Baofeng Wang

Upregulation of LRIG1 suppresses malignant glioma cell growth by attenuating EGFR activity

Activated epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target for a variety of solid tumors, particularly malignant gliomas. A recently discovered transmembrane glycoprotein, LRIG1, antagonizes the activity of epidermal growth factor receptor family receptor tyrosine kinases and acts as a negative feedback loop of EGFR and proposed tumor suppressors. The aim of this study was to investigate the impact of LRIG1 on the biological features of glioma cells and the possible mechanisms of enhanced apoptosis induced by upregulation of LRIG1. We observed that the expression of LRIG1 was decreased, while the expression of EGFR was increased in the majority of astrocytomas, and the ratio of EGFR/LRIG1 was increased by sixfold in tumors versus corresponding non-neoplastic tissue. Upregulation of LRIG1, followed by a decrease of EGFR on the cytomembrane of the cells, induced cell apoptosis and cell growth inhibition, and further reversed invasion in glioma cell lines and primary glioma cells. Our study now clearly indicates that LRIG1 indeed affects cell fate and biology behaviors of the cells in vitro by inhibiting phosphorylation of downstream MAPK and AKT signaling pathway, and the elevated release level of caspase-8 might contribute to the enhanced apoptosis in LRIG1 transfected glioma cells. Taken together, these findings provide us with an insight into LRIG1 function, and we conclude that LRIG1 evolved in gliomas as a rare feedback negative attenuator of EGFR and could offer a novel therapeutic target to treat patients with malignant gliomas.

Down-regulation of LRIG2 expression by RNA interference inhibits glioblastoma cell (GL15) growth, causes cell cycle redistribution, increases cell apoptosis and enhances cell adhesion and invasion in vitro

The leucine-rich and immunoglobulin-like domains (LRIG) gene family contains LRIG1, 2 and 3. LRIG1 is a negative regulator of EGFR, but little is known about the function of LRIG2. To determine the role of LRIG2 in the progression of glioma, we performed RNA interference-mediated knockdown of LRIG2 in a human glioma cell line (GL15). Down-regulation of LRIG2 expression resulted in: rapid EGF-mediated loss of EGFR; decreased proliferation; G0/G1 arrest; increased spontaneous apoptosis; enhanced cell adhesion and increased invasion capability of GL15 cells in vitro. These findings indicate that LRIG2 possesses distinct functions compared with LRIG1 and validate the attractiveness of LRIG2 as a target in glioma therapy.

Inhibition of LRIG3 gene expression via RNA interference modulates the proliferation, cell cycle, cell apoptosis, adhesion and invasion of glioblastoma cell (GL15)

LRIG3 (leucine-rich repeats and immunoglobulin-like domains, LRIG) gene is both under-and over-expressed in human cancers and its role on tumor growth is not fully clarified. Here, we used a human U6 promoter-driven DNA template approach to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block LRIG3 gene expression in the human glioma cell line GL15. Specific knockdown of LRIG3 by shRNA resulted in significantly increase of the GL15 invasion and adhesion activity in vitro and markedly promoted cell growth. LRIG3 repression also induced increment of the proportion of G0/G1 cells and inhibited apoptosis in GL15 cells. Our results demonstrated that RNAi against LRIG3 could effectively down regulate LRIG3 gene expression. LRIG3 might be involved in the regulation of EGFR signaling, and serve as a tumor suppressor gene in the pathogenesis of glioma.

RNA interference therapy for glioblastoma

Importance of the field: Despite numerous advances made during the last decade in brain tumor therapy, the prognosis of glioblastoma has not improved and these tumors inevitably recur with no effective treatment. Thus, any new therapeutic strategy to target this most malignant tumor will be of significant benefit. RNAi is a powful gene silencing method that might be used in combination with other agents to improve the efficacy of glioblastoma treatment. Areas covered in this review: Recent progress and challenges of pre-clinical and clinical research of RNAi therapy for glioblastoma. The review covers literature from 2003 to 2009. What the reader will gain: The principle of RNA interference therapy, three categories of RNAi triggers, different RNAi delivery system and pre-clinical and clinical studies that are currently underway to evaluate the validity of RNAi as a potential therapeutic strategy against glioblastoma are discussed. Take home message: RNA inference therapy combined with other therapeutics may offer therapeutic potential for glioblastoma multiforme. Further studies are required to develop more efficient and specific delivery systems, select suitable gene targets, optimize treatment dose and administration schedule, evaluate the efficacy of combination treatment strategies, establish a validated clinical response measure system etc.

Downregulation of LRIG1 expression by RNA interference promotes the aggressive properties of glioma cells via EGFR/Akt/c-Myc activation

The LRIG1 [leucine-rich repeats and immunoglobulin-like domains (LRIG)] gene is not universally downregulated in human cancers, and its role in tumorigenesis and the development of glioma has not been well addressed. In this study, we used short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block LRIG1 gene expression in the GL15 human glioma cell line. Specific downregulation of LRIG1 by shRNA resulted in significantly enhanced capabilities of proliferation, inhibition of apoptosis and invasion in the GL15 cells. LRIG1 repression induced marked activation of epidermal growth factor receptor (EGFR), protein kinase B (Akt) and c-Myc signaling molecules. Our results demonstrated that RNAi against LRIG1 may effectively downregulate LRIG1 gene expression. LRIG1 functions as a tumor suppressor in the pathogenesis of glioma via EGFR/Akt/c-Myc activation.

A role for LRIG1 in the regulation of malignant glioma aggressiveness

The molecular mechanisms that drive the development and aggressive progression of malignant astrocytic tumors remain obscure. Recently, in the search for endogenous negative regulators of EGF receptor, LRIG1 was cloned and characterized as a putative tumor suppressor gene often downregulated in various human tumors, including astrocytic tumors. Although several studies have implicated the function of LRIG1 in the inhibition of tumorigenesis, its precise role and potential underlying mechanisms remain obscure. Therefore, we generated a full-length expression vector to overexpress LRIG1 in the U251 malignant glioma cell line. Introduction of exogenous LRIG1 into glioma cells inhibited cell proliferation manifested by MTT and soft agar clone assay in vitro and subcutaneously tumor xenografts. On the other hand, LRIG1 overexpression inhibited glioma growth by significantly changing the expression pattern of cyclins, resulting in delayed cell cycle. Employing transwell invasion and wound scratch assay and gelatin zymography, LRIG1 inhibited U-251 MG cell invasion and migration by attenuating MMP2 and MMP9 production. Under ligand-stimulated conditions, p-ERK levels did not change, whereas p-AKT levels were inhibited in cells with LRIG1 upregulation, indicating that LRIG1 exerts more inhibiting effects on the PI3K/AKT pathway. Our findings suggest that LRIG1 restricted glioma aggressiveness by inhibiting cell proliferation, migration and invasion. Restoration of LRIG1 to glioma cells could offer a novel therapeutic strategy.

Soluble LRIG2 ectodomain is released from glioblastoma cells and promotes the proliferation and inhibits the apoptosis of glioblastoma cells in vitro and in vivo in a similar manner to the full-length LRIG2.

The human leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family contains LRIG1, 2 and 3, encoding integral membrane proteins with an ectodomain, a transmembrane domain and a cytoplasmic tail. LRIG1 negatively regulates multiple receptor tyrosine kinases signaling including the epidermal growth factor receptor (EGFR) and is a proposed tumor suppressor. The soluble LRIG1 ectodomain is demonstrated to be shed naturally and inhibit the progression of glioma. However, little is known regarding the functions of LRIG2. In oligodendroglioma, LRIG2 expression is associated with poor survival, suggesting that LRIG2 might have different functions compared with LRIG1. Since soluble LRIG1 ectodomain has a similar function to the full-length LRIG1, we hypothesize that the different roles exerted by LRIG2 and LRIG1 result from the difference of their ectodomains. Here, we addressed the functions of LRIG2 and LRIG2 ectodomain in the proliferation and apoptosis of glioma and the possible underlying mechanisms. Firstly, we found that LRIG2 expression levels positively correlated with the grade of glioma. Further, we demonstrated for the first time that soluble LRIG2 ectodomain was capable of being released from glioblastoma cells and exerted a pro-proliferative effect. Overexpression of LRIG2 ectodomain promoted the proliferation and inhibited the apoptosis of glioblastoma cells in vitro and in vivo in a similar manner to the full-length LRIG2. Both full-length LRIG2 and LRIG2 ectodomain were found to physically interact with EGFR, enhance the activation of EGFR and its downstream PI3 K/Akt pathway. To our knowledge, this is the first report demonstrating that soluble LRIG2 ectodomain is capable of being released from glioblastoma cells and exerts a similar role to the full-length LRIG2 in the regulation of EGFR signaling in the progression of glioblastoma. LRIG2 ectodomain, with potent pro-tumor effects, holds promise for providing a new therapeutic target for the treatment of glioblastoma.

Association of expression of Leucine-rich repeats and immunoglobulin-like domains 2 gene with invasiveness of pituitary adenoma

The Leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2) gene expression in pituitary adenoma and its correlation with tumor invasiveness were studied. The expression of LRIG2 mRNA and protein in human pituitary adenoma obtained surgically was detected by RT-PCR (39 cases) and immunohistochemical staining (30 cases). It was found that LRIG2 was mostly localized at the nucleus of the pituitary adenoma cells. Its expression was significantly higher in the invasive cases than in the non-invasive cases. LRIG2 protein was positive in 14 cases out of 21 cases of invasive adenoma, but only 2 cases were positive in 9 cases of non-invasive adenoma. The positive expression rate of LRIG2 mRNA was 91.3% in invasive cases (total 23 cases) and 62.5% in non-invasive cases (total 16 cases), respectively. LRIG2 gene is overexpressed in invasive pituitary adenoma. It may play an important role in pituitary adenoma invasiveness and further studies are necessary to elucidate the mechanism under this phenomenon.SummaryThe Leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2) gene expression in pituitary adenoma and its correlation with tumor invasiveness were studied. The expression of LRIG2 mRNA and protein in human pituitary adenoma obtained surgically was detected by RT-PCR (39 cases) and immunohistochemical staining (30 cases). It was found that LRIG2 was mostly localized at the nucleus of the pituitary adenoma cells. Its expression was significantly higher in the invasive cases than in the non-invasive cases. LRIG2 protein was positive in 14 cases out of 21 cases of invasive adenoma, but only 2 cases were positive in 9 cases of non-invasive adenoma. The positive expression rate of LRIG2 mRNA was 91.3% in invasive cases (total 23 cases) and 62.5% in non-invasive cases (total 16 cases), respectively. LRIG2 gene is overexpressed in invasive pituitary adenoma. It may play an important role in pituitary adenoma invasiveness and further studies are necessary to elucidate the mechanism under this phenomenon.

LRIG3 modulates proliferation, apoptosis and invasion of glioblastoma cells as a potent tumor suppressor.

Leucine-rich repeats and immunoglobulin-like domains (LRIG) 3 gene is mapped to chromosome 12q13.2, a region that is frequently deleted in a subset of glioblastoma multiforme (GBM). It has been reported that perinuclear LRIG3 staining correlated with low WHO grade of glioma and better survival of the patients. However, the relationship between LRIG3 and glioma is not very clear. The purpose of this study is to demonstrate the impacts of LRIG3 on biological characteristics of glioma and its possible mechanisms. We found that transduction of LRIG3 into glioblastoma cells inhibited cell growth in vitro and in vivo, promoted cell apoptosis, and restrained cell invasion and migration. Further studies demonstrated that LRIG3 negatively regulated the epidermal growth factor receptor (EGFR) signaling pathway. Inhibition of EGFR could reduce the effects of LRIG3 knockdown on cell proliferation and EGFR signaling pathway. In conclusion, LRIG3 functions as a tumor suppressor by attenuating EGFR signaling pathway and the restoration of LRIG3 may offer therapeutic potential against malignant gliomas.

LRIG1 enhances cisplatin sensitivity of glioma cell lines.

LRIG family shares similar structures that include a signal peptide, an extracellular region consisting of a leucine-rich repeat domain and three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. After activation of EGFR, the extracellular LRR domain and immunoglobulin-like domains of LRIG1 can bind to the extracellular parts of EGFR, resulting in recruitment of c-Cbl to the cytoplasmic domains, and induction of EGFR degradation. This study investigated the effects of overexpression of leucine-rich repeats and LRIG1 on cisplatin (CDDP) sensitivity in the glioma cell line U251 and explored the possible mechanisms mediating this effect. We found that CDDP could inhibit the growth of U251 cell line and induced activation of the EGFR. Overexpression of LRIG1 increased the inhibitory effect of CDDP on the U251 cell line via the inhibition of proliferation and induction of apoptosis. The mechanisms underlying the effect of the combined treatment of LRIG1 and CDDP could be that LRIG1 blocked CDDP-induced EGFR activation and regulated the apoptosis proteins. These findings suggest that upregulation of LRIG1 expression enhances the CDDP sensitivity in the glioma cell line U251.