PDGF-BB/PDGFRβ induces tumour angiogenesis via enhancing PKM2 mediated by the PI3K/AKT pathway in Wilms’ tumour

doi:10.21203/rs.3.rs-2480642/v1

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PDGF-BB/PDGFRβ induces tumour angiogenesis via enhancing PKM2 mediated by the PI3K/AKT pathway in Wilms’ tumour

https://doi.org/10.21203/rs.3.rs-2480642/v1

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published 13 Jul, 2023

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Platelet-derived growth factor receptor-β (PDGFRβ) is an important member of the type III receptor tyrosine kinase family, which is involved in Wilms’ tumour (WT) metastasis and aerobic glycolysis. The role of PDGFRβ in tumour angiogenesis has not been fully elucidated. Here, we examined the effect of PDGFRβ on angiogenesis in WT. First, the NCBI database was used to integrate three datasets, GSE2712, GSE11151, and GSE73209, and to screen differentially expressed genes. The R language was used to analyse the correlation between PDGFRB and vascular endothelial growth factor (VEGF )in WT. The results showed that PDGFRB, encoding PDGFRβ, was upregulated in WT, and its level was correlated with VEGFA expression. Next, PDGFRβ expression was inhibited by small interfering RNA (siRNA) or activated with the exogenous ligand PDGF-BB. The expression and secretion of the angiogenesis elated factor VEGFA in WT G401 cells were detected using Western blotting and ELISA, respectively. The effects of conditioned medium from G401 cells on endothelial cell viability, migration, invasion, total length of tube, and the number of fulcrums were investigated. To further explore the mechanism of PDGFRβ in the angiogenesis of WT, the expression of VEGFA was detected after blocking the phosphatidylinositol-3-kinase (PI3K) pathway and inhibiting the expression of PKM2, a key enzyme of glycolysis. The results indicated that PDGFRβ regulated the process of tumour angiogenesis through the PI3K/Akt/PKM2 pathway. Therefore, this study provides a novel therapeutic strategy to target PDGFRβ and PKM2 to inhibit glycolysis and anti-angiogenesis, thus, developing a new anti-vascular therapy.

Anti-vascular therapy

Endothelial cells

PDGFRβ

PKM2

VEGFA

Wilms’ tumour

Wilms’ tumour (WT), also known as nephroblastoma, is the most common renal tumour in children. It is a triphasic embryonal neoplasm comprising varying proportions of the blastema, stroma, and epithelium [1, 2]. Current treatments for WT include nephrectomy, chemotherapy, and radiotherapy. With treatment advances, the overall survival (OS) of localised, metastatic, and recurrent WT is over 90%, 75%, and 50%, respectively [3, 4]. Although the majority of patients with WT have a good prognosis, 20% relapse after first-line therapy, mainly within two years of diagnosis, with a long-term survival rate of only 50% [5, 6]. Haematogenous metastases to the lung, liver, bone, or other sites are present in approximately 10–12% of patients with WT. The lungs are by far the most common site of metastasis [7]. It is difficult to achieve further development by simply enhancing existing therapies; therefore, finding new targeted therapies is an urgent task.

Angiogenesis is the most crucial process in invasive tumour growth and metastasis. Tumours can stimulate the production of new blood vessels by releasing hypoxia-induced angiogenic factors, such as vascular endothelial growth factor (VEGF), which allows them to acquire nutrients and oxygen for survival and overcome proliferation limitations in the hypoxic microenvironment [8]. high microvessel density are associated with poor prognosis in WT [9]. Yasemin Ozluk et al. [10] found a significant association between angiogenesis and OS in patients with WT. The primary tumour tissues of WT express VEGF, and its high expression is closely related to the risk of invasion, metastasis, and recurrence. Effective VEGF blockade therapy achieved good histological and experimental WT xenografts and vascular regression while reducing the incidence and size of metastases [11, 12]. Bevacizumab was the first U.S. Food and Drug Administration (FDA)-approved anti-vascular drug that specifically blocks VEGF and prevents VEGF from interacting with its receptors on the surface of endothelial cells such as Vascular endothelial growth factor receptor-1 and Vascular endothelial growth factor receptor-2 [13]. After treatment of patients with WT with bevacizumab combined with anti-vascular agents, such as vincristine, the disease is stable or even almost complete remission is observed, but patients will eventually relapse [14, 15]. In contrast, in paediatric patients with WT, bevacizumab was associated with an increased risk of pneumothorax [16]. Given that tumour angiogenesis drives rapid tumour growth and increased invasiveness, it is necessary to investigate further the molecular mechanisms involved and seek new therapeutic targets to limit recurrence.

Platelet-derived growth factor receptor β (PDGFRβ), a transmembrane glycoprotein dimer, is an important member of the type III receptor tyrosine kinase (RTK) family. PDGF-BB is a ligand of PDGFR-β and is associated with angiogenesis. Enhanced PDGF/PDGFR signalling has also been observed in various diseases, such as cancer, pulmonary fibrosis, and atherosclerosis [17]. Several studies have supported this vessel-stabilising function and tumour-promoting effect of PDGFRβ signalling in pericytes. In addition to maintaining vascular homeostasis in pericytes, PDGFRβ is highly expressed in a variety of tumours, such as colorectal cancer, ovarian carcinoma, and hepatocellular carcinoma. The PDGFRβ expression is increased in the perivascular area of colorectal cancer, and the inpatient heterogeneity of PDGFRβ expression is also high [18-20]. In a xenograft mouse model of human WT [21], PDGFRβ was found in the blood vessels of recurrent tumours. PDGFRβ is found in the cytoplasm and membrane of WT patient-derived xenografts [22]. These studies suggest that PDGFRβ expression may be a part of the early events that lead to metastasis. Ligand-activated PDGFRβ can initiate many signalling pathways, such as the RAS-Raf, PLCγ-PKC, and PI3K/AKT pathways, thereby regulating tumour proliferation, metastasis, invasion, and angiogenesis [23]. PDGF-BB can promote the proliferation, migration, and angiogenesis of EPC by activating the PDGFRβ/PI3K/AKT signalling pathway [24].

Angiogenesis and metabolism are important processes that support tumour growth and development. In contrast to normal tissues, the low-glucose environment caused by the high level of glycolysis in tumour cells leads to the upregulation of VEGF expression in tumour tissues, thereby promoting tumour angiogenesis [25]. Our previous study [26] found that PDGFRβ is closely related to aerobic glycolysis in WT G401 cells, and PKM2 is a key enzyme in regulating aerobic glycolysis by PDGFRβ. Therefore, we hypothesised that PDGFRβ promotes tumour angiogenesis by regulating aerobic glycolysis in G401 cells.

In this study, we investigated the effect of PDGFRβ on angiogenesis in WT and whether this effect was mediated by aerobic glycolysis. PDGFRβ mediates PKM2 through the PI3K/AKT pathway to promote tumour angiogenesis.

2.1 Bioinformatic tools

The data of GSE2712, GSE73209 and GSE11151 chip were obtained by using “Wilms’ tumour” as the key word in the database. The PDGFRβ gene expression in the database was analysed by the NCBI data. We used NCBI (https://www.ncbi.nlm.nih.gov/) in the database dataset GSE2712 PDGFRB for VEGFA and correlation analysis. The GGStatsplot package in the R 4.0.3 software was used to draw the correlation map. The ClusterProfiler package (version:3.18.0) in R was used to analyse the GO function of potential targets and to enrich the KEGG pathway. Potential protein-protein interactions (PPIs) were analysed using the STRING database (http://string-db.org/).

2.2 Cell culture and reagents

The human WT cell line G401 cells and human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Gaithersburg, MD, USA) containing 10% foetal bovine serum (FBS; OriCell) and 1% penicillin-streptomycin solution (Beyotime). The cultures were placed in a humidified incubator at 37°C and 5%CO₂.

PDGF‐BB (15ng/ml) was purchased from PEPROTECH. DASA‐58 (30mM) (S7928) and C3K (0.15μM) (S8616) were purchased from Selleck. LY-294002 (30μM) was bought from APExBIO. Lactate (10mM) (V900136) was purchased from Sigma-Aldrich. The working concentrations of all the reagents were selected according to the manufacturer's instructions.

2.3 siRNA transfection

Small interfering RNA (siRNA) targeting PDGFRβ (si‐PDGFRβ) was designed and synthesised by RIB BIO (Guangzhou, China). Negative control siRNA (NC) was used as the control. When the density of adherent cells reached 40% in a six-well plate, the indicated siRNA was transfected into the cells using CP Reagent. After culturing at 37°C in a humidified atmosphere containing 5% CO₂ for 48 h, cells were used for subsequent assays.

The siRNA sequences are as follows:

si‐NC sense, 5′‐UUCUCCGAACGUGUCACGUTT‐3′,

antisense, 5′‐ACGUGACACGUUCGGAGAATT‐3′,

si‐PDGFRβ sense, 5′‐GUGAGAAGCAAGCCCUUAUTT‐3′,

antisense, 5′‐AUAAGGGCUUGCUUCUCACTT‐3′.

2.4 Preparation of conditioned culture medium for tumour cells

A total of 48 h after transfection with siRNA, G401 cells were cultured in fresh DMEM for another 24 h. The cell supernatant was collected, centrifuged at 14,000 rpm for 20 min, filtered with a 0.22μm filter, and transferred to a refrigerated tube, which was stored at -20℃ and -80℃ for a short and long time, respectively.

2.5 Western blotting

A total of 48 h after transfection, each well was added to each DMEM medium. After 24 h of culture, the cell supernatant was collected as a tumour-conditioned medium, and the remaining cells were harvested using a cell scraper for total protein isolation. Western blotting was performed as previously described by Guo et al. [26]. The corresponding primary antibody catalogue numbers and dilutions were as follows: PDGFRβ (1:1000, ab32570, Abcam), p-PDGFRβ (1:1000, ab218534, Abcam), VEGFA (1:1000, ab52917, Abcam), and β-actin (1:100,000, AC026, ABclonal).

2.6 Cell counting kit‑8 (CCK‑8) assay

HUVECs were seeded in 96-well plates at a density of 2000 cells/well, cultured overnight, and incubated in fresh medium containing different groups of tumour-conditioned medium for 24 h. A total of 10 μL Cell Counting Kit-8 reagent (Boster, Wuhan, China) was added to each well and the cells were cultured for 2 h. Optical density (OD) was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.7 Enzyme-linked immunosorbent assay (ELISA)

The supernatant of the G401 cells was collected in a centrifuge tube and centrifuged at 14,000 rpm for 20 min to remove cell debris and impurities. The supernatant was sampled for ELISA to measure extracellular levels of VEGFA. An ELISA assay was performed according to the manufacturer’s instructions using a QuantiCyto® Human VEGF ELISA kit (NeoBioscience, EHC108.96, Shenzhen, China).

2.8 Migration and invasion assays

Migration and invasion assays were performed in 24-well transwell chambers with a pore size of 8 μm (Corning Life Sciences, New York, USA), whereas invasion assays were performed by coating the upper chamber with Matrigel in advance (BD Biosciences). Ten thousand HUVECs were added to each well in the upper chamber, and 800 μl of tumour-conditioned medium from different groups was added to the lower chamber. After 24 h of co-culture, the upper chamber was removed, fixed with paraformaldehyde for 30 min, and stained with crystal violet for 20 min. The remaining dye in the chamber was carefully wiped with a cotton swab. After air drying the chamber, images were obtained using a microscope (Leica 200 ).

2.9 Matrigel tube formation assay

For the tube formation assay, 50 μl of Matrigel (BD Biosciences, San Jose, CA, USA) was added to each well of a 96-well plate. After polymerisation at 37°C for 30 min, HUVECs were cultured in tumour conditioned medium from different groups, with 25,000 cells/well. After incubation for 5 h in a humidified incubator at 37°C with 5% CO₂, tubules were visualised under an inverted microscope (Leica 100 ) and analysed using ImageJ.

2.10 Wound healing assay

HUVECs were cultured in a six-well plate, and they reached at least 90% confluency. A sterilized 200 μL pipette tip was used to create a wound. The wells were then washed twice with PBS to eliminate cell debris and replenished with the tumour-conditioned medium of different groups. Images were obtained with a microscope（Leica）at 200 magnification for 0 and 24 h and analysed using ImageJ.

2.11 Statistical analysis

All experiments were performed in triplicates, and the data were analyzed with the SPSS 25.0 IBM (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 9.0 (GraphPad Inc., La Jolla, CA, USA). The data are presented as the mean ± SD and were evaluated using one-way analysis of variance (ANOVA) or Student's t-test. Statistical significance was set at P < 0.05.

3.1 The expression of PDGFRβ was up-regulated in WT

We first downloaded the corresponding datasets from the CCLE (https://sites.broadinstitute.org/ccle) and GEPIA2 databases (http://gepia2.cancer-pku.cn/#index). The expression of PDGFRB in normal and cancerous tissues was analysed online. These results suggest that PDGFRB may play a crucial role in tumour progression (Fig1 a,b). To determine the roleof PDGFRβ in WT tumourigenesis, we comprehensively analysed the expression of PDGFRβ in WT, and the relevant data sets were downloaded from NCBI data, and 12,341 genes were screened by integrating GSE2712, GSE73209, and GSE11151 datasets. The ggplot package of the R 4.0.3 software was used to make the volcano map, according to logFC≥1 and P value <0.05 showing that the differentially expressed genes (DEGs) were up-regulated, while logFC≤-1 and P value <0.05 indicated that other genes were not significantly DEGs. Differentially expressed genes were screened. A total of 5,784 DEGs were screened (Fig1 c and Supplementary Table S1), of which 1,617 genes were upregulated and 3,081 genes were downregulated. The results explicitly showed that PDGFRB encoding PDGFRβ was upregulated in the WT. To determine whether PDGFRβ is involved in angiogenesis in WT, we used NCBI (https://www.ncbi.nlm.nih.gov/) in the dataset GSE2712 PDGFRB between VEGFA and correlation analysis. As expected, the results of the datasets showed that PDGFRB expression was positively correlated with angiogenesis-related genes in WT (Fig1 d).

3.2 Effect of PDGFRβ overexpression on angiogenesis of WT induced by PDGF-BB

VEGFA protein expression and extracellular secretion were detected by western blot assay and ELISA, respectively, after G401 cells were stimulated with exogenous PDGF-BB. Upregulation of PDGFRβ promoted VEGFA protein expression (Fig2 a) and extracellular secretion (Fig2 b). The CCK-8 assay showed that the upregulation of PDGFRβ in G401 cells significantly promoted the proliferation of HUVECs (Fig2 c). We further evaluated the effect of PDGFRβ on the migration and invasion of HUVECs using wound healing, transwell migration, and transwell invasion assays. The wound healing rate in the PDGF-BB group was significantly higher than that in the MOCK group (Fig2 f). The number of cells that invaded and migrated simultaneously was significantly higher than that in the MOCK group (Fig2 d, e), indicating that the upregulation of PDGFRβ promoted the migration and invasion of HUVECs. To further explore the effect of PDGFRβ on angiogenesis, we performed in vitro vascular experiments, and the results showed that PDGFRβ overexpression promoted vascular formation in HUVECs (Fig2 g).

3.3 Effect of PDGFRβ inhibition on tumour angiogenesis in WT

Inhibition of PDGFRβ by siRNA reduces both intracellular and extracellular VEGFA levels in G401 cells. The CCK-8 assay showed that inhibition of PDGFRβ reduced the viability of HUVECs. Following PDGFRβ inhibition, the number of invading and migrating cells decreased relative to that in the NC group (Fig3 d-e). The wound healing assay results confirmed that the wound healing ability of HUVECs was suppressed after PDGFRβ inhibition (Fig3 f). In addition to proliferation and migration, we evaluated the effect of PDGFRβ inhibition on HUVEC angiogenesis in G401 cells. In the in vitro angiogenesis assay, it was observed that the total length of the tube and number of fulcrum by HUVECs in the Si-PDGFRβ group was lower than that in the HUVECS-NC group (Fig3 g).

3.4 PDGFRβ affects WT angiogenesis by mediating PKM2 expression.

Our previous study showed that PDGFRβ is closely related to aerobic glycolysis. Inhibition of PDGFRβ can significantly inhibit aerobic glycolysis, and PKM2 is a key enzyme in the regulation of aerobic glycolysis [26]. To explore the effect of glycolysis on angiogenesis, we treated G401 cells with the glycolysis inhibitor 2-DG and found that angiogenesis in HUVECs was significantly reduced (Fig3 g). Therefore, we hypothesised that PDGFRβ regulates tumour angiogenesis by mediating aerobic glycolysis. Lactic acid, the end product of aerobic glycolysis, plays an important role in angiogenesis. PKM2, a key enzyme in glycolysis, can promote tumour blood vessel growth by participating in VEGF, a key molecule in tumour blood vessel formation. To explore the downstream factors influencing PDGFRβ in regulating angiogenesis, lactate solution and 30 mM PKM2 agonist DASA-58 were used for stimulation after inhibition of PDGFRβ. CCK-8 results showed that activation of PKM2 could reverse the inhibition of HUVEC activity after PDGFRβ inhibition. However, lactate stimulation did not reverse the effect of PDGFRβ on HUVEC activity (Fig4 a). To explore the effect of PKM2 on tumour angiogenesis, the Western blot assay was used to detect the expression of angiogenation-related factor VEGFA in G401 cells after treatment with PKM2 agonist (DASA-58) and inhibitor (C3K). We found that, compared with the MOCK group, the expression level of VEGFA protein was increased in the DASA-58 agonist group, while the expression level of the C3K histone inhibitor was significantly decreased (Fig4 b). At the same time, we verified that PKM2 activation significantly promoted the extracellular secretion of VEGF (Fig3 d). When PDGFRβ was inhibited, PKM2 activation reversed both intracellular and extracellular VEGFA levels in G401 cells (Fig4 c,e). The results of transwell invasion (Fig4 f), migration assay (Fig4 g) and wound healing assay (Fig4 h) also indicated that PKM2 activation could reverse the effect of PDGFRβ inhibition on the migration and invasion ability of HUVECs. PKM2 activation also reversed the effect of PDGFRβ inhibition on HUVEC tube formation (Fig4 i). Taken together, these results suggest that PDGFRβ affects WT angiogenesis by mediating PKM2 expression.

3.5 PDGFRβ regulates WT angiogenesis by mediating PKM2 axis through PI3K/AKT pathway

KEGG pathway analysis of the integrated DEG revealed that the PI3K-AKT signalling pathway was closely related to the occurrence and development of WT (Fig5 a). The protein-protein interconnect (PPI) network string database revealed that PDGF-BB/PDGFRβ was linked to PKM2 and VEGFA via PI3K and AKT (Fig5 b). Based on these results, we hypothesised that PDGFRβ affects angiogenesis by mediating PKM2 through the PI3K/AKT pathway. G401 cells were pre-treated with 30 μM LY-294002 (PI3K inhibitor) and 0.15μM C3K (PKM2 inhibitor) for 6 h. A total of 15 ng/ml PDGF-BB was added, and the cells were cultured for 24 h. Western blotting results showed that PDGF-BB could not promote angiogenesis in WT after adding PI3K and PKM2 inhibitors to inhibit the PI3K/AKT pathway and PKM2 expression, respectively (Fig5 c). Therefore, we concluded that PDGF-BB/PDGFRβ regulates WT angiogenesis through the PI3K/AKT/PKM2/VEGFA axis.

The anti-angiogenesis therapy is based on the inhibition of known angiogenesis signalling pathways, and several kinds of antibodies targeted to promote angiogenesis signalling tyrosine kinase inhibitors are included in the FDA approved medicines list. However, anti-VEGF monotherapy can lead to recurrence by enhancing tumour invasiveness and drug resistance [27–29]. Therefore, a better understanding of the tumour vascular phenotype and the specific mechanisms of tumour angiogenesis is necessary to identify bottlenecks in the process of anti-vascular therapy and, thus, seeking new targeted therapies to limit tumour development is important to prevent compensation by other potential angiogenic mechanisms.

A study of the pathological structure of WT showed that there was vascular tortuosity, capillary clumping, and bleeding at the tumour site. However, these features are absent in normal kidneys [30]. Angiogenesis in WT is driven by angiogenic growth factors, primarily VEGFA [31]. VEGFA exists in the plasma and tissues of patients with WT, and the level of VEGFA is closely related to patient prognosis [32]. Studies have demonstrated that highly specific anti-VEGF therapy can effectively inhibit primary tumour growth and experimental anaplastic WT metastasis [33–35]. Most antiangiogenic therapies have been developed for the VEGF/VEGFR family; however, due to resistance and other compensatory pathways resulting in poor efficacy, other targets need to be sought, including PDGFs/PDGFRs. In WT, PDGF-AA and PDGFRα were overexpressed in 50% and 30% of cases, respectively. Their expression is associated with a good prognosis [36]. PDGFRβ is highly expressed in various tumours, and inhibition of PDGFRs can inhibit cancer proliferation, metastasis, and invasion. Our previous study showed that PDGFRβ affects the invasion and migration of WT G401 cells [26, 37]. Most studies have focused on the therapeutic effects of PDGFRβ on tumour cells; however, the mechanism of anti-angiogenesis has not been clarified.

The mechanisms underlying the role of PDGFRβ in angiogenesis in WT have not been fully elucidated. In this study, we first observed that PDGFRB was upregulated in WT by biosynthesis analysis, analysed the correlation between PDGFRB and VEGF, and found a correlation between PDGFRB and VEGF. Therefore, we hypothesised that PDGFRβ plays a role in regulating angiogenesis in WT. In the absence of aberrant ligand and receptor activation, the kinase domain of PDGFRs becomes self-quiescent. PDGFs bind and activate different PDGFRs to perform different functions [38]. Data from the indirect co-culture model further supported our hypothesis. First, we added the exogenous ligand PDGF-BB and found that PDGFRβ overexpression increased both intracellular and extracellular VEGFA levels in G401 cells. Second, overexpression of PDGFRβ promoted HUVEC proliferation, migration, invasion, and tube formation in vitro. In contrast, the knockdown of PDGFRβ with siRNA reduced the levels of intracellular and extracellular VEGFA in G401 cells and inhibited the biological phenotype of HUVECs. This indicates that PFGFRβ is involved in angiogenesis in WT.

In the past, it was believed that "glycolytic transition" occurred before angiogenesis, and aerobic glycolysis promoted tumour angiogenesis in many ways [39]. Lactic acid, a tumour metabolite, is one of the first metabolites to develop. Lactic acid output from aerobic glycolysis of tumours promotes acidification of the tumour environment, which facilitates metastasis, angiogenesis, and immunosuppression. It also triggers the stabilisation of hypoxia-inducible factor-1α (HIF-1α) and subsequently increases the expression of VEGF [40]. Our previous studies have also confirmed that PDGFRβ regulates aerobic glycolysis in WT and promotes glucose consumption and lactate production in G401 cells. However, surprisingly, after PDGFRβ knockdown and stimulation of G401 cells with lactic acid, HUVEC viability could not be reversed. Therefore, we focused on key enzymes involved in glycolysis. In the WT, PKM2 is the key enzyme of PDGFRβ that regulates the aerobic glycolytic pathway. Studies have shown that PKM2 not only acts as a rate-limiting enzyme to control the glycolysis rate of tumour cells but also regulates HIF-1α-induced VEGF secretion and promotes tumour angiogenesis in an NF-κB/p65-dependent manner [41]. The specificity of PKM2 in regulating angiogenesis after PDGFRβ knockdown was confirmed by measuring VEGFA levels in G401 cells. It is confusing that under normoxic conditions, the protein level of HIF-1α is upregulated after PDGFRβ knockdown, but the expression of VEGFA is decreased. Therefore, we suspect that tumour cells may promote the expression of HIF-1α through other compensatory mechanisms.

The PI3K pathway is one of the most frequently enhanced oncogenic pathways in human cancers and plays a central role in several signalling cascades that regulate biological processes, such as cell growth, survival, proliferation, motility, and angiogenesis [42]. PI3K/AKT is an important signal transduction pathway involved in glucose metabolism. PI3K/AKT directly or indirectly upregulates glycolysis through post-translational modifications of metabolic enzymes, such as phosphorylation and glycosylation. It also increased glucose uptake and lactate production. Studies have shown that AKT regulates glucose transporter expression (GLUT1) and stimulates phosphofructokinase activity and PKM2 expression after activation by PI3K signalling [43, 44]. We screened out the key signalling pathway that regulates the occurrence and development of WT through biosynthesis analysis, namely, the PI3K/AKT pathway, which is consistent with the previous experimental results of our group, indicating that the PI3K/AKT pathway plays an important role in WT.

Based on the above findings, we combined PDGF-BB with the PI3K (LY-294002) and PKM2 (C3K) inhibitors and found that PDGF-BB could not promote angiogenesis in WT under the condition of pre-blocking the PI3K/AKT pathway and inhibiting PKM2 (Fig. 5c). Therefore, we conclude that PDGFRβ can regulate angiogenesis by activating the PI3K/AKT pathway to mediate PKM2, which provides a new target for the anti-vascular therapy of WT. The focus of this study was to analyse the role of PDGFRβ in angiogenesis in WT, and its molecular mechanism is still a preliminary exploration. The exact molecular mechanism and downstream effectors between glycolysis and angiogenesis in WT need to be further explored and analysed.

Statements & Declarations

Funding :

Program for Youth Innovation in Future Medicine, Chongqing Medical University

Competing Interests :

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions :

Sang botao was responsible for the experimental design of the study; Experimental operation; Results analysis and paper writing. Wang Changdong and Liu Xing were responsible for the experimental design of the study. Guo Jiaqi was responsible for the results analysis. Lai Jiayi was responsible for the bioinformatics analysis. Wu Xiangmei was responsible for the experimental design and results analysis of the study.

Data Availability :

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

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PDGF-BB/PDGFRβ induces tumour angiogenesis via enhancing PKM2 mediated by the PI3K/AKT pathway in Wilms’ tumour

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