IL13RA2 promotes progression of infantile haemangioma by activating glycolysis and the Wnt/β-catenin signaling pathway

Background Interleukin 13 receptor subunit alpha 2 (IL13RA2) plays an essential role in the progression of many cancers. However, the role of IL13RA2 in infantile haemangioma (IH) is still unknown. Materials and Methods IL13RA2 expression in IH tissues was analyzed using western blot, qRT-PCR, and immunofluorescence. The role of IL13RA2 in haemangioma-derived endothelial cells (HemECs) was determined following knockdown or overexpression of IL13RA2 using CCK-8, colony formation, apoptosis, wound healing, tubule formation, Transwell, and western blot. Results IL13RA2 expression was upregulated in IH tissues. IL13RA2 overexpression promoted proliferation, migration, and invasion of HemECs and induced glycolysis, which was confirmed with a glycolysis inhibitor. Specifically, IL13RA2 interacted with β-catenin and activated the Wnt/β-catenin pathway in HemECs, which were involved in the above-mentioned effects of IL13RA2. Conclusions These findings revealed that targeting IL13RA2 is a potential therapeutic approach for IH.


Introduction
Infantile haemangioma (IH) is a benign vascular tumors [1].IH develops in the first few weeks after birth and proliferates rapidly in the first year of life [2].Propranolol, a β-blocker, is the primary drug used for the treatment of IH; however, it has many side effects, such as bradycardia and hypotension [3].The combination of β blockers and laser treatment is advantageous for the treatment of refractory haemangioma as it reduces the required dosage of oral propranolol and associated adverse reactions [4,5].Unfortunately, due to tumor location, size, and rate of proliferation, a minority of IH cases are associated with severe complications such as disfigurement, vision loss, ulceration, and danger to lives [6].Hence, further clarification of mechanisms underlying IH pathogenesis is important to minimize the development of IH and associated complications.
Most tumor cells utilize glucose principally through aerobic glycolysis to meet their energy supply even with plenty of oxygen [11].Restraint of glycolysis through targeting PFKFB3 inhibits IH progression [12].In addition, OTUB1 regulates the angiogenesis in IH via mediating glycolysis [13].Hence, inhibiting aerobic glycolysis might be a meritorious pattern for IH treatment.
Wnt/β-catenin pathway is activated in various malignancies and is known to potentiate tumor recurrence [14].A previous study reported that renin acted as renin receptor to promote proliferation of IH cells via the Wnt pathway [15].Dai et al. reported that luteolin, a flavonoid, inhibited IH through targeting Frizzled-6 via the Wnt pathway [16].Inhibition of the Wnt/β-catenin pathway will bring about outstanding results in the treatment of IH.
In our study, IL13RA2 expression was observably up-regulated in IH.IL13RA2 was found to mediate the proliferation, migration, and invasion of haemangioma-derived endothelial cell shaemangioma (HemECs).Knockdown of IL13RA2 inhibited glycolysis and the Wnt/β-catenin pathway.Our findings suggest that IL13RA2 is a therapeutic target for IH.

Bioinformatics analyses
GSE43742 was obtained from the Gene Expression Omnibus.Gene Set Enrichment Analysis (GSEA) was conducted with GSE43742 using the GSEA software with hallmark gene sets.

Tissue specimens
Four proliferating and four involuting haemangioma tissues were collected from patients.Normal group was four foreskin tissues.Written informed consent was obtained from all patients.This study was approved by the Ethics Committee of 970th Hospital of the People's Liberation Army.All methods were performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards.

Isolation of endothelial cells
We established primary cultures of HemECs from four proliferating IH tissues as described previously [17].Tissues were digested with 0.2% collagenase type 1, centrifuged, resuspended.HemECs were selectively isolated using anti-CD31-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany).HemECs were cultured in endothelial cell growth medium-2 supplemented with 20% fetal bovine serum (FBS) on fibronectin-coated plates at 37°C in a 5% CO 2 environment.

Colony formation assay
HemECs (2000/well) were seeded in 6-well plates. 2 weeks later, the colonies were fixed with methanol, stained with crystal violet, and captured by light microscope.

Apoptosis assay
HemECs were suspended in binding buffer and stained with Annexin V-FITC apoptosis detection kit (C1062M, Beyotime, Jiangsu, China).The rate of apoptosis was analysed by flow cytometry (BD Bioscience, Franklin Lakes, USA).

Transwell assay
HemECs in serum-free medium were added to the upper chamber, while medium containing 10% FBS was added to the lower chamber.After incubation for 24 h, migratory and invading cells were stained with crystal violet and captured by light microscope.For invasion assay, Matrigel (BD Biosciences) was used.

Tubules formation assay
Matrigel (BD Biosciences) was added into 96-well plate and allowed to polymerise for 20 min at 37°C.HemECs were plated on 96-well plate and incubated for 12 h.The plate was observed under a microscope.ATP, glucose, and lactate measurements ATP, glucose, and lactate levels were measured using appropriate kits (BC0305, Solarbio; MAK083-1KT, Sigma-Aldrich St. Louis, USA; ab65331, Abcam).Samples were analysed using a microplate reader (Bio-Rad).

Seahorse extracellular flux assay
Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were detected using Seahorse XF24 analyser (Seahorse, Santa Clara, USA).For ECAR, a Seahorse XF Cell Mito Stress Test kit was used.For OCR, a Seahorse XF Glycolysis Stress Test kit (Agilent Technologies, Santa Clara, USA) was used.

Statistical analysis
Statistical significance was calculated using one-way analysis of variance, followed by Tukey's post hoc test.All statistical analyses were undertaken using Prism GraphPad 7 Software.p < 0.05 was considered statistically significant.

IL13RA2 expression is upregulated in IH
We analyzed the microarray GSE43742 and found that IL13RA2 expression was remarkably higher in HemECs than in human dermal microvascular endothelial cells (HDMVECs) (Fig. 1A).Next, we evaluated IL13RA2 expression in four proliferating haemangioma tissues, four involuting haemangioma tissues, and normal tissues.IL13RA2 expression was remarkably increased in IH tissues (Figs.1B-1D).Mesenchymal cells are often mixed with endothelial cells isolated from IH tissues.Hence, we assessed the expression of endothelial cell markers, CD31 and vWF and mesenchymal cell marker, α-SMA.Immunofluorescence staining of α-SMA, CD31, and vWF are shown in Fig. 1E.

IL13RA2 activates glycolysis
Through GESA, IL13RA2 was found to induce glycolyses in IH (Figs. 4A and 4B).Aggravated glycolysis has been recognised in malignant phenotypes of tumor cells [18].Therefore, we investigated the impact of IL13RA2 on glycolysis in HemECs.We found that ECAR was declined in sh-IL13RA2-transfected cells and increased in IL13RA2 vector-transfected cells (Fig. 4C).Additionally, The OCR of cells was enhanced by sh-IL13RA2 and decreased by IL13RA2 vector (Fig. 4D).IL13RA2 knockdown decreased ATP, lactate, and glucose consumption levels; whereas IL13RA2 overexpression increased ATP, lactate, and glucose consumption levels (Figs.4E-4G).To further assess the effects of IL13RA2 on glycolysis, we investigated key glycolytic kinases levels.Protein levels of GLUT1, HK2, LDHA, and PDK1 were inhibited by IL13RA2 knockdown and enhanced by IL13RA2 overexpression (Fig. 4H).Next, we explored whether activation of glycolysis by IL13RA2 facilitates cell migration and invasion.HemECs were treated with 2-DG (a glycolysis inhibitor) at 5 mM for 24 h.In IL13RA2-knockdown cells, 2-DG did not alter cell migration and invasion.IL13RA2 overexpression induced migration and invasion were reversed by 2-DG (Figs.4I  and 4J).

Discussion
Although most cases of IH are benign, it can also result in serious complications, such as functional damage and disfigurement [19].Therefore, the discovery of novel therapeutic targets for IH is necessary.More and more studies have found that IL13RA2 expression is related with tumor progression.IL13RA2 is enhanced in multitudinous patients with glioblastoma [20].Targeting IL13RA2 is a potential therapeutic approach for glioblastoma [21].Plasma IL13RA2 levels are linked to overall survival of patients with glioblastoma [22].In glioblastoma, high IL13RA2 expression predicts worse prognosis [23].IL13RA2 overexpression was related to poor survival of breast cancer brain metastases [24] and promoted migration of thyroid cancer cells [9].IL13RA2 knockdown confered invasive and metastatic abilities to hepatocellular carcinoma cells throng activating ERK pathway [25].In our study, we analysed GSE43742 dataset and found that IL13RA2 expression was remarkably higher in HemECs than that in HDMVECs.Analogously, Mao et al. revealed that CASZ1 expression is enhanced in glioma by analysing GSE22891 and GSE21354 [26].Small interfering RNA and shRNA have been used widely to silence gene expression in cells [27,28].In our study, pcDNA3.1-IL13RA2or shIL13RA2 was transfected to HemECs to overexpress or silence IL13RA2, respectively.IL13RA2 expression was remarkably increased in IH tissues.IL13RA2 overexpression promoted proliferative, migration, invasion, and tubule formation capacities of HemECs.Our results were consistent with a previous study that reported that OIP5-AS1 knockdown halts the proliferative, migration, invasion, and angiogenetic capacities of HemECs [29].
The Wnt/β-catenin pathway is a common oncogenic pathway that is activated in various cancers [14,39].βcatenin transfer into the nucleus is a key components of the Wnt pathway, leads to the upregulation or downregulation of specific genes [40].Thomann et al. have reported that low blood vessel density and β-catenin levels in regressed hepatic haemangioma [41].Ilan et al. demonstrated that nuclear β-catenin is decreased in VEGF-knockdown EOMAcells and increased in VEGF-overexpressed human umbilical vein endothelial cells [42].Similarly, in our study, β-catenin levels in nuclear and cytoplasmic fractions were downregulated by IL13RA2 knockdown and upregulated by IL13RA2 overexpression.Cyclin D1 and c-Myc are downstream oncogenes of the Wnt/β-catenin pathway [43].Our data confirmed that β-catenin, c-Myc, and cyclin D1 expression were decreased following IL13RA2 knockdown and increased following IL13RA2 overexpression.A rescue experiment with XAV-939 indicated that the effect of IL13RA2 on IH progression was mediated via the Wnt/βcatenin pathway.Collectively, these findings suggest that IL13RA2 promotes malignant progression of IH by activating the Wnt/β-catenin pathway.
The mechanism of IL13RA2 activation of glycolysis and Wnt/β-catenin pathway affecting HemECs migration and invasion is not clear and in-depth.Further studies will address these limitations.

Conclusions
IL13RA2 promotes the progression of IH by activating glycolysis and the Wnt/β-catenin pathway.IL13RA2 is a potential therapeutic target for IH.

FIGURE 1 .
FIGURE 1. IL13RA2 expression is upregulated in IH. (A) IL13RA2 expression in HDMVEs and HemECs from patients in the GSE43742 cohort.(B-D) IL13RA2 mRNA and protein expression in tissues from patients.(E) The results of immunofluorescence staining.*p < 0.05.

FIGURE 3 .
FIGURE 3. IL13RA2 promotes migration, invasion, and tubules formation abilities of HemECs.(A) Cell migration was detected by wound healing assay.(B) and (C) Cell migration and invasion were measured by Transwell.(D) Tubules formation in HemECs.(E) E-cadherin, Ncadherin, and Snail expression were detected by western blot.*p < 0.05.