway in DHCR24 knockdown or DHCR24-overexpressing UM-UC-3 cells. C and D. ELISA of VEGFC secretion in DHCR24 knockdown or DHCR24-overexpressing UM-UC-3 cells. E–H, qRT‒PCR analysis of VEGFC expression and ELISA analysis of VEGFC secretion in DHCR24-overexpressing UM-UC-3 or T24 cells with or without treatment with LY294002. I, Representative images and quantification of tube formation and migration of HLECs treated with CM from DHCR24-overexpressing UM-UC-3 cells with or without αVEGFC treatment. Scale bars, 100 μm. J, Quantification of bioluminescence of the popliteal metastatic LNs (n = 12) in mice of the indicated groups. K, The table shows the popliteal LN-metastatic rate in different groups (n = 12). L–N, Representative IHC images and quantification of DHCR24 and VEGFC expression and LYVE1-indicated lymphatic vessel density in footpad tumor tissues from the indicated mice. Scale bar, 50 μm. Significant differences were assessed through one-way ANOVA, followed by the Dunnett test in C, E–J, M, and N; two-tailed Student t test in D; and the χ2 test in K. Error bars , SD. *, P < 0.05; **, P < 0.01.

Figure 5.

DHCR24 activates the TBK1/PI3K/Akt signaling pathway to promote VEGFC secretion by bladder cancer. A and B, Western blot analysis was performed to evaluate the expression of associated genes in the PI3K/Akt signaling pathway in DHCR24 knockdown or DHCR24-overexpressing UM-UC-3 cells. C and D. ELISA of VEGFC secretion in DHCR24 knockdown or DHCR24-overexpressing UM-UC-3 cells. E–H, qRT‒PCR analysis of VEGFC expression and ELISA analysis of VEGFC secretion in DHCR24-overexpressing UM-UC-3 or T24 cells with or without treatment with LY294002. I, Representative images and quantification of tube formation and migration of HLECs treated with CM from DHCR24-overexpressing UM-UC-3 cells with or without αVEGFC treatment. Scale bars, 100 μm. J, Quantification of bioluminescence of the popliteal metastatic LNs (n = 12) in mice of the indicated groups. K, The table shows the popliteal LN-metastatic rate in different groups (n = 12). L–N, Representative IHC images and quantification of DHCR24 and VEGFC expression and LYVE1-indicated lymphatic vessel density in footpad tumor tissues from the indicated mice. Scale bar, 50 μm. Significant differences were assessed through one-way ANOVA, followed by the Dunnett test in C, E–J, M, and N; two-tailed Student t test in D; and the χ2 test in K. Error bars , SD. *, P < 0.05; **, P < 0.01.

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VEGFC is a downstream regulator of the PI3K/Akt signaling pathway that catalyzes the process of lymphangiogenesis to regulate tumor LN metastasis (38). The observation that DHCR24 significantly enhanced the lymphangiogenesis of bladder cancer prompted us to hypothesize that DHCR24 might activate the TBK1/PI3K/Akt signaling pathway to stimulate VEGFC secretion and promote the lymphangiogenesis and LN metastasis of bladder cancer. To verify our hypothesis, qRT-PCR analysis and Western blot analysis were conducted and showed that VEGFC levels were significantly reduced in DHCR24-silenced bladder cancer cells and elevated in DHCR24-overexpressing cells (Supplementary Fig. S7C–S7L). Consistently, the results of ELISA revealed that knocking down DHCR24 strikingly decreased VEGFC secretion, whereas overexpressing DHCR24 exhibited the opposite effect (Fig. 5C and D; Supplementary Fig. S7G and S7H). Furthermore, VEGFC expression mediated by DHCR24 overexpression was remarkably attenuated after treatment with the TBK1/PI3K/Akt signaling pathway inhibitor (Fig. 5EH), indicating that DHCR24 activates the TBK1/PI3K/Akt signaling pathway to promote VEGFC secretion in bladder cancer cells.

DHCR24 drives lymphangiogenesis and LN metastasis of bladder cancer via a VEGFC–dependent mechanism

We then examined whether VEGFC secretion was required for DHCR24-mediated lymphangiogenesis and LN metastasis of bladder cancer. A VEGFC–neutralizing antibody, αVEGFC, significantly impaired the ability of DHCR24 overexpression to induce the tube formation and migration of HLECs (Fig. 5I; Supplementary Fig. S7M). Moreover, the in vivo footpad–popliteal LN metastasis model showed that treatment with αVEGFC markedly inhibited the ability of DHCR24 to enhance the metastasis of bladder cancer cells to the popliteal LNs, as indicated by IVIS analysis (Fig. 5J). The popliteal LN-metastatic ratio in DHCR24-overexpressing tumor-bearing mice was reduced by treatment with αVEGFC (Fig. 5K). IHC revealed that DHCR24 overexpression markedly increased the density of microlymphatic vessels in the footpad tumors of mice compared with control mice, whereas αVEGFC administration markedly attenuated these effects (Fig. 5LN). Together, our results demonstrate that DHCR24 promotes VEGFC secretion to induce lymphangiogenesis and LN metastasis in bladder cancer.

Therapeutic effect of DHCR24 in PDXs from LN-metastatic bladder cancer

Because DHCR24 plays a vital role in promoting the LN metastasis of bladder cancer, we further established PDX models using LN-metastatic bladder cancer tissues to determine the therapeutic effect of DHCR24 inhibition. When the tumors of PDXs reached 200 mm3, the mice were randomly divided into two groups and intratumorally injected with sh-DHCR24 or sh-NC (Fig. 6A). The results showed that treatment with sh-DHCR24 significantly suppressed the growth of tumor volume in PDX models compared with the controls (Fig. 6BD). Moreover, analysis of molecules downstream of DHCR24 in PDXs revealed that DHCR24 silencing in PDX tumors markedly reduced the expression of TBK1 compared with than in the control group (Supplementary Fig. S8A and S8B). Importantly, inhibition of DHCR24 significantly decreased TBK1 expression, VEGFC secretion and LYVE1–indicated microlymphatic vessel density (MLD) in tumor tissues, as indicated by immunofluorescence staining (Fig. 6E; Supplementary Fig. S8C–S8F). Collectively, these results demonstrate that silencing DHCR24 inhibits the lymphangiogenesis of bladder cancer and that targeting DHCR24 might serve as a potential treatment for LN-metastatic bladder cancer.

Figure 6.

Inhibition of DHCR24 suppresses tumor growth in PDXs from LN-metastatic bladder cancer. A, Schematic illustration of the construction of the PDX model. B–D, Images and quantification of the tumor volume in mice treated with sh-DHCR24 or sh-NC (n = 6 per patient). E, Representative images of immunofluorescence staining of DHCR24, TBK1, and VEGFC expression and LYVE1–indicated lymphatic vessel density in the tumor tissues from PDXs. F and G, qRT‒PCR analysis of TBK1 expression in bladder cancer tissues versus NATs and LN-positive versus LN-negative bladder cancer tissues (n = 296). H and I, Kaplan‒Meier survival analysis of OS (H) and DFS (I) in patients with bladder cancer with high versus low TBK1 expression. The cutoff value is the median. J–L, Representative IHC images and correlation analysis of DHCR24, TBK1, and VEGFC expression and LYVE1–indicated lymphatic vessel density in both intratumoral and peritumoral regions of bladder cancer tissues (n = 296). Scale bars, 50 μm. H&E, hematoxylin and eosin.The significant difference was assessed through two-tailed Student t test in C and D; the nonparametric Mann–Whitney U test in F and G; and the χ2 test in K and L. **, P < 0.01.

Figure 6.

Inhibition of DHCR24 suppresses tumor growth in PDXs from LN-metastatic bladder cancer. A, Schematic illustration of the construction of the PDX model. B–D, Images and quantification of the tumor volume in mice treated with sh-DHCR24 or sh-NC (n = 6 per patient). E, Representative images of immunofluorescence staining of DHCR24, TBK1, and VEGFC expression and LYVE1–indicated lymphatic vessel density in the tumor tissues from PDXs. F and G, qRT‒PCR analysis of TBK1 expression in bladder cancer tissues versus NATs and LN-positive versus LN-negative bladder cancer tissues (n = 296). H and I, Kaplan‒Meier survival analysis of OS (H) and DFS (I) in patients with bladder cancer with high versus low TBK1 expression. The cutoff value is the median. J–L, Representative IHC images and correlation analysis of DHCR24, TBK1, and VEGFC expression and LYVE1–indicated lymphatic vessel density in both intratumoral and peritumoral regions of bladder cancer tissues (n = 296). Scale bars, 50 μm. H&E, hematoxylin and eosin.The significant difference was assessed through two-tailed Student t test in C and D; the nonparametric Mann–Whitney U test in F and G; and the χ2 test in K and L. **, P < 0.01.

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Clinical relevance of the DHCR24/hnRNPA2B1/TBK1/VEGFC axis in patients with bladder cancer

Because DHCR24 mediates the UBC9/hnRNPA2B1/TBK1/VEGFC axis to promote the LN metastasis of bladder cancer, we further investigated the clinical relevance of this regulatory axis in a cohort of 296 patients with bladder cancer. We found that both TBK1 and UBC9 were overexpressed in bladder cancer tissues compared with NATs (Fig. 6F; Supplementary Fig. S8G). LN-metastatic bladder cancer tissues had higher expression levels of TBK1 and UBC9 than those without LN metastasis (Fig. 6G; Supplementary Fig. S8H). Moreover, TBK1 and UBC9 overexpression was positively associated with shorter DFS and OS in patients with bladder cancer (Fig. 6H and I; Supplementary Fig. S8I and S8J). Furthermore, correlation analysis showed that DHCR24 expression was positively correlated with the expression of TBK1 and UBC9 (Supplementary Fig. S8K and S8L). Importantly, IHC revealed that higher DHCR24 expression levels were accompanied by higher TBK1 and VEGFC expression and a higher MLD in bladder cancer tissues (Fig. 6JL). Taken together, our results demonstrate that the DHCR24/UBC9/TBK1/VEGFC axis is widely involved in the lymphangiogenesis and LN metastasis of bladder cancer.

Abnormal translational control is crucial for inducing uncontrolled synthesis of oncogenic proteins, which is crucial for the distant dissemination of cancer cells (39–41). However, the underlying key mediator and precise mechanism remain unknown. Herein, we revealed that a high abundance of SUMOylation widely participates in oncogenic mRNA translation initiation and identified a key SUMOylation regulator, DHCR24, that controls translation initiation complex assembly to promote bladder cancer LN metastasis. DHCR24 mediates the conjugation of hnRNPA2B1 with SUMO2 modifiers at the lysine 108 residue, which determines the location of hnRNPA2B1 in the 3′-UTR of TBK1 mRNA. Subsequently, the SUMOylated hnRNPA2B1 in the 3′-UTR of TBK1 mRNA physically binds with eIF4G1 on the 5′ m7G cap, fostering TBK1 mRNA circularization and eIF4F initiation complex assembly to enhance the translation efficiency of TBK1 mRNA, resulting in lymphangiogenesis and LN metastasis of bladder cancer. It has been widely acknowledged that the interaction between PABPC1 in the 3′ poly (A) tail and eIF4G1 in the 5′ m7G cap to modulate mRNA circularization and translation initiation is the principal mechanism for mRNA translational control under physiological conditions (42). Thus, our findings uncover a novel mechanism in oncogenic mRNA translation initiation and suggest that targeting the translation machinery by inhibiting DHCR24-induced hnRNPA2B1 SUMOylation is a potential therapeutic strategy for LN-metastatic bladder cancer.

DHCR24 has been previously reported as the key synthetase in cholesterol synthesis and plays important roles in the hypermetabolism, chemotherapy resistance, invasion and distant metastasis of various malignancies (43–45). Blocking DHCR24 to inhibit cholesterol biosynthesis significantly reduces the hematogenous dissemination of various cancers. Previous studies on DHCR24 have focused mainly on its lipid anabolic function in tumor progression, whereas it has been proposed that its potential functions are distinct from the metabolic regulation of cholesterol-metabolizing enzymes, including DHCR24, displayed during cancer dissemination (46). In this study, we identified a novel functional role of DHCR24 in promoting bladder cancer lymphangiogenesis and LN metastasis instead of hematogenous dissemination by serving as a central driver to form a SUMO2/hnRNPA2B1/eIF4G1 feed forward loop rather than a cholesterol metabolic enzyme. DHCR24 directly anchored to the 3′-UTR of TBK1 mRNA and recruited SUMOylated hnRNPA2B1 to foster TBK1 mRNA circularization and eIF4F translation initiation complex assembly. Moreover, the interaction of DHCR24 with the 3′-UTR of TBK1 mRNA exhibited a significant prolonging effect on the half-life of TBK1 mRNA in a SUMOylated hnRNPA2B1-independent manner, thus constituting a feed forward loop to enhance TBK1 expression and stimulate the lymphangiogenesis of bladder cancer. These results highlight a previously uncharacterized pattern for the DHCR24-induced SUMO2/hnRNPA2B1/eIF4G1 feed forward loop to possess prolymphangiogenic function in a cholesterol biosynthesis-independent manner and support DHCR24 as a promising therapeutic target for LN-metastatic bladder cancer.

We and others have previously demonstrated that dysregulated SUMOylation modification is widely involved in the lymphatic vasculature, of which, hnRNPA2B1 was characterized as the crucial SUMO conjugation substrate triggering lymphatic metastasis and poor prognosis in bladder cancer (14, 47). However, hnRNPA2B1 SUMOylation is also widely found in other physiological phenomena, and this functional heterogeneity is implicated in the recognition of distinct SIMs on its various binding partners (14, 15, 27). Thus, the regulator driving hnRNPA2B1 SUMOylation via specific SUMO isoforms and SIMs needs further exploration to clarify the functional role of SUMOylated hnRNPA2B1 in LN-metastatic bladder cancer and develop effective therapeutic targets. In the present study, we identified an inducer of SUMO2 conjugation on hnRNPA2B1, DHCR24, which was markedly upregulated in LN-metastatic bladder cancer. DHCR24-mediated SUMOylation of hnRNPA2B1 was reciprocally recognized and recruited by DHCR24 to the 3′-UTR of TBK1 mRNA through the SUMO2 interaction motif (LLPLSLIFD) on DHCR24 to induce lymphangiogenesis and LN metastasis of bladder cancer. Therefore, our study reveals the potential mechanism underlying the SUMO2 modification of hnRNPA2B1 in promoting LN metastasis of bladder cancer, suggesting that DHCR24 is an effective therapeutic target for the blockade of specific hnRNPA2B1 SUMOylation in patients with LN-metastatic bladder cancer.

TBK1 is a vital member of the IκB kinase family that plays a crucial role in promoting the progression of tumors (48). Previous studies have reported that overexpression of TBK1 engages the inhibition of programmed cell death to induce oncogenic transformation and enhance tumor metastasis (49, 50). Moreover, TBK1 depletion attenuates the epithelial–mesenchymal transition of tumor cells and exhibits a significant effect in suppressing tumor metastasis (37). Although the oncogenic role of TBK1 has been widely explored in various cancers, it is still unclear how TBK1 is dynamically regulated in bladder cancer and its biological role in the lymphangiogenesis and LN metastasis of bladder cancer. Here, we found that TBK1 was overexpressed in LN-metastatic bladder cancer, which was mediated by the DHCR24-induced SUMO2/hnRNPA2B1/eIF4G1 feed forward loop. Moreover, we demonstrated that DHCR24-induced TBK1 overexpression facilitated the lymphangiogenesis and LN metastasis of bladder cancer via direct TBK1-mediated phosphorylation of Akt to promote the secretion of VEGFC. Our results reveal the prolymphangiogenic role of TBK1 in bladder cancer and elucidate the precise regulatory mechanism of TBK1, providing a prospective therapeutic target for bladder cancer LN metastasis.

In summary, our findings demonstrate that DHCR24 not only promotes the SUMO2 modification of hnRNPA2B1 to foster TBK1 mRNA circularization but also directly anchors to the 3′-UTR of TBK1 mRNA to increase its stability, thus provoking TBK1 expression and facilitating the lymphangiogenesis and LN metastasis of bladder cancer. The systematic elucidation of DHCR24 in inducing the SUMO2/hnRNPA2B1/eIF4G1 feed forward loop to promote bladder cancer lymphangiogenesis supports DHCR24 as a potential therapeutic target for LN metastasis in bladder cancer.

W. He reports grants from National Natural Science Foundation of China during the conduct of the study. No disclosures were reported by the other authors.

Y. Zhao: Investigation, project administration. J. Chen: Resources, data curation. H. Zheng: Formal analysis, visualization, methodology. Y. Luo: Investigation, methodology. M. An: Resources, software. Y. Lin: Methodology, project administration. M. Pang: Formal analysis, validation. Y. Li: Data curation, formal analysis. Y. Kong: Visualization, methodology. W. He: Conceptualization, resources. T. Lin: Conceptualization, funding acquisition, project administration. C. Chen: Conceptualization, funding acquisition, writing–review and editing.

The authors thank Prof. J.X. Zhang of the Department of Medical Statistics and Epidemiology, Sun Yat-sen University, for statistical advice and research comments. This study was funded by the National Key Research and Development Program of China (grant no. 2022YFA1305500 and 2018YFA0902803); the Distinguished Young Scholars of the National Natural Science Foundation of China (grant no.32322023); the National Natural Science Foundation of China (grant no. 82202276, 82072639, 81802530, 81871945, 81702951, 81672395, 81672807, 81702417, and 81701715); the Natural Science Foundation of Guangdong Province (grant no. 2022A1515012288 and 2021A1515010355); the Regional Joint Project of the Natural Science Foundation of Guangdong Province (key project; grant no. 2022B1515120086); the Guangdong Science and Technology Department (grant no. 2020A1515010815, 2018A030313564, 2017A020215072, 2016A030313340, 2016A030313296, 2017A030313880, and 2017A030310200); the Science and Technology Program of Guangzhou (grant no. 2023A04J2206, 202002030388, and 201803010049); Medical Scientific Research Foundation of Guangdong Province, China (grant no. A2022117).

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

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