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
et al. (2014) 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. PLoS ONE 9(10): e111419. doi:10.1371/journal.pone.
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
Qungen Xiao 0
Yihu Tan 0
Yang Guo 0
Hongkuan Yang 0
Feng Mao 0
Ruifan Xie 0
Baofeng Wang 0
Ting Lei 0
Antonio Paolo Beltrami, University of Udine, Italy
0 Department of Neurosurgery and Sino-German Neuro-Oncology Molecular Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan , P.R. China
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.
Glioblastoma multiforme (GBM) is by far the most common
and lethal type of brain cancer. Despite the recent improvements
in surgery, radiation therapy and cytotoxic chemotherapy, the
prognosis for GBM remains grim, with a median survival time of
only 1215 months after diagnosis . Thus, the development of
novel efficacious therapies is greatly warranted to improve the
poor prognosis of patients afflicted with GBM.
Substantial research effort has focused on the identification of
genetic alterations in GBMs that might help response to specific
therapies. The most common genetic alteration associated with
GBM is the amplification of the epidermal growth factor receptor
(EGFR), with a frequency of about 50% .The ligand-binding
triggered the activation of amplified EGFR, resulting in enhanced
downstream signaling controlling pleiotropic cellular responses,
such as cell proliferation and survival . Owing to the vital role of
the EGFR activation in glioblastoma progression, the
understanding of its endogenous regulators has been a subject of intense
In the research on the negative regulators of EGFR, the human
leucine-rich repeats and immunoglobulin-like domains (LRIG)
gene family was found . The mammalian LRIG gene family is
composed of three paralogous genes, namely LRIG1, LRIG2 and
LRIG3, which encode integral membrane proteins, with a signal
peptide, an extracellular part consisting of 15 leucine-rich repeats
(LRR) with cysteine-rich N- and C-terminal flanking domains and
three immunoglobulin-like domains, followed by a
transmembrane domain and a cytoplasmic tail . LRIG1, the best-studied
LRIG family member, negatively regulates the signaling pathways
mediated by ERBB [5,6], MET  and RET  receptor tyrosine
kinases, and is suggested to be a tumor suppressor . LRIG1 is
down-regulated and associated with a favorable prognosis in many
cancers [10,11,12,13]. Inhibition of EGFR signaling by LRIG1
results from a physical interaction between the extracellular
domain of both proteins, inducing the recruitment of E3 ubiquitin
ligases, follow by internalization and enhanced lysosomal
degradation of the protein complex [5,6]. Recently, soluble LRIG1
ectodomain is demonstrated to be released naturally by proteolytic
shedding and suppress EGF signaling without any apparent EGFR
protein downregulation . Moreover, soluble extracellular part
of mouse Lrig1 is capable of inhibiting glioma growth in vitro and
in vivo irrespective of EGFR status . LRIG3 appears to have a
similar role to LRIG1 in the progression of glioma [16,17,18].
However, little is known regarding the molecular and
developmental functions of mammalian LRIG2. Recently, it was found
that Lrig2-deficient mice were protected against PDGFB-induced
glioma . In addition, LRIG2 expression is associated with poor
survival in oligodendroglioma  and squamous cell carcinoma
of the uterine cervix . Noteworthy, we previously demonstrate
that downregulation of LRIG2 inhibits glioblastoma cell growth
in vitro . To date, the data suggest that LRIG2 might promote
the genesis of tumors and have different, possibly opposing,
functions compared with LRIG1. The mechanism underlying the
different roles played by LRIG2 and LRIG1 in the progression of
tumors has not been explored yet. Based on the findings that
soluble LRIG1 ectodomain has the similar functions to the
fulllength LRIG1 and full-length LRIG2 plays different roles
compared to the full-length LRIG1, it is reasonable to hypothesize
that the LRIG2 ectodomain may give rise to the special role
played by LRIG2.
In this study, we studied the functions of the full-length LRIG2
and LRIG2 ectodomain in the progression of glioblastoma to gain
insight into the mechanism underlying the role of LRIG2. We
firstly investigated the relationship between the expression level of
LRIG2 and the grade of glioma. Further, we generated
glioblastoma cells with stable expressions of the full-length LRIG2
and LRIG2 ectodomain and investigated the effects of full-length
LRIG2 and LRIG2 ectodomain on the proliferation and apoptosis
of glioblastoma in vitro and in vivo. We then explored the
possible mechanisms underlying the effects. Strikingly, we
demonstrated for the first time that the soluble LRIG2 ectodomain
was capable of being secreted by glioblastoma cells and exerted a
pro-proliferative effect. Both full-length LRIG2 and LRIG2
ectodomain promoted the proliferation and inhibited the apoptosis
of glioblastoma in vitro and in vivo probably through enhancing
the EGFR activation and its downstream PI3K/Akt pathway. To
our knowledge, this is the first report showing that the soluble
LRIG2 ectodomain, which can be released from glioblastoma
cells, positively regulates the growth of glioblastoma and
EGFRmediated PI3 K/Akt signaling in a similar manner to the
Materials and Methods
TCGA Data and Glioma Sample Description
For expression analysis according to WHO grade, gene
expression data of glioblastoma multiforme (GBM) and brain
low-grade glioma (LGG) were downloaded from the public TCGA
data repositories (https://tcga-data.nci.nih.gov/tcga/
tcgaDownload. jsp) (date of download: December 2013), which
include 27 cases of LGG and 466 cases of GBM. Fresh clinical
glioma samples of different grade (20 cases of LGGs and 20 cases
of high-grade gliomas (HGGs)) were obtained from the
Department of Neurosurgery, Tongji Hospital after informed consent
from the patients according to the Declaration of Helsinki.
Diagnosis and grading was according to the WHO 2007 criteria
. The collection and usage of patient tumor tissue samples was
approved by the appropriate local ethics committees and the
consents of all the patients were written and approved
(Institutional Review Board, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, IRB ID:
Antibodies and Reagents
The mouse anti-Flag monoclonal antibody (Sigma F1804) was
purchased from Sigma-Aldrich (St.Louis, MO, USA). The Ki-67
(Abcam ab16667) and PCNA (Abcam ab92552) rabbit
monoclonal antibodies were purchased from Abcam (Cambridge Science
Park) and the anti-LRIG2 rabbit polyclonal antibody was
generously provided by Prof. Hakan Hedman (Umea University,
Sweden). The following antibodies used in western blotting were
diluted in TBST/5%BSA: Anti-EGFR 1:1000 (Cell signaling
4267), anti-pEGFR 1:1000 (Cell signaling 2220), anti-Akt 1:1000
(Cell signaling 9272), anti-pAkt 1:1000 (Cell signaling 4058),
anticMyc 1:1000 (Cell signaling 5605), anti-cyclin B1 1:1000 (Cell
signaling 4138), anti-cyclin D1 1:1000 (Cell signaling 2978),
anticyclin E 1:200 (Santa Cruz, sc-481), anti-Bcl-2 1:1000 (Cell
signaling 2870), anti-Bax 1:1000 (Cell signaling 5023), and
anticaspase-3 1:1000 (Cell signaling 9662). Mitochondrial membrane
potential assay kit with JC-1 was purchased from Beyotime
Institute of Biotechnology (Jiangsu, China).
Cell Lines and Cell Culture
The U87 and U251 human glioblastoma cell lines, purchased
from American Type Culture Collection, were cultured in
Dulbeccos modified Eagles medium (DMEM) supplemented
with 10% (v/v) fetal bovine serum (FBS) (Hyclone, USA) in a
humidified incubator with 5% CO2 at 37uC as previously
Expression Constructs and Stable Transduction
To generate glioblastoma cells with stable expressions of the
full-length LRIG2 or the ectodomain of LRIG2, two lentiviral
expression constructs, pLVX-puro-36FLAG-LRIG2 and
pLVXpuro-36FLAG-LRIG2ecto, were generated by subcloning
fulllength LRIG2 (LRIG2) and ectodomain of LRIG2 (LRIG2ecto)
from pEGFP-LRIG2 (gifted by Hakan Hedman) into
pLVX-puro36FLAG. Lentiviral particles were produced by transfecting
293 T cells with pLVX-puro (Con),
pLVX-puro-36FLAGLRIG2 (LRIG2) or pLVX-puro-36FLAG-LRIG2ecto
(LRIG2ecto) using Lenti-X lentiviral Expression Systems (Clontech)
according to the manufacturers instructions. The glioblastoma cell
lines U87 and U251 were infected with the respective lentivirus
and selected with culture medium containing 1 mg/ml puromycin
for 2 weeks. The stably transduced cell populations were pooled
without clonal selection.
Detection and elimination of Soluble LRIG2 Ectodomain
in the Conditioned Medium
U87 glioblastoma cells stably transduced with control,
fulllength LRIG2 or LRIG2 ectodomain expressing vetor (26106
cells/group) were cultured in DMEM with 10% FBS for 48 h then
maintained in 14 ml DMEM without FBS for another 48 h. The
conditioned medium were collected and filtered through a
0.45 mm Millex filter (Millipore Corporation, USA). The filtered
conditioned medium were subjected to Amicon Ultra-4 10 K
centrifugal filter devices (Millipore Corporation, USA) spinning at
4,0006g for approximately 20 minutes to concentrate the
proteins. The concentrated conditioned mediums were added
with protease inhibitor (Sigma P8340) and 56 loading buffer,
boiled at 100uC for 5 minutes and subjected to Western blotting
for the detection of soluble LRIG2 ecdomain. The anti-Flag
monoclonal antibody was used to detect the Flag-tagged proteins.
To investigate the proliferative effects of soluble LRIG2
ectodomain in the conditioned medium on glioblastoma cells,
the conditioned medium were filtered through a 0.45 mm Millex
filter, followed by immunoprecipitation using Anti-Flag M2
Affinity Gel (Sigma A2220) to eliminate the soluble LRIG2
ectodomain as per the manufacturers instructions. The
supernatant before immunoprecipitation and the resulting soluble LRIG2
ectodomain-free supernatant after immunoprecipitation were both
used in the cell proliferation assay.
The glioma samples of human and nude mice were fixed in a
phosphate-buffered 4% formaldehyde solution and processed into
paraffin blocks using standard methods. Paraffin sections with a
thickness of 4 mm were incubated with anti-LRIG2 (1:100),
antiKi-67 (1:200), anti-PCNA (1:200), and anti-caspase3 (1:100)
antibodies at 4uC overnight as previously described .
Cytoplasmic immunoreactivity was scored artificially in four
different categories: 0 for no or very faint immunoreactivity, 1
for weak immunoreactivity, 2 for moderate immunoreactivity, and
3 for intense immunoreactivity. Percentage scores were assigned as
follows: ,5%, 0; 525%, 1; 2650%, 2; 5175%, 3; .75%, 4.
Cells were fixed with 4% paraformaldehyde for 1 h at 4uC,
permeabilized with 0.25% Triton X-100 for 5 min, blocked with
10% bovine serum albumin for 1 h and incubated with primary
antibodies overnight at 4uC. After washing with PBS, cells were
incubated with FITC or CY3-labeled secondary antibodies (1:100,
ProteinTech, USA) and subjected to immunofluorescence
microscopy using appropriate filter.
After cells were cultured with DMEM containing 10% FBS for
48 h, RNA isolation and quantitative RT-PCR were performed as
described previously . DNA primer sequences of LRIG2 and
LRIG2ecto were designed as follows: sense,
Western Blotting Analysis and Immunoprecipitation
Western blotting analysis and immunoprecipitation were
performed as described . All the antibodies were diluted at
optimal concentrations based on multiple experimental
Cell Proliferation Assay
Cells were seeded into 96-well plate with 56103 cells in 200 ml
suspension per well in triplicate and maintained in complete
culture medium for 7 days. Every day, CCK8 was added and
incubate for 2 h at 37uC. The absorbance of each well was
measured with Microplate Reader using a wavelength of 450 nm.
Soft Agar Colony Formation Assay
To perform anchorage-independent cell proliferation assay,
sixwell plates were first covered with a layer of 0.6% agar made in
10% FBS DMEM and 46104 cells per well were embedded in
triplicates into 0.3% top agar gel containing DMEM
supplemented with 10% FBS and incubated in a humidified chamber for 2
weeks. The six-well plates were inspected, and 30 randomly
selected fields were photographed under a 106 microscopic lens
and a 66 optical lens (606 magnification). Colonies were then
counted, and the mean number of colonies per field was
calculated. All experiments were done in triplicate.
Flow Cytometry Method (FCM)-assessed Apoptosis
The extent of spontaneous apoptosis was determined with an
Annexin V-FITC/Propidium Iodide Kit (KeyGEN Biotech,
Nanjing, China) according to the manufacturers instructions as
previously described .
Measurement of Mitochondria Membrane Potential
Changes in the mitochondrial membrane potential were
measured by flow cytometry using the mitochondrial membrane
potential assay kit with JC-1 as described . Briefly, the cells
were collected after 48 h of incubation by trypsinization and
centrifugation. The cell pellets were then washed twice with PBS.
0.5 ml JC-1 dye was added to the cell pellet and incubated at 37uC
in dark. The staining solution was removed by centrifugation and
cells were washed twice by JC-1 staining buffer. To assess the
mitochondria membrane potential transition, cells were examined
for each sample on an FL-1 versus FL-2 dot plot on a FACS
Calibur and the data were analyzed using Cell Quest software
(Becton Dickinson, USA).
In Vivo Tumor Growth Study
Female 6- to 8- week-old BALB/c nude mice (n = 15) (Wuhan
Laboratory Animal Center) were housed under specific
pathogenfree conditions in a temperature- and humidity-controlled
environment. All animal experiments were conducted in
accordance with the Institutional Animal Care and Use Committee
guidelines and the animal protocol used in this study was approved
by local ethics committee (Ethical Committee of Tongji Hospital,
Tongji Medical College, Huazhong University of Science and
Technlogy, IRB ID: 2011A01). Briefly, U87 glioblastoma cells
stably transduced with control vector, full-length LRIG2 or
LRIG2 ectodomain were harvested and resuspended in PBS
(26107/ml), respectively. A total of 26106 cells (in 100 ml of PBS)
were injected subcutaneously into the right flank of each mouse
(n = 5 per group). Tumor measures were taken every five days with
calipers and volume was calculated as (width)26(length)6(p/6). At
the end of the experiment, mice were euthanized and tumors were
surgically harvested, measured, and fixed in 4% paraformaldehyde
overnight and analyzed by immunohistochemistry.
Statistical analyses were performed using SPSS 13.0 for
Windows software (SPSS Inc., Chicago, IL, US). Significance
between HGGs and LGGs in the immunohistochemical staining
analysis was determined using chi-square test. Significance of
others analyses was performed by One Way Anova followed by
Bonferroni post-test. The level of statistical significance was
defined as P,0.05.
Figure 1. LRIG2 expression positively correlates with the WHO grade of glioma. (A) Microarray-based gene expression data of brain low
grade gliomas (LGGs) and glioblastoma multiform (GBMs) were downloaded from public TCGA website. The expression level of LRIG2 gene in GBM
was much higher compared with that in LGG (P,0.001). (B) Human gliomas of different grade were immunostained for the LRIG2 protein.
Representative images of each grade of glioma were present (scale bar, 100 mm).
Figure 3. The detection of soluble LRIG2 ectodomain in the supernatants and its proliferative effects on glioblastoma cells. (A) Cells
of U87-Con, U87-LRIG2 and U87-LRIG2ecto were cultured in DMEM for 48 h, the conditional cell culture supernatants were harvested, concentrated
and subjected to western blot using an anti-Flag antibody. A representative blot from three independent experiments is shown. (B) After cells were
cultured in DMEM for 48 h, the supernatants were harvested, filtered through 0.45 mm filter and subjected to immunoprecipitation to eliminate the
expression of Flag-fusion proteins. The supernatants with or without immunoprecipitation were concentrated and subjected to western blotting.
Representative image was shown. (C) The conditioned mediums after or before immnoprecipitation were subjected to proliferation assay. Statistical
analysis was present (*P,0.05 vs con).
Expression of LRIG2 mRNA and protein positively
correlates with the grade of glioma
To investigate the expression levels of LRIG2 in different grade
of glioma, we firstly used public TCGA data repositories as our
primary source of samples. The level 1 data of gene expression,
including 27 cases of LGGs and 466 cases of GBMs, were
downloaded and analyzed. The results revealed that the LRIG2
gene expression level of GBM (WHO IV) was markedly higher
than that of LGG (WHO I&II) (P,0.001) (Figure 1A). To further
confirm the results, we collected the fresh human glioma samples
of different grade to investigate the expression levels of LRIG2
proteins by immunnohistochemical staining. Of the investigated
20 LGGs (WHO I, II), the LRIG2 immunoreactivity (IR) of 14
cases were scored as 0 or 1 and 6 cases scored as 2 or 3. Of the 20
high-grade gliomas (HGGs, WHO III, IV), the LRIG2 IR of 5
cases were scored as 0 or 1 and 15 cases scored as 2 or 3
(Figure 1B). There was a significant difference in LRIG2 IR
between low-grade gliomas and high-grade gliomas (P = 0.004),
which indicated that the expression level of LRIG2 protein in
HGGs was much higher compared to that in LGGs.
Establishment of glioblastoma cell lines stably expressing
the full-length LRIG2 and LRIG2 ectodomain
LRIG1, which has been extensively studied, is proved to be a
tumor suppressor  and the soluble LRIG1 ectodomain has also
been found to act as a negative regulator of tumor growth . To
observe the effects of LRIG2 and LRIG2 ectodomain
overexpression on glioblastoma growth in vitro and in vivo and elucidate
the underlying mechanisms, we subcloned a human full-length
LRIG2 cDNA and LRIG2 ectodomain cDNA into
pLVX-puro36FLAG mammalian expression vectors respectively and
transduced stably into U87 and U251 glioblastoma cells with
pLVXpuro as the control vector. The domain organizations of the
fulllength LRIG2 and LRIG2 ectodomain were presented as
Figure 2A. Figure 2B showed that the Flag-tagged full-length
LRIG2 and LRIG2 ectodomain were successfully expressed. To
assess the expression levels of corresponding mRNA, we assessed
mRNA transcript levels in the stably transduced cells. LRIG2 and
LRIG2ecto transduced U87 or U251 cells showed robustly
increase in mRNA levels when compared with the corresponding
control cells (Figure 2C). As assessed by immunofluorescence
microscopy, Flag staining was observed in both cell lines
transduced with LRIG2 and LRIG2 ectodomain (Figure 2D),
which was in line with the results of western blotting. Taken
together, glioblastoma cell lines stably expressing full-length
LRIG2 and LRIG2 ectodomain were successfully established.
The soluble LRIG2 ectodomain is released from
glioblastoma cells and promotes the proliferation of
As previous study demonstrated that soluble LRIG1
ectodomains could be released from the full-length LRIG1 , we
wanted to address whether soluble LRIG2 ectodomains could be
released from glioblastoma cells. U87 glioblastoma cells were
transduced with expression vectors encoding full-length LRIG2
carrying an N-terminal Flag epitope or LRIG2 ectodomain with a
Flag tag respectively as illustrated in Figure 2A, and cultured in
DMEM without FBS for 48 h. The conditioned cell culture
supernatants were harvested, concentrated and subjected to
western blotting using an anti-Flag antibody. Strikingly, the
antiFlag-reactive soluble protein was detected in the cell culture
supernatants of cells with full-length LRIG2 overexpression,
suggesting that the flag tagged protein fragment was released
from the surface of glioblastoma cells. Strikingly, the molecular
weight of the band corresponds to the predicted molecular weight
of the entire LRIG2 ectodomain (Figure 3A). Consequently, we
for the first time demonstrated that the soluble LRIG2 ectodomain
was capable of being secreted by glioblastoma cells. Further, we
evaluated the effects of the soluble LRIG2 ectodomain in the
conditioned medium on the proliferation of glioblastoma cells.
The results showed that the conditioned medium harvested from
LRIG2 and LRIG2ecto overexpressing cells could significantly
promote the proliferation of U87 cells (Figure 3C). After the
Flagfusion soluble LRIG2 ectodomain were down-regulated from the
supernatants, the effects of conditioned medium on the
proliferation of U87 cells were abrogated (Figure 3B, 3C), which
indicated that the soluble LRIG2 ectodomain in the conditioned
medium exerted proliferative effects on the glioblastoma cells.
Collectively, we demonstrated for the first time that the soluble
LRIG2 ectodomain could be released from the glioblastoma cells
and promote the proliferation of glioblastoma cells in vitro.
Full-length LRIG2 and LRIG2 ectodomain overexpressions
promote the growth of glioblastoma cells in vitro
Previously, we demonstrated that overexpression of full-length
LRIG1 inhibited the growth of glioblastoma cells in vitro [18,26].
The soluble LRIG1 ectodomain was proved to inhibit the glioma
cell proliferation recently . However the effect of
overexpression of full-length LRIG2 or LRIG2 ectodomain on the growth of
glioblastoma cells has not been addressed yet. Firstly, we
demonstrated that both LRIG2 and LRIG2ecto overexpressions
markedly promoted the growth of U87 and U251 glioblastoma
cells (Figure 4A). Secondly, we investigated the expression levels of
Ki-67, a proliferation marker closely correlated with the
proliferation of glioblastoma cells, in stably transduced U87 and U251
cells. The results revealed that the percentages of Ki-67 positive
cells of LRIG2 and LRIG2ecto overexpressing U87 cells were
(91.7467.05)% and (90.5966.72)% respectively, which were
significantly higher than that of U87 control cells
(79.1964.29)% (P,0.05). In line with the results of U87 cells,
the percentages of Ki-67 positive cells of LRIG2 and LRIG2ecto
overexpressing U251 cells were also significantly increased
compared to the U251 control cells (Figure 4B). Further, we
investigated whether LRIG2 or LRIG2ecto altered glioblastoma
anchorage-independent growth, a property that mimics
tumorigenesis in vivo. Using soft agar colony formation assays, we found
that gliobloastoma cells of LRIG2 or LRIG2ecto overexpression
formed more and bigger colonies compared to the control cells
(Figure 4C), indicating that overexpression of LRIG2 or
LRIG2ecto enhances the ability of anchorage-independent growth.
Taken together, these results revealed that full-length LRIG2 as
well as LRIG2 ectodomain promoted the growth of glioblastoma
cells in vitro.
Full-length LRIG2 and LRIG2 ectodomain overexpressions
inhibit the spontaneous apoptosis of glioblastoma cells
through mitochondrial pathway in vitro
It has become apparent that tumor growth depends not only on
the rate of cell proliferation but also on the rate of apoptosis .
Previously, we demonstrated that downregulation of LRIG2
expression by RNA interference increased spontaneous apoptosis
of glioblastoma cells in vitro . Congruently, by using flow
cytometric analysis with Annexin V/PI staining, we revealed that
the spontaneous apoptotic rates of LRIG2 overexpressing U87
and U251 cells were significantly decreased compared to the
corresponding control cells (Figure 5A), indicating that LRIG2
overexpression inhibited the spontaneous apoptosis of
glioblastoma cells. Most strikingly, we for the first time demonstrated that
overexpression of LRIG2 ectodomain could also significantly
decrease the spontaneous apoptotic rates of U87 and U251
glioblastoma cells (Figure 5A). To further explore the mechanisms
of LRIG2 or LRIG2ecto mediated inhibition of apoptosis, we
examined the mitochondrial membrane potential (MMP),
depolarization of which is an early event in the mitochondrial pathway
of apoptosis, using JC-1 red/green fluorescence measurement by
flow cytometry . The results revealed that the ratios of JC-1
FL-2/FL-1 of glioblastoma cells transduced with LRIG2 or
LRIG2ecto were robustly increased compared to the
corresponding control cells (Figure 5B), which indicated that LRIG2 and
LRIG2ecto overexpressions both stabilized the MMP of
glioblastoma cells. Collectively, we demonstrated that full-length LRIG2
and LRIG2 ectodomain overexpressions inhibited the
spontaneous apoptosis of glioblastoma cells through mitochondrial pathway
by stabilizing the mitochondria membrane potential.
Figure 7. LRIG2 as well as LRIG2ecto interacts with EGFR in glioblastoma cells. (A) Confocal immunofluorescence laser microsopy showed
colocalization of LRIG2 and EGFR in LRIG2 overexpresing U87 and U251 glioblastoma cells. Cells were stained with anti-LRIG2 and anti-EGFR
antibodies, followed by using Cy3- and FITC-conjugated secondary antibodies. Yellow of merge panel indicates regions of colocalization. Scale bar,
50 mm. (B) Lysates from cells overexprssing LRIG2 or LRIG2ecto were immunoprecipitated with purified mouse anti-Flag monoclonal antibodies or
control mouse IgG, and immunoprecipitates were blotted with antibodies to EGFR or Flag.
Full-length LRIG2 and LRIG2 ectodomain overexpressions
promote the growth of tumor xenograft in vivo
We next determined whether the effects exerted by LRIG2 and
LRIG2ecto overexpressions on the growth and apoptosis of
glioblastoma cells in vitro could be demonstrated in vivo. Stably
transduced glioblastoma cells were injected subcutaneously into
the female 6- to 8- week-old nude mice and the tumor size of
xenografts were measured every five days from postimplantation
day 15. Consistent with the cell-based in vitro studies, tumor
growth of LRIG2 and LRIG2ecto overexpression groups were
strikingly promoted compared to the control group (Figure 6A, B)
(**P,0.01). The xenografts were surgically removed and
subjected to immunohistochemistry staining for Ki-67, PCNA, and
caspase3. Similar to the results obtained from in vitro studies, the
expression levels of Ki-67 and PCNA, two markers of cell
proliferation activity, in LRIG2 and LRIG2ecto groups were both
significantly increased and the levels of caspase3, a marker of
apoptosis, were both markedly inhibited compared to the control
group (Figure 6C). These results further demonstrated that LRIG2
and LRIG2ecto overexpressions promoted the growth of tumor
xenograft by enhancing the proliferation and inhibiting the
apoptosis in vivo.
indicated cells were starved for 24 h and cultured in complete medium for 48 h. The cell lysates were exacted and examined by western blotting for
the cell cycle and apoptosis associated proteins. Analysis of quantification of the bands intensity was shown in the lower panel (*P,0.05 vs con).
Full-length LRIG2 and LRIG2 ectodomain each can
physically interact with EGFR in glioblastoma cells
To explore the possible mechanisms underlying the
forementioned effects of full-length LRIG2 and LRIG2 ectodomain on the
glioblastomas, we examined whether LRIG2 or LRIG2ecto could
interact with EGFR. Firstly, to determine the subcellular locations
of full-length LRIG2 and possible sites of LRIG2-EGFR
interaction, we examined the localization of LRIG2 and EGFR
in LRIG2 overexpressing U251 and U87 cells by confocal
immunofluorescence laser microscopy. As illustrated in Figure 7A,
LRIG2 was found largely in the intracellular cytoplasm as well as
in the plasma membrane and EGFR exhibited a similar
distribution. Merging of images demonstrated that LRIG2 and
EGFR signals overlap at the similar peripheral and intracellular
locations, suggesting that these are sites of LRIG2-EGFR
interaction. To further confirm the results, we precipitated Flag
tagged LRIG2 from extracts of LRIG2-overexprssing cells using
Flag antibody and detected the precipitates by western blotting
using EGFR antibody. Consistent with the immunofluorescence
results, this analysis revealed that LRIG2 physically interacted
with EGFR (Figure 7B). Most strikingly, we found that the LRIG2
ectodomain was also capable of interacting physically with EGFR
(Figure 7B), which indicates that the full-length LRIG2 interacts
with EGFR through the recognition of the ectodomain of LRIG2
Full-length LRIG2 and LRIG2 ectodomain enhances EGFR
activation and its downstream PI3K/Akt pathway
Based on the above results that LRIG2 and LRIG2ecto were
capable of physically interacting with EGFR, we further explored
the effects of LRIG2 and LRIG2ecto on the activation of EGFR
and its downstream pathways. Stably transduced cells were
cultured in DMEM with 10% FBS for 48 h, then the cell lysates
were extracted and subjected to western blotting. The results
revealed that the levels of phosphorylated EGFR (pEGFR) and
phosphorylated Akt (pAkt) in LRIG2 and LRIG2ecto
overexpressing cells were markedly increased compared to the
corresponding control cells (Figure 8A). As expected, the expression
levels of c-Myc, a transcription factor that triggers cell proliferation
and regulated by PI3 K/Akt pathway , were also increased in
LRIG2 and LRIG2ecto overexpressing cells (Figure 8A). The cell
cycle proteins cyclin B1, cyclin D1, and cyclin E, which were
closely associated with cell proliferation, were also expectedly
enhanced. The anti-apoptotic protein Bcl-2 was increased and the
pro-apoptotic proteins Bax and cleaved caspase3 were decreased
(Figure 8B). To further confirm the effects of LRIG2 and
LRIG2ecto on the activation of EGFR and the downstream
PI3 K/Akt pathway, cells were cultured in DMEM without FBS
for 24 h, followed by EGF stimulation. U87 cells overexpressing
LRIG2 or LRIG2ecto exhibited not only increased levels of total
and phosphorylated EGFR but also enhanced Akt activation with
or without EGF stimulation (Figure 9A). U251 cells
overexpressing LRIG2 or LRIG2ecto revealed strikingly increased levels of
total EGFR with or without EGF stimulation, modestly increased
phosphorylated EGFR and Akt activation with EGF stimulation
for 5 minutes (Figure 9A). Next, we used gefitinib (10 mM) to
inhibit the phosphorylation of EGFR followed by detecting the
downstream PI3 K/Akt pathway and results revealed that
inhibition of phosphorylation of EGFR abrogated the effects of
LRIG2 and LRIG2ecto overexpressions on the Akt activation
(Figure 9B), which further demonstrated that LRIG2 or
LRIG2ecto overexpression enhanced the activation of PI3 K/Akt
pathway through stabilizing the EGFR and enhancing the EGFR
phosphorylation. Combining the forementioned results, we suggest
that upregulation of LRIG2 or LRIG2ecto lead to stabilization of
EGFR and activation of EGFR, thereby enhancing the
downstream PI3K/AKT pathway.
In the present study, we revealed that the expression levels of
LRIG2 transcript in glioblastomas and LRIG2 protein in HGGs
were both significantly higher compared to that in LGGs and we
for the first time demonstrated that LRIG2 expression levels
positively correlated with the grade of glioma, which predicted
that LRIG2 might serve as a negative prognostic factor associated
with poor glioblastoma survival. This mirrors to some extent the
situation in oligodendrogliomas  and early-stage squamous cell
carcinoma of the uterine cervix , where high expression of
LRIG2 is associated with poor survival, whereas LRIG1
expression negatively correlates with tumor grade [10,11,12] and
associates with better survival in numerous tumors [11,12],
indicating that the functions of LRIG2 and LRIG1 may be
different in the progression of tumors.
To further confirm the role of LRIG2 in glioblastoma and
explore the possible underlying mechanisms, we established
glioblastoma cells with stable expressions of the full-length LRIG2.
In line with the forementioned results, we showed that
overexpression of full-length LRIG2 promoted the proliferation and
inhibited the apoptosis of glioblastoma cells in vitro and in vivo.
Combined with our previous reported finding that downregulation
of LRIG2 inhibits glioblastoma cell growth in vitro , our
results provided compelling evidences in support of the proposal
that full-length LRIG2 served as a tumor promoter in
glioblastoma. Congruently, it is recently reported that, by using an animal
model of PDGF-induced glioma, the Lrig2E122/2 mice showed
increased spontaneous mortality, reduced growth rate and
protection against PDGFB-induced glioma and Lrig2E12+/+
mice developed gliomas at a higher frequency and of higher
malignancy than Lrig2E122/2 mice, which indicated a
protumoregenic role of Lrig2 in the progression of oligodentroglioma.
. Collectively, data to date suggest that LRIG2 in human and
Lrig2 in mouse both play critical roles in the genesis and
progression of glioma. In previous study, we demonstrated that
LRIG1 inhibited the proliferation and promoted the apoptosis of
glioblastoma cells in vitro and in vivo [18,26], suggesting that
LRIG1 exerts as a tumor suppressor in glioblastoma, which is in
contrast to the results of LRIG2 herein presented. However, the
mechanism underlying the function difference between LRIG2
and LRIG1 remain largely unexplored.
LRIG proteins share the same domain organization: an
ectodomain consisting of 15 leucine-rich repeats (LRR) with
cysteine-rich N- and C-terminal flanking domains and three
immunoglobulin-like domains, a transmembrane domain and a
cytoplasmic tail . It has been reported that a recombinant
fragment of the LRIG1 ectodomain causes growth inhibition of a
panel of carcinoma cells . Noteworthy, soluble LRIG1
ectodomain has recently been demonstrated to be shed naturally
from the full-length LRIG1 and suppress cell proliferation .
cultured with 10% FBS addition with gefitinib (10 mM) or DMSO for 48 h. The total lysates were extracted and subjected to western blotting. The
representative images of three independent experiments were present and quantification of the bands intensity was shown in the right panel (*P,
0.05 vs con).
Moreover, this soluble LRIG1 ectodomain has been shown to
inhibit the genesis and progression of glioma in vivo .
However, little is known regarding the functions of LRIG2
ectodomain. In the present study, we for the first time
demonstrated that the soluble LRIG2 ectodomain was capable
of being released from glioblastoma cells and soluble 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, which suggested that LRIG2 ectodomain
played a similar role to the full-length LRIG2 in the progression of
glioblastoma. The herein present function of LRIG2 ectodomain
in progression of glioblastoma was opposite to the full-length
LRIG1 [18,26] and LRIG1 ectodomain [14,30], while in line with
full-length LRIG2, which leads to the proposal that LRIG2
ectodomain may contribute to the function difference of the
fulllength LRIG2 and full-length LRIG1 in glioblastoma progression.
To our knowledge, the soluble counterparts of transmembrane
proteins can be released mainly by two distinct mechanisms:
proteolytic processing and microvesicle shedding . Soluble
LRIG1 ectodomain has been demonstrated to be secreted from
the full-length LRIG1 through the proteolytic processing , the
mechanism underlying the release of the soluble LRIG2
ectodomain is not addressed and deserves further study.
Further, in order to provide more compelling evidence to
support our proposal, we explored the possible mechanisms
underlying the effects of full-length LRIG2 and LRIG2
ectodomain on the proliferation and apoptosis of glioblastoma.
Accumulating evidence indicates that LRIG1 functions as a tumor
suppressor in humans by negatively regulating tyrosine kinase
receptors of the EGFR family [9,32]. The mechanisms by which
LRIG1 restricts growth factor signaling include three different
ways, such as enhancing liand-induced EGFR ubiquitylation and
degradation [5,6], promoting Met degradation in a
lysosomedependent and cbl-independent manner , and inhibiting
binding of GDNF with Ret . Nevertheless, LRIG1 ectodomain
has also been demonstrated to suppress EGF signaling without any
apparent downregulation of EGFR levels [14,30]. It is not known
yet that whether or not the full-length LRIG2 and LRIG2
ectodomain exert the similar roles in the regulation of EGFR
signaling. Remarkably, in the present study we for the first time
demonstrated that full-length LRIG2 and LRIG2 ectodomain
both could physically interact with EGFR, increase the level of
EGFR and enhance the activation of EGFR and its downstream
PI3K/Akt pathway, resulting in increment of pro-proliferative and
anti-apoptotic proteins and attenuation of pro-apoptotic proteins.
Besides the EGFR signaling, the PDGF-induced immediate-early
gene induction of Fos and Egr2 has also recently been
demonstrated to be regulated by Lrig2 in primary MEF cells.
However, Lrig2 just regulated PDGF-induced immediate-early
gene induction and had no effects on the PDGFR protein levels or
phosphorylation events of PDGFR, Akt in MEF cells . The
tumor promoting effects of LRIG2 on receptor tyrosine kinases
(RTKs) might be prominent in tumor cells and subtle in
nontumor cells. Most notably, the results herein present suggest that
LRIG2 ectodomain plays a similar role to the full-length LRIG2 in
the regulation of EGFR signaling pathways, whereas LRIG1 and
LRIG1 ectodomain exert the opposite functions. Such
observations further strengthen the proposal that LRIG2 ectodomain may
be responsible for the function difference of LRIG2 and LRIG1 in
the progression of glioblastoma.
Based on our finding that soluble LRIG2 ectodomain is capable
of being released from full-length LRIG2, it is reasonable to
speculate that the soluble LRIG2 ectodomain can release from the
glioblastoma cells as an effective pro-growth factor in the
microenvironment, interact with EGFR in an autocrine or
paracrine way and enhance the EGFR signaling to promote the
growth of glioblastoma. The amino acid sequence of human
LRIG2 was 47% identical to human LRIG1, whereas the
ectodomains of LRIG2 and LRIG1 were highly conserved .
Therefore the mechanisms underlying the function differences
between LRIG2 ectodomain and LRIG1 ectodomain in the
progression of glioblastoma are needed to be further addressed in
Taken together, we report for the first time that LRIG2
expression levels positively correlate with the grade of glioma.
Most strikingly, we demonstrate for the first time that the soluble
LRIG2 ectodomain is capable of being released from glioblastoma
cells. Both full-length LRIG2 and LRIG2 ectodomain potently
promote the proliferation and inhibited the apoptosis of
glioblastoma cells in vitro and in vivo through physically interacting with
EGFR, stabilizing EGFR, and enhancing EGFR activation and its
downstream PI3 K/Akt pathway. Going forward, it will be
important to determine how LRIG2 ectodomain is released from
glioblastoma cells, whether this kind of release is universal, and
how LRIG2 ectodomain interact with EGFR and enhance the
activation of EGFR. We believe that LRIG2 ectodomain may
provide a promising therapeutic target in the treatment of
glioblastoma multiform in the future.
The authors would like to thank Prof. Feng Wan and Prof. Fei Ye for their
dedicated revision of the manuscript. We also thank Xueyan Wan,
Shiqiang Wu, and Min Zhao for the help in the analysis of TCGA data.
Ran Li is acknowledged for his assistance in the collection of human glioma
Conceived and designed the experiments: BW DG TL. Performed the
experiments: QX YT YG. Analyzed the data: QX HY FM RX.
Contributed to the writing of the manuscript: QX BW DG.
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