LRIG1 modulates aggressiveness of head and neck cancers by regulating
EGFR-MAPK-SPHK1 signaling and extracellular matrix remodeling
Jim Jinn-Chyuan Sheu,1,2,3 Chun-Chung Lee,4 Chun-Hung Hua,5 Chuan-i Li,4 Ming-Tsung Lai,6,7 Shan-Chih Lee,8 Jack Cheng,1 Carmen Chan,1 Steven Chun-Chin Chao,1 Jen-Yang Chen,4 Jang-Yang Chang,4 Chia-Huei Lee4
1Human Genetic Center, China Medical University Hospital, Taichung, Taiwan; 2School of
Chinese Medicine, China Medical University, Taichung, Taiwan; 3Department of Health and
Nutrition Biotechnology, Asia University, Taichung, Taiwan; 4National Institute of Cancer
Research, National Health Research Institutes, Miaoli, Taiwan; 5Department of
Otolaryngology, China Medical University Hospital, Taichung, Taiwan; 6Department of
Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan; 7School of Medicine
and 8College of Medical Science and Technology, Chung Shan Medical University, Taichung,
Taiwan
Running title: LRIG1 downregulates EGFR-MAPK-SPHK1 signaling and MMPs.
Chia-Huei Lee, PhD
National Institute of Cancer Research, National Health Research Institutes, R2, R1211, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan. Tel: +(886)-37-246-166, ext. 31740
Fax: +(886)-37-463
Abstract
Epidermal growth factor receptor (EGFR) overexpression and chromosome 3p deletion are two frequent events in head and neck cancers. We previously mapped the smallest region of recurrent copy-number loss at 3p12.2-p14.1. LRIG1, a negative regulator of EGFR, was found located at 3p14, and its copy-number loss correlated with poor clinical outcome. Inducible expression of LRIG1 in head and neck cancer TW01 cells, a line with low LRIG1 levels, suppressed cell proliferation in vitro and tumor growth in vivo. Using gene expression profiling, quantitative RT-PCR, chromatin immunoprecipitation, and Western blot analysis, we demonstrated that LRIG1 modulates extracellular matrix (ECM) remodeling and the EGFR-MAPK-SPHK1 transduction pathway by suppressing expression of EGFR ligands/activators, MMPs, and the signaling molecule SPHK1. By contrast, knockdown of endogenous LRIG1 in head and neck cancer TW06 cells, a line with normal LRIG1 levels, significantly enhanced cell proliferation, migration, and invasiveness. In addition, LRIG1 suppression promoted cell morphology changes to be more spindle cell shape with SNAI2 expression increased and E-cadherin expression reduced. These tumor-promoting effects could be abolished by specific MAPK or SPHK1inhibitors. Our data demonstrate that LRIG1 acts as a potent tumor suppressor for head and neck cancers and that downregulation of LRIG1 in cancer cells enhances EGFR-MAPK-SPHK1 signaling and ECM remodeling activity, leading to malignant phenotypes of head and neck cancers.
Keywords: LRIG1, head and neck cancers, EGFR, MAPK, SPHK1, ECM remodeling.
Introduction
Nasopharyngeal carcinoma (NPC) and oral squamous cell carcinoma (OSCC) are two major subtypes of head and neck cancers. Genetic susceptibility, viral infection, and chemical exposure have been suggested as risk factors for the development of these human malignancies (Pai and Westra, 2009). Enhanced genome instability with gross chromosomal abnormalities has been directly correlated with tumor recurrence and metastasis of NPC (Fang et al., 2009; Sheu et al., 2009b), leading to high mortality and a low five-year overall survival, which have not been improved in decades (Maseki et al., 2012). An understanding of the molecular mechanisms involved in the development of advanced head and neck cancers will help in clinical management and drug discovery. Genome-wide surveys and clinical association studies have identified two recurrent copy-number aberrations, 3p deletion (3p12.3-p14.2) and 3q amplification (3q26.2-q26.32), with prognostic value for metastatic NPC (Sheu et al., 2009b). Notably, chromosome 3p loss is also a genetic hallmark in other types of head and neck cancers (Jin et al., 2005; Koy et al., 2008), and has been defined as a crucial early event in NPC progression (Huang et al., 2004).
These findings suggest that potent tumor suppressors may be encoded within the 3p deletion region, 3p12.3-p14.2.
LRIG1, a negative regulator of the EGFR mapping to 3p14, encoding a transmembrane protein containing fifteen leucine-rich repeats (LRRs) and three Ig-like domains in its extracellular region, has been proposed as a tumor suppressor gene (Hedman et al., 2002; Sheu et al., 2009b) and shown to be downregulated in several tumor types, including renal cell carcinoma (Thomasson et al., 2003), advanced cervical cancer (Lindstrom et al., 2008), glioma (Stutz et al., 2008), and breast cancer (Ljuslinder et al., 2009; Miller et al., 2008). In addition, LRIG1 downregulation correlates with higher tumor grade and a worse outcome in human squamous cell carcinomas (Jensen et al., 2008) and breast cancer (Krig et al., 2011). Currently, the tumor suppressive function of LRIG1 is mainly attributed to its inhibitory effect on EGFR signaling. In the past few years, it has been shown that LRIG1 binds to the EGFR and attenuates EGFR signaling through both receptor degradation and catalytic inhibition (Gur et al., 2004). Moreover, soluble ectodomains of LRIG1 are sufficient to cause decreased EGFR activity as a result of shedding of the LRR region and Ig-like domains (Goldoni et al., 2007; Yi et al., 2011). Ectopic expression of LRIG1 on the other hand, inhibits cancer cell motility and invasiveness of EGFR (+) prostate cancer cells (Laederich et al., 2004) and EGFRvIII (+) glioblastoma cells (Stutz et al., 2008; Ye et al., 2009). Besides ErbB family, the functions of other receptor tyrosine kinases, such as Met and Ret, have been
reported to be regulated by LRIG1 via a cbl-independent degradation mechanism (Ledda et al., 2008; Shattuck et al., 2007). These findings imply that LRIG1 may play an anti-metastasis role in human cancers.
Although constitutive activation or overexpression of EGFR is frequently found in head and neck cancers (Abhold et al., 2012; Sheu et al., 2009a), the molecular mechanisms underlying the functional roles of LRIG1 in Head and neck cancers development still remain elusive. In the present study, we demonstrated that LRIG1 expression is significantly lower in NPC and OSCC tumors than in their non-tumor counterparts and these lower levels are prognostic for poor survival in NPC patients. Using a Tet-off inducible system in NPC cells, we demonstrated the anti-cancer effects both in vivo and in vivo triggered by LRIG1 induction. Using cDNA microarrays, we investigated the signaling network regulated by LRIG1 expression in head and neck cancers and validated the results by chromatin-immunoprecipitation (ChIP), antibody array and Western blotting. To mimic the clinical condition, we also examined the oncogenic effects of LRIG1 loss in NPC cells by siRNA knockdown. Finally, we provide evidence that malignant behaviors of the LRIG1 knockdown cells can be greatly decreased by treatment with specific inhibitors of components of the EGFR-MAPK-SPHK1 signaling pathway.
Results
LRIG1 downregulation correlates with EGFR activation and poor clinical outcome in human head and neck cancers
Our previous study found a significant association between LRIG1 deletion and poor clinical outcome in NPC patients (Sheu et al., 2009b). To examine LRIG1 protein levels, we performed immunohistochemistry on tissue microarrays containing 44 metastatic NPC tumor samples. LRIG1 immunoreactivity was very intense in the adjacent non-tumor epithelium, but only weakly positive in 65.9% (29 of 44) and undetectable in 34.1% (15 of 44) NPC samples (Figure 1a). Although no association was found between LRIG1 loss and total EGFR levels (data not shown), NPC tumors with undetectable LRIG1 had higher phospho-EGFR levels than those with positive LRIG1 staining (86.7 % vs. 13.8 %, P<0.001, Figure 1a), support for a negative regulatory role of LRIG1 in EGFR signaling. Kaplan-Meier survival analysis indicated a significant association (p = 0.038) between low levels of LRIG1 and poor clinical outcome in NPC patients, as patients with positive LRIG1 staining exhibited a longer median survival time (108.6 months) than those with undetectable LRIG1 (33.8 months) (Figure 1b). In agreement with these findings for NPC, cDNA microarray data from OSCC samples showed reduced LRIG1 expression in tumors compared to the adjacent non-tumor epithelium (P < 0.001) (Figure 1c).
blot analysis. Figure S1 shows that LRIG1 was markedly downregulated in all 5 NPC and 4 OSCC cells tested compared to the non-transformed nasopharyngeal NP460hTert and oral CGHNK6 cell lines. These results indicate that LRIG1 downregulation is common in head and neck cancer patients, along with a bad prognosis. LRIG1 deletion may therefore provide oncogenic signals promoting the development of head and neck cancers.
LRIG1 induction in NPC cells reduces cell growth in vitro and tumorigenicity in vivo
To characterize the role of LRIG1 in head and neck cancers, we established stable Tet-off inducible clones in TW01 cells, in which LRIG1 expression is normally barely detectable (Figure S1).Western blots confirmed that LRIG1 expression could be turned on/off under the control of the tetracycline-response element in two clones, TW01-LG1/a and TW01-LG1/b (Figure 2a). Figure 2b shows that LRIG1 induction significantly reduced the proliferation rate of TW01-LG1/a cells as early as 24 hr after the start of induction. Cell cycle analysis ( Figure S2a) and annexin V staining (Figure S2b) indicated a tendency of G2/M block triggered by LRIG1 expression, which led to accumulation of cells in the sub-G1 population and cell death. To determine whether LRIG1 controls tumor growth in vivo, we used a xenograft model in immunocompromised (nu/nu) mice injected subcutaneously with TW01-LG1/a cells and found that, compared to the non-induced group, LRIG1-expresssing tumors showed a
much slower growth rate ( Figure 2c) and lower tumor weight (p = 0.008, Figure 2d).
LRIG1 plays negative regulatory roles in oncogenic signaling by downregulation of MMPs, EGFR ligands, and SPHK1
To identify cancer-related genes and pathways targeted by LRIG1,we performed time-course microarray experiments to compare the expression profiles of LRIG1-induced and non-induced TW01-LG1/a cells. Data analysis revealed that a significant transcriptional change occurred 36 hr after LRIG1 turn on. Venn diagram analysis showed 46 genes overlapping with either an ascending (upregulated genes) or a descending (downregulated genes) trend in mRNA expression levels during the period of LRIG1 induction (Figure 3a). These differentially expressed genes were cross-validated by quantitative RT-PCR (Table 1). Functional annotation analysis using the PANTHER classification system (http://www.pantherdb.org) indicated that genes involved in extracellular matrix (ECM) remodeling, such as those coding for MMP-1, MMP-3, MMP-10, MMP-12, MMP-13, ITGA2, LAMB3, AMIGO2, and GJB5, accounted for the major group of LRIG targets (Figure 3a and Table 1). Subsequent Western blot analysis using an anti-MMP antibody array demonstrated that several MMPs, including 1, 2, 9, 10, and MMP-13, have a reducing trend in protein level upon LRIG1 turned-on, whereas levels of TIMP2, an MMP inhibitor, were significantly increased (Figure 3b). Since phospho-MAPK can be
translocated into the cell nucleus and act as a transcriptional regulator (Chang et al., 2007), we examined whether phospho-MAPK was directly responsible for the reduced expression of LRIG1 targets. A chromatin-immunoprecipitation (ChIP) analysis using anti-phospho-MAPK antibodies revealed that LRIG1 induction significantly attenuates the binding of phospho-MAPK to promoter regions of MMP-1, MMP-3, and MMP-10 (Figure 3c). This finding suggests a novel role of LRIG1 in transcriptional regulation by inactivation of signaling molecules, such as phospho-MAPK.
The microarray results also revealed that the transcription of EGFR ligands/activators, such as amphiregulin (AREG), neuregulin 1 (NRG1), transforming growth factor-alpha (TGFA), and heparin-binding EGF-like growth factor (HBEGF), was reduced in response to LRIG1 induction ((Figure 3a). As shown in Figure 4a, consistent with previous findings (Gur et al., 2004; Laederich et al., 2004), Western blotting showed that LRIG1 induction was accompanied in a time-dependent manner by enhanced EGFR degradation and reduced phosphorylation of EGFR and MAPK. Expression of sphingosin kinase 1 (SPHK1), a downstream target of MAPK (Pitson et al., 2003), was also reduced by LRIG1 induction and, notably, expression of an epithelial-to-mesenchymal transition (EMT) marker, SNAI2, was also reduced (Figure 4a), supporting our finding from microarray (Figure 3a and Table 1). Subsequent cell morphology analyses revealed a spread-out squamous cell shape in LRIG1 turned-on cells (LRIG1 on) compared to the spindle-like shape of LRIG1 turned-off cells
(LRIG1 off) ( Figure 4b), suggesting that restored LRIG1 expression induced cell differentiation. These results imply that LRIG1 may regulate ECM remodelingand support a negative regulatory role of LRIG1 in EGFR signaling.
Based on our data, we proposed a model for the role of LRIG1 in a positive feedback inhibitory loop for EGFR-MAPK-SPHK1 signaling and ECM remodeling (Figure 4c). In the presence of LRIG1, activation of EGFR and MAPK is attenuated, thus reducing the transcription of several EGFR ligands/activators by decreasing the levels of phospho-MAPK or other unidentified transcriptional factors, resulting in further attenuation of EGFR signaling. In addition, decreased expression of MMPs reduces ECM remodeling activity and inhibits the release of EGFR ligands/activators sequestered by the ECM, which also leads to inhibition of EGFR signaling. Furthermore, decreased SPHK1 levels may reduce the production of S1P, which binds to S1PRs, G-protein-coupled receptors that can transactivate other receptor type tyrosine kinases (RTKs), including EGFR (Shida et al., 2004). Thus, increased LRIG1 expression may not only attenuate EGFR-MAPK signaling, but also trigger a positive feedback inhibitory loop to further downregulates EGFR-MAPK-SPHK1 signaling. These findings suggest MAPK and SPHK1 as potent therapeutic targets for tumors with LRIG1 loss.
inhibitors
According to the proposed model, the aggressiveness of cancer cells caused by LRIG1 loss might be suppressed by treatment with MAPK or SPHK1 inhibitors. To test this possibility, we used specific siRNA (siLRIG1) to knockdown LRIG1 expression in TW06 cells, which showed the highest LRIG1 levels of the head and neck cancer cell lines tested (Figure S1). Western blotting confirmed the robust effect of siLRIG1 on LRIG1 expression and that levels of phospho-EGFR, phospho-MAPK, and SNAI2 were all increased by LRIG1 knockdown, while the amount of SPHK1 was also slightly increased (Figure 5a). In addition, increased SNAI2 levels and decreased E-cadherin levels seen ( Figure 5a) support the cell morphological observations that cells with low LRIG1 expression have a spindle-like shape, while those with high LRIG1 levels have a squamous cell-like shape (Fig 4b). The proliferation rate rose to 155-170% of that of controls on Day 3 after LRIG1 knockdown (Figure 5b). An anchorage-independent growth assay showed that more and larger colonies were formed by cells transfected with siLRIG1 than those transfected with scrambled control (Figure 5c), suggesting that LRIG1 downregulation led to acquisition of survival advantages by head and neck cancer cells. Interestingly, these effects were significantly decreased by treatment with the MAPK inhibitor U0126 or the SPHK1 inhibitor SK1-I (Figure 5b and 5c). Trans-well migration (Figure 5d) and invasiveness (Figure 5e) were also significantly enhanced by LRIG1 knockdown and these effects were both almost completely abolished by
U0126 or SK1-I. The inhibitory effect of SK1-I in all experiments was dose-dependent, suggesting the key role of SPHK1 signaling in NPC cells. It should be noted that, at the concentrations used, U0126 or SK1-I had only a minor or undetectable cytotoxic effect on TW06 cells (Figure S4). Together, these results support that cross-talk among EGFR, MAPK, and SPHK1 regulated by LRIG1 and demonstrate the association of LRIG1 downregulation with aggressiveness of head and neck cancers.
Discussion
The present study has provided the first evidence that LRIG1 down-regulation promotes malignant characteristics in head and neck cancers by enhancing EGFR-MAPK-SHPK1 signaling and the associated ECM remodeling. Our data also demonstrated that LRIG1 downregulation serves as a prognostic marker for aggressive NPC (Figure 1b). A Tet-off inducible system revealed that SPHK1 and MMPs were the direct downstream targets of LRIG1 that LRIG1 induction associates with low SPHK1 and MMPs expression (Figure 3 and Table 1). In addition, LRIG1 was shown to regulate transcription through reduction of phospho-MAPK and possibly other transcriptional factors (Figure 4a and 4c). Increased expression of LRIG1 changed gene expression profiles, leading to a dynamic shift in the cellular signaling network from oncogenic to tumor-suppressing (Figure 3a). Interestingly, this transcriptional regulation resulted in a positive-feedback inhibitory loop, e.g. reduced expression of EGFR ligands, to further attenuate EGFR signaling (Figure 4c). These findings might explain why LRIG1 knockdown provided advantages for tumor growth in mice, with larger tumors being seen in the LRIG1-off group than the LRIG1-on group (Figure 2c and 2d).
SPHK1 catalyses the conversion of sphingosine to sphingosine-1-phosphate (S1P), a bioactive lipid molecule which mediates S1P signaling through binds to its receptors. A handful of studies have shown that various types of human cancer acquire survival advantage,
aggressiveness, and chemotherapy resistance through non-oncogenic addiction to S1P signaling transduction pathway (Pyne and Pyne, 2010; Pyne et al., 2012). With over-expression / activation of SPHK1 in cancer cells, SPHK1 has been cited as an emerging target for cancer therapy (Pyne et al., 2011; Shida et al., 2008). Activation of EGFR-MAPK signaling is one mechanism for increasing SPHK1 activity, both at the level of expression and phosphorylation (Pitson et al., 2003). In addition, hyperactivation of EGFR signaling has been confirmed to be a crucial event in the development of head and neck cancers by various approaches, including functional genomics, quantitative phosphoproteomics, and clinical association studies (Ma et al., 2003; Ruan et al., 2011; Sheu et al., 2009a). S1P signaling can activate G protein-coupled S1P receptors (S1PRs) that subsequently transactivates EGFR in the presence of MMPs and HBEGF (Shida et al., 2004). Our findings uncovered a novel reciprocal mechanism of signaling cross-talk between EGFR-MAPK-SPHIK1 and S1PR signaling in head and neck cancers, in which LRIG1 plays a key role in regulating this signaling network (Figure 4b).
The signaling strength and duration of activation of EGFR receptors are controlled by multiple circuits of positive and/or negative feedback regulation. One such mechanism involves increased MMP activity in the ECM which triggers ECM remodeling, which is accompanied by local release of ECM-sequestered growth factors, cytokines, and receptor tyrosine kinase ligands without de novo protein synthesis (Gaide Chevronnay et al., 2012;
Munger and Sheppard, 2011). In head and neck cancers, EGFR amplification and LRIG1 (chromosome 3p) deletion are pathogenic events that are frequently observed in tumor tissues (Abhold et al., 2012; Jin et al., 2005; Sheu et al., 2009b). Clinically, both EGFR overexpression and LRIG1 downregulation correlate with a worse clinical outcome (Burtness, 2005); biologically, LRIG1 acts as a negative regulator to suppress EGFR-MAPK-SPHK1 signaling and ECM remodeling by positive-feedback inhibitory regulation (Figure 4c). Our study shows that low levels of EGFR negative regulators, for example by LRIG1 deletion, could elicit full oncogenic signaling, resulting in a permissive environment for tumor onset and progression.
In addition to its role as a negative regulator for EGFR, LRIG1 was recently identified as a potent stem cell marker for the non-cycling, long-lived populations located at the interfollicular epidermis (Jensen et al., 2009; Watt and Jensen, 2009) or crypt base of the intestine (Powell et al., 2012; Wong et al., 2012). Genetic ablation of LRIG1 disturbs epithelial homeostasis and increases the proliferative capacity of stem cells (Ordonez-Moran and Huelsken, 2012), which may contribute to tumor formation and invasion. These findings raise the possibility that LRIG1-/- cells harbor a mesenchymal phenotype to promote cancer
progression and metastasis. Our results showed that expression of SNAI2, an EMT marker, was induced in LRIG1 knockdown cells, while expression of the epithelium marker E-cadherin was dramatically reduced (Figure 5a). In agreement with this finding, cell
morphology analyses showed that LRIG1 controls cell shape/architecture (Figure 4b). Since SPHK1-S1P signaling (Milara et al., 2012), MMPs, and ECM remodeling (Lochter et al., 1997; Radisky and Radisky, 2010; Song et al., 2000) have also been linked to EMT, it will be interesting to investigate how LRIG1 regulates EMT in promoting aggressive phenotypes, such as metastasis and resistance to radio-/chemo-therapy. Furthermore, differential subcellular protein localization or compensatory feedback mechanisms might account for the LRIG1 protein upregulation seen in some tumor types (Hedman and Henriksson, 2007). This suggests that the subcellular localization of LRIG1 may determine its function in oncogenesis. The significance of the specific subcellular localization of LRIG1 needs further investigation to fully elucidate the role of LRIG1 in human cancer.
In summary, our study suggests novel functions for LRIG1 in regulating cellular mobility and metastasis via EGFR-MAPK-SPHK1 signaling and ECM remodeling. Through transcriptional regulation and signaling transaction, LRIG1 is involved in a positive feedback loop for cell growth signaling which may determine cancer development, cell differentiation, and apoptosis. Inhibitors of MAPK or SPHK1 signaling components are suggested as candidate drugs for treating patients whose tumors show chromosome 3p / LRIG1 loss.
Materials and methods
Cell lines and reagentsThe inhibitors U0126 and SK1-I (BML-258) were purchased from Sigma (St Louis, MO, USA) and ENZO Life Sciences (Farmingdale, NY, USA), respectively. Two established human nasopharyngeal carcinoma cell lines, TW01 and TW06 (Lin et al., 1990), were used in this study and were cultured in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal calf serum at 37℃ under 5% CO2. Immunohistochemistry and Western blots were performed using antibodies against human LRIG1 (Abcam, Cambridge, UK), EGFR, p-EGFR, or SNAI2 (Cell Signaling, Danvers, MA, USA), MAPK and phospho-MAPK (pMAPK) (Merk Millipore, Billerica, MA, USA), SPHK1 (Abnova, Taipei, Taiwan), or GAPDH or -tubulin (Santa Cruz, CA, USA). ECM proteins including MMP-1, -2, -3, -8, -9, -10, and -13 and their inhibitors TIMP-1, -2, and -4 were measured using an antibody array (RayBio MMP human array 1, RayBiotech, GA, USA) following the manufacturer’s protocol and quantified on an ImageQuant LAS4000 (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
Human tumor tissues and immunohistochemistry
Thetissue microarrays used in this study containing samples of 44 metastatic NPC tissues and two normal oral epithelia have been described previously (Sheu et al., 2009b). Four representative cores from each formalin-fixed paraffin-embedded tumor were arranged on
tissue microarrays and immunohistochemistry was performed using anti-LRIG1 polyclonal antibody (Abcam) at a dilution of 1:100.
Statistical analysis and clinical correlation
Clinical information about the NPC samples analyzed in this study, including tumor site, clinical stage, treatment history, recurrent status, and five year survival, was collected from the clinical notes. The overall survival time was defined as the number of months between diagnosis and death or between diagnosis and the most recent follow-up. All calculations and statistical analyses were carried out using the SAS/STAT software package (SAS Institute) and plotted as survival curves using the Kaplan-Meier method. With this method, P values < 0.05 were considered significant.
Quantitative real-time PCR
The PCR primers were designed by the Universal Probe Library Assay Design Center
(https://www.roche-applied-science.com). Quantitative real time PCR was carried out on an
ABI 7900HT sequence detection system (Applied Biosystems, Carlsbad, CA, USA) using a FastStart Universal Probe Master Kit (Roche Applied Science, Indianapolis, IN, USA). Fold changes in expression of the target genes in the experimental sample relative to that in the control sample were calculated by normalization to ACTB or GAPDH levels.
Transfection and siRNA knockdown
LRIG1 was knocked down in TW06 cells using Dharmacon ON-TARGET plus SMART pool (Dharmacon, Lafayette, CO, USA), with ON-TARGET plus siCONTROL non-targeting pool as the control. The cells were transiently transfected with 100 nM siRNA using Turbofect (Fermentas/Thermo Scientific, Waltham, MA, USA), then the medium was replaced 8 h after transfection and the cells incubated for a further 16-24 h, then were either reseeded for functional assays or lyzed for immunoblotting analysis.
Generation of stable LRIG1-inducible clones
An inducible system was generated in TW01 cells by transfecting cells with the tetracycline-controlled transactivator (tTA) expression vector. Full length LRIG1 cDNA (tagged with V5 at the C-terminus) was cloned into a pBI inducible vector (Clontech, Mountain View, CA, USA), and these expression vectors were subsequently introduced into TW01 cells. For controls, the empty vector was introduced into TW01-tTA cells. Two TW01 transfectants, TW01-LG1/a and TW01-LG1/b, were generated by selection with G418 and hygromycin. Ectopic expression of LRIG1 was controlled by tetracycline-responsive element in the promoter, with the gene being turned on in doxycycline-free medium and turned off in doxycycline-containing medium. Cell clones were validated by Western blot analysis and immunostaining.
Tumor xenografts in nude mice
LRIG1–inducible TW01-LG1/a cells were injected subcutaneously into athymic nu/nu mice (5 x 106 cells per injection, two injection sites per mouse, five mice per group). The LRIG1 turned-off control mice were injected intraperitoneally with doxycycline (125 g/mouse) every day to suppress LRIG1 expression, while the LRIG1 turned-on group was injected with the same volume of phosphate-buffered saline. Tumor volume was measured every 3 days and the tumors were excised and weighed on day 43.
Microarray analysis
Cellular RNA was harvested using an RNeasy kit (Qiagen, MD, USA) according to the manufacturer's instruction. A WT cDNA Synthesis Kit and WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA) were used to generate amplified and labeled sense cDNA. The cDNA was hybridized to the Affymetrix Human Gene 1.0 ST Array according to the standard Affymetrix protocol. The washing and staining steps were carried out using a GeneChip Fluidics station FS 450 and slides were scanned with an Affymetrix Gene Chip scanner 3,000 7G system. Raw data were imported and analyzed using GeneSpring GX (Agilent Technologies, CA, USA) to generate gene lists of differentially expressed (fold change in expression >2). The microarray data have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) and are accessible through GEO with the accession number GSE37349 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?
acc=GSE37349).
Chromatin immunoprecipitation (ChIP) assay
LRIG1-inducible TW01 cells after gene turn on or turn off were fixed by addition of 1% formaldehyde to the culture medium and cells lyzed on ice with RIPA buffer (Sigma) containing protease inhibitors (Roche, Basel, Switzerland). Genomic DNA was fragmented by sonication to sizes ranging from 200 bp to 500 bp, then 1 g of the fragments was incubated overnight at 4oC with gentle rotation with 2 L of anti-phospho-MAKP antibodies (ab50011 from Abcam) and 50 L of protein A/G beads (Sigma), then the protein-DNA complexes on the beads were washed with RIPA buffer before elution with 1% SDS and 50 mM NaHCO3. ChIP-PCR for the promoter regions of candidate genes was performed using the primers shown in Supplementary Table 1.
Cell viability and proliferation assays
Cells (1x 104 cells per well) were seeded overnight in 24-well plates, then placed in medium for the indicated time, then cell numbers were quantified using the MTT assay. Briefly, MTT was added to the cells at a final concentration of 0.5 mg/ml, then the cells were incubated at 37 °C for 3 h, then lysed with DMSO, and the amount of formazan in each well determined colorimetrically at 570 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The data from three independent experiments, each with four replicates, were
averaged.
Morphology assay
Collagen coated dish was prepared for morphology assay as described previously (Chang et al., 2002). Briefly, Tissue culture dishes (BD Biosciences, CA, USA) were coated with 5 g/ml of collagen type I (BD Biosciences) in 0.02 N glacial acetic acid at 4℃ overnight, then aspirated out the coating solution. The coated plate was washed with PBS once before use. TW01-LG1/a cells were seeded at a density of 2 x 106 cells in 10-cm plates. After adhesion, cells were cultured in doxycycline-containing (LRIG1 off) or doxycycline-free medium (LRIG1 on). Phase-contrast pictures were taken every 24 h after doxycycline induction.
Soft agar assay
Cells were resuspended in culture medium containing 0.3% agarose at a density of 1 x 105 cells in 6-well plates, then were overlaid with 0.6% agarose and fed with fresh medium weekly for 3 weeks. After fixation and staining with violet blue, colonies were photographed and quantified using MetaMorph software (Molecular Devices). The experiment was performed three times, each time with 4 replicates.
Cell migration and invasion assays
BD Biosciences); for the cell invasion assay, the chambers were coated with Matrigel (BD Biosciences). Cells were plated at a density of 5 x 104 cells per chamber in a 24-well plate in serum-free DMEM in the top chamber according to the manufacturer’s instructions and DMEM containing 10% FCS was placed in the bottom chamber as the chemoattractant. After 24 h, cells that had migrated into, or invaded, the bottom chambers were stained with crystal violet and counted in four random fields using MetaMorph software (Molecular Devices). Data were averaged for three independent experiments, each with four replicates.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgement
The authors thank Dr. Ming-Chi Hung (MD Anderson Cancer Center, TX) and Dr. Yosef Yarden (Weizmann Institute, Israel) for their critical comments on this study, which was supported by grants from the National Science Council, Taiwan (NSC99-2320-B-400-009, NSC100-2325-B-400-014, and NSC101-2320-B-039-006), the National Health Research Institutes, Taiwan (CA-101-PP-01), the China Medical University Hospital, Taiwan (DMR99-047), and the Department of Health, Taiwan (DOH101-TD-C-111-005).
References
Abhold EL, Kiang A, Rahimy E, Kuo SZ, Wang-Rodriguez J, Lopez JP et al (2012). EGFR kinase promotes acquisition of stem cell-like properties: a potential therapeutic target in head and neck squamous cell carcinoma stem cells. PLoS One 7: e32459.
Burtness B (2005). The role of cetuximab in the treatment of squamous cell cancer of the head and neck. Expert Opin Biol Ther 5: 1085-93.
Chang H, Shyu KG, Lin S, Wang BW, Liu YC, Lee CC (2002). Cell adhesion induces the plasminogen activator inhibitor-1 gene expression through phosphatidylinositol 3-kinase/Akt activation in anchorage dependent cells. Cell Commun Adhes 9: 239-47.
Chang SJ, Wang TY, Lee YH, Tai CJ (2007). Extracellular ATP activates nuclear translocation of ERK1/2 leading to the induction of matrix metalloproteinases expression in human endometrial stromal cells. J Endocrinol 193: 393-404.
Fang CY, Lee CH, Wu CC, Chang YT, Yu SL, Chou SP et al (2009). Recurrent chemical reactivations of EBV promotes genome instability and enhances tumor progression of nasopharyngeal carcinoma cells. Int J Cancer 124: 2016-25.
Gaide Chevronnay HP, Selvais C, Emonard H, Galant C, Marbaix E, Henriet P (2012). Regulation of matrix metalloproteinases activity studied in human endometrium as a paradigm of cyclic tissue breakdown and regeneration. Biochim Biophys Acta 1824: 146-56. Goldoni S, Iozzo RA, Kay P, Campbell S, McQuillan A, Agnew C et al (2007). A soluble ectodomain of LRIG1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity. Oncogene 26: 368-81.
Gur G, Rubin C, Katz M, Amit I, Citri A, Nilsson J et al (2004). LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation. EMBO J 23: 3270-81. Hedman H, Henriksson R (2007). LRIG inhibitors of growth factor signalling - double-edged swords in human cancer? Eur J Cancer 43: 676-82.
Hedman H, Nilsson J, Guo D, Henriksson R (2002). Is LRIG1 a tumour suppressor gene at chromosome 3p14.3? Acta Oncol 41: 352-4.
Huang Z, Desper R, Schaffer AA, Yin Z, Li X, Yao K (2004). Construction of tree models for pathogenesis of nasopharyngeal carcinoma. Genes Chromosomes Cancer 40: 307-15.
Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S et al (2009). Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4: 427-39.
Jensen KB, Jones J, Watt FM (2008). A stem cell gene expression profile of human squamous cell carcinomas. Cancer Lett 272: 23-31.
Jin Y, Jin C, Lv M, Tsao SW, Zhu J, Wennerberg J et al (2005). Karyotypic evolution and tumor progression in head and neck squamous cell carcinomas. Cancer Genet Cytogenet 156: 1-7.
Koy S, Plaschke J, Luksch H, Friedrich K, Kuhlisch E, Eckelt U et al (2008). Microsatellite instability and loss of heterozygosity in squamous cell carcinoma of the head and neck. Head Neck 30: 1105-13.
Krig SR, Frietze S, Simion C, Miller JK, Fry WH, Rafidi H et al (2011). Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERalpha-positive breast cancer. Mol Cancer Res 9: 1406-17.
Laederich MB, Funes-Duran M, Yen L, Ingalla E, Wu X, Carraway KL, 3rd et al (2004). The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases. J Biol Chem 279: 47050-6.
Ledda F, Bieraugel O, Fard SS, Vilar M, Paratcha G (2008). Lrig1 is an endogenous inhibitor of Ret receptor tyrosine kinase activation, downstream signaling, and biological responses to GDNF. J Neurosci 28: 39-49.
Lin CT, Wong CI, Chan WY, Tzung KW, Ho JK, Hsu MM et al (1990). Establishment and characterization of two nasopharyngeal carcinoma cell lines. Lab Invest 62: 713-24.
Lindstrom AK, Ekman K, Stendahl U, Tot T, Henriksson R, Hedman H et al (2008). LRIG1 and squamous epithelial uterine cervical cancer: correlation to prognosis, other tumor markers, sex steroid hormones, and smoking. Int J Gynecol Cancer 18: 312-7.
Co-incidental increase in gene copy number of ERBB2 and LRIG1 in breast cancer. Breast Cancer Res 11: 403.
Lochter A, Galosy S, Muschler J, Freedman N, Werb Z, Bissell MJ (1997). Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype in mammary epithelial cells. J Cell Biol 139: 1861-72.
Ma BB, Poon TC, To KF, Zee B, Mo FK, Chan CM et al (2003). Prognostic significance of tumor angiogenesis, Ki 67, p53 oncoprotein, epidermal growth factor receptor and HER2 receptor protein expression in undifferentiated nasopharyngeal carcinoma--a prospective study. Head Neck 25: 864-72.
Maseki S, Ijichi K, Tanaka H, Fujii M, Hasegawa Y, Ogawa T et al (2012). Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3beta/snail signalling pathway. Br J Cancer 106: 1196-204.
Milara J, Navarro R, Juan G, Peiro T, Serrano A, Ramon M et al (2012). Sphingosine-1-phosphate is increased in patients with idiopathic pulmonary fibrosis and mediates epithelial to mesenchymal transition. Thorax 67: 147-56.
Miller JK, Shattuck DL, Ingalla EQ, Yen L, Borowsky AD, Young LJ et al (2008). Suppression of the negative regulator LRIG1 contributes to ErbB2 overexpression in breast cancer. Cancer Res 68: 8286-94.
Munger JS, Sheppard D (2011). Cross talk among TGF-beta signaling pathways, integrins, and the extracellular matrix. Cold Spring Harb Perspect Biol 3: a005017.
Ordonez-Moran P, Huelsken J (2012). Lrig1: a new master regulator of epithelial stem cells. EMBO J 31: 2064-6.
Pai SI, Westra WH (2009). Molecular pathology of head and neck cancer: implications for diagnosis, prognosis, and treatment. Annu Rev Pathol 4: 49-70.
Pitson SM, Moretti PA, Zebol JR, Lynn HE, Xia P, Vadas MA et al (2003). Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J 22: 5491-500.
pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 149: 146-58.
Pyne NJ, Pyne S (2010). Sphingosine 1-phosphate and cancer. Nat Rev Cancer 10: 489-503. Pyne NJ, Tonelli F, Lim KG, Long JS, Edwards J, Pyne S (2012). Sphingosine 1-phosphate signalling in cancer. Biochem Soc Trans 40: 94-100.
Pyne S, Bittman R, Pyne NJ (2011). Sphingosine kinase inhibitors and cancer: seeking the golden sword of Hercules. Cancer Res 71: 6576-82.
Radisky ES, Radisky DC (2010). Matrix metalloproteinase-induced epithelial-mesenchymal transition in breast cancer. J Mammary Gland Biol Neoplasia 15: 201-12.
Ruan L, Li XH, Wan XX, Yi H, Li C, Li MY et al (2011). Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics. Proteome Sci 9: 35.
Shattuck DL, Miller JK, Laederich M, Funes M, Petersen H, Carraway KL, 3rd et al (2007). LRIG1 is a novel negative regulator of the Met receptor and opposes Met and Her2 synergy. Mol Cell Biol 27: 1934-46.
Sheu JJ, Hua CH, Wan L, Lin YJ, Lai MT, Tseng HC et al (2009a). Functional genomic analysis identified epidermal growth factor receptor activation as the most common genetic event in oral squamous cell carcinoma. Cancer Res 69: 2568-76.
Sheu JJ, Lee CH, Ko JY, Tsao GS, Wu CC, Fang CY et al (2009b). Chromosome 3p12.3-p14.2 and 3q26.2-q26.32 are genomic markers for prognosis of advanced nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev 18: 2709-16.
Shida D, Kitayama J, Yamaguchi H, Yamashita H, Mori K, Watanabe T et al (2004). Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells. FEBS Lett 577: 333-8.
Shida D, Takabe K, Kapitonov D, Milstien S, Spiegel S (2008). Targeting SphK1 as a new strategy against cancer. Curr Drug Targets 9: 662-73.
metalloproteinases is an important step in the epithelial-mesenchymal transformation of the endocardial cushions. Dev Biol 227: 606-17.
Stutz MA, Shattuck DL, Laederich MB, Carraway KL, 3rd, Sweeney C (2008). LRIG1 negatively regulates the oncogenic EGF receptor mutant EGFRvIII. Oncogene 27: 5741-52. Thomasson M, Hedman H, Guo D, Ljungberg B, Henriksson R (2003). LRIG1 and epidermal growth factor receptor in renal cell carcinoma: a quantitative RT--PCR and immunohistochemical analysis. Br J Cancer 89: 1285-9.
Watt FM, Jensen KB (2009). Epidermal stem cell diversity and quiescence. EMBO Mol Med 1: 260-7.
Wong VW, Stange DE, Page ME, Buczacki S, Wabik A, Itami S et al (2012). Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling. Nat Cell Biol 14: 401-8.
Ye F, Gao Q, Xu T, Zeng L, Ou Y, Mao F et al (2009). Upregulation of LRIG1 suppresses malignant glioma cell growth by attenuating EGFR activity. J Neurooncol 94: 183-94.
Yi W, Holmlund C, Nilsson J, Inui S, Lei T, Itami S et al (2011). Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1. Exp Cell Res 317: 504-12.
Figure legends
Figure 1. LRIG1 expression in head and neck tumors and clinical association analysis. (a) Immunohistochemistry for LRIG1 (upper panel) and phospho-EGFR (pEGFR, lower panel) was performed on tissue microarray sections containing nasopharyngeal carcinoma samples and normal nasal epithelium. The bar chart on the right shows the percentage of cells with weak or high staining for pEGFR in the LRIG1 (+) and LRIG1 (-) samples. (b) Overall survival of LRIG1 (+) and LRIG1 (-) NPC patients compared using Kaplan-Meier survival analysis. (c) LRIG1 expression in 28 OSCC tumors and the matched non-tumor tissues (GSE37991).
Figure 2. LRIG1 expression inhibits tumor cell growth in vitro and in vivo. (a) Western blots confirms the regulation of LRIG1 expression by the Tet-off vector system with (D+) or without (D-) doxycycline. NP460 and TW01 parental cells were used as the positive and negative control, respectively. (b) In vitro growth analysis of TW01-LG1/a cells under LRIG1 turned-off and turned-on conditions. Upper panels: Representative images showing the inhibitory effect of LRIG1 expression on NPC cell growth. Lower panels: Cell numbers were assessed by the MTT assay and expressed as a percentage of the numbers at time zero.. The data represent the mean ± SD values for three independent experiments, each with four replicates. (c-d) A tumor xenograft experiment was performed on athymic nu/nu mice by
subcutaneously injecting LRIG1 inducible TW01-LG1/a cells. Induction of LRIG1 expression was performed as described in Materials and methods. Measuring tumor volume with time (c) and tumor weight on day 43 (d). Data indicate mean tumor weight (mean ± SD, n = 6). The right panels of d show representative photographs of tumors taken on day 40.
Figure 3. Transcriptional targets of LRIG1. (a) Hierarchical clustering of differentially expressing genes at 24, 36, and 48 h following LRIG1 turn-on. The level of gene expression at time zero (LRIG1 turned-off) was assigned as the basal level for each gene. Red indicates upregulation and blue downregulation of gene expression. (b) Protein levels of MMPs (upper panel) and TIMPs (lower panel) after LRIG1 induction in TW01-LG1/a cells measured using metalloproteinase antibody arrays. The intensity of each spot was quantified using an ImageQuant LAS4000 and normalized to that of the positive controls (P). Right panels: template for the Matrix Metalloproteinase Antibody Array. “M” indicates MMP, “T” TIMP, “P” positive control, and “N” negative control. LRIG1 non-expressing TW01-LG1/a cells (0 hr) were used as the 100% level in all experiments. (c) ChIP data in LRIG1 turned-on and turned-off cells were validated by ChIP-qPCR. Upon LRIG1 induction in TW01-LG1/a cells, the content of phospho-MAPK decreased, thus reduced the binding capability towards the promoter region of MMP1, MMP3 and MMP10.
Western blot analysis showing that LRIG1 enhances EGFR degradation, but inhibits the phosphorylation of EGFR and MAPK and the expression of SPHK1 and SNAI2. Cells were lysed at the indicated times after turn on of LRIG1 expression and equal amounts of proteins analyzed by Western blotting with antibodies against LRIG1, EGFR, phospho-EGFR (pEGFR), MAPK, phospho-MAPK (pMAPK), SPHK1, or SNAI2, using GAPDH as the loading control. (b) NPC cells develop distinct morphologies when induced LRIG1 expression for 24 hours in collagen coated culture dishes. TW01-LG1/a cells with LRIG1 expression turned on for 24 h (LRIG1 on) or off (LRIG1 off) were grown on collagen coated dishes as described in Materials and methods. Phase-contrast images at 400x magnification. (c) Model outlining the role of LRIG1 in a positive feedback inhibitory loop in the regulation of EGFR-MAPK-SPHK1 signaling and ECM remodeling. Sph, sphingosine; S1P, sphingosine 1-phosphate; S1PR, sphingosine 1-phosphate receptor.
Figure 5. LRIG1 knockdown enhances the oncogenic properties of head and neck cancer cells. (a) Western blot analysis of LRIG1, EGFR, pEGFR, MAPK, pMAPK, SPHK1, and SNAI2 in scrambled control- and siLRIG1-transfected TW06 cells, using tubulin as the loading control. (b-e) Oncogenic properties of U0126 (5M), SK1-I (5M), and SK1-I (10M)-treated LRIG1 siRNA transfected cells, using scrambled siRNA-transfected cells as the control. (b) Twenty-four hours after siRNA transfection, cells were re-seeded at equal
densities and the number of cells 24 or 48 hr later expressed as a percentage of the number of seeded cells. (c) Transforming ability determined by colony formation in soft agar. The number of colonies is expressed as a percentage of the number for cells transfected with scrambled siRNA. (d and e) Migration (d) and invasiveness (e) of LRIG1 knockdown cells evaluated by the Trans-well and Matrigel-coated Trans-well assay, respectively. Upper panels: representative results. Lower panels: Number of cells in the experimental groups that migrated through Trans-well expressed as a percentage of that in the control group. Results are from at least three independent experiments (mean ± SD), each with four replicates.
