Leucine-rich Repeats And Immunoglobulin-like Domains 1 Research Articles

LRIG1 engages ligand VISTA and impairs tumor-specific CD8+ T cell responses.

Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell-specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1+ CD62Lhi PD-1low) and a reciprocal increase in progenitor and memory-like CTLs (TCF1+ PD-1+). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8+ CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.

Aryl Hydrocarbon Receptor Defect Attenuates Mitogen-Activated Signaling through Leucine-Rich Repeats and Immunoglobulin-like Domains 1 (LRIG1)-Dependent EGFR Degradation.

Aryl hydrocarbon receptor (AHR) genomic pathway has been well-characterized in a number of respiratory diseases. In addition, the cytoplasmic AHR protein may act as an adaptor of E3 ubiquitin ligase. In this study, the physiological functions of AHR that regulate cell proliferation were explored using the CRISPR/Cas9 system. The doubling-time of the AHR-KO clones of A549 and BEAS-2B was observed to be prolonged. The attenuation of proliferation potential was strongly associated with either the induction of p27Kip1 or the impairment in mitogenic signal transduction driven by the epidermal growth factor (EGF) and EGF receptor (EGFR). We found that the leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), a repressor of EGFR, was induced in the absence of AHR in vitro and in vivo. The LRIG1 tends to degrade via a proteasome dependent manner by interacting with AHR in wild-type cells. Either LRIG1 or a disintegrin and metalloprotease 17 (ADAM17) were accumulated in AHR-defective cells, consequently accelerating the degradation of EGFR, and attenuating the response to mitogenic stimulation. We also affirmed low AHR but high LRIG1 levels in lung tissues of chronic obstructive pulmonary disease (COPD) patients. This might partially elucidate the sluggish tissue repairment and developing inflammation in COPD patients.

Lrig1expression identifies airway basal cells with high proliferative capacity and restricts lung squamous cell carcinoma growth

BackgroundLung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Methods and resultsUsing an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression.ConclusionTogether, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

LRIG1 is a conserved EGFR regulator involved in melanoma development, survival and treatment resistance

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-negative regulator of receptor tyrosine kinase (RTK) signaling and a tumor suppressor in several cancers, but its involvement in melanoma is largely unexplored. Here, we aim to determine the role of LRIG1 in melanoma tumorigenesis, RTK signaling, and BRAF inhibitor resistance. We find that LRIG1 is downregulated during early tumorigenesis and that LRIG1 affects activation of the epidermal growth factor receptor (EGFR) in melanoma cells. LRIG1-dependent regulation of EGFR signaling is evolutionary conserved to the roundworm C. elegans, where negative regulation of the EGFR-Ras-Raf pathway by sma-10/LRIG completely depends on presence of the receptor let-23/EGFR. In a cohort of metastatic melanoma patients, we observe an association between LRIG1 and survival in the triple wild-type subtype and in tumors with high EGFR expression. During in vitro development of BRAF inhibitor resistance, LRIG1 expression decreases; and mimics LRIG1 knockout cells for increased EGFR expression. Treating resistant cells with recombinant LRIG1 suppresses AKT activation and proliferation. Together, our results show that sma-10/LRIG is a conserved regulator of RTK signaling, add to our understanding of LRIG1 in melanoma and identifies recombinant LRIG1 as a potential therapeutic against BRAF inhibitor-resistant melanoma.

Cell-Autonomous Role of EGFR in Spontaneous Duodenal Tumors in LRIG1 Null Mice

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a tumor suppressor that negatively regulates the ERBB family, including epidermal growth factor receptor (EGFR), and other receptor tyrosine kinases (RTKs).1,2 Lrig1CreERT2/CreERT2 (functionally Lrig1 null; hereafter Lrig1Cre) mice spontaneously develop duodenal tumors with increased EGFR and ERBB2-3 expression and increased ERK1/2 activity by immunoblotting.3 These Lrig1 null tumors arise over expanded Brunner glands that exhibit increased expression of the EGFR ligands amphiregulin and betacellulin.

CSIG-04. PAN-RTK INHIBITION OF sLRIG1 MEDIATES AXL DOWNREGULATION IN GLIOBLASTOMA

Abstract INTRODUCTION Aberrant regulation of receptor tyrosine kinase (RTK) activity is characteristic of Glioblastoma (GBM). However, RTK-based targeted therapies have been largely unsuccessful in GBM patients, partially due to the complexity and redundance of RTK signaling. LRIG1 (Leucine-rich Repeats and ImmunoGlobulin-like domains 1) is an important endogenous inhibitor of epidermal growth factor receptor (EGFR) during health and disease, however its mechanism of action is poorly understood. We previously showed that the soluble form of LRIG1 potently inhibits GBM growth in vivo, irrespective of EGFR expression level and status, suggesting the involvement of other RTKs. Here, we aimed to shed light on the molecular mechanisms underlying its anti-cancer activity. MATERIAL AND METHODS We generated a recombinant human soluble LRIG1 protein (rh-sLRIG1) by expressing LRIG1 ectodomain in insect cells via baculovirus infection and subsequent His-tag purification. rh-sLRIG1 was used to treat patient-derived GBM stem-like cells, classical GBM cell lines and patient-derived 3D tumor organoids. Using different cell-based assays, cell proliferation, invasion, cell morphology, as well as protein expression and protein-protein interactions were investigated. RESULTS We find that recombinant sLRIG1 efficiently reduced proliferation, invasion and viability of GBM cells and patient-derived organoids, and modulated cytoskeleton proteins and cell shape. In line with previous data, the effect of recombinant sLRIG1 was independent of EGFR expression. Interestingly sLRIG1 impacted multiple RTKs including AXL, by direct protein downregulation, while EGFR expression was not affected. At the molecular level, we find that sLRIG1 interfereed with AXL dimerization, while no protein interaction with EGFR was detected. CONCLUSION We identify AXL as a novel LRIG1 target and provide evidence that sLRIG1-mediated RTK downregulation requires direct protein-protein interaction. These data pave the way for a potential therapeutic application of recombinant sLRIG1 in the inhibition of growth factor signaling in GBM.

LRIG1‑2 and LMO7 immunoreactivity in vulvar squamous cell carcinoma: Association with prognosis in relation to HPV‑DNA and p16INK4a status.

The present study was conducted to investigate the possible prognostic value of molecular markers LRIG1-2 and LIM domain 7 protein (LMO7) in vulvar squamous cell carcinoma (VSCC) and their possible correlation to human papilloma virus (HPV)- and p16INK4a-status of the tumors. Patients diagnosed with VSCC at the University Hospital of Umeå, Sweden, during the years 1990–2013 were selected. Tumor blocks were retrieved from tissue archives and clinical data were collected from the records of patients. HPV-PCR analysis, HPV genotyping and immunohistochemistry were performed. In total, 112 patients were included. Forty percent of the tumors were HPV-positive, 27% were p16INK4a-positive and 23% were positive for both HPV and p16INK4a (considered HPV-driven). HPV-positivity and p16INK4a-positivity were associated with prolonged disease-free survival (DFS) in Kaplan-Meier survival analysis. Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) immunoreactivity was not significantly associated with survival. High leucine-rich repeats and immunoglobulin-like domains 2 (LRIG2) immunoreactivity was associated with a prolonged overall survival (OS) (P=0.001). By analyzing HPV-negative cases only, it was determined that high LRIG2 immunoreactivity was associated with both favorable OS (P=0.008) and DFS (P=0.031). LRIG2 immunoreactivity was also an independent prognostic factor in multivariate analysis of OS (P=0.002, HR=0.41; 95% CI, 0.24-0.71). High immunoreactivity with LMO7-1250 antibody was associated with survival benefits in the whole cohort (OS; P=0.011) although DFS was only prolonged in HPV-negative and not HPV-driven tumors (P=0.038 and 0.042, respectively). The present study indicated that LRIG2 and LMO7 may be useful prognostic markers in VSCC, particularly for patients without HPV-driven tumors or with advanced tumors at diagnosis. In contrast to earlier observations regarding other types of squamous cell carcinoma, LRIG1 was not a significant prognostic factor in VSCC.

Downregulation of microRNA-4295 enhances cisplatin-induced gastric cancer cell apoptosis through the EGFR/PI3K/Akt signaling pathway by targeting LRIG1.

Gastric cancer (GC) is one of the leading causes of cancer-associated mortality worldwide. The aim of the present study was to investigate the mechanism of microRNA-4295 (miR-4295), which regulates cisplatin (DDP)-induced apoptosis in GC cells through the leucinerich repeats and immunoglobulin-like domains 1 (LRIG1)-mediated epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Two cell lines were selected, one with the highest expression of miR-4295 and one with the lowest expression of LRIG1, for the experiments. The half maximal inhibitory concentration of DDP in the human GC MKN-28 and MKN-45 cell lines was calculated, and mitochondrial membrane potentials of the GC cells were detected by tetramethylrhodamine, ethyl ester, perchlorate staining. The proliferation and apoptosis of GC cells with or without DDP treatment were assessed by MTT assay and plate colony formation, as well as flow cytometry and TUNEL staining. Western blot analysis and reverse transcription-quantitative polymerase chain reaction were employed to determine the expression of EGFR/PI3K/Akt signaling pathway-related genes and apoptosis-related genes. LRIG1 was identified as a target gene of miR-4295. The expression of miR-4295 was upregulated, and the expression of LRIG1 was downregulated in GC cells. Furthermore, DDP enhanced the decrease in miR-4295 expression and the increase in LRIG1 expression in GC cells. miR-4295 promoted the proliferation and inhibited the DDP-induced apoptosis of GC cells without DDP treatment. In addition, miR-4295 increased the expression levels of EGFR, PI3K, Akt, p-PI3K and p-Akt, suggesting that miR-4295 promotes the activation of the EGFR/PI3K/Akt signaling pathway by targeting LRIG1. miR-4295 targeted and negatively regulated LRIG1 expression to activate the EGFR/PI3K/Akt signaling pathway, thereby promoting the proliferation of the GC cells and inhibiting the apoptosis of the GC cells induced by DDP. Therefore, miR-4295 may be a novel therapeutic target in patients with GC.

Downregulation of leucine-rich repeats and immunoglobulin-like domains 1 by microRNA-20a modulates gastric cancer multidrug resistance.

Multidrug resistance (MDR) significantly restricts the clinical efficacy of gastric cancer (GC) chemotherapy, and it is critical to search novel targets to predict and overcome MDR. Leucine‐rich repeats and immunoglobulin‐like domains 1 (LRIG1) has been proved to be correlated with drug resistance in several cancers. The present study revealed that LRIG1 was overexpressed in chemosensitive GC tissues and decreased expression of LRIG1 predicted poor survival in GC patients. We observed that upregulation of LRIG1 enhanced chemosensitivity in GC cells. Interestingly, miR‐20a, which was overexpressed in GC MDR cell lines and tissues, was identified to regulate LRIG1 expression by directly targeting its 3′ untranslated region. We also found that inhibition of miR‐20a suppressed GC MDR, and upregulation showed opposite effects. Moreover, we demonstrated that the miR‐20a/LRIG1 axis regulated GC cell MDR through epidermal growth factor receptor (EGFR)‐mediated PI3K/AKT and MAPK/ERK signaling pathways. Finally, LRIG1 expression in human GC tissues is inversely correlated with miR‐20a and EGFR. Taken together, the newly identified miR‐20a/LRIG1/EGFR link provides insight into the MDR process of GC, and targeting this axis represents a novel potential therapeutic strategy to block GC chemoresistance.

A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a tumor suppressor and a negative regulator of several receptor tyrosine kinases. The molecular mechanisms by which LRIG1 mediates its tumor suppressor effects and regulates receptor tyrosine kinases remain incompletely understood. Here, we performed a yeast two-hybrid screen to identify novel LRIG1-interacting proteins and mined data from the BioPlex (biophysical interactions of ORFeome-based complexes) protein interaction data repository. The putative LRIG1 interactors identified in the screen were functionally evaluated using a triple co-transfection system in which HEK293 cells were co-transfected with platelet-derived growth factor receptor α, LRIG1, and shRNAs against the identified LRIG1 interactors. The effects of the shRNAs on the ability of LRIG1 to down-regulate platelet-derived growth factor receptor α expression were evaluated. On the basis of these results, we present an LRIG1 protein interaction network with many newly identified components. The network contains the apparently functionally important LRIG1-interacting proteins RAB4A, PON2, GAL3ST1, ZBTB16, LRIG2, CNPY3, HLA-DRA, GML, CNPY4, LRRC40, and LRIG3, together with GLRX3, PTPRK, and other proteins. In silico analyses of The Cancer Genome Atlas data sets revealed consistent correlations between the expression of the transcripts encoding LRIG1 and its interactors ZBTB16 and PTPRK and inverse correlations between the transcripts encoding LRIG1 and GLRX3. We further studied the LRIG1 function–promoting paraoxonase PON2 and found that it co-localized with LRIG1 in LRIG1-transfected cells. The proposed LRIG1 protein interaction network will provide leads for future studies aiming to understand the molecular functions of LRIG1 and the regulation of growth factor signaling.

LRIG1 negatively regulates RET mutants and is downregulated in thyroid cancer.

Papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC) are characterized by genomic rearrangements and point mutations in the proto-oncogene RET. Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a suppressor of various receptor tyrosine kinases, including RET. LRIG1 expression levels are associated with patient survival in many cancer types. In the present study, we investigated whether the oncogenic RET mutants RET2A (C634R) and RET2B (M918T) were regulated by LRIG1, and the possible effects of LRIG1 expression in thyroid cancer were investigated in three different clinical cohorts and in a RET2B-driven mouse model of MTC. LRIG1 was shown to physically interact with both RET2A and RET2B and to restrict their ligand-independent activation. LRIG1 mRNA levels were downregulated in PTC and MTC compared to normal thyroid gland tissue. There was no apparent association between LRIG1 RNA or protein expression levels and patient survival in the studied cohorts. The transgenic RET2B mice developed pre-cancerous medullary thyroid lesions at a high frequency (36%); however, no overt cancers were observed. There was no significant difference in the incidence of pre-cancerous lesions between Lrig1 wild-type and Lrig1-deficient RET2B mice. In conclusion, the findings that LRIG1 is a negative regulator of RET2A and RET2B and is also downregulated in PTC and MTC may suggest that LRIG1 functions as a thyroid tumor suppressor.

Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma

Recently, a genome-wide association study showed that a single nucleotide polymorphism (SNP) —rs11706832—in intron 2 of the human LRIG1 (Leucine-rich repeats and immunoglobulin-like domains 1) gene is associated with susceptibility to glioma. However, the mechanism by which rs11706832 affects glioma risk remains unknown; additionally, it is unknown whether the expression levels of LRIG1 are a relevant determinant of gliomagenesis. Here, we investigated the role of Lrig1 in platelet-derived growth factor (PDGF)-induced experimental glioma in mice by introducing mono-allelic and bi-allelic deletions of Lrig1 followed by inducing gliomagenesis via intracranial retroviral transduction of PDGFB in neural progenitor cells. Lrig1 was expressed in PDGFB-induced gliomas in wild-type mice as assessed using in situ hybridization. Intriguingly, Lrig1-heterozygous mice developed higher grade gliomas than did wild-type mice (grade IV vs. grade II/III, p = 0.002). Reciprocally, the ectopic expression of LRIG1 in the TB107 high-grade human glioma (glioblastoma, grade IV) cell line decreased the invasion of orthotopic tumors in immunocompromised mice in vivo and reduced cell migration in vitro. Concomitantly, the activity of the receptor tyrosine kinase MET was downregulated, which partially explained the reduction in cell migration. In summary, Lrig1 is a haploinsufficient suppressor of PDGFB-driven glioma, possibly in part via negative regulation of MET-driven cell migration and invasion. Thus, for the first time, changes in physiological Lrig1 expression have been linked to gliomagenesis, whereby the SNP rs11706832 may affect glioma risk by regulating LRIG1 expression.

LRIG1 inhibits hypoxia-induced vasculogenic mimicry formation via suppression of the EGFR/PI3K/AKT pathway and epithelial-to-mesenchymal transition in human glioma SHG-44 cells.

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-negative regulator of the epidermal growth factor receptor (EGFR) signaling pathway. The aim of this study was to investigate the underlying mechanism of LRIG1 in the regulation of vasculogenic mimicry (VM) formation in glioma cells. We constructed an enhanced green fluorescent protein plasmid (pEGFP) system, pEGFP-C1-LRIG1, for overexpression of LRIG1, and transfected it into human glioma cell line SHG-44. Under hypoxic conditions induced by CoCl2, we investigated the effects of LRIG1 overexpression on VM formation and VM-dependent malignant behaviors including migration, invasion, and proliferation. Additionally, we explored the effects of LRIG1 on the expression levels of major components of the EGFR/PI3K/AKT pathway as well as E-cadherin and vimentin. We found that LRIG1 overexpression is able to inhibit hypoxia-induced VM formation, migration, invasion, and proliferation. Furthermore, LRIG1 overexpression counteracts hypoxia-induced increase in the expression of phosphorylated EGFR (pEGFR), PI3K (pPI3K), and AKT (pAKT) and reverts hypoxia-induced alteration in E-cadherin and vimentin expression levels. In LRIG1 knockdown SHG-44 cells, however, hypoxia-induced VM formation and alteration in E-cadherin and vimentin expression levels were exacerbated. These results suggest that the inhibitory effects of LRIG1 are most likely mediated by suppression of the EGFR/PI3K/AKT pathway and epithelial-mesenchymal transition (EMT) process. Our findings provide compelling evidence implicating LRIG1 in glioma pathophysiology, suggesting that gene therapy using LRIG1 may serve as a treatment for this disease.

LRIG1, human EGFR inhibitor, reverses multidrug resistance through modulation of ABCB1 and ABCG2

In our previous study, we have found that leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) can improve the chemosensitivity in U251 cells whereas the role of LRIG1 in multidrug resistance (MDR) remains unknown. Here, we reported that LRIG1 can reverse MDR by inhibiting epidermal growth factor (EGF) receptor (EGFR) and secondary inhibiting ATP-binding cassette, sub-family B member 1(ABCB1) and ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2). Our data showed that the expression of LRIG1 was significantly higher in O6-methylguanine DNA methyltransferase (MGMT) Promoter Methylation positive glioblastoma tissues compared to MGMT Promoter Methylation negative glioblastoma tissues. In addition, we found that LRIG1 expression was significantly decreased in MDR cells U251/TMZ compared to U251cells. Our results demonstrated that over-expression of LRIG1 can reverse the MDR. The expression of ABCB1 and ABCG2 were markedly suppressed when LRIG1 was over-expressed, supporting the negative relationship between LRIG1 level and ABCB1 and ABCG2 level in human specimen. Furthermore, we found that LRIG1 downregulated ABCB1 and ABCG2 through suppressing EGFR expression. In case of EGFR knockdown, the effect of LRIG1 on regulating MDR, ABCB1 and ABCG2 was partially compromised. Our results, for the first time, showed that LRIG1 can reverse MDR in glioblastoma, by negatively regulating EGFR and secondary suppressing the levels of ABCB1 and ABCG2.

Loss of Lrig1 Leads to Expansion of Brunner Glands Followed by Duodenal Adenomas with Gastric Metaplasia

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-ErbB negative regulator and intestinal stem cell marker down-regulated in many malignancies. We previously reported that 14 of 16 Lrig1-CreERT2/CreERT2 (Lrig1(-/-)) mice developed duodenal adenomas, providing the first in vivo evidence that Lrig1 acts as a tumor suppressor. We extended this study to a larger cohort and found that 49 of 54 Lrig1(-/-) mice develop duodenal adenomas beginning at 3 months. Most adenomas were histologically low grade and overlaid expanded Brunner glands. There was morphologic and biochemical blurring of the boundary between the epithelium and Brunner glands with glandular coexpression of ErbB2, which is normally restricted to the epithelium, and the Brunner gland marker Mucin6. Some adenomas were high grade with reduced Brunner glands. At age 4 to 5 weeks, before adenoma formation, we observed enhanced proliferation in Brunner glands and, at 2 months, an increase in the size of the Brunner gland compartment. Elevated expression of the epidermal growth factor receptor (Egfr) ligands amphiregulin and β-cellulin, as well as Egfr and phosphorylated Egfr, was detected in adenomas compared with adjacent normal tissue. These adenomas expressed the gastric-specific genes gastrokine1 and mucin5ac, indicating gastric metaplasia. Moreover, we found that a subset of human duodenal tumors exhibited features of LRIG1(-/-) adenomas, including loss of LRIG1, gastric metaplasia (MUCIN5AC and MUCIN6), and increased amphiregulin and Egfr activity.

Adenovirus-mediated LRIG1 expression enhances the chemosensitivity of bladder cancer cells to cisplatin.

Cisplatin (cis-diaminodichloroplatinum, CDDP) is one of the most effective chemotherapeutic agents that has been widely used in the treatment of many malignancies, including muscle invasive bladder cancer. However, development of CDDP resistance in cancer cells is a major obstacle to the effective treatment of bladder cancer. Therefore, the development of chemosensitizers to overcome the acquired resistance to chemotherapeutic agents is crucial. Previous studies have confirmed that the epidermal growth factor receptor (EGFR) and its signaling pathways are important in the chemoresistance of cancer cells against CDDP‑induced cell apoptosis. In a preliminary study we showed that leucine-rich repeats and immunoglobulin-like domains1 (LRIG1) is the natural ligand of EGFR, and that the extracellular leucine-rich repeat (LRR) domain and immunoglobulin-like domains of LRIG1 were able to bind to the extracellular domain of EGFR, resulting in the downregulation of EGFR expression. Based on these findings, we hypothesized that LRIG1 may enhance the chemosensitivity of bladder cancer cells to CDDP. In the present study, LRIG1 was overexpressed by the adenovirus vector to determine the effect of LRIG1 on chemosensitivity in the T24 bladder cancer cell line and explored the possible mechanisms. The results showed that CDDP inhibited the growth of the T24 cell line and induced activation of EGFR. Overexpression of LRIG1 increased the inhibitory effect of CDDP on the T24 cell line, which may be associated with inactivation of the EGFR signaling pathway, followed by the decrease of Bcl-2 expression and a concomitantly induced expression of Bax. Based on these results, we concluded that the upregulation of LRIG1 expression inhibited the EGFR signaling pathway, activated the mitochondrial pathway of apoptosis and eventually increased the sensitivity of bladder cancer cells to CDDP.

Effect of microRNA-19a down-regulation on invasiveness of glioma cells and the potential mechanism

Objective To explore whether down-regulation of microRNA(miR)-19a could inhibit invasion of glioma cells and the potential mechanism. Methods The transfection efficiency was determined by real-time fluorescent quantitative polymerase chain reaction(FQ-PCR).The expression of leucine-rich repeats and immunoglobulin-like domains 1(LRIG1)was detected by Western blotting and the invasion ability of U87 cells was evaluated by Transwell assay at 48 h post-transfection.Luciferase reporter assay was performed to identify the interaction between miR-19a and LRIG1 at 72 h post-transfection. Results FQPCR demonstrated that the expression of miR-19a was drastically decreased by(78. 2±5. 1)% following miR-19a inhibition(P<0. 01).The number of invasive cells in antago miR-19a group was obviously less than that in control group(25. 9±3. 9 vs.85. 3±6. 1, P<0. 01).Following down-regulation of miR-19a, the luciferase activity of wild reporter was increased by(4. 89±0. 26) fold(P< 0. 01)compared to that of control, whereas the luciferase activity of mutant reporter was almost unaffected.The number of invasive cells in experimental group(antagomiR-19a+ siLRIG1) was significantly greater than in control group(antagomiR-19a+ scramble)(47. 8±3. 1 vs.22. 7±4. 2,P<0. 05). Conclusion Down-regulation of miR-19a could suppress invasion of U87cells, which may be mediated by LRIG1. Key words: Glioma; MicroRNA-19a; Leucine-rich repeats and immunoglobulin-like domains 1; Invasion

MiR-20a mediates temozolomide-resistance in glioblastoma cells via negatively regulating LRIG1 expression

AimsResistance to temozolomide (TMZ) is a major obstacle in the treatment of glioblastoma multiforme (GBM). MiRNAs is considered as an important modulator of drug resistance in many cancers. Here, we aimed to elucidate the relationship between miR-20a, its predicted target genes leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) and TMZ resistance in GBM. Main methodsReal-time PCR or western blot was used to measure the levels of miR-20a and LRIG1. The cell viability was obtained to investigate the sensitivity of U251 cells and TMZ-resistant U251 (U251/TMZ) cells to TMZ. MiR-20a inhibitor or miR-20a mimic was used to down-regulate or up-regulate miR-20a expression. The interaction between miR-20a and its predicted target gene LRIG1 was confirmed by 3′-UTR dual-luciferase reporter assay. RNAi was used to knockdown LRIG1 expression. A xenograft tumor model was used to investigate the in vivo antitumor activity. Key findingsMiR-20a was highly expressed and LRIG1 lowly expressed in U251/TMZ cells. Knockdown of miR-20a by treatment with miR-20a inhibitor restored sensitivity of U251/TM cells to TMZ in vivo and in vitro, whereas overexpression of miR-20a by treatment with miR-20a mimic resulted in increased TMZ resistance. The levels of LRIG1 were inversely related to miR-20a levels. And the luciferase reporter assays showed that miR-20a directly targeted the 3′UTR of LRIG1. In addition, functional knock-down of LRIG1 by gene specific siRNA reversed the effect of miR-20a inhibitor. SignificanceMiR-20a mediated TMZ-resistance in glioblastoma cells through negatively regulating LRIG1 expression, which suggesting that miR-20a and LRIG1 would be potential therapeutic targets for glioma therapy.

LRIG1 improves chemosensitivity through inhibition of BCL-2 and MnSOD in glioblastoma.

We have reported that Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) can improve the chemosensitivity in U251 cells. However, the underlying mechanisms how LRIG1 improves the chemosensitivity remain unknown. In this study, we reported that LRIG1 can improve the chemosensitivity through the inhibition of B cell lymphoma 2 (BCL-2) and manganese superoxide dismutase (MnSOD). In addition, we showed that the expression level of LRIG1 was significantly negatively correlated with BCL-2 and MnSOD expression in glioma. Our research demonstrated that overexpression of LRIG1 can enhance the chemosensitivity. BCL-2 and MnSOD were inhibited in LRIG1 overexpressing cells. On the other hand, when BCL-2 and MnSOD were knocked down, the chemosensitivity of U251 cells decreased, and the effect of LRIG1 in regulating chemosensitivity was compromised. For the first time, our results showed that LRIG1 can enhance chemosensitivity in glioblastoma by inhibition of BCL-2 and MnSOD.

Expression and prognostic significance of leucine-rich repeats and immunoglobulin like domains 1 in pituitary adenomas

Objective To investigate the expression and prognostic significance of leucine-rich repeats and immunoglobulin like domains 1 (LRIG1) in pituitary adenomas.Methods Immunohistochemical streptavidin avidin-peroxidase (SP) method was used to detect the expression of LRIG1 in 74 cases of pituitary adenomas tissues and 20 cases of normal brain tissues,and the relationship between LRIG1 expression and patient prognosis was analyzed.Results The positive expression rates of LRIG1 in normal brain tissue and pituitary adenomas were 100.0％ and 54.1 ％,respectively,with a significant difference (P ＜ 0.05).Meanwhile,the positive rates of LRIG1 expression in non-invasive pituitary adenomas was 66.7％,which is significantly higher than that in invasive pituitary adenomas (20.0％).Kaplan-Meier survival analysis showed that LRIG1 and tumor type was associated with patients' prognosis (P ＜ 0.05).Cox multivariate survival analysis showed LRIG1 could be as independent prognostic factor.Conclusion LRIG1 is involved in the formation and development of pituitary adenomas,and patients with low expression of LRIG1 have poorer prognosis. Key words: Leucine-rich repeats and immunoglobulin like domains 1; Pituitary adenomas; Prognosis