LRIG1 regulates invasion and migration of human gliomas through SNAI2 and E-cadherin CURRENT STATUS: REVIEW

The particular molecular mechanisms that activates invasion and migration of human gliomas remains obscure. Our previous study has indicated the function of leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) in the inhibition of the cell invasion and downstream genes snail homolog 2(SNAI2) and E-cadherin were involved in this process. In this study we give an insight into the role SNAI2 and E-cadherin played in this process and the relationship between them in glioma tissue specimen. We employed a full length expression plasmid to overexpress LRIG1 in the malignant glioma cell line U251. Introduction of exogenous LRIG1 into U251 significantly inhibited cell invasion and metastasis detected by transwell assay and scratch test, respectively. On the other hand, LRIG1 overexpression leads to reduced SNAI2 expression and elevated E-cadherin expression, manifested by qRT-PCR and western blot, which was consistent with the results in glioma tissue specimens. Further research revealed that 4-chloro-DL-phenylalanine (PCPA), a small molecule inhibitor of SNAI2, can significantly promote the inhibitory effect of cell invasion and migration caused by overexpressed LRIG1. Our data suggested that LRIG1 as a tumor suppressor restricted glioma invasion and migration by regulating SNAI2 and E-cadherin axis and low expression of LRIG1 is associated with poor prognosis in glioma. In conclusion, restoration of LRIG1 in glioma cells could be a novel therapeutic strategy.


Introduction
Gliomas are the most common primary intracranial tumors which cause significant mortality and morbidity in spite of the advances in diagnostic imaging, microneurosurgical techniques, radiation therapy, and chemotherapy. Tumor invasion and migration are the major contributors to the death in glioma patients. Our previous study has indicated the function of LRIG1 in the inhibition of the cell invasion, but the particular molecular mechanisms remains obscure. LRIG1 is a type 1 transmembrane protein whose extracellular domain contains 15 leucine-rich repeats (LRRs) and three immunoglobulin (Ig)-like domains, mainly function as regulators of growth factor signaling [1] . LRIG1 has been identified as a tumor suppressor in human cancers, such as prostate cancer [2] , non-small cell lung cancer [3] , thyroid cancer [4] as well as gliomas [5] . The SNAI2 gene encoding Slug, an 3 oncogenic transcriptional repressor acting as a key regulator of cell migration, has been found overexpressed in many cancers such as leukemia, lung cancer, esophageal cancer, colorectal cancer, prostate cancer, breast cancer and ovarian cancer [6] . Cadherin family are a group of cell-surface adhesion molecules which played important roles in intercellular adhesion and E-cadherin is the major subtype of cadherin family expressed in epithelial tissues. E-cadherin is a key component of the adherens junctions which are indispensable in cell adhesion and maintaining cell epithelial phenotype [7] . E-cadherin is often inactivated or functionally inhibited, which resulting in tumor development or progression [8] .The mechanism of glioma invasion and migration is an intricate program called epithelial-to-mesenchymal transition (EMT), in which process SNAI2 and E-cadherin play an important role [9] . Glioma cells undergo a series of molecular and conformational changes shifting towards mesenchymal traits, which include cytoskeletal re-patterning, extracellular matrix (ECM) remodeling, and stem-like trait acquisition [10] . It has been reported that LRIGI1 regulates EMT in melanoma cells [11] and basal-like breast cancer cells [12] to inhibit cell invasion, whether this is present in gliomas is still unknown. We found in our study that low expression of LRIG1 is associated with poor prognosis in glioma and LRIG1 inhibits glioma invasion and migration by regulating downstream SANI2-E-cadherin axis.

Materials And Methods
Clinical specimen and data collection. Human glioma specimens were collected from the inpatients in Department of Neurology, Renmin Hospital of Wuhan University from 2015 to 2017. This study was reviewed and approved by Medical Ethics Committee of Wuhan University. Written informed consent was obtained from each patient included. Normal contral brain tissue that corresponds to glioma tissues were taken from patients undergoing surgical treatment for craniocerebral trauma. Immunohistochemistry (IHC) and evaluation. The IHC was performed as described previously [13] . LRIG1-positive cells displayed brownish blue granules on the cytoplasm. According to the percentage of immunoreactive cells and intensity of the staining, cell were divided into four parts from + to ++++ through immunohistochemical scores (IHC scores). The percentage was rated on a scale of 0-4 4 as follows: 0, < 5%; 1, 5-25%; 2, 26-50%; 3, 51-75%; and 4, 76-100%. The evaluation of the staining intensity was rated and scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. By multiplying the percentage and intensity score we obtained the IHC score. The final grouping criteria was as follows: IHC scores 0-3 were considered as group +, scores 4-6 were considered as group ++, scores 7-9 were considered as group +++ and scores 10-12 were considered as group ++++. These scores were independently determined by two independent senior pathologists. Group + and group + + were considered as low expression, group +++ and group ++++ were considered as high expression. RNA isolation and qRT-PCR. Total RNA were extracted from fresh frozen glioma tissues or glioma cell line using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. One µg of total RNA was used as a template for reverse transcription using ReverTra Ace-A (Toyobo, Osaka, Japan), after the determination of the amount of total RNA by ultraviolet (UV) spectrophotometry.

Results
Low expression of LRIG1 is associated with poor prognosis in glioma. We employed IHC to detect the expression of LRIG1 in 78 glioma specimens, 40 normal brain tissue specimens were used as a control group. IHC results showed that LRIG1 was significantly down-regulated in glioma tissues compared with the control group ( Figure 1A and 1B). To avoid possible false positive results, we used qRT-PCR to further verify the expression of LRIG1 mRNA in another independent group of fresh frozen glioma tissues and the control group, a total of 30 glioma tissues and 20 normal controls.
Consistently, Results of qRT-PCR corroborated what was achieved by IHC, exhibiting that LRIG1 was markedly reduced in glioma tissues in comparison with normal controls ( Figure 1C). Subsequently, clinicopathological characteristics and LRIG1 expression in 78 cases of glioma tissues were performed with Chi-square Test. The results displayed that expression of LRIG1 was remarkably associated with tumor WHO grade and relapse, but no significant correlation can be identified between LRIG1 expression and other clinicopathological characteristics comprising age, sex and histological subtype (Table 1). Kaplan-Meier survival analysis revealed that reduced LRIG1 dramatically led to inferior overall survival ( Figure 1D) and disease-free survival ( Figure 1E) in glioma. Furthermore, multivariate COX regression analysis was performed suggesting that LRIG1 expression was an independent prognostic factor in glioma (Table 2), in addition to tumor WHO grade and relapse. In conclusion, the data demonstrated down-regulated LRIG1 was significantly linked with poor prognosis in glioma.
The LRIG1 expression plasmid pLRIG1-GFP and empty control pEGFP-N1 were transfected into U251 cells. Fluorescence microscopy (488nm, Nikon, Japan) showed transfected cells pLRIG1-GFP-U251 and pEGFP-N1-U251 emitted green fluorescence, and U251 cells emitted none 24h after transfection (Figure 2A). Cells were selected by culture medium containing G418 48h after transfection and neomycin-resistant clones were generated after 3 weeks. LRIG1 mRNA level was significantly up regulated in pLRIG1-GFP-U251 cells compared with controls, as shown by qRT-PCR ( Figure 2B). Further analysis by western blot showed that LRIG1 protein level in pLRIG1-GFP-U251 cells increased accordingly ( Figure 2C).

LRIG1 regulates downstream genes SNAI2 and E-cadherin in glioma. LRIG1, SNAI2 and E-
cadherin protein level in 30 cases of fresh frozen glioma tissues and 20 cases normal controls were evaluated by western blot analysis. LRIG1 and E-cadherin were down regulated while SNAI2 was up regulated in glioma tissue (p<0.05) ( Figure 3A). SNAI2 and E-cadherin mRNA level were evaluated by qRT-PCR in four groups of U251 cells, which were p-EGFP-N1, p-EGFP-N1+PCPA, pLRIG1-GFP, pLRIG1-GFP+PCPA U251 cells, respectively. The results indicated that LRIG1 can down regulated SNAI2 mRNA level in glioma (p<0.05) and PCPA treatment has no effect on the mRNA level of SNAI2 ( Figure 3B). LRIG1 can also up regulated E-cadherin mRNA level, and PCPA has a positive effect on this process (p<0.05) ( Figure 3C). SNAI2 and E-cadherin protein level in four groups of U251 cells abovementioned were evaluated by western blot analysis ( Figure 3D). The results indicated that LRIG1 can down regulated SANI2 protein level in glioma (p<0.05) ( Figure 3E). LRIG1 can also up regulated E-cadherin protein level, and PCPA has a positive effect on this process (p<0.05) ( Figure 3F). There result indicated that LRIG1 modulate SANI2-E-cadherin axis in glioma. healing speed of each image are presented as the mean ± standard deviation (SD). One way ANOVA was employed to analyze the significant difference (P<0.001). The number of invaded cells were 420±40, 310±32, 198±43 and 99±29 from front to back and the migrated cells were 386±35, 295±29, 220±33, 86±25.The results show that LRIG1 inhibits invasion, migration and speed of scratch healing, and PCPA, the small molecule inhibitor of SNAI, has obvious promotive effect on such a process. In summary, we believe that LRIG1 inhibits the invasion and migration of glioma by regulating the SANI2-E-cadherin axis.

Discussion
LRIG1 is a cell surface transmembrane protein, according to the existing research, it mainly has the following functions: an endogenous feedback regulator of receptor tyrosine kinases (RTKs), a tumor suppressor, an intestinal stem cell marker and a modulator of different cellular phenotypes [14,15] . It has been reported to be a tumor suppressor gene in malignant glioma, involved in the proliferation [16] , radioresistance [17] , chemosensitivity [18] of glioma cells. LRIG1 inhibits hypoxiainduced vasculogenic mimicry formation via suppression of the EGFR/PI3K/AKT pathway [19] . Xie R, et al reported that down regulation of LRIG1 RNA interference promotes the aggressive properties of glioma cells via EGFR/Akt/c-Myc activation [20] , other than that, no more detailed reports. Our previous study have focused on the role LRIG1 played in glioma invasion inhibition, however the molecular mechanism involved is still unknown. In this study we revealed low expression of LRIG1 is associated with poor prognosis in glioma and indicated higher WHO grade, relapse rate and shorter survival. To our knowledge, the invasive growth and migration of gliomas to surrounding normal brain tissues is an important reason why gliomas are extremely prone to relapse. Tumor relapse was the chief reason for poor prognosis of glioma, is mainly attributed to glioma stem cells (GSCs) and epithelial-mesenchymal transition (EMT). The latest report states another transmembrane protein containing leucine-rich repeat, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), promotes EMT by activating the Wnt/β-catenin pathway and predicts poor prognosis of glioma [21] .
Many proteins or microRNAs regulate glioma cell invasion, and the detailed mechanism is by modulating EMT [22][23][24] . EMT is regulated by key transcription factors, including SNAI2. We showed in 9 our data that SNAI2 and E-cadherin were involved in the inhibition of cell invasion and migration by LRIG1, and this provides guidance and direction for us to further study its detailed mechanism. EMT mainly involving an E-cadherin to N-cadherin shift, led to tumor invasion or migration and therapeutic resistance. E-cadherin is a transmembrane glycoprotein which connects epithelial cells together at adherens junctions. E-cadherin functional loss has frequently been associated with poor prognosis and survival in patients of various cancers [25] . In our study, E-cadherin loss was detected in both tissue specimens and cell lines and the ability of cell invasion and migration rises sharply with the loss of Ecadherin,.
In conclusion, we demonstrated for the first time that LRIG1 is down-regulated in glioma tissues, resulting in the activation of SANI2 and loss of E-cadherin, lead to malignant prognosis of gliomas.
LRIG1regulated invasion and migration of gliomas by modulating SNAI2-E-cadherin axis, EMT may play a decisive role in this process but more details still need further study.     The LRIG1 expression plasmid pLRIG1-GFP and pEGFP-N1 were transfected into U251 cells.