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LRIG proteins in glioma: Functional roles, molecular mechanisms, and potential clinical implications
Journal of the Neurological Sciences 383 (2017) 56–60
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Review Article
LRIG proteins in glioma: Functional roles, molecular mechanisms, and potential clinical implications
MARK
Feng Mao, Baofeng Wang, Qungen Xiao, Fangling Cheng, Ting Lei, Dongsheng Guo⁎ Department of Neurosurgery and Sino-German Neuro-Oncology Molecular Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
A R T I C L E I N F O
A B S T R A C T
Keywords: LRIG proteins Glioma Molecular mechanisms Functional roles Prognosis
Gliomas are the most common intracranial tumors of the nervous system. These tumors are characterized by unlimited cell proliferation and excessive invasiveness. Despite the advances in diagnostic imaging, microneurosurgical techniques, radiation therapy, and chemotherapy, significant increases in the progression free survival of glioma patients have not been achieved. Improvements in our understanding of the molecular subtypes of gliomas and the underlying alterations in specific signaling pathways may impact both the diagnosis and the treatment strategies for patients with gliomas. Growth factors and their corresponding receptor tyrosine kinases are associated with oncogenesis and development of tumors in numerous human cancer types, including glioma. Leucine-rich repeats and immunoglobulin-like domains (LRIG) are integral membrane proteins which contain three vertebrate members including LRIG1, LRIG2 and LRIG3. They mainly function as regulators of growth factor signaling. Specifically, LRIG1 has been identified as a tumor suppressor in human cancers. In contrast, LRIG2 appears to function as a tumor promoter, while LRIG3 appears to have a function similar to that of LRIG1. In the present review, we summarize the functional roles, molecular mechanisms, and clinical perspectives of LRIG proteins in gliomas and propose that these proteins may be useful in the future as targets for treatment and prognostication in glioma patients.
1. Introduction Gliomas are the most common primary intracranial tumor which originate from neuroepithelial tissue. According to the World Health Organization (WHO) classification of nervous system tumors, gliomas are divided into four grades. Grade I tumors are considered benign and have a favorable prognosis, while grade IV tumors, also named glioblastoma multiforme (GBM), are the most common and aggressive malignant primary brain tumor, accounting for 50–60% of all gliomas [1]. In patients who present with GBM, the median survival is only 12–15 months despite debulking surgery, adjuvant radiotherapy, and chemotherapy [2]. Although tumor grade and histopathology have formed the basis of diagnosis and treatment of gliomas, recent advances in our understanding of the molecular mechanisms of gliomas have begun to translate to improvements in accurate classification and potentially promising treatment for gliomas [3,4]. Genomic studies have identified molecular characteristics, including DNA copy number, gene expression patterns, and DNA methylation aberrations, in gliomas [5]. These analyses demonstrated that glioblastomas frequently acquire gains of
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chromosomes 7 and 19; losses of chromosomes 10 and 13; mutations of phosphatase and tensin homolog (PTEN), tumor protein 53 (p53), neurofibromatosis type 1 (NF1), and isocitrate dehydrogenase 1 (IDH1) and IDH2; amplifications of platelet-derived growth factor receptoralpha (PDGFRA) and epidermal growth factor receptor (EGFR, including amplification of EGFR variants); and deletions of cyclin dependent kinase inhibitor 2A/B (CDKN2A/B) and MGMT [5–8]. Defining the genomic heterogeneity among glioblastoma and the molecular subclasses within these tumors have enabled stratification of patients, ultimately offering treatment benefits for these patients [5]. Over the last two decades, the mutational activation and amplification of receptor tyrosine kinases (RTKs), such as EGFR, PDGFR, and hepatocyte growth factor receptor (HGFR, c-MET), have been found to play important roles in the initiation and progression of malignant gliomas [9–11]. Despite the fact that EGFR is amplified in approximately 50% of high-grade malignant astrocytoma cases [12], the clinical benefit of EGFR tyrosine kinase inhibitors (TKIs) has been limited for this disease [13], perhaps due to poor central nervous system (CNS) penetration of many of these agents [14] and redundancy in RTK signaling in gliomas [5].
Corresponding author at: 1095# Jiefang Avenue, Wuhan 430030, China. E-mail address: [email protected] (D. Guo).
http://dx.doi.org/10.1016/j.jns.2017.10.025 Received 25 July 2017; Received in revised form 26 September 2017; Accepted 17 October 2017 Available online 18 October 2017 0022-510X/ © 2017 Elsevier B.V. All rights reserved.
Journal of the Neurological Sciences 383 (2017) 56–60
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3. LRIG gene family and glioma
A better understanding of the regulating molecular mechanisms would almost certainly improve our understanding of the undesirable prognosis for glioma patients. The leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family has been implicated in the regulation of RTK signaling [15]. Here, we provide a review of the studies that have been conducted on the LRIG gene family over the past decade, describe the correlation between the LRIG gene family and glioma, and discuss the possible molecular mechanisms underlying this disease. We also discuss the potential and prospect of employing LRIGs as therapeutic targets for glioma in clinical practice.
3.1. LRIG1 and glioma LRIG1, which was the first LRIG protein gene discovered, has been the most extensively studied of the LRIG gene family members [15]. This gene is located on chromosome 3p14 [26], and deletion of this region is closely associated with prognosis in several cancers. Recently, a genome-wide association study was performed via analysis of 12,496 cases, including GBM and non-GBM tumors and 18,190 controls [27]. This study revealed a single nucleotide polymorphism (SNP; rs11706832) localized in intron 2 of LRIG1 at 3p14.1 in non-GBM tumors, providing support for genetic susceptibility for GBM [27]. In an immunohistochemical study including 404 patients with astrocytic brain tumors, perinuclear staining of LRIG1 negatively correlated with WHO histological grade and positively correlated with the prognosis of glioma patients [28]. A comparative study between fresh astrocytoma specimens and surrounding non-tumor tissues from clinical surgical resections showed that LRIG1 expression was significantly reduced in tumor tissue compared to the corresponding surrounding non-tumor tissues [29]. These findings suggest that LRIG1 plays a role in the development and prognostication of astrocytic tumors. In-depth studies of LRIG1 revealed the molecular mechanisms underlying its anticancer functions (Fig. 1). Similar to Drosophila Kekkon1, EGF ligand binds to EGFR to induce synthesis of the human LRIG1 protein, which then physically binds to EGFR via its extracellular LRRs and Ig-like domains [30]. LRIG1 enhances ligand-induced receptor ubiquitination, and EGFR degradation involves recruitment of c-Cbl, which is an E3 ubiquitin ligase [30]. Recent studies found that the extracellular domain of the LRIG1 (sLRIG1) protein can be cleaved by a disintegrin and metalloproteinase 17 (ADAM17) and can inhibit EGFR signaling [31]. Intriguingly, several studies demonstrated that decreased activity of EGFR was not associated with LRIG1-mediated ubiquitination of RTKs [32,33], suggesting that LRIG1 functions in this situation via an RTK ubiquitination-independent mechanism. In a different cell context, disturbance of LRIG1 expression affects different intracellular signaling pathways of EGFR. For example, LRIG1 downregulation mainly affects the EGFR/Akt/c-Myc pathway to promote invasion of glioma cells [34], while LRIG1 overexpression affects the EGFR/MAPK pathway to inhibit in vivo glioma growth [35]. A recent study by Johansson et al. showed that LRIG1 suppresses the growth of glioma independently of EGFR status [36]. Based on the LINGO1 (also named LERN1, leucine-rich repeat neuronal protein 1) structure described previously [37], the three-dimensional crystal structure of the LRIG1 extracellular domain (ECD) protein that had been expressed in baculovirus-infected insect cells was developed. Intriguingly, this study failed to detect any binding of LRIG1 or LRIG1 ectodomain with EGFR, and the phosphorylation of EGFR and Erk1/2 remained unchanged [38]. In addition to inhibition of wild-type EGFR, LRIG1 also affects EGFRvIII, which possesses persistent kinase activity irrespective of ligand regulation [39]. LRIG1 enhances the degradation of EGFRvIII independent of the E3 ubiquitin ligase c-Cbl, thereby inhibiting glioma cell proliferation, invasion, and survival [40]. Other studies found that LRIG1 inhibits other RTKs, such as c-MET [41], the glial cell line-derived neurotrophic factor (GDNF)/Ret and PDGFRα [42,43]. Although recent studies of the molecular mechanisms underlying the functions of multiple RTK receptors partially explain the anti-tumor function of LRIG1, additional studies are in progress.
2. Introduction of the LRIG gene family The LRIG gene family was discovered because of research on oncogenic EGFR, which is closely related to the development of cancer. In a screen to identify downstream genes regulated by the EGFR, researchers discovered the Kekkon-1 gene in Drosophila [16]. Kekkon-1 encodes a single-pass transmembrane protein that features a cell adhesion molecule with leucine-rich repeats (LRRs) and one immunoglobulin-like (Ig-like) domain [16]. Kekkon-1 is produced following EGF stimulation and binds to the EGFR to regulate its activity, thereby forming a negative feedback loop in the EGFR signaling pathway [16]. Based on evolutionary conservation, researchers surmised that a negative feedback involving an EGFR-responsive protein similar to Kekkon-1 may also exist in mammalians. From the literature review, the research team led by Professor Håkan Hedman of Sweden found a study describing a Kekkon-1 paralogous gene in mice, called Lig-1, which functions in neural differentiation and development [17]. Thus, they attempted to identify genes similar to Drosophila Kekkon-1 and mouse Lrig-1 in humans [15]. After repeated screening and biological information analysis of the human gene bank, the researchers discovered three LRIG genes that were similar in structure to the Drosophila Kekkon-1 and mouse Lrig-1 genes. They were first named LIG; however, after negotiation with the Gene Nomenclature Committee, LIG was replaced with LRIG to distinguish from the lig gene which encodes the E. coli DNA ligase. The human LRIG gene family contains three members, which were LRIG1, LRIG2, and LRIG3, according to their order of discovery [15,18,19]. The LRIG polypeptides have a similar structure: a signal peptide, 15 tandem LRRs, 3 Ig-like domains, a transmembrane segment, and an intracellular domain. LRIG proteins may occur in different subcellular structures, such as in the cell membrane, cytoplasm, perinucleus, and nucleus at the cellular level [20], and have differential expression in mouse and human tissues, suggesting that these proteins are important for many cell types and organs [18]. LRIG1 has been suggested to maintain epidermal stem cells in a quiescent state [21] and to control intestinal stem cells homeostasis [22,23]. LRIG2 mutations are associated with congenital urofacial syndrome [24], and deletion of LRIG3 in mice results in cranio-facial and inner ear defects [25]. In addition, the expression of LRIG proteins is closely related to a variety of human cancers, including glioma. The individual functional roles of LRIG proteins in glioma are summarized in Table 1 and will be discussed below. Table 1 Functions of LRIG protein in glioma cells.
Apoptosis Proliferation Migration and (or) invasion Tumor growth in vivo
LRIG1
LRIG2
LRIG3
↑29,34 ↓29,31,34,35,36 ↓29,34,35 ↓35,36
↓46,48 ↑46,48 ↓48 ↑46
↑51,52 ↓51,52 ↓51,52 ↓52
3.2. LRIG2 and glioma LRIG2 is located on chromosome 1p13, a locus that harbors abnormalities [44] in many human tumors. Deletions of chromosomes 1p and 19q positively correlate with prognosis in patients with oligodendrogliomas [45]. The subcellular localization pattern of LRIG2 in
↑ indicates promote or increase. ↓ means inhibit or decrease. Numbers indicated the associated reference.
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Fig. 1. Classical molecular mechanisms underlying the anticancer functions of LRIG1 protein in glioma. LRIG1 is postulated to physically bind to EGFR via its extracellular LRRs and Ig-like domains and to inhibit the downstream signaling pathways, including MAPK and AKT. LRIG1 inhibits cell cycle progression, proliferation, and invasion and promotes apoptosis.
functions [52], although this finding has been challenged by other studies, suggesting that when LRIG1 and LRIG3 are simultaneously expressed in cells, they have opposing functions [53]. Together, these findings suggest that the functions of LRIG proteins may be more complex than we thought and therefore warrant further study.
different types of gliomas is different. For example, LRIG2 is located within the cell membrane, perinucleus, and nucleus in astrocytomas [46], whereas, this protein is expressed at extremely low levels in the nucleus in oligodendrogliomas [47]. In an immunohistochemical study of 63 patients with oligodendroglial brain tumors, high expression of cytoplasmic LRIG2 correlated with poor prognosis for these patients [47]. Analysis of a large sample from the TCGA database revealed that LRIG2 expression levels positively correlate with the WHO histological grade of gliomas [46]. In Ntv-a transgenic glioma mouse models in which tumors are induced by intracranial injection of constructed PDGFB, the tumor malignancy and formation rate of glioma were lower in mice lacking LRIG2 [43]. These data suggest that LRIG2 may be a cancer-promoting gene that is closely correlated with the occurrence and development of glioma. In addition, LRIG2 overexpression has biological effects including promotion of proliferation and inhibition of apoptosis in glioma cells [46] and LRIG2 downregulation results in decreased invasion capability in glioma cells [48]. Similar to LRIG1, an sLRIG2 also occurs for LRIG2 [46]. LRIG2 and sLRIG2 physically interact with EGFR, thereby enhancing EGFR downstream signal transduction and regulating the expression of cell cycle and apoptosis-related proteins [46]. Recently, the transmembrane protein Lrig2 was found to negatively regulate ADAMmediated shedding of Neogenin at the membrane of the cortical neurons, suggesting that that regulation of ectodomain shedding may constitute a new mechanism underlying LRIG-dependent regulation of membrane proteins, including RTKs [32]. Although LRIG1 and LRIG2 share a similar structure of the extracellular domain, these proteins may have different interaction activities, and these differences may partly explain the different functions of LRIG1 and LRIG2.
3.4. LRIG1 has potential applications in the treatment of glioma An understanding of the correlation between the LRIG gene family and glioma is still in the early phase, but its potential for translation to the treatment of gliomas has emerged in recent studies. LRIG1 sensitizes glioma cells to chemotherapeutics, such as cisplatin and temozolomide [40,54–56], and enhances the radiosensitivity of radioresistant human glioma U251-MG cells [57]. Previously, we demonstrated that overexpression of LRIG1 inhibits subcutaneous glioma growth in nude mice [35]. The injection of microbeads carrying encapsulated sLRIG1producing cells into an intracranial glioma model inhibits the growth of glioma, reduces the formation of tumor blood vessels, and prolongs the survival of tumor-bearing mice [36]. These preliminary studies indicate that LRIG1 can be used alone or in combination with other chemotherapeutics as a potential new method for the treatment of gliomas; however, additional studies are needed before these findings can be translated for human patients.
4. Summary Great advances have been made in our understanding of the etiology and molecular subclassification of gliomas. Although many unresolved questions remain, genetic studies may provide information to allow us to overcome the resistance to treatment by glioma cells and to improve the prognosis for glioma patients. As discussed above, LRIG proteins play important roles in modulating a wide range of RTK signaling pathways that influence tumor glioma cell growth, motility, and apoptosis. With a more definitive view of LRIG1-mediated tumor suppression, it will be possible to design therapies that inhibit the oncogenic signaling pathways activated by the loss of LRIG1. Although experimental and clinical data are based on a few cell lines or specific subtypes of glioma, the molecular mechanisms underlying the function of LRIG proteins in glioma are being uncovered and will lead to a better understanding of the development and progression of gliomas and ultimately strategies for improved treatment options.
3.3. LRIG3 and glioma LRIG3 was the last discovered gene in the LRIG gene family. This gene is located on chromosome 12q13.2, and multiple genes from the locus 12q13-q15 have been found to harbor abnormalities in human malignant gliomas [49]. A distinct molecular subtype of glioblastomas harbors upregulated expression of multiple genes at 12q13–15 [50]. Perinuclear staining of LRIG3 is negatively correlated with the glioma proliferation index and WHO classification in astrocytoma patients and is an independent prognostic factor [28]. Suppression of LRIG3 expression using RNA interference enhances the invasiveness, promotes proliferation, and inhibits apoptosis of glioma cells [51], while overexpression of LRIG3 inhibits cell invasion, decreases proliferation, and promotes cell apoptosis of glioma cells [52]. In vivo studies demonstrated that LRIG3 inhibits subcutaneous glioma growth in nude mice via inhibition of EGFR signaling [52]. Thus, LRIG3 and LRIG1 appear to have similar tumor suppressor
Disclosure statement The authors report no conflict of interest. 58
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Acknowledgments [25]
This work was supported by grants from the National Natural Science Foundation of China (81372711, 81001116, 81702480).
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Review Article
LRIG proteins in glioma: Functional roles, molecular mechanisms, and potential clinical implications
MARK
Feng Mao, Baofeng Wang, Qungen Xiao, Fangling Cheng, Ting Lei, Dongsheng Guo⁎ Department of Neurosurgery and Sino-German Neuro-Oncology Molecular Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
A R T I C L E I N F O
A B S T R A C T
Keywords: LRIG proteins Glioma Molecular mechanisms Functional roles Prognosis
Gliomas are the most common intracranial tumors of the nervous system. These tumors are characterized by unlimited cell proliferation and excessive invasiveness. Despite the advances in diagnostic imaging, microneurosurgical techniques, radiation therapy, and chemotherapy, significant increases in the progression free survival of glioma patients have not been achieved. Improvements in our understanding of the molecular subtypes of gliomas and the underlying alterations in specific signaling pathways may impact both the diagnosis and the treatment strategies for patients with gliomas. Growth factors and their corresponding receptor tyrosine kinases are associated with oncogenesis and development of tumors in numerous human cancer types, including glioma. Leucine-rich repeats and immunoglobulin-like domains (LRIG) are integral membrane proteins which contain three vertebrate members including LRIG1, LRIG2 and LRIG3. They mainly function as regulators of growth factor signaling. Specifically, LRIG1 has been identified as a tumor suppressor in human cancers. In contrast, LRIG2 appears to function as a tumor promoter, while LRIG3 appears to have a function similar to that of LRIG1. In the present review, we summarize the functional roles, molecular mechanisms, and clinical perspectives of LRIG proteins in gliomas and propose that these proteins may be useful in the future as targets for treatment and prognostication in glioma patients.
1. Introduction Gliomas are the most common primary intracranial tumor which originate from neuroepithelial tissue. According to the World Health Organization (WHO) classification of nervous system tumors, gliomas are divided into four grades. Grade I tumors are considered benign and have a favorable prognosis, while grade IV tumors, also named glioblastoma multiforme (GBM), are the most common and aggressive malignant primary brain tumor, accounting for 50–60% of all gliomas [1]. In patients who present with GBM, the median survival is only 12–15 months despite debulking surgery, adjuvant radiotherapy, and chemotherapy [2]. Although tumor grade and histopathology have formed the basis of diagnosis and treatment of gliomas, recent advances in our understanding of the molecular mechanisms of gliomas have begun to translate to improvements in accurate classification and potentially promising treatment for gliomas [3,4]. Genomic studies have identified molecular characteristics, including DNA copy number, gene expression patterns, and DNA methylation aberrations, in gliomas [5]. These analyses demonstrated that glioblastomas frequently acquire gains of
⁎
chromosomes 7 and 19; losses of chromosomes 10 and 13; mutations of phosphatase and tensin homolog (PTEN), tumor protein 53 (p53), neurofibromatosis type 1 (NF1), and isocitrate dehydrogenase 1 (IDH1) and IDH2; amplifications of platelet-derived growth factor receptoralpha (PDGFRA) and epidermal growth factor receptor (EGFR, including amplification of EGFR variants); and deletions of cyclin dependent kinase inhibitor 2A/B (CDKN2A/B) and MGMT [5–8]. Defining the genomic heterogeneity among glioblastoma and the molecular subclasses within these tumors have enabled stratification of patients, ultimately offering treatment benefits for these patients [5]. Over the last two decades, the mutational activation and amplification of receptor tyrosine kinases (RTKs), such as EGFR, PDGFR, and hepatocyte growth factor receptor (HGFR, c-MET), have been found to play important roles in the initiation and progression of malignant gliomas [9–11]. Despite the fact that EGFR is amplified in approximately 50% of high-grade malignant astrocytoma cases [12], the clinical benefit of EGFR tyrosine kinase inhibitors (TKIs) has been limited for this disease [13], perhaps due to poor central nervous system (CNS) penetration of many of these agents [14] and redundancy in RTK signaling in gliomas [5].
Corresponding author at: 1095# Jiefang Avenue, Wuhan 430030, China. E-mail address: [email protected] (D. Guo).
http://dx.doi.org/10.1016/j.jns.2017.10.025 Received 25 July 2017; Received in revised form 26 September 2017; Accepted 17 October 2017 Available online 18 October 2017 0022-510X/ © 2017 Elsevier B.V. All rights reserved.
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3. LRIG gene family and glioma
A better understanding of the regulating molecular mechanisms would almost certainly improve our understanding of the undesirable prognosis for glioma patients. The leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family has been implicated in the regulation of RTK signaling [15]. Here, we provide a review of the studies that have been conducted on the LRIG gene family over the past decade, describe the correlation between the LRIG gene family and glioma, and discuss the possible molecular mechanisms underlying this disease. We also discuss the potential and prospect of employing LRIGs as therapeutic targets for glioma in clinical practice.
3.1. LRIG1 and glioma LRIG1, which was the first LRIG protein gene discovered, has been the most extensively studied of the LRIG gene family members [15]. This gene is located on chromosome 3p14 [26], and deletion of this region is closely associated with prognosis in several cancers. Recently, a genome-wide association study was performed via analysis of 12,496 cases, including GBM and non-GBM tumors and 18,190 controls [27]. This study revealed a single nucleotide polymorphism (SNP; rs11706832) localized in intron 2 of LRIG1 at 3p14.1 in non-GBM tumors, providing support for genetic susceptibility for GBM [27]. In an immunohistochemical study including 404 patients with astrocytic brain tumors, perinuclear staining of LRIG1 negatively correlated with WHO histological grade and positively correlated with the prognosis of glioma patients [28]. A comparative study between fresh astrocytoma specimens and surrounding non-tumor tissues from clinical surgical resections showed that LRIG1 expression was significantly reduced in tumor tissue compared to the corresponding surrounding non-tumor tissues [29]. These findings suggest that LRIG1 plays a role in the development and prognostication of astrocytic tumors. In-depth studies of LRIG1 revealed the molecular mechanisms underlying its anticancer functions (Fig. 1). Similar to Drosophila Kekkon1, EGF ligand binds to EGFR to induce synthesis of the human LRIG1 protein, which then physically binds to EGFR via its extracellular LRRs and Ig-like domains [30]. LRIG1 enhances ligand-induced receptor ubiquitination, and EGFR degradation involves recruitment of c-Cbl, which is an E3 ubiquitin ligase [30]. Recent studies found that the extracellular domain of the LRIG1 (sLRIG1) protein can be cleaved by a disintegrin and metalloproteinase 17 (ADAM17) and can inhibit EGFR signaling [31]. Intriguingly, several studies demonstrated that decreased activity of EGFR was not associated with LRIG1-mediated ubiquitination of RTKs [32,33], suggesting that LRIG1 functions in this situation via an RTK ubiquitination-independent mechanism. In a different cell context, disturbance of LRIG1 expression affects different intracellular signaling pathways of EGFR. For example, LRIG1 downregulation mainly affects the EGFR/Akt/c-Myc pathway to promote invasion of glioma cells [34], while LRIG1 overexpression affects the EGFR/MAPK pathway to inhibit in vivo glioma growth [35]. A recent study by Johansson et al. showed that LRIG1 suppresses the growth of glioma independently of EGFR status [36]. Based on the LINGO1 (also named LERN1, leucine-rich repeat neuronal protein 1) structure described previously [37], the three-dimensional crystal structure of the LRIG1 extracellular domain (ECD) protein that had been expressed in baculovirus-infected insect cells was developed. Intriguingly, this study failed to detect any binding of LRIG1 or LRIG1 ectodomain with EGFR, and the phosphorylation of EGFR and Erk1/2 remained unchanged [38]. In addition to inhibition of wild-type EGFR, LRIG1 also affects EGFRvIII, which possesses persistent kinase activity irrespective of ligand regulation [39]. LRIG1 enhances the degradation of EGFRvIII independent of the E3 ubiquitin ligase c-Cbl, thereby inhibiting glioma cell proliferation, invasion, and survival [40]. Other studies found that LRIG1 inhibits other RTKs, such as c-MET [41], the glial cell line-derived neurotrophic factor (GDNF)/Ret and PDGFRα [42,43]. Although recent studies of the molecular mechanisms underlying the functions of multiple RTK receptors partially explain the anti-tumor function of LRIG1, additional studies are in progress.
2. Introduction of the LRIG gene family The LRIG gene family was discovered because of research on oncogenic EGFR, which is closely related to the development of cancer. In a screen to identify downstream genes regulated by the EGFR, researchers discovered the Kekkon-1 gene in Drosophila [16]. Kekkon-1 encodes a single-pass transmembrane protein that features a cell adhesion molecule with leucine-rich repeats (LRRs) and one immunoglobulin-like (Ig-like) domain [16]. Kekkon-1 is produced following EGF stimulation and binds to the EGFR to regulate its activity, thereby forming a negative feedback loop in the EGFR signaling pathway [16]. Based on evolutionary conservation, researchers surmised that a negative feedback involving an EGFR-responsive protein similar to Kekkon-1 may also exist in mammalians. From the literature review, the research team led by Professor Håkan Hedman of Sweden found a study describing a Kekkon-1 paralogous gene in mice, called Lig-1, which functions in neural differentiation and development [17]. Thus, they attempted to identify genes similar to Drosophila Kekkon-1 and mouse Lrig-1 in humans [15]. After repeated screening and biological information analysis of the human gene bank, the researchers discovered three LRIG genes that were similar in structure to the Drosophila Kekkon-1 and mouse Lrig-1 genes. They were first named LIG; however, after negotiation with the Gene Nomenclature Committee, LIG was replaced with LRIG to distinguish from the lig gene which encodes the E. coli DNA ligase. The human LRIG gene family contains three members, which were LRIG1, LRIG2, and LRIG3, according to their order of discovery [15,18,19]. The LRIG polypeptides have a similar structure: a signal peptide, 15 tandem LRRs, 3 Ig-like domains, a transmembrane segment, and an intracellular domain. LRIG proteins may occur in different subcellular structures, such as in the cell membrane, cytoplasm, perinucleus, and nucleus at the cellular level [20], and have differential expression in mouse and human tissues, suggesting that these proteins are important for many cell types and organs [18]. LRIG1 has been suggested to maintain epidermal stem cells in a quiescent state [21] and to control intestinal stem cells homeostasis [22,23]. LRIG2 mutations are associated with congenital urofacial syndrome [24], and deletion of LRIG3 in mice results in cranio-facial and inner ear defects [25]. In addition, the expression of LRIG proteins is closely related to a variety of human cancers, including glioma. The individual functional roles of LRIG proteins in glioma are summarized in Table 1 and will be discussed below. Table 1 Functions of LRIG protein in glioma cells.
Apoptosis Proliferation Migration and (or) invasion Tumor growth in vivo
LRIG1
LRIG2
LRIG3
↑29,34 ↓29,31,34,35,36 ↓29,34,35 ↓35,36
↓46,48 ↑46,48 ↓48 ↑46
↑51,52 ↓51,52 ↓51,52 ↓52
3.2. LRIG2 and glioma LRIG2 is located on chromosome 1p13, a locus that harbors abnormalities [44] in many human tumors. Deletions of chromosomes 1p and 19q positively correlate with prognosis in patients with oligodendrogliomas [45]. The subcellular localization pattern of LRIG2 in
↑ indicates promote or increase. ↓ means inhibit or decrease. Numbers indicated the associated reference.
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Fig. 1. Classical molecular mechanisms underlying the anticancer functions of LRIG1 protein in glioma. LRIG1 is postulated to physically bind to EGFR via its extracellular LRRs and Ig-like domains and to inhibit the downstream signaling pathways, including MAPK and AKT. LRIG1 inhibits cell cycle progression, proliferation, and invasion and promotes apoptosis.
functions [52], although this finding has been challenged by other studies, suggesting that when LRIG1 and LRIG3 are simultaneously expressed in cells, they have opposing functions [53]. Together, these findings suggest that the functions of LRIG proteins may be more complex than we thought and therefore warrant further study.
different types of gliomas is different. For example, LRIG2 is located within the cell membrane, perinucleus, and nucleus in astrocytomas [46], whereas, this protein is expressed at extremely low levels in the nucleus in oligodendrogliomas [47]. In an immunohistochemical study of 63 patients with oligodendroglial brain tumors, high expression of cytoplasmic LRIG2 correlated with poor prognosis for these patients [47]. Analysis of a large sample from the TCGA database revealed that LRIG2 expression levels positively correlate with the WHO histological grade of gliomas [46]. In Ntv-a transgenic glioma mouse models in which tumors are induced by intracranial injection of constructed PDGFB, the tumor malignancy and formation rate of glioma were lower in mice lacking LRIG2 [43]. These data suggest that LRIG2 may be a cancer-promoting gene that is closely correlated with the occurrence and development of glioma. In addition, LRIG2 overexpression has biological effects including promotion of proliferation and inhibition of apoptosis in glioma cells [46] and LRIG2 downregulation results in decreased invasion capability in glioma cells [48]. Similar to LRIG1, an sLRIG2 also occurs for LRIG2 [46]. LRIG2 and sLRIG2 physically interact with EGFR, thereby enhancing EGFR downstream signal transduction and regulating the expression of cell cycle and apoptosis-related proteins [46]. Recently, the transmembrane protein Lrig2 was found to negatively regulate ADAMmediated shedding of Neogenin at the membrane of the cortical neurons, suggesting that that regulation of ectodomain shedding may constitute a new mechanism underlying LRIG-dependent regulation of membrane proteins, including RTKs [32]. Although LRIG1 and LRIG2 share a similar structure of the extracellular domain, these proteins may have different interaction activities, and these differences may partly explain the different functions of LRIG1 and LRIG2.
3.4. LRIG1 has potential applications in the treatment of glioma An understanding of the correlation between the LRIG gene family and glioma is still in the early phase, but its potential for translation to the treatment of gliomas has emerged in recent studies. LRIG1 sensitizes glioma cells to chemotherapeutics, such as cisplatin and temozolomide [40,54–56], and enhances the radiosensitivity of radioresistant human glioma U251-MG cells [57]. Previously, we demonstrated that overexpression of LRIG1 inhibits subcutaneous glioma growth in nude mice [35]. The injection of microbeads carrying encapsulated sLRIG1producing cells into an intracranial glioma model inhibits the growth of glioma, reduces the formation of tumor blood vessels, and prolongs the survival of tumor-bearing mice [36]. These preliminary studies indicate that LRIG1 can be used alone or in combination with other chemotherapeutics as a potential new method for the treatment of gliomas; however, additional studies are needed before these findings can be translated for human patients.
4. Summary Great advances have been made in our understanding of the etiology and molecular subclassification of gliomas. Although many unresolved questions remain, genetic studies may provide information to allow us to overcome the resistance to treatment by glioma cells and to improve the prognosis for glioma patients. As discussed above, LRIG proteins play important roles in modulating a wide range of RTK signaling pathways that influence tumor glioma cell growth, motility, and apoptosis. With a more definitive view of LRIG1-mediated tumor suppression, it will be possible to design therapies that inhibit the oncogenic signaling pathways activated by the loss of LRIG1. Although experimental and clinical data are based on a few cell lines or specific subtypes of glioma, the molecular mechanisms underlying the function of LRIG proteins in glioma are being uncovered and will lead to a better understanding of the development and progression of gliomas and ultimately strategies for improved treatment options.
3.3. LRIG3 and glioma LRIG3 was the last discovered gene in the LRIG gene family. This gene is located on chromosome 12q13.2, and multiple genes from the locus 12q13-q15 have been found to harbor abnormalities in human malignant gliomas [49]. A distinct molecular subtype of glioblastomas harbors upregulated expression of multiple genes at 12q13–15 [50]. Perinuclear staining of LRIG3 is negatively correlated with the glioma proliferation index and WHO classification in astrocytoma patients and is an independent prognostic factor [28]. Suppression of LRIG3 expression using RNA interference enhances the invasiveness, promotes proliferation, and inhibits apoptosis of glioma cells [51], while overexpression of LRIG3 inhibits cell invasion, decreases proliferation, and promotes cell apoptosis of glioma cells [52]. In vivo studies demonstrated that LRIG3 inhibits subcutaneous glioma growth in nude mice via inhibition of EGFR signaling [52]. Thus, LRIG3 and LRIG1 appear to have similar tumor suppressor
Disclosure statement The authors report no conflict of interest. 58
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Acknowledgments [25]
This work was supported by grants from the National Natural Science Foundation of China (81372711, 81001116, 81702480).
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