Autocrine motility factor receptor promotes the malignancy of glioblastoma by regulating cell migration and invasion

ABSTRACT Objective One of the important causes of death in cancer patients is malignant metastasis, invasion, and metastasis of tumor cells. Metastasis is also the most basic physiological characteristics and pathogenesis of various tumors. Previously published studies have suggested that autocrine motor factor receptor (AMFR) is the key regulator of tumor cell migration and invasion. Meanwhile, AMFR is highly expressed in esophageal tumors, gastrointestinal tumors, and bladder cancer, and it is also involved in its pathogenesis. However, the role of AMFR in glioblastoma has not been reported. Methods In order to study the role of AMFR in the cell migration and invasion of glioblastoma, AMFR was silenced using siRNA and overexpressed using cDNA. Immunoblotting analysis and real-time quantitative polymerase chain reaction (PCR) were employed to assess the expression of AMFR. We conducted wound healing assay, cell migration assay, and tumorsphere formation assay to detect the invasion and metastatic ability of glioblastoma. Results This study found that the level of AMFR expression was significantly correlated with the malignant degree of glioma tissue in clinic samples. AMFR silencing decreased cell migration and invasion of LN229. Overexpression of AMFR significantly increased cell migration and invasion of U251. Conclusion This study suggests that AMFR could be used as a therapeutic strategy for the clinical treatment of glioblastoma.


Introduction
There is a poor prognosis associated with glioblastoma, which is a tumor of the central nervous system (CNS) and the most malignant among all types of astrocytic tumors [1].Several factors contribute to the poor prognosis of glioma patients, including the highly invasive nature of the tumor cells [2].The invasion of tumor cells into the parenchyma results in incomplete surgical resection, which is extremely resistant to chemotherapy.The clinic, however, does not currently offer anti-invasive treatments [3].To develop novel therapeutic strategies, it is essential to clarify the details of glioblastoma invasion's molecular pathogenesis.
It is important to identify factors that contribute to invasion and metastasis of tumor cells, since locomotion plays a key role in invasive and metastatic capabilities.It has previously been shown that the secreted 55 kDa cytokine autocrine motility factor (AMF) can stimulate tumor angiogenesis [4,5], ascites formation [6], apoptotic resistance [7], cell proliferation [8] and cell motility [9].There is an association between all of the above factors and tumor aggression, invasion, and metastasis.In addition to melanoma cells, AMF can promote the spread of other cancer cells as well [10,11] and induce cell motility in the ligand-receptor manner through interacting with its 78 kDa glycoprotein receptor, which is a seven-transmembrane domain glycoprotein [12], also known as AMF receptor (AMFR).
AMFR is a cytokine receptor, which is a transmembrane protein that transmits a signal into the cell upon ligand binding [13].In terms of structure, AMFR belongs to the seventransmembrane protein family, while it can be recognized as AMF ligand by its extracellular domain.The AMF-AMFR interaction triggers a pertussis toxin-sensitive G protein, leading to the stimulation of small Rho-like GTPase, Rac1, and RhoA through the increase in Rho GDP dissociation inhibitor beta expression.Subsequently, the activation of c-Jun N-terminal kinase 1 (JNK1) and c-Jun N-terminal kinase 2 (JNK2) results in actin fiber rearrangement, which can directly regulate the cancer cell motility, invasion, as well as metastasis [14,15].
By interacting with AMF and AMFR, a positive feedback loop is created, resulting in the dynamic transformation of tumor cells into the aggressive forms [16].Through the above pathway, AMF-AMFR further affects the movement, migration, and invasion ability of tumor cells [17][18][19].Therefore, patients with malignant tumors, such as glioblastoma, are expected to benefit from the important therapy targeting AMF-AMFR pathway.Previously published study has shown that the mRNA level of AMF expression in glioblastomas is significantly higher than that in astrocytomas, resulting in a shorter survival in glioblastoma patients with AMF-positive population compared with AMFnegative group [10].However, the relationship between AMFR and glioblastoma is still largely unknown.
In this study, we first explored the mRNA and protein levels of AMFR expression in glioma tissue chips using immunohistochemistry technology and in glioblastoma LN229, U251, U373 cell lines via real-time PCR and immunoblotting assays.Then, we explored the role of AMFR in invasion ability of glioblastoma LN229, U251, U373 cell lines by assessing the performance of silencing or overexpression AMFR on the ability to migrate in these cell lines.As a result of the present study, an AMFRtargeted therapeutic strategy is described for treating glioblastoma.

Immunohistochemistry
A total of 68 cases of human different malignant glial tumor tissues were purchased from Alena Biotechnology (Xi'an, China).These cases of tissue chip immunohistochemistry experiments were also commissioned to Alena Biotechnology.Due to mistakes and losses in the process of specimen preparation, a total of 68 cases could be used for immunohistochemistry chip analysis in this experiment.

Cell culture
Human glioblastoma LN229 cells, U251 cells, and U373 cells were purchased from the TransGen Biotech (Beijing, China) and then cultured in Dulbecco's modified Eagle's medium (DMEM).The DMEM was added to heat-inactivated fetal bovine serum (FBS, 10%) and penicillin/streptomycin.The cultures were incubated with an incubator at 37°C with 5% CO 2 , and the medium was replaced every 2 days.

RNA and cDNA interference
Adenovirus-AMFR shRNA and adenovirus-AMFR cDNA were purchased from the Weizhen Biotechnology (Shandong, China).The LN229 cells or U251 cells seeded onto 6-well plates after 48 h of incubation were infected with 5 μL adenovirus-AMFR shRNA or adenovirus-AMFR cDNA.

Wound healing and cell migration assay
The LN229 cells or U251 cells infected with adenovirus-AMFR shRNA or adenovirus-AMFR cDNA.The detailed procedures were similar with previously published study [20].Briefly, the cell lines were cultured in 24-well plates and transwell cell chambers for wound healing assay and cell migration assay, respectively.In order to calculate the relative mobility, we used the formula: The relative mobility = (distance between the edges of migrated scratches/distance between the edges of initial scratches) × 100%.For cell migration assay, phase-contrast images were acquired using an inverted microscope (CKX41, Olympus) at 200× magnification.Then the migrating cells were counted within at least three random fields.

Cell proliferation activity detection
Thiazolyl blue tetrazolium bromide (MTT), a yellow dye, was used to detect the proliferative activity of cells by MTT assay.LN229, U251 and U373 cells were diluted in filled with DMEM (containing 10% FBS) and then plated in the 96-well plates, and the cultured cells were not added to the blank control.Each well was added with 200 µl MTT solution at 0.5 µg/µl and then continued cultured for 4 h.Finally, remove the cell culture medium and add 150 μl dimethyl sulfoxide (DMSO).The crystallization was fully dissolved by slow oscillation.Multiple wells were set, and the optical density (OD) values for each sample were detected at 492 nm.Three independent repeated experiments were completed.

Statistical analysis
Data for each group are represented as the mean values ± standard errors of means (SEM) from at least three independent repeated experiments.Unpaired Students' t-tests were employed to determine the statistical significance of the data by employing GraphPad Prism.The statistical significance of a difference was determined by a two-sided P-value of 0.05.

Expression of AMFR in glioblastoma tissues
Numerous studies have suggested that the expression levels of AMF are upregulated in malignant tumors patients and correlated with a poor prognosis [11,21].Therefore, we measured the protein level of AMFR expression in glioblastoma tissues.In total, we collected 68 glioma samples from patients that ranged in age from 9 years to 66 years.Analysis of immunohistochemical results of glioma with different malignant degrees are shown in Table 1.Represent results are shown in Figure 1.Based on the result, we found that AMFR was expressed in glioblastoma tissues (Figure 1(a-c).Moreover, we found that the highest expression of AMFR was found in grade IV gliomas (Figure 1d).Further analysis suggested that the protein level of AMFR protein was positively correlated with the malignant degree of glioma tissue (Figure 1e).

AMFR stimulates glioblastoma cell migration and proliferation
To explore the role of AMFR in glioblastoma cell migration and proliferation, human glioblastoma cell lines LN229, U251, and U373 were used.Here, we detected the migrate and proliferate ability of these three glioblastoma cell lines.Our results shown that the LN229 cell line had the fasted migrate and proliferate rate, and U251 had the slowest proliferate and migrate rate (Figure 2(ac).Then, the mRNA level and protein level of AMFR expression in these glioblastoma cell lines were explored.The results demonstrated that the mRNA and protein expression of AMFR were different among three cell lines.The highest mRNA and protein level of AMFR expression appeared in LN229, whereas the lowest ones appeared in U251 Figure 2(d, e).This result was consistent with the cell proliferation and cell migration ability of glioma cell lines, that is, the more AMFR expression, the stronger the migration potential and proliferation ability of glioma cell lines.

Knockdown of AMFR decreases glioblastoma cell migration and invasion
In order to verify that AMFR acts as an autocrine stimulator of glioblastoma cell migration in vitro, we silenced AMFR expression.Initially, LN229 cells were transduced with shRNA targeting AMFR (shRNA group) or with non-silencing control shRNA (shControl group).The efficiency of shRNA targeting knockdown of AMFR protein was achieved in LN229 cells by immunoblotting (Figure 3a).LN229 knockdown sublines showed significantly reduced glioma cells migration Figure 3(b, c) and invasion capability Figure 3(d,  e) compared with shControl group.

Overexpression of AMFR increases glioblastoma cell migration and invasion
To analyze the effect of the AMFR protein on glioma cells migration capability, we up-regulated AMFR expression.Initially, U251 cells were transduced with cDNA targeting AMFR (cDNA group) or with empty vector (Vector group).Stable over-

Discussion
In this study, we found that AMFR was crucial for glioma cell migration, especially in high-grade glioma.The level of AMFR expression was significantly correlated with the malignant degree of glioma tissues and migration of glioblastoma cell lines.Silencing AMFR expression decreased glioma cell migration capability.Meanwhile, over-expression of AMFR increased glioma cell migration capability.It has been reported that the AMF and AMFR expression levels are increased in many different types of tumor tissues, especially in metastatic or migratory tumors, such as endometrial cancer [22] and prostate cancer [23].Although studies have shown that AMF-AMFR has relationship with glioblastoma cell migration, whether AMFR is related to the malignancy of the glioma is still unknown.Here, we collected 68 samples including normal and glioma tissues.We analyzed and classified the AMFR staining results of these samples.Similar to other tumors, the AMFR content was positively correlated with the degree of tumor progression.
Previously published studies have suggested that AMF-AMFR is related with migration of tumor cells [17][18][19].Our results are consistent with the above studies.The mRNA and protein levels of AMFR expression in LN229 cell line, which had the fastest rate of proliferation and migration, were highest among LN229, U251, U373 cell lines.To further study the relationship between AMFR expression and glioblastoma cell migration, AMFR shRNA was used to silence AMFR in LN229 because it has the highest level of AMFR expression, and AMFR cDNA was used to overexpress AMFR in U251 for the reason that it exhibits the lowest expression of AMFR among three cell lines.Our results showed that silencing AMFR in LN229 decreased the cell migration ability, and overexpression of AMFR in U251 increased the cell migration ability.
Recent studies have confirmed that AMF-AMFR binding signaling pathway is closely related to local invasion of various malignant tumor cells, tumor distant metastasis and lymph node metastasis [24][25][26][27][28]. AMFR acts as a cell surface receptor of AMF.AMF stimulates the AMFR to produce a G-protein, which is sensitive to the laryngopharyngeal toxin, and then activates inositol triphosphate, protein kinase C (PKC), phosphorylated receptors, thus further affecting the motility and migration ability of cells [17,19,29].The migration ability of cell movement is an important index of tumor cell invasion and plays a key role in tumor cell invasion.Therefore, we detected the level of AMFR expression in both human glioma samples and glioblastoma cell lines.Our results show that the AMFR expression was positively correlated with the degree of tumor progression and glioblastoma cell migration.These findings are consistent with the fact that the high AMF-AMFR expression is closely related to the invasion of various tumor cells and poorer survival, verifying the bioinformatics analyses and the previously published study by using in situ hybridization [30].
Regarding the mechanisms underlying AMFRmediated the malignancy of glioblastoma cell migration and invasion, there may be attributed to several aspects.It is well known that AMFR is also an important E3 ubiquitin ligase beyond the receptor.Previously published study has suggested that AMFR promotes cell proliferation, motility, and invasion by the regulation of both epidermal growth factor receptor (EGFR) and extracellular-signal-regulated kinase (ERK) activation.Mechanically, AMFR stimulates ERK activation via the mitogen-activated protein kinases (MAPKs) endogenous negative regulator dualspecificity phosphatase 1 (DUSP1) degradation to involve in EGFR-mediated hepatocellular carcinoma cell proliferation and invasion [31].The clinical evidence also showed that high levels of AMFR are associated with poor outcomes in both estrogen receptorspositive (or ER + ) and ER-negative (ER -) breast cancer patients [32].Numerous studies have indicated that AMFR mainly influences cell signaling pathways such as cell cycle, cell metabolism, immunity, and autophagy [33,34].Moreover, dysfunction of AMFR has been suggested to be involved in many other diseases, such as allergic asthma, epileptic seizures, coronary atherosclerotic heart disease, and pulmonary fibrosis [35,36].For example, AMFR was involved in the development of allergic asthma by enhancing lung alveolar macrophage-derived granulocytemacrophage colony-stimulating factor (GM-CSF) production [37].Pharmacological induction of AMFR by acetaminophen treatment increases functional excitatory amino acid transporter 2 (EAAT2) oligomer levels and relieves the temporal lobe epilepsy in mice [38].Meanwhile, other studies also indicated that the AMFR gene polymorphisms were significantly correlated to the coronary atherosclerotic heart disease individuals [39,40].For the immunological function of AMFR on host immune responses, it was recruited to the endoplasmic reticulum (ER) and then interacted with stimulator of interferon genes (STING) during the herpes simplex virus 1 (HSV-1) infection [41,42].

Conclusion
Collectively, our findings indicate that AMFR expression was positively correlated with the degree of tumor progression.AMFR silencing can inhibit glioblastoma invasion, and AMFR overexpression can accelerate glioblastoma invasion.All these results suggest that the AMF-AMFR signaling pathway may be a potential molecular target for inhibiting glioblastoma progression.

Figure 1 .Figure 2 .
Figure 1.The expression level of AMFR was positively correlated with the malignant degree of glioma tissue.(a) The result of AMFR staining on II grade glioma.(b) The result of AMFR staining on III grade glioma.(c) The result of AMFR staining on IV grade glioma.Scale bar: 500 μm for (a-c).(d) The statistical chart of distribution of AMFR on II-IV grade glioma.(e) The statistical diagram of (c).IHC experiment evaluation criteria: staining results on the chip were judged as negative (-) without staining, weak positive (+) with light brown, positive (++) with brown, and strong positive (+++) with brown.

Figure 3 .
Figure 3. Knockdown of AMFR decreases cell migration and invasion of LN229.(a) The representative graph (upper) and statistical chart (below) of expression level of AMFR in LN229 after using scramble shRNA (shControl group) and AMFR shRNA (shRNA group).(b) The representative graph of wound healing assay of human glioblastoma cell line LN229.Scale bar: 100 μm.(c) The statistical chart of (b).(d) The representative graph of migration assay of cell line LN229.The migrating cells were indicated by white arrows.(e) The statistical chart of (d).Scale bar: 100 μm.*P < 0.05, **P < 0.01, *P < 0.001.

Figure 4 .
Figure 4. Overexpression of AMFR increases migration and invasion of U251.(a) The representative graph (upper) and statistical chart (below) of expression level of AMFR in U251 after using empty vector (Vector group) and AMFR cDNA (cDNA group).(b) The representative graph of wound healing assay of human glioblastoma cell line U251.Scale bar: 100 μm.(c) The statistical chart of (b).(d) The representative graph of migration assay of cell line U251.The migrating cells were indicated by white arrows.(e) The statistical chart of (d).Scale bar: 100 μm.*P < 0.05, **P < 0.01, *P < 0.001.

Table 1 .
Pathological examination results of paraffin tissue chip.
IHC experiment evaluation criteria: staining results on the chip were judged as negative (-) without staining, weak positive (+) with light brown, positive (++) with brown, and strong positive (+++) with brown.