Carcinogenesis vol.29 no.4 pp.738–746, 2008
doi:10.1093/carcin/bgn037
Advance Access publication February 14, 2008
GLI1 repression of ERK activity correlates with colony formation and impaired
migration in human epidermal keratinocytes
Graham W.Neill1,, Wesley J.Harrison1, Mohammed
S.Ikram1, Tomos D.L.Williams1, Lucia S.Bianchi1,
Sandeep K.Nadendla1, Judith L.Green1,3, Lucy Ghali1,4,
Anna-Maria Frischauf2, Edel A.O’Toole1, Fritz Aberger2
and Michael P.Philpott1
1
Centre for Cutaneous Research, Institute of Cell and Molecular Science,
Barts and The London School of Medicine and Dentistry, 4 Newark Street,
London E1 2AT, UK and 2Department of Molecular Biology, University of
Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria
3
Present address: Division of Parasitology, MRC National Institute for
Medical Research, London NW7 1AA UK
4
Present address: Department of Biomedical Sciences, School of Health and
Social Sciences, Middlesex University, Enfield EN3 4SA UK
To whom correspondence should be addressed. Tel: þ44 207 882 2343;
Fax: þ44 207 882 7172;
Email: [email protected]
Basal cell carcinoma (BCC) of the skin is a highly compact, nonmetastatic epithelial tumour type that may arise from the aberrant propagation of epidermal or progenitor stem cell (SC)
populations. Increased expression of GLI1 is a common feature
of BCC and is linked to the induction of epidermal SC markers in
immortalized N/Tert-1 keratinocytes. Here, we demonstrate that
GLI1 over-expression is linked to additional SC characteristics
in N/Tert-1 cells including reduced epidermal growth factor receptor (EGFR) expression and compact colony formation that is
associated with repressed extracellular signal-regulated kinase
(ERK) activity. Colony formation and repressed ERK activity
remain evident when EGFR is increased exogenously to the basal
levels in GLI1 cells revealing that ERK is additionally inhibited
downstream of the receptor. Exposure to epidermal growth factor
(EGF) to increase ERK activity and promote migration negates
GLI1 colony formation with cells displaying an elongated, fibroblast-like morphology. However, as determined by Snail messenger RNA and E-cadherin protein expression this is not associated
with epithelial–mesenchymal transition (EMT), and GLI1 actually
represses induction of the EMT marker vimentin in EGF-stimulated cells. Instead, live cell imaging revealed that the elongated
morphology of EGF/GLI1 keratinocytes stems from their being
‘stretched’ due to migrating cells displaying inefficient cell–cell detachment and impaired tail retraction. Taken together, these data
suggest that GLI1 opposes EGFR signalling to maintain the epithelial phenotype. Finally, ERK activity was predominantly negative
in 13/14 BCCs (superficial/nodular), indicating that GLI1 does not
routinely co-operate with ERK to induce the formation of this
common skin tumour.
the formation of BCC, primarily due to loss of function mutations of
the Ptc1 tumour suppressor gene (2–4). The downstream effectors of
SHH signalling include members of the GLI family of transcription
factors, GLI1 and GLI2, that are consistently up-regulated in BCC
compared with normal skin (5,6). However, increased GLI1 expression is not always associated with loss of PTC1 strongly supporting
the hypothesis that GLI1 function is central to BCC formation (7).
Indeed, targeted expression of either GLI1 or GLI2 to the basal layer
of murine epidermis induces BCC even in the presence of wild-type
Ptc1 alleles (8,9).
Tumour formation may arise from the accumulation of specific
mutations in stem cells (SCs) that permit their uncontrolled proliferation and neoplastic transformation (10). HH/GLI signalling is
increasingly implicated in the development of a number of malignancies and this may be linked to its role in regulating SC populations (5).
Whether human BCCs arise from the aberrant proliferation of epidermal/follicular SCs remains to be established but this theory is supported by evidence from transgenic mouse models (11,12).
A number of studies have identified downstream targets of GLI
proteins that may be relevant to tumour formation; these include
platelet-derived growth factor receptor alpha (PDGFRa), FoxM1,
Bcl-2, members of the Wnt/b-catenin family as well as a number of
growth factors and cell cycle proteins (13–19). Studies on the mechanisms regulating GLI proteins have focused mainly on GLI1 and
these are mainly by analogy to components that regulate the Drosophila homologue of GLI, termed Cubitus interruptus (20). However,
HH-related factors regulating GLI activity have been identified in
mammalian cells and include MIM/BEG4, intraflagellar transport
proteins and a mutant form of PTC1 (21–24). With regard to nonHH mechanisms of control, GLI1 activity is subject to regulation by
retinoic acid, Dyrk1 and protein kinase C (PKC) isoforms (25–27),
and recent studies have shown that GLI activity is dependent upon
PKCd/extracellular signal-regulated kinase (ERK), phosphatidylinositol
3-kinase (PI3-K)/protein kinase B (AKT) and Ras-MEK (ERK Kinase)/
AKT signalling (28–30).
We have recently shown that GLI1 induces the expression of various putative epidermal SC markers including K15 and K19 in immortalized N/Tert-1 keratinocytes and that the induction of specific
target genes is regulated by EGF signalling (31). Here, we present
further evidence that GLI1 induces SC characteristics in N/Tert-1
cells including compact colony formation and reduced epidermal
growth factor receptor (EGFR) expression. Colony formation is associated with repressed ERK activity and highlights the presence of
a novel feedback mechanism to modulate GLI1 activity and promote
the epithelial phenotype in human epidermal keratinocytes (HEKs).
Material and methods
Introduction
Basal cell carcinoma (BCC) is the most common malignancy in humans, the incidence of which is increasing globally by up to 10% per
annum and with an estimated lifetime risk of 30% in western society.
Although BCC can be treated by radiotherapy or surgery, its high
prevalence is a cause of significant morbidity (1). Aberrant activation
of the Sonic hedgehog (SHH) signalling pathway is associated with
Abbreviations: BCC, basal cell carcinoma; EGFR, epidermal growth factor
receptor; EMT, epithelial–mesenchymal transition; ERK, extracellular signalregulated kinase; HEK, human epidermal keratinocyte; JNK, c-jun N-terminal
kinase; mRNA, messenger RNA; PBS, phosphate-buffered saline; PDGFRa,
platelet-derived growth factor receptor alpha; qPCR, quantitative polymerase
chain reaction; RT, room temperature; SC, stem cell; SHH, Sonic hedgehog.
Plasmid construction
The coding sequence of EGLI1 was cloned into pEGFP-C3 (Clontech,
Mountain View, CA) to create pEGLI1. This vector used as a template to create
pEGLI1mNLS using the Site-Directed Mutagenesis Kit (Stratagene, La Jolla,
CA). Retroviral vectors were created by subcloning the CMV-EGLI1/
EGLImNLS regions into SIN-IP-EGFP (Enhanced Green Fluorescent Protein)
after removal of the CMV-EGFP region and similar to that described previously (32). The vector encoding EGFR (cloned into pBABE-puro) was created
by Matthew Meyerson (33) and obtained through Addgene (http://www.
addgene.org/11011), and pBP-DN-MEK-EE was kindly provided by David
Beach (Barts and The London School of Medicine and Dentistry).
Cell culture and retroviral production
N/Tert-1 keratinocytes were kindly provided by James Rheinwald (Harvard
Medical School, Boston, MA) and cultured in defined serum-free medium
(Gibco/Invitrogen, Paisley, UK). Retroviral particles were produced as denoted
previously using the Phoenix packaging cell line (32). N/Tert-pBP, N/Tert-EGFR
Ó The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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GLI1 regulation of EGFR/ERK signalling
and N/Tert-DN-MEK-EE cells were created by retroviral transduction of N/
Tert-1 cells with pBABE-puro, pBP-EGFR, pBP-DN-MEK-EE particles, respectively, and selection with puromycin at 1 lg/ml (Sigma–Aldrich, St. Louis, MO).
N/Tert-1-pBP/EGFR/DN-MEK-EE cells were seeded 18 h prior to retroviral
transduction with EGFP, EGLI1 or EGLI1mNLS particles. Post-transduction,
cells were incubated for 24 h in defined serum-free medium-containing commercial growth supplement (þGS), then starved for 8 h (GS) before the
addition of recombinant EGF (10 ng/ml, Sigma–Aldrich), AG1478 (1 lM),
U0126 (10 lM) or SP600125 (10 lM) with untreated cells maintained GS.
All cells were fixed or harvested 48 h post-transduction. Pharmacological
agents were purchased from Merck KGaA (Darmstadt, Germany).
Western blotting
Protein lysates were prepared in boiling Buffer A (50 mM Tris–HCl, pH 8.0,
2% sodium dodecyl sulphate, 1 mM Na3OV4) and quantified using the DC
Protein Assay (Bio-Rad, Laboratories, Hercules, CA). Samples (5–25 lg) were
separated on sodium dodecyl sulphate–polyacrylamide gels and transferred to
Hybond-C nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK)
according to the standard protocols. Primary antibodies used were vinculin
(Sigma–Aldrich), GLI1-C18 and EGFR SC-03 (both Santa Cruz Biotechnology, Santa Cruz, CA), E-cadherin and vimentin (both BD BiosciencesPharmingen, San Diego, CA) and ERK, phospho-ERK, MEK, phospho-MEK,
AKT, phospho-AKT, c-Jun and phospho-c-Jun (all Cell Signalling Technology,
Danvers, MA). Secondary horseradish peroxidase-linked antibodies were obtained commercially (DAKO, Glostrup, Denmark) and immunodetection performed with enhanced chemiluminescence (ECLþ) reagent (GE Healthcare).
Immunofluorescent labelling
After fixing in 4% paraformaldehyde for 10 min at room temperature (RT),
cells (on glass coverslips) were washed 2 phosphate-buffered saline (PBS)
prior to permeabilisation with 0.1% TritonX-100 (in PBS) for 4 min at RT. To
analyse substructures, cells were then washed 1 PBS and blocked in 0.1%
BSA/0.1% sodium azide in PBS (Buffer B) for 20 min prior to incubation for
45 min (RT) with E-cadherin or vinculin antibodies diluted to 2 lg/ml in Buffer
B. After washing 3 2 min in Buffer B, cells were incubated with Alexa Fluor
564 secondary antibody (Molecular Probes, Invitrogen, Paisley, UK) diluted
1:400 in Buffer B for 30 min at RT. Finally, cells were washed 1 in Buffer B,
counterstained with DAPI for 5 min, washed 3 2 min in Buffer B, rinsed in
distilled water and mounted on glass slides with Vectashield (Vector Laboratories, Peterborough, UK). To analyse the actin cytoskeleton, cells were fixed
and permeabilized as described above, washed 1 PBS then exposed to phalloidin–Tetramethyl Rhodamine Iso-Thiocyanate (TRITC) (Sigma–Aldrich) at
5 lg/ml in Buffer B for 45 min, counterstained with 4#-6-Diamidino-2-phenylindole (DAPI) for 5 min, washed 3 2 min in Buffer B, rinsed in distilled
water and mounted on glass slides as described above.
Cell imaging
Phase contrast and EGFP fluorescence was visualized with a Nikon Eclipse
TE2000-S microscope with a T-FL Epi-Fl attachment. Live cell imaging (190
pictures captured every 5 min) was performed with the same microscope using
MetaMorph Offline software (version 6.1). Immunofluorescent labelling was
visualized with a DM5000B microscope and images captured using IM50
software (Leica Microsystems, Wetzlar, Germany).
Quantitative polymerase chain reaction
Total RNA was extracted using Trizol (Invitrogen) with first-strand synthesis
performed using the Reverse Transcription System according to the manufacturer’s instructions (Promega, Madison, WI). Quantitative polymerase chain
reaction (qPCR) was carried out using DyNAmoTM SYBRÒ Green qPCR kit
(Finnzymes, Espoo, Finland) with reactions performed in triplicate. The
primers used were Ptc1-F 5#-CTCCCAAGCAAATGTACGAGCA-3# and
Ptc1-R 5#-TGAGTGGAGTTCTGTGCGACAC-3#, Snail-1-F 5#-ACCCCACATCCTTCTCACTG-3# and Snail-1-R 5#-TACAAAAACCCACGCAGACA3#, Snail-2-F (Slug) 5#-CTTTTTCTTGCCCTCACTGC-3# and Snail-2-R
5#-GCTTCGGAGTGAAGAAATGC-3#, vimentin-F 5#-AAGAGAACTTTGCCGTTGAA-3# and vimentin-R 5#-GTGATGCTGAGAAGTTTCGT-3#,
b-actin-F 5#-GTTTGAGACCTTCAACACCCC-3# and b-actin-R 5#-GTGGCCATCTCTTGCTCGAAGTC-3#. The melting curve graph of the PCR
product indicated that the data generated were from a single product and
confirmed by running on a 1.5% agarose gel. Induction values (v) were calculated using the formula v 5 2DDCt where Ct represents the mean threshold
cycle of replicate analyses, DCt represents the difference between the Ct values
of the target gene and the reference gene b-actin and DDCt is the difference
between the DCt values of the target gene for each sample compared with the
DCt mean of the reference sample (i.e. EGFP cells).
Immunohistochemistry
BCC paraffin sections were analysed immunohistochemically using an Elite
Universal Vectastain ABC kit (Vector laboratories) and liquid diaminobenzidene (BioGenex, San Ramon, CA). Briefly, 4 lm sections were de-paraffinized
and rehydrated using xylene and a graded series of ethanol (100, 96 and 70%)
for 1 min each prior to rinsing in distilled water. Antigen retrieval was performed by immersion in preheated sodium citrate buffer (10 mM sodium
citrate, 0.05% Tween 20, pH 6.0) followed by microwaving for a further 5
min and cooling for 20 min. Sections were incubated with anti-phospho-ERK
antibody (#9106 Cell Signalling Technology) diluted 1:300 in PBS buffer at
37oC for 1 h. After applying biotinylated secondary antibody, endogenous
peroxidase activity was quenched using 0.3% hydrogen peroxide in methanol
for 20 min and the rest of the procedure was carried out as per the manufacturer’s instructions.
Results
GLI1 induces compact colony formation in immortalized
keratinocytes
To clarify the terminology used below, N/Tert-1 keratinocytes retrovirally expressing EGFP will be referred to as EGFP cells and those
expressing an EGFP–GLI1 fusion protein will be referred to as
EGLI1. In contrast to control EGFP cells that cultured as single cells
or loosely associated colonies (Figure 1A, top), EGLI1 induced compact colony formation with many cells displaying a high nuclear to
cytoplasmic ratio (Figure 1A, middle). As the fluorescent signal was
generally diffuse within cells, we sought to determine if the subcellular localization of EGLI1 was relevant to the phenotype by manipulating the fact that GLI1 is subject to nuclear-cytosolic shuttling
(27,34,35). Potential GLI1 nuclear localization motifs were identified
using the PSORT predictive program (http://psort.nibb.ac.jp/); such
motifs are characterized by clusters of basic residues (Figure 1B).
Mutation of the residues arginine and lysine within three putative
motifs produced a mutant EGLI1 protein (EGLI1mNLS—mutant nuclear localization sequence) that was localized exclusively to the cytosol (Figure 1B, bottom) and displays no transcriptional activity (31).
As no difference was observed for the morphology of EGLI1mNLS
cells compared with EGFP cells, this reveals that nuclear localization
of EGLI1 is required to promote colony formation. EGLI1mNLS cells
were also generally more fluorescent than EGLI1 cells and an increase
in protein level was confirmed by western blot analysis (Figure 1C).
GLI1 promotes cell–cell adhesion and impairs keratinocyte migration
To investigate how EGLI1 promotes colony formation, the expression
pattern of E-cadherin was determined to assess the presence of
adherens junctions that mediate cell–cell adhesion. In EGFP cells,
E-cadherin expression was predominantly perinuclear, reflecting the
fact that N/Tert-1 cells do not form compact colonies but are loosely
associated when cultured in this medium. In contrast, E-cadherin was
redistributed to the cell membrane in EGLI1 cells. Thus, enhanced
cell–cell adhesion may account for increased colony formation
(Figure 2A, top). EGLI1 colonies also displayed increased cortical
actin bundling that helps stabilize adherens junctions (Figure 2A,
middle) (36). Evidence that EGLI1 impairs cell migration was
provided by reduced levels of vinculin-containing focal adhesions
(Figure 2A, bottom) and confirmed by live cell imaging (supplementary Movies 1 and 2, available at Carcinogenesis Online). No difference was observed for the level of E-cadherin, b-actin or vinculin
between EGFP and EGLI1 samples (Figure 2B).
GLI1 colony formation is associated with reduced ERK activity
As inhibition of EGFR–ERK signalling impairs cell migration by
inducing cell clustering, a compact morphology and redistribution
of E-cadherin to the cell membrane in HeLa cells (37), EGLI1 colony
formation may be linked with negative regulation of EGFR–ERK
signalling. EGLI1 cells displayed reduced levels of EGFR and ERK
activity as determined by its phosphorylation status. The activity of
the upstream activator of ERK, MEK (ERK kinase/mitogen-activated
protein kinase kinase), was also reduced in EGLI1 cells, suggesting that
reduced ERK activity stems from reduced MEK activity (Figure 3A).
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Fig. 1. The effect of GLI1 on cell morphology is dependent upon its nuclear localization in N/Tert-1 keratinocytes. (A) In contrast to EGFP cells that display
a well spread and migratory phenotype (top panel), GLI1 expressed as an EGFP fusion protein (EGLI1) induces compact colony formation with many cells
displaying a high nuclear/cytoplasmic ratio (denoted by arrows, middle panel). Colony formation is not observed in cells expressing a mutant GLI1 protein
EGLI1mNLS that is localized exclusively to the cytosol (bottom panel). (B) Amino acid sequence of GLI1 denoting three putative Nuclear Localization Signals
(bold font) with the residues that were subject to mutagenesis underlined. (C) Western blot analysis of EGLI1 and EGLI1mNLS protein levels.
To determine if colony formation stems from reduced MEK–ERK
signalling through loss of the upstream cell surface receptor, EGFR
was exogenously expressed in EGLI1 cells. In comparison with
EGLI1 cells, no difference was observed in the morphology of
EGFR/EGLI1 cells with colony formation still evident (Figure 3B,
top). However, despite an increase in EGFR expression comparable
with EGFP cells, the level of ERK activity in EGFR/EGLI1 cells was
still below the basal levels, suggesting that GLI1 represses ERK
downstream of the receptor (Figure 3C).
We and others have recently shown that GLI1 transcriptional activity and target gene identity are regulated by EGFR–ERK signalling
(28,31). Therefore, GLI1 repression of ERK highlights the presence
of a negative feedback loop to modulate GLI1 activity and the potency
of target gene induction in keratinocytes. AKT has also recently been
shown to regulate GLI1 (29) but its activity remained constant between EGFP and EGLI1 cells, revealing the absence of a feedback
loop between GLI1 and AKT (Figure 3A).
Crosstalk between MEK and c-jun N-terminal kinase (JNK) signalling has been shown in neuronal cells and JNK regulates cell adhesion/migration in part through control of the c-Jun transcription factor
(part of the AP-1 complex) (38,39). We were unable to assess JNK
activity by analysis of endogenous phospho-JNK expression but the
activity of its target c-Jun was reduced in EGLI1 cells (Figure 3A).
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Interestingly, the characteristics of keratinocytes isolated from
c-Jun-null mice resemble those observed for EGLI1 cells including
impaired migration, loss of focal adhesion formation and increased
cortical actin bundling. In addition, both c-Jun- and EGFR-null mice
display the ‘Eye-open at Birth’ phenotype and a link between these
pathways has been established as reduced Egfr messenger RNA
(mRNA) and protein expression is observed in c-Jun-null keratinocytes (40). To further delineate between a possible role for MEK–
ERK and JNK–c-Jun in EGLI1 morphology, control EGFP cells were
exposed to the MEK inhibitor U0126 or the JNK inhibitor SP600125.
Both inhibitors induced colony formation (Figure 3B, bottom) and, as
for EGLI1, the expression of phospho-ERK and phospho-c-Jun was
reduced in both EGFP/U0126 and EGFP/SP600125 cells, revealing
potential crosstalk between MEK and JNK signalling (Figure 3D). In
support of this, MEK activity was reduced in EGFP/SP600125 cells
but, as discussed above, it was not possible to determine if JNK
activity was reduced in EGFP/U0126 cells (data not shown). Regarding EGFR, its expression was not reduced in EGFP/U0126 or EGFP/
SP600125 cells (Figure 3D), suggesting that GLI1 repression of
EGFR does not stem from reduced c-Jun activity.
Although GLI1 and murine c-Jun-null keratinocytes share certain
phenotypic similarities as detailed above, colony formation has not
been described for c-Jun-null cells, suggesting that this is not the
GLI1 regulation of EGFR/ERK signalling
Fig. 3. GLI1 colony formation is associated with reduced pathway activation
downstream of EGFR. (A) The expression of EGFR and activity of various
downstream components including MEK, ERK and c-Jun are reduced in
EGLI1 cells, whereas AKT activity remains constant. (B) Exogenous
expression of EGFR does not negate EGLI1 colony formation (top panel).
Colony formation is observed in EGFP cells exposed to the MEK inhibitor
U0126 (10 lM) and the JNK inhibitor SP600125 (10 lM) (bottom panel).
(C) Western blot analysis showing reduced ERK activity in EGLI1 cells
when EGFR levels are maintained exogenously at the basal levels. (D)
Western blot analysis denoting ERK and c-Jun activity and EGFR expression
in EGFP/U0126 and EGFP/SP600125 cells.
Fig. 2. GLI1 promotes cell–cell adhesion and impairs keratinocyte
migration. (A) Immunofluorescent analysis of cellular substructures reveals
that compared with control EGFP cells, EGLI1 induces the formation of
E-cadherin-containing adherens junctions (top panel), promotes cortical actin
bundling (middle panel) and inhibits the formation of vinculin-containing focal
adhesions (bottom panel); bar, 10 lM. (B) Western blot analysis of
E-cadherin, b-actin and vinculin protein expression in EGFP and EGLI1 cells.
mechanism promoting colony formation in GLI1 cells. In contrast,
reduced ERK activity is observed in GLI1 colonies as well as colonies
induced by exposure of control cells to the inhibitors U1026 (MEK) or
SP600125 (JNK), suggesting that low ERK activity is integral to
colony formation, although other pathways may be involved.
GLI1 impairs ERK-mediated migration
As exogenous expression of EGFR was insufficient to activate ERK to
a level comparable with that of control EGFP levels (Figure 3C),
EGLI1 cells were exposed to EGF to stimulate ERK activity and
promote cell migration (41). Although colony formation was negated,
many cells displayed distinct morphological changes characterized
principally by an elongated and fibroblast-like phenotype. In contrast,
there was no discernable difference in the morphology of EGFP cells
upon exposure to EGF (Figure 4A, top). Activation of EGFR mediated
the change in EGLI1 cell morphology as this was negated when cells
were pre-incubated with the EGFR inhibitor, AG1478, prior to EGF
exposure (data not shown). Western blot analysis confirmed that in
EGF/EGLI1 cells, the expression of phospho-ERK was comparable
with, if slightly higher, than that of control EGFP cells (Figure 4B,
lanes 1 and 4). However, this was consistently below that of EGF/
EGFP cells (n 5 5) revealing that EGLI1 attenuates full pathway
activation upon ligand stimulation (Figure 4B, lanes 3 and 4). To
determine if the morphology of EGF/EGLI1 cells was dependent
upon activation of the canonical MEK–ERK pathway, EGLI1 was
co-expressed with a constitutively active MEK mutant, DN-MEKEE. The phenotype of DN-MEK-EE/EGLI1 cells (Figure 4A, bottom)
was similar to EGF/EGLI1 cells (Figure 4A, top), thus confirming that
activation of ERK (Figure 4C, lane 4) induces morphological changes
in EGLI1 cells.
EGF does not co-operate with GLI1 to induce epithelial–
mesenchymal transition
A recent study has shown that in epithelial RK3E cells GLI1 induces
the expression of Snail-1, a zinc finger transcription factor that represses E-cadherin and that is associated with the phenomenon termed
epithelial–mesenchymal transition (EMT) (42). EMT is linked to tumour invasion and metastasis, and in cultured cells can be associated
with transformed epithelial cells adopting a fibroblast-like morphology through activation of receptor tyrosine kinases such as EGFR,
fibroblast growth factor receptor and c-Met (43). Therefore, due to
their morphology and from previous studies, the possibility that EGF
co-operates with EGLI1 to induce characteristics of EMT in keratinocytes was explored.
As documented above, no difference was observed for the expression of E-cadherin between EGFP or EGLI1 cells, suggesting that
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Fig. 4. GLI1 does not co-operate with EGF to induce EMT. (A) Exposure of EGLI1 cells to EGF or co-expression of an active MEK mutant, DN-MEK-EE (right
panels), induces distinct morphological changes that are not observed in EGFP cells (left panels). (B) Western Blot analysis showing increased but attenuated
phospho-ERK expression in EGF/EGLI1 cells (lane 4) compared with EGFP (lane 1) and EGF/EGFP (lane 3) cells, respectively. (C) Western blot analysis
showing increased ERK activity induced by co-expression of DN-MEK-EE in EGFP and EGLI1 cells. (D) Western blot analysis denoting the effect of EGF upon
vimentin and E-cadherin expression in EGFP and EGLI1 keratinocytes (N/Tert-1 and HEK). (E) qPCR analysis showing Ptc1, Snail-1 and Snail-2 mRNA levels in
EGFP and EGLI1 N/Tert-1 cells in the presence and absence of EGF. Each bar represents mean fold induction relative to EGFP (arbitrary value of 1) with ±SD
(n 5 3). (F) qPCR analysis showing vimentin mRNA levels in EGFP and EGLI1 HEKs in the presence and absence of EGF. Each bar represents mean fold
induction relative to EGFP (arbitrary value of 1) with ±SD (n 5 3).
EGLI1 alone does not induce EMT in keratinocytes (Figure 2B).
Furthermore, exposure to EGF did not alter E-cadherin expression
in EGFP or EGLI1 cells such that its expression remained constant
in all four samples (Figure 4D) and, despite inducing high levels of
Ptc1 mRNA, no increase was observed for Snail-1 or Snail-2 mRNA
in EGLI1 or EGF/EGLI1 cells (Figure 4E). The expression of vimentin that is an established marker of EMT was also investigated (43).
Whereas exposure to EGF induced a marked increase of vimentin
expression in EGFP cells (Figure 4D, lanes 1 and 2), only a slight
increase was observed in EGLI1 cells (Figure 4D, lanes 3 and 4). We
also investigated if EGLI1 (±EGF) induces characteristics of EMT in
primary HEKs. As for N/Tert-1 cells, no change was observed for the
expression of E-cadherin in EGFP, EGF/EGFP, EGLI1 or EGF/EGLI1
HEKs (Figure 4D). In contrast to N/Tert-1 cells, vimentin was not
readily detected in HEKs and this probably reflects our previous
observation that N/Tert-1 cells display higher basal levels of EGFR
(31). However, a weak band was detected in EGF/EGFP cells (Figure
4D, lane 2). Further analysis by qPCR revealed that the basal level of
vimentin mRNA was 5-fold lower in EGLI1 compared with control
EGFP primary cells and that, although its expression was induced by
EGF in both cell types, this was less pronounced in EGLI1 (8-fold)
than EGFP primary cells (16-fold) (Figure 4F). Ultimately, the level
of vimentin mRNA was only slightly higher in EGF/EGLI1 cells than
control EGFP cells (Figure 4F, columns 1 and 4), thus accounting for
why vimentin protein was not observed in the former (Figure 4D, lane
4). Therefore, as opposed to promoting EMT, our data suggest that
GLI1 actually inhibits EMT (as denoted by vimentin expression) and
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opposes loss of the epithelial phenotype by repressing EGFR signalling and target gene expression.
GLI1 impairs keratinoycte migration
Having discounted EMT as a mechanism accounting for EGF/EGLI1
cell morphology, the cells were then analysed by live cell imaging.
This revealed that, in contrast to EGFP cells, when stimulated by EGF,
EGLI1 cells adopted an elongated phenotype through two distinct
mechanisms: (i) upon contact two or more migrating cells could not
detach from each other such that they were effectively being
‘stretched’ in different directions and (ii) single cells displayed impaired tail retraction (supplementary Movies 3 and 4, available at
Carcinogenesis Online). Thus, impaired cell–cell detachment and tail
retraction contribute to the morphology of EGF/EGLI1 cells observed
by phase contrast microscopy (Figure 4A, top). As for EGLI1 cells
(Figure 2A, bottom), few vinculin-containing focal adhesions were
observed in EGF/EGLI1 cells. Indeed, in elongated cells with impaired tail retraction, and despite the leading edge being evident, there
was a complete absence of focal adhesion formation (Figure 5A, top).
As there was no difference in the level of vinculin expression between
cell types (Figure 5B), this suggests that GLI1 either directly inhibits
the localization of vinculin at focal adhesions or indirectly through
a general loss of focal adhesion formation. In both EGF/EGFP and
EGF/EGLI1 cells, E-cadherin was predominantly perinuclear but in
the latter expression was also observed at areas of cell–cell contact
(Figure 5A, bottom). Therefore, the inability of migrating EGF/
EGLI1 cells to detach efficiently may be due to impaired disassembly
GLI1 regulation of EGFR/ERK signalling
Fig. 5. GLI1 impairs keratinocyte migration. (A) EGF/EGLI1 cells display
a loss of vinculin-containing focal adhesions (arrows denote the leading
edge) (top panel). E-cadherin-containing adherens junctions (denoted by an
arrow) are still evident in migrating EGF/EGLI1 cells, thus accounting for
the ‘stretched’ phenotype (bottom panel); bar, 10 lM. (B) Western blot
analysis of vinculin and E-cadherin expression in EGF/EGFP and EGF/
EGLI1 cells.
of adherens junctions. E-cadherin expression was constant between
samples (Figure 5B). These data reveal that EGLI1 impairs cell migration induced by EGF. In addition, although reduced ERK activity
correlates with and may help promote EGLI1 colony formation, impaired cell–cell detachment is still evident in the presence of EGF (i.e.
when ERK is activated), suggesting that EGLI1 may promote cell–
cell adhesion via ERK-independent mechanisms. Alternatively, adherens junction assembly/disassembly may be regulated spatiotemporally and depend upon whether GLI1 or EGFR signalling
predominates in individual cells.
ERK activity is rarely observed in BCC
In contrast to loss of PTC1 function or increased expression of SHH,
the most consistent event in BCC is increased expression of GLI1 (7).
To determine if reduced ERK activity is also a feature of BCC, we
analysed 14 tumour samples (3 superficial and 11 nodular/micronodular) by immunohistochemistry. Internal controls for staining efficacy
were provided by the detection of phospho-ERK in the epidermis, hair
follicle, sebaceous glands, sweat glands and/or lymphatics. ERK activity was predominantly negative in the tumour masses of 13/14
samples with only small areas displaying staining that was restricted
to short stretches of palisading cells as well as a few cells within
tumour masses (Figure 6A–D). Intriguingly, in the one nodular sample displaying infiltrative characteristics, phospho-ERK was absent in
the tumour mass but expressed in adjacent infiltrating cells (Figure
6E). Only 1/14 BCC samples (nodular) displayed significant levels of
nuclear phospho-ERK expression that was present throughout one
large tumour island, although expression was absent in multiple, adjacent but smaller tumour islands (Figure 6F).
Discussion
Here, we present data showing that the over-expression of GLI1 in N/
Tert-1 keratinocytes attenuates EGFR–ERK signalling, induces compact colony formation and impairs cell migration upon growth factor
stimulation. GLI1 stabilizes adherens junctions and inhibits the formation of focal adhesions that are required for efficient cell migration.
Indeed, in the presence of EGF, GLI1 cells displayed impaired migration through enhanced cell–cell adhesion, reduced focal adhesions
and impaired tail retraction. The morphologies induced by EGLI1
(±EGF) were also observed in HEKs although the effect on colony
Fig. 6. ERK activity is predominantly absent in human BCC. (A–D)
Analysis of phospho-ERK expression by immunohistochemistry revealed an
overall lack of staining in nodular tumour islands but infrequent expression
was observed in short stretches of peripheral palisading cells (denoted by
arrows). (E) Nuclear phospho-ERK expression was observed in infiltrating
cells (denoted by arrows) emanating from one large nodular tumour but not
in the tumour mass itself. (F) A large area of nuclear phospho-ERK
expression was observed in one nodular tumour but not in the surrounding
smaller tumour islands.
formation was less pronounced as, in contrast to N/Tert-1 cells, primary keratinocytes inherently culture as compact colonies. Colony
formation was not observed upon expression of a mutant protein,
EGLI1mNLS, which is localized exclusively to the cytosol suggesting
that the phenotype is dependent upon GLI1 nuclear localization and
hence transcriptional activity. GLI1 residues 380–420 that encompass
the NLS (Figure 1B) do not encompass the degron sequences recently
shown to regulate GLI1 stability (44). Therefore, the observation that
EGLI1mNLS is more stable than EGLI1 may be accounted for by
recent studies showing that Cubitus interruptus is targeted for nuclear
degradation by the HH target HIB/Rdx in Drosophila and that GLI2
and GLI3 stability is regulated by the murine homologue of HIB/Rdx,
SPOP (45,46).
GLI1 regulation of EGFR signalling
How GLI1 represses EGFR expression remains to be identified but
our data suggest that this is not through reduced c-Jun activity (40).
However, c-Jun regulation of EGFR may be specific to murine keratinocytes or it may actually be independent of its phosphorylation
status at residue serine 63 and involve another region of the protein;
this would account for the loss of EGFR regulation in c-Jun-null cells.
Theoretically, EGFR repression highlights a mechanism whereby
GLI1 attenuates all pathways activated by this receptor and this may
be linked to its role in maintaining SC populations (see subsection
GLI1 and epidermal stem cell populations below). Furthermore, the
fact that GLI1 additionally represses ERK activity downstream of
EGFR highlights a cumulative and potent mechanism by which
EGFR–ERK signalling is negatively regulated. The exact level at
which this occurs has not been determined but MEK activity was
markedly reduced in GLI1 cells and whether this is due to negative
regulation of the classical Ras–Raf–MEK cascade requires further
743
G.W.Neill et al.
investigation. In contrast to our results, Xie et al. (13) have reported
previously that GLI1 induces ERK2 activity in the pluripotent mesenchymal cell line, C3H10T1/2, and that this may be through the upregulation of PDGFRa . Despite high GLI1 levels and potent induction of Ptc1 mRNA in N/Tert-1 cells (Figure 4F), we did not observe
an induction of PDGFRa mRNA by qPCR analysis (G. Neill and
M. Philpott, unpublished data). Moreover, despite high GLI1 levels
and potent induction of mPtc1 mRNA (60-fold) in C3H10T1/2 cells,
neither an increase in the expression of mPDGFRa mRNA nor a discernable increase of phoshpo-ERK expression were observed in cells
cultured in 10 or 0.5% foetal calf serum (G. Neill and M. Philpott,
unpublished data). Therefore, our data reveal the absence of
a PDGFRa–ERK signalling axis and further studies may be required
to address this anomaly.
GLI1 and epidermal SC populations
Characteristics of epidermal SC populations include a compact morphology, high nuclear to cytoplasmic ratios and reduced motility
(47–49). In addition, reduced EGFR expression and strong cell–substrate
adhesion have been documented as defining SC parameters and recently a novel marker, Lrig1, was identified and shown to maintain SC
quiescence by repressing EGFR expression and downstream signalling including ERK activity (50,51). As well as repressing EGFR
expression, GLI1 also reduces the proliferation of N/Tert-1 keratinocytes and this may reflect a state akin to SC quiescence. Moreover,
GLI1 colonies do not readily dissociate from culture dishes upon
trypsinisation, revealing that GLI1 also enhances cell–substrate adhesion (G. Neill, unpublished observations).
Recently, we have shown that GLI induces the expression of a number of epidermal SC markers and that this is negated in the presence of
EGF (31). The fact that GLI1 represses EGFR expression may provide
a mechanism by which HH signalling maintains SC identity by dampening pathway activation. To exit the SC niche, EGFR signalling must
be activated by either increased expression of EGFR and/or exposure
to the appropriate ligand. Accordingly, we have shown that a combination of HH/GLI and EGFR–MEK–ERK signalling may control the
fate of hair follicle outer root sheath cells that have exited the SC
niche (31). Likewise, in the murine brain, SHH controls SC niches and
co-operates with EGF to regulate precursor proliferation and lineage
commitment (52,53). Studies have also shown that HH positively
regulates EGFR signalling; for example, Egfr mRNA expression
was completely repressed in embryonic mouse cortex cells exposed
to the HH inhibitor cyclopamine (53) and SHH was recently shown to
activate EGFR in HaCat cells to enhance matrix invasion (54). However, whether these phenomena were dependent upon GLI expression
is unclear.
GLI1 and EGFR signalling in neoplasia
Cancer SCs i.e. SCs that have lost their normal proliferative control
mechanisms are considered to play an important role in tumour propagation (55). The fact that HH/GLI signalling is implicated in the
maintenance of SC pools from various tissues suggests that this pathway may contribute to the cancer SC phenotype (10) and is supported
by recent studies showing strong pathway activation in malignant
mammary and glioma SCs (56,57).
As discussed, reduced EGFR–ERK signalling is characteristic of
epidermal SCs (50,51). Regarding BCC, Nazmi et al. (58) observed
reduced or absent EGFR expression in all 22 BCC samples examined
when compared with normal epidermis, and Krahn et al. (59) observed weak or absent EGFR in 10/16 BCC samples. More recently,
small reductions in Egfr mRNA and protein levels were described in
BCC versus normal skin (60,61). As it is suggested that BCC be
derived from a population of pluripotent hair follicle SCs (11,12), this
may reflect the source of BCC in that tumours with reduced EGFR
represent those of a SC origin, whereas those with higher levels represent progenitor cells that have exited the SC niche. Paradoxically,
however, the level of EGFR is not a good determinant of downstream
signalling potency as low expression may stem from ubiquitin-
744
mediated receptor degradation as a result of ligand-induced pathway
activation and, reciprocally, higher expression may indicate a lack of
pathway activation (62). This important point was highlighted in a recent study of squamous cell carcinomas (63). Similarly, despite comparable protein levels, Rittie et al. (61) observed that the activity of
EGFR was .2-fold higher in squamous cell carcinoma compared
with BCC and normal skin. As such, we analysed ERK activity in
a panel of human BCC samples and found that it was predominantly
absent in 13/14 patient samples; this supports the results of Rittie et al.
(61) who have recently shown that ERK (and AKT) activity is absent
in BCC. As BCCs consistently display high levels of GLI1 expression,
this reveals that the development or progression of these tumours is
unlikely to arise from the co-operation of GLI1 and ERK activities.
Indeed, our in vitro data suggest that the absence of ERK activity in
BCC may be due to increased GLI1 activity and this may also account
for why these tumours manifest as slow-growing, highly compact
epithelial cell masses that are rarely metastatic. Interestingly, in one
sample strong nuclear phospho-ERK expression was observed in infiltrating tumour cells derived from a nodular tumour mass suggesting
that ERK activation may promote the transformation towards a more
aggressive BCC subtype (Figure 6E). Moreover, as phospho-ERK was
observed in short stretches of palisading cells (Figure 6C and D), this
may highlight cells that are susceptible to becoming infiltrative, but
whether conversion to an infiltrative phenotype is co-dependent upon
GLI and ERK warrants further investigation, especially considering
recent evidence showing that active mutants of Ras, MEK and AKT
induce the nuclear accumulation of GLI1 in cancer cell lines (30).
Although we have discussed that GLI1 regulation of EGFR-ERK
signalling may represent a mechanism by which the HH pathway
maintains epidermal SCs and that BCCs may represent their aberrant
proliferation, an alternative explanation may be that loss of ERK
activity represents an oncogene-induced stress response (activated
by high GLI levels) to limit tumour growth and invasion. This was
recently highlighted in human embryonic fibroblasts that responded to
the presence of oncogenic Raf by increasing the expression of several
genes that suppressed pathway activation (64), and whether or not this
mode of ‘tumour suppression’ accounts for reduced ERK activity in
BCC may be addressed by global array analysis.
In comparison with BCC, other tumours associated with HH signalling are generally more aggressive including those of the prostate,
breast, lung and brain (5,6). Such tissues may be more susceptible
to oncogenic transformation as indicated by the ‘one-step’ immortalization of primary prostate epithelial cells expressing GLI1 or c-Myc
(65,66). In addition, these tumours often display enhanced EGFR
signalling through mutational activation or gene amplification, the
latter being highlighted in a study of high-grade gliomas (67,68).
Finally, we found no evidence that GLI (±EGF) induces EMT in
keratinocytes as judged by E-cadherin, Snail and vimentin expression,
with the latter providing further evidence that GLI1 attenuates EGFR
signalling to maintain the epithelial phenotype. This is in contrast
with a recent study in RK3E cells where GLI1 has been shown to
repress E-cadherin through the induction of Snail (42); these differences may reflect differing GLI1 expression levels as well as the fact
that RK3E cells represent a rat kidney cell line immortalized with the
adenoviral E1A protein that inhibits various cell cycle regulators (69).
However, although Li et al. (42) state that GLI1 repression of
E-cadherin in RK3E cells (as well as in infiltrating keratinocytes of
an inducible GLI mouse model) correlates with the loss of E-cadherin
reported in human infiltrative BCC (70), this does not correlate with
the fact that E-cadherin is expressed in superficial and nodular BCC
(70,71), subtypes that are known to express high levels of GLI1 and
GLI2 (7,32,72). Indeed, currently there is no distinct molecular or
genetic basis for infiltrative BCC or whether enhanced invasion is
dependent upon GLI1. Analysis of N/Tert-1 cells co-expressing
GLI1 and E1A may provide a clue as to how keratinocytes are transformed and whether this represents a good model to study BCC formation and progression.
In summary, we show that GLI1 induces an epithelial, compact,
non-migratory phenotype in HEKs that is associated with reduced
GLI1 regulation of EGFR/ERK signalling
ERK activity. This recapitulates several features of human BCC and
thus provides a good model to further investigate the mechanisms that
contribute to GLI1-mediated keratinocyte transformation including
those that regulate the cell cycle.
Supplementary material
Supplementary Movies 1–4 can be found at http://carcin.
oxfordjournals.org/
Funding
The Medical Research Council; St Bartholomew’s and The Royal
London Charitable Foundation.
Acknowledgements
We thank David Kelsell for critical reading of the manuscript and members of
the Centre for Cutaneous Research for their support and helpful discussions.
We also thank Charlotte Proby (Centre for Cutaneous Research) and Rino
Cerio (Barts and The London) for BCC subclassification, and John Priestley,
Gary Warnes, Fozia Chaudry and Samantha Matthew (Institute of Cell and
Molecular Science, Queen Mary, University of London) for their technical
expertise.
Conflict of Interest Statement: None declared.
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Received August 24, 2007; revised December 19, 2007;
accepted January 26, 2008