ABCF1/CXCL12/CXCR4 Enhances Glioblastoma Cell Proliferation, Migration, and Invasion by Activating the PI3K/AKT Signal Pathway

Abstract Glioblastoma (GBM) is the most prevalent and fatal form of brain tumor, which is associated with a poor prognosis. ATP-binding cassette subfamily F member 1 (ABCF1) is an E2 ubiquitin-conjugating enzyme, which is implicated in regulating immune responses and tumorigenesis. Aberrant E3 ubiquitylation has been evidenced in GBM. However, the role of ABCF1 in GBM needs to be further explored. The expression of ABCF1, CXC chemokine ligand 12 (CXCL12), and CXC chemokine receptor 4 (CXCR4) in GBM tissues was examined by the GEPIA tool, real-time PCR and Western blotting. HMC3, U251MG, and LN-229 cells were cultured and transfected with shRNA targeting ABCF1 and ABCF1 plasmids. The proliferative, migrative, and invasive ability of cells was detected. Western blotting was used to detect the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (AKT). We observed that GBM tissues had higher ABCF1, CXCL12, and CXCR4 expression levels. The expression levels of CXCL12 and CXCR4 were enhanced by ABCF1 overexpression, which were significantly reversed by silence of ABCF1 in GBM cells. Silencing ABCF1 or CXCR4 inhibition weakened the capacity of GBM cell growth, migration, and invasion, while ectopic ABCF1 expression or CXCL12 treatment enhanced the cellular function of GBM cells. Furthermore, p-PI3K and p-AKT protein levels were downregulated by ABCF1 knockdown or CXCR4 blockade, which were prompted by ABCF1 overexpression or CXCL12 supplement. The ABCF1-CXCL12-CXCR4 axis was identified as a key player in GBM cell survival and metastasis by activating the PI3K/AKT signaling pathway in GBM cells.


Introduction
Glioblastoma (GBM) is one of the deadliest cancers in human beings, and its incidence accounts for more than 60% of all brain tumors [1].Although there are a variety of clinical treatments, including surgical treatment, radiotherapy, and chemotherapy [2][3][4], the prognosis of GBM is still very poor, and the average survival time of GBM patients is between 12 and 15 months owing to limited effective treatment options [5].Therefore, it is crucial to comprehend the molecular mechanisms underlying GBM in full depth and to develop more potent treatment interventions.
ATP-binding cassette subfamily F member 1 (ABCF1) is a kind of ATP-binding cassette transporter [6], which is essential for murine embryo development [7].Increasing research studies have shown that ABCF1 was involved in the development and progress of multiple malignancies.In Ewing's sarcoma, ABCF1 mRNA acts as a sponge and binds to IGF2BP3 to partially inhibit its function [8].In hepatocellular carcinoma (HCC), ABCF1 was markedly overexpressed, which not only improved the drug resistance of HCC cells to chemotherapeutic drugs but also facilitated in the transition of the epithelial matrix and the malignant development of HCC [9].In ovarian cancer, the effective sensitivity to chemotherapy was found to be linked to the downregulated expression of ABCF1 [10].Additionally, ABCF1 expression was increased in breast cancer tissues [6].Until now, the function of ABCF1 in GBM is rarely reported, and more research is required to determine its underlying mechanisms.
Prior studies have shown that ectopic ABCF1 expression promoted the expression of SOX4 [9].Aberrate SOX4 expression facilitates the production of CXC chemokine ligand 12 (CXCL12) in HCC [11].It is wellestablished that CXCL12 and its receptor, CXC chemokine receptor (CXCR4), are the key players in tumorigenesis, which regulate tumor cell survival, tumor cell metastasis, angiogenesis, and other biological processes [12][13][14].Upon binding with CXCL12, CXCR4 triggers the activation of multiple signal transduction pathways, which thus participate in regulating the growth and metastasis of tumor cells [15].
We aimed to explore the role of ABCF1 and the CXCL12-CXCR4 pathway in regulating cellular functions of GBM cells.The capability of GBM cell viability, migration, and invasion was evaluated.The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway of GBM cells mediated by the ABCF1/CXCL12/CXCR4 axis was also validated.Thus, the present research provides new insights for the treatment of GBM.

Patients and Sample Collection
This study was approved by the Ethics Committee of Guangyuan Central Hospital (Approval No.: GYZXLL202080), and informed consent was given in writing from each patient.The diagnosis of gliomas was based on histologically results [16].Patients who received radiation or chemotherapy before surgery were excluded.A total of 10 GBM tissues and 10 adjacent normal brain tissues from GBM patients were collected.All samples were rapidly frozen after surgical excision and preserved at −80°C until used.

Bioinformatics Analysis
The transcript levels of ABCF1, CXCL12, and CXCR4 in GBM tissues and normal brain tissues were analyzed by the Gene Expression Profiling Interactive Analysis (GEPIA), which is a webbased tool (http://ualcan.path.uab.edu/) to provide fast and customizable gene expression datasets obtained from TCGA and GTEx data [17].

Cell Culture and Transfection
In this study, neural stem cells (NSCs) and human GBM cell lines (LN-229 and U251MG) were purchased from American type culture collection (Manassas, USA).NSCs, U251MG, and LN-229 cells were maintained in DMEM (Thermo Fisher Scientific, Waltham, USA) with 10% FBS (Thermo Fisher Scientific) and 100 U/mL of penicillin/streptomycin (Thermo Fisher Scientific).
Real-Time PCR Using Trizol reagent (TaKaRa Biotechnology, Dalian, China), total RNA was isolated from brain tissues and harvested cells as previously described [19].The cDNA was generated using Pri-merScript reagent Kit (TaKaRa Biotechnology).Real-time PCR was carried out using StepOnePlus (Applied Biosystems, Foster City, CA, USA).The 2(-Delta Delta CT) method was used to calculate relative gene expression, which was then normalized to βactin [20].The list of primer sequences used was included in Table 1.

Colony Formation Assay
After trypsin digestion, cells were inoculated into 12-well plates and cultured for 14 days at 37°C.Then, cells were fixed with methanol, and stained with crystal violet for 10 min.The number of cell colonies stained with crystal violet was photographed and counted.

Transwell Migration and Invasion Test
Cell migration and invasion test was detected using a 24-well Transwell chamber.For the cell invasion test, the Transwell chamber was coated with Matrigel.Then, cells were seeded into the upper chamber with or without Matrigel treatment.The lower chamber was added with DMEM containing 10% FBS.After 24-h incubation, the cells in the lower chamber were fixed and stained with crystal violet.Under an inverted microscope, the cells on the lower chamber were photographed and counted.

Cell Viability Assay
Cell viability was assessed using an MTT kit (Sigma) following the manufacturer's instruction [21].Cells were digested, and 5 × 10 3 cells mixed in 0.2 mL culture medium were inoculated in 96well plate per well.For 4-h incubation, 0.01 mL of MTT at a dose of 5 mg/mL was added in 96-well plate at 37°C.After 3 days, the OD value was detected by a microplate reader at 490 nm.

Western Blotting
The collected cells were lysed in the RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific).A BCA protein detection kit (Thermo Fisher Scientific) was utilized to evaluate the concentrations of the supernatant [22].Protein was loaded and separated by SDS-PAGE and transferred to PVDF membranes.After blocking, the blots were incubated with primary antibodies.Second antibodies (Thermo Fisher Scientific) were added for 1-h incubation.Following that, ECL was applied to develop the blots.The primary antibodies used in Western blotting were present in Table 2.

Statistical Analysis
In this study, the GraphPad Prism software was used for all data analysis and visualization.The mean ± standard deviation was employed to express the data.To ascertain the statistical difference, the Student's t test or one-way analysis of variance was applied.p < 0.05 was considered statistically significant.

ABCF1, CXCL12, and CXCR4 Are Highly Expressed in GBM
The increased expression of ABCF1, CXCL12, and CXCR4 in GBM tissues was found by the GEPIA tool (Fig. 1a).Further analysis of ABCF1, CXCL12, and CXCR4 mRNA expression was performed by real-time PCR.In comparison to normal tissues, the higher expression of ABCF1, CXCL12, and CXCR4 in GBM tissues was validated (Fig. 1b).There were positive correlations between the expression of ABCF1 and the expression of CXCL12 as well as between the expression of ABCF1 and the expression of CXCR4 in GBM tissues (Fig. 1c).The protein levels of ABCF1, CXCL12, and CXCR4 in GBM tissues were higher than those in normal tissues (Fig. 1d).The protein levels of ABCF1, CXCL12, and CXCR4 were also increased in human GBM cell lines (U251MG and LN-229) compared to human NSCs (Fig. 1e).These data suggested that ABCF1, CXCL12, and CXCR4 are highly expressed in GBM.
The Cellular Functions of GBM Are Suppressed by Silencing ABCF1 or CXCR4 Inhibition LN-229 and U251MG cells were transfected with shABCF1 or treated with the CXCR4 inhibitor, AMD3100, to determine the effects of ABCF1 and the CXCL12-CXCR4 axis on the cellular function of GBM.After ABCF was knocked down, the expression of ABCF was significantly reduced in LN-229 and U251MG cells.Silencing ABCF1 or AMD3100 treatment decreased the expression of CXCL12 and CXCR4 in LN-229 and U251MG cells (Fig. 2a).According to MTT assay, knockdown of ABCF1 or treatment with AMD3100 reduced GBM cell viability (Fig. 2b).Colony formation assay demonstrated that silencing ABCF1 or AMD3100 treatment had the ability to suppress GBM cell proliferation (Fig. 2c).Moreover, the migrative and invasive capacity of GBM cells was decreased after silencing ABCF1 or treating with AMD3100 (Fig. 2d).

The Cellular Functions of GBM Are Promoted by ABCF1 Overexpression or CXCL12 Treatment
To further investigate the role of ABCF1 and the CXCL12-CXCR4 signaling pathway in the cellular function of GBM, LN-229 and U251MG cells were transfected with ABCF1 plasmid or treated with recombinant CXCL12 proteins.The expression of ABCF1 in LN-229 and U251MG cells was upregulated after transfection with ABCF1 plasmid.An increase in CXCL12 and CXCR4 expression in GBM cells was showed in both the ABCF1 overexpression group and the CXCL12 treatment group in relation to control groups (Fig. 3a).We also observed that GBM cell viability was promoted by ABCF1 overexpression or CXCL12 treatment (Fig. 3b).The number of cell colonies in both the ABCF1 overexpression group and the CXCL12 treatment group was increased, suggesting that GBM cell proliferation was enhanced by ABCF1 overexpression or CXCL12 treatment (Fig. 3c).Furthermore, the migrative and invasive capability of GBM cells was enhanced by ABCF1 overexpression or CXCL12 treatment (Fig. 3d).Collectively, the ABCF1-CXCL12-CXCR4 axis had a regulatory role in the growth, migration, and invasion of GBM cells.

ABCF1/CXCL12/CXCR4 Enhances the Tumorigenesis of Glioblastoma
The Effect of ABCF1 on GBM Cells Is Mediated by CXCL12 We next explore whether the effect of ABCF1 on GBN cells is mediated by the CXCL12-CXCR4 axis.We observed that the decreased levels of CXCL12 and CXCR4 in the shABCF1 group were restored by the CXCL12 supplement (Fig. 4a).Additionally, MTT and colony formation assays showed that CXCL12 treatment reversed the decreased proliferation of GBM cell induced by ABCF1 knockdown (Fig. 4b, c).Moreover, Transwell assays revealed that the decreased migrative and invasive capacities of GBM cells in the shABCF1 group were restored upon CXCL12 stimulation (Fig. 4d).These data indicated that CXCL12 is vital for the ABCF1-mediated functions of GBM cell survival and metastasis.
The PI3K/AKT Signaling Pathway of GBM Cells Is Regulated by ABCF1/CXCL12/CXCR4 Axis According to the findings that CXCL12-CXCR4 axis-mediated cell growth and metastasis are related to the activity of the PI3K/AKT signaling pathway [23], we performed Western blotting experiments for further validation.As shown in Figure 2a, the expression of ABCF1 was decreased in the shABCF1 group in comparison to the shNC group.After ABCF1 was knocked down in LN-229 and U251MG cells, the protein levels of p-PI3K and p-AKT were decreased (Fig. 5a).We also observed that both ectopic ABCF1 expression and CXCL12 treatment enhanced the levels of phosphorylated PI3K and phosphorylated AKT proteins in GBM cells (Fig. 5b).Furthermore, CXCL12 supplement in LN-229 and U251MG cells with ABCF1 knockdown could restore the protein levels of phosphorylated PI3K and phosphorylated AKT (Fig. 5c).In conclusion, the ABCF1-CXCL12-CXCR4 axis modulated the activation of the PI3K/AKT signaling pathway, thereby regulating the cellular function of GBM cells.

Discussion
GBM is the most prevalent and fatal form of brain tumor, which is associated with a poor prognosis [1,5].GBM, which may offer new knowledge into developing the treatment for GBM patients [24,25].In this study, we identified that GBM tissues had higher ABCF1, CXCL12, and CXCR4 expression levels.ABCF1 promoted the expression of CXCL12 and CXCR4 in GBM cells.Silencing ABCF1 or CXCR4 blockade weakened the survival and metastasis of GBM cells, while overexpression of ABCF1 or CXCL12 supplement enhanced the cellular function of GBM cells.Furthermore, we found that ABCF1-CXCL12-CXCR4 axis regulated the activation of the PI3K/AKT signaling pathway in GBM cells.ABCF1 is an E2 ubiquitin-conjugating enzyme, which is involved in regulating immune responses and tumorigenesis [6,9,26].Ubiquitylation is a posttranslational modification that can be reversed.In GBM, abnormal E3 ubiquitylation has been evidenced, which has been linked to the growth and progression of the disease [27].Previous study has shown that ABCF1 expression was elevated in HCC, which was correlated with reduced overall survival of HCC patients [26].Consistent with a previous report, we also observed an increase in ABCF1 expression in GBM tissues.Additionally, ABCF1 is thought to regulate the translation of inflammatory cytokines such as TNF and IL-6 [28,29].A significantly decreased level of TNF in blood serum was observed in mice with ABCF1 knockdown compared to wild-type controls.In GBM cells, silence of ABCF1 reduced the expression of CXCL12 and its receptor, CXCR4, which was augmented by ABCF1 overexpression.These results suggested that CXCL12 and CXCR4 expression in GBM cells are modulated by ABCF1 expression.Moreover, we found that silencing ABCF1 significantly inhibited the proliferation, migration, and invasion of GBM cell, while ectopic ABCF1 expression promoted GBM cell growth and metastasis.ABCF1/CXCL12/CXCR4 Enhances the Tumorigenesis of Glioblastoma Thus, we discovered that ABCF1 controlled the expression of CXCL12 and CXCR4 to influence GBM cell survival and metastasis.In recent years, the CXCL12/CXCR4 signaling pathway has emerged as a key player in inflammatory process and tumorigenesis [13,15].CXCR4 is the sole receptor for the CXCL12 chemokine family and is abundantly expressed in immune cells, stromal cells, and cancer cells [30].In bladder cancer, CXCR4 is overexpressed in tumor cells and the CXCL12-CXCR4 axis enhances tumor cell mobility [31,32].We found that CXCL12 and CXCR4 are highly expressed in GBM tissues.CXCR4 blockade weakened the abilities of GBM cell growth and metastasis, whereas CXCL12 supplement upregulated the cellular functions of GBM cells.Engagement of CXCR4 leads to the activation of G proteins and several downstream signaling cascades such as calcium release, MAPK, and NF-κB proteins [15].In ovarian cancer, CXCR4 induces MAPK activation and ovarian cancer cell proliferation [33].The PI3K/AKT signaling pathway is essential for the development and progress of GBM [34].We found that CXCL12 treatment promoted the activation of the PI3K/ AKT signaling pathway in GBM cells, which was suppressed by CXCR4 blockade.These data indicated that the activation of the PI3K/AKT signaling pathway was modulated by CXCL12-CXCR4 axis in GBM cells.

Numerous gene abnormalities have been discovered in
In summary, ABCF1 and the CXCL12-CXCR4 axis were identified as key players in the growth and metastasis of GBM cells.Besides, the ABCF1-CXCL12-CXCR4 axis facilitated the cellular functions of GBM cells by activating the PI3K/AKT signaling pathway in GBM cells.Therefore, by gaining a deeper understanding of the ABCF1-CXCL12-CXCR4 axis, this report may bring about new therapeutic interventions for treating GBM patients.

Fig. 1 .
Fig. 1.ABCF1, CXCL12, and CXCR4 are highly expressed in GBM. a The RNA expression of ABCF1, CXCL12, and CXCR4 in GBM tissues (n = 163) and normal tissues (n = 207) was obtained from GEPIA tool.b Real-time PCR showing the mRNA expression of ABCF1, CXCL12, and CXCR4 in GBM tissues (n = 10) and paired normal tissues (n = 10).c The correlations between the expression of ABCF1 and CXCL12 as well as the expression of ABCF1 and CXCR4 in GBM tissues (n = 10).d Western blotting showing the protein levels of ABCF1, CXCL12, and CXCR4 in GBM tissues (n = 10) and paired normal tissues (n = 10).e Western blotting showing the protein levels of ABCF1, CXCL12, and CXCR4 in human GBM cell lines (U251MG and LN-229) (n = 3) and neural stem cells (NSCs) (n = 3).(*) represents p < 0.05 and (**) represents p < 0.01 versus the normal tissue group or NSCs.The original image of WB showed in the supplementary figure (for all online suppl.material, see https://doi.org/10.1159/000533130).

Fig. 2 .
Fig. 2. The cellular functions of GBM are suppressed by silencing ABCF1 or CXCR4 inhibition.a-d U251MG and LN-229 cells were transfected with shABCF1 or treated with the CXCR4 inhibitor, AMD3100.a Western blotting showing the protein levels of ABCF1, CXCL12, and CXCR4 in the shNC, shABCF1, control, and AMD3100 treatment groups (n = 3).b MMT assay showing cell viability of U251MG and LN-229 cells in the shNC, shABCF1, control, and AMD3100 treatment groups (n = 3).c Colony for-mation assay showing the ability of cell proliferation of U251MG and LN-229 cells in the shNC, shABCF1, control, and AMD3100 treatment groups (n = 3).d Transwell migration and invasion assay showing the ability of cell migration and invasion of U251MG and LN-229 cells in the shNC, shABCF1, control, and AMD3100 treatment groups (n = 3).(**) represents p < 0.01 versus the shNC group and (^^) represents p < 0.01 versus the control group.The original image of WB showed in the supplementary figure.

Fig. 3 .
Fig. 3.The cellular functions of GBM cells are promoted by ABCF1 overexpression or CXCL12 treatment.a-d U251MG and LN-229 cells were transfected with ABCF1 plasmid or treated with recombinant CXCL12 proteins.a Western blotting showing the protein levels of ABCF1, CXCL12, and CXCR4 in the NC, ABCF1 overexpression, control, and CXCL12 treatment groups (n = 3).b MMT assay showing cell viability of U251MG and LN-229 cells in the NC, ABCF1 overexpression, control, and CXCL12 treatment groups (n = 3).c Colony formation assay showing the ability of cell proliferation of U251MG and LN-229 cells in the NC, ABCF1 overexpression, control, and CXCL12 treatment groups (n = 3).d Transwell migration and invasion assay showing the ability of cell migration and invasion of U251MG and LN-229 cells in the NC, ABCF1 overexpression, control, and CXCL12 treatment groups (n = 3).(**) represents p < 0.01 versus the NC group and (^^) represents p < 0.01 versus the control group.The original image of WB showed in the supplementary figure.

Fig. 4 .
Fig. 4. The effect of ABCF on GBM cells is mediated by CXCL12.a-d U251MG and LN-229 cells were transfected with shABCF1 and treated with recombinant CXCL12 proteins.a Western blotting showing the protein levels of ABCF1, CXCL12, and CXCR4 in the shNC, shABCF1, and shABCF1 with CXCL12 treatment groups (n = 3).b MMT assay showing cell viability of U251MG and LN-229 cells in the shNC, shABCF1, and shABCF1 with CXCL12 treatment groups (n = 3).c Colony formation assay showing the ability of cell proliferation of U251MG and LN-229 cells in the shNC, shABCF1, and shABCF1 with CXCL12 treatment groups (n = 3).d Transwell migration and invasion assay showing the ability of cell migration and invasion of U251MG and LN-229 cells in the shNC, shABCF1, and shABCF1 with CXCL12 treatment groups (n = 3).(**) represents p < 0.01 versus the shNC group and (^^) represents p < 0.01 versus the shABCF1 group.The original image of WB showed in the supplementary figure.

Fig. 5 .
Fig. 5.The PI3K/AKT signaling pathway of GBM cells is regulated by ABCF1/CXCL12/CXCR4 axis.a U251MG and LN-229 cells were transfected with shABCF1 or treated with the CXCR4 inhibitor, AMD3100.Western blotting showing the protein levels of phosphorylated PI3K and AKT in the shNC, shABCF1, control, and AMD3100 treatment groups (n = 3).(**) represents p < 0.01 versus the shNC group and (^^) represents p < 0.01 versus the control group.b U251MG and LN-229 cells were transfected with ABCF1 plasmid or treated with recombinant CXCL12 proteins.Western blotting showing the protein levels of phosphorylated PI3K and AKT in the NC, ABCF1 overexpression, control, and CXCL12 treatment groups (n = 3).(**) represents p < 0.01 versus the NC group and (^^) represents p < 0.01 versus the control group.c U251MG and LN-229 cells were transfected with shABCF1 and treated with recombinant CXCL12 proteins.Western blotting showing the protein levels of phosphorylated PI3K and AKT in the shNC, shABCF1, and shABCF1 with CXCL12 treatment groups (n = 3).(**) represents p < 0.01 versus the shNC group and (^^) represents p < 0.01 versus the shABCF1 group.The original image of WB showed in the supplementary figure.

Table 1 .
Primers used in real-time PCR

Table 2 .
Primary antibodies used in