1
MAP4K3/GLK promotes lung cancer metastasis by phosphorylating and
activating IQGAP1
Huai-Chia Chuang1, Chih-Chi Chang
1, Chiao-Fang Teng
1, Chia-Hsin Hsueh
1, Li-Li
Chiu1, Pu-Ming Hsu
1, Ming-Ching Lee
2, Chung-Ping Hsu
2, Yi-Rong Chen
3,
Yi-Chung Liu4, Ping-Chiang Lyu
5, and Tse-Hua Tan
1,6
1Immunology Research Center, National Health Research Institutes, Zhunan 35053,
Taiwan.
2Division of Thoracic Surgery, Department of Surgery, Taichung Veterans General
Hospital, Taichung, 40705, Taiwan.
3Institute of Molecular and Genomic Medicine, National Health Research Institutes,
Zhunan 35053, Taiwan.
4Institute of Population Sciences, National Health Research Institutes, Zhunan 35053,
Taiwan.
5Institute of Bioinformatics and Structural Biology, National Tsing-Hua University,
Hsinchu 30013, Taiwan.
6Department of Pathology & Immunology, Baylor College of Medicine, Houston,
Texas 77030, USA.
Correspondence should be addressed to Tse-Hua Tan, email: [email protected], Tel:
+866-37-206-166 ext. 37601
The authors have declared that no conflict of interest exists.
Running title: MAP4K3/GLK promotes lung cancer metastasis via IQGAP1
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ABSTRACT
Overexpression of the serine/threonine kinase GLK/MAP4K3 in human lung cancer
is associated with poor prognosis and recurrence, however, the role of GLK in cancer
recurrence remains unclear. Here, we report that transgenic GLK promotes tumor
metastasis and cell migration through the scaffold protein IQ motif-containing
GTPase-activating protein 1(IQGAP1). GLK transgenic mice displayed enhanced
distant metastasis. IQGAP1 was identified as a GLK interacting protein; two
proline-rich regions of GLK and the WW domain of IQGAP1 mediated this
interaction. GLK and IQGAP1 co-localized at the leading edge including filopodia
and lamellipodia of migrating cells. GLK directly phosphorylated IQGAP1 at Ser-480
enhancing Cdc42 activation and subsequent cell migration. GLK-induced cell
migration and lung cancer metastasis were abolished by IQGAP1 depletion.
Consistently, human NSCLC patient tissues displayed increased phospho-IQGAP1
which correlated with poor survival. Collectively, GLK promotes lung cancer
metastasis by binding to, phosphorylating, and activating IQGAP1.
Statement of Significance: Findings show the critical role of the GLK-IQGAP
cascade in cell migration and tumor metastasis, suggesting it as a potential biomarker
and therapeutic target for lung cancer recurrence.
Keywords: GLK/MAP4K3, IQGAP1, Cell migration, Lung cancer metastasis, Cdc42
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INTRODUCTION
More than 90% of human cancer death is associated with tumor metastasis (1,2).
Cancer cell migration contributes to tumor metastasis (3). Understanding the
fundamental mechanisms of cancer cell migration should help the development of
novel therapeutic approaches for treating cancer metastasis.
IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein
that promotes multiple aspects of cell migration (4). For example, IQGAP1 weakens
cell-cell adhesion, induces cytoskeletal rearrangement, and degrades extracellular
matrix (5-7). Upon phosphorylation by PKC-ε at Ser-1443, IQGAP1 undergoes a
conformational change and becomes activated (8). The Rho family GTPases Cdc42
and Rac1 directly interact with IQGAP1 and localize to the leading edge of migrating
cells, leading to actin meshwork formation and cell migration (9). The regulation of
cell migration by the IQGAP1/Cdc42/Rac1 system suggests that this protein complex
is involved in tumor progression (10). Further studying the molecular mechanisms
involved in the control of IQGAP1 activity shall provide important insights into the
regulation of cancer cell migration and metastasis.
The serine/threonine protein kinase GLK (also named MAP4K3) is a member
of the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family (11).
As upstream regulators of the MAP kinase cascades, GLK and other MAP4Ks
activate c-Jun N-terminal kinase (JNK) in response to environmental stress and
proinflammatory cytokines in cultured cell lines (12-17). One MAP4K family kinase,
HGK (MAP4K4), is a critical regulator of cell migration, cancer invasion, and cell
adhesion (18-22). HPK1 (MAP4K1) regulates cell apoptosis, cell growth, and
cytokine production through binding to multiple adaptor proteins including members
of the Grb2 family, Nck family, Crk family, and SLP-76 family (23). GLK regulates
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mTOR signaling, cell growth, apoptosis, and autophagy (24-27). GLK also
upregulates NF-κB signaling by activating PKC-θ in T cells, leading to T-cell
activation (28,29). GLK signaling in T cells specifically enhances IL-17A
transcription by inducing the AhR-RORγt complex (30,31). The increase of GLK
protein levels in T cell is correlated with the disease severity of several human
autoimmune diseases (28,31-33). Moreover, GLK protein levels are increased in
tissues of human lung cancer and hepatoma (34,35); GLK overexpression in the
cancer tissue is correlated with cancer recurrence and poor recurrence-free survival
rates (34,35). GLK overexpression in cancer cell lines may be due to the
downregulation of microRNA let-7c and microRNA-199a-5p, which target the
3’-untranslated region of GLK (36,37). However, the role of GLK in cancer
recurrence remains unclear. Here we report a novel GLK-targeted protein, IQGAP1,
which mediates GLK-induced cell migration and lung cancer metastasis.
METHODS
Plasmids and reagents.
The plasmids expressing 3xFlag-tagged or HA-tagged human GLK cDNA (NCBI
accession number: NM_003618) were generated by subcloning the cDNA insert from
the Flag-tagged clone into the vector pCMV6-AN-3DDK or pCMV6-AC-HA
(OriGene,). The plasmid expressing Myc-tagged human IQGAP1 was purchased from
Addgene (#30118). The plasmid expressing GLK kinase-dead (K45E) mutant was
generated using the PCR-based site-directed mutagenesis from the 3xFlag-tagged
GLK clone, by mutating Lys-45 to glutamic acid at the ATP-binding domain of this
kinase, as described (12,38). The plasmids expressing GLK (P436/437A), GLK
(P478/479A), IQGAP1 (∆WW), IQGAP1 (S480A), IQGAP1 (S480D), and IQGAP1
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(S480E) were generated by mutating the indicated residue on the 3xFlag-tagged GLK
and Myc-tagged IQGAP1plasmids. The plasmids expressing CFP-fused GLK and
monomeric GFP (mGFP)-fused GLK were generated by subcloning the GLK cDNA
insert from the HA-tagged GLK plasmid into the vector pCMV6-AC-mCFP and
pCMV6-AN-mGFP (OriGene), respectively. The plasmids expressing YFP-fused
IQGAP1 and Tomato-fused IQGAP1 were generated by subcloning the IQGAP1
cDNA insert from the Myc-tagged IQGAP1 plasmid into the vector pCMV6-AC-YFP
(OriGene) and ptd-Tomato-C1 (Clontech), respectively. The GLK or IQGAP1shRNA
expression plasmids were obtained from the National RNAi Core Facility (Academia
Sinica, Taiwan). For pervanadate treatment, pervanadate was freshly prepared by
mixing H2O2 and Na3VO4 as described (39), and cells were then incubated with a
final concentration of 25 μM pervanadate for 1 h at 37°C. G-LISA® Activation Assay
Biochem kits for Cdc42 or Rac1 were purchased from Cytoskeleton, Inc. Anti-Flag
agarose beads (M2) and anti-Myc agarose beads (9E10) were purchased from Sigma.
The primary antibodies used in this study were anti-IQGAP1 (BD Biosciences),
anti-HA, anti--Actin (Sigma) and anti--Tubulin (Sigma). Both homemade
anti-GLK antibody (α-GLK-N) (30) and homemade anti-GLK monoclonal antibody
(mAb clone C3) (30) were used in this study.
Human subjects
We collected primary lung tumor specimens from seven non-small cell lung cancer
(NSCLC) patients who underwent first pulmonary resection in the Division of
Thoracic Surgery at Taichung Veterans General Hospital, Taiwan. Every patient
provided written informed consent approved by the hospital’s Institutional Review
Board (approval number: CF13082). All experiments were performed in accordance
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with the guidelines and protocols approved by the Institutional Review Board,
Taichung Veterans General Hospital, Taiwan. Tumor types and stages of individual
specimens were determined according to the American Joint Committee on Cancer
Staging Manual. All specimens, including tumor tissues and paired normal adjacent
tissues of NSCLC patients, were taken at the time of surgical resection. Portions of
samples were freshly fixed with formaldehyde and then embedded with paraffin.
Follow-up data were collected from chart reviews and confirmed by thoracic
surgeons.
Human lung cancer and normal adjacent tissue array slides (#CC5, #CCA4, and
#CCN5) were purchased from SUPER BIO CHIPS. The company provided certified
documents that all human lung-tissue samples were collected with patients’ informed
consents. The pulmonary tissue array contained 68 normal adjacent tissues and 109
tumor tissues (including small cell carcinoma, NSCLC, mucoepidermoid carcinoma,
and carcinosarcoma).
We analyzed the GLK-IQGAP1 complex in 177 pulmonary samples from tissue
arrays and in 7 pulmonary resection samples from NSCLC patients. We analyzed
IQGAP1 phosphorylation in 6 pulmonary resection samples from NSCLC patients
Cell lines and transfection.
The murine lung cancer (luciferase-expressing Lewis lung carcinoma, LLC/luc;
PerkinElmer # BW119267), human lung cancer (HCC827, H661, H1299; ATCC), and
human normal lung (NL20; ATCC) cell lines were cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100
mg/ml streptomycin (Invitrogen). The human embryonic kidney cell line HEK293T
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was maintained in DMEM medium containing 10% FBS, 100 units/ml penicillin, and
100 mg/ml streptomycin. Cell lines at passages greater than ten were not used for the
experiments in this study. All cells were free of mycoplasma contamination and
grown at 37°C in a humidified atmosphere of 5% CO2 in air. Plasmids were
transfected into cells using polyethylenimine reagents. All cell lines used were tested
and confirmed to be negative for mycoplasma.
Generation of polⅡ-GLK transgenic mice and IQGAP1 knockout mice.
PolⅡ-GLK Tg mice and PolⅡ-GLKE351K
Tg mice in C57BL/6 background were
generated using pronuclear microinjection by NHRI Transgenic Mouse Core. A
full-length human GLK coding region (wild-type or E351K mutant) was placed
downstream of the RNA polymerase II (PolⅡ) promoter (40) (Figure 1a). IQGAP1
knockout mice in C57BL/6 background were generated using embryo microinjection
of TALEN mRNA by NHRI Transgenic Mouse Core. The nucleotide (nt) 161 guanine
of the IQGAP1 exon 1 was deleted in the mutated allele. All animal experiments were
performed in the AAALAC-accredited animal housing facilities at National Health
Research Institutes (NHRI). All mice were used according to the protocols and
guidelines approved by the Institutional Animal Care and Use Committee of NHRI.
Purification of primary lung epithelial cells.
The mice were sacrificed by CO2 asphyxiation. The lung from the chest was excised
and cut into small fragments, followed by placing the lung fragments into the
dispase-containing culture dish at room temperature. After 10 min, lung fragments
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were homogenized by gentleMACS Dissociator (Miltenyi Biotec) to obtain single-cell
suspension. Mouse lung epithelial cells were negatively selected by MACS®
Separation. The cells were incubated with biotin-conjugated antibodies (anti-CD45,
anti-CD11b, anti-CD11c, anti-CD16/32, anti-CD19, anti-F4/80, and anti-TER-119) at
4°C for 15 min. Ten μl of Streptavidin MicroBeads were added into cell suspension at
4°C for 15 min. Cell suspension was applied onto the column, and then the effluent
fraction containing epithelial cells was collected.
Immunohistochemistry.
Tissue sections were deparafinized, and then treated for antigen retrieval by
incubating the slides in boiling buffer (pH 6.0) at 85°C for 10 min. Nonspecific
binding was sequentially blocked with 3% H2O2 for 10 min and Immunoblock-Ultra
V block for 5 min. Tissue sections were incubated with anti-proliferating cell nuclear
antigen (PCNA) (1:200; GeneTex) or anti-EGFRdel
antibodies (1:200; Cell Signaling)
at 4°C overnight, and then incubated with horseradish peroxidase (HRP)-conjugated
secondary antibodies. The protein signals were detected using the HRP substrate
3,3'diaminobenzidine (DAB) (Ultravision Quanto Detection System; Thermo,
TL-060-QHL). For negative controls, primary antibodies were replaced with 2%
normal serum. Tissue sections were also counterstained with Mayer’s hematoxylin.
Time-lapse super-resolution live cell imaging.
For monitoring subcellular localization of GLK-mGFP and IQGAP1-Tomato in
migrating cells, 2×104 cells were seeded into 8-chamber slides 24 h after transfection.
After a further 24 h of incubation, cells were traced using Nikon’s Structured
Illumination Microscope (N-SIM) performed on an Eclipse Ti inverted microscope
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equipped with a Plan Apo x60 water immersed objective and time-lapse live-cell
imaging systems (Nikon). Motile transfected (mGFP- and Tomato-positive) cells
were followed in time-lapse recording for 10 h at an interval of 10 min. The images
were acquired and analyzed with the NIS Elements software (Nikon).
Liquid chromatography-mass spectrometry.
The mass spectrometry was performed as previously described (41,42). Briefly,
protein samples were separated by SDS-PAGE and stained with silver. Specific
protein bands were excised, destained and digested with trypsin. The resulting peptide
mixtures were analyzed by loading on the nanoAcquity system (Waters) connected to
an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific) equipped
with a nanospray interface (Proxeon).
In situ proximity ligation assay (PLA) technology.
Cells seeded on sterile cover slides were co-transfected with Flag-tagged GLK and
Myc-tagged IQGAP1 expression plasmids, followed by fixation, permeabilization,
and blocking. In situ PLA assays were performed using the Duolink In Situ-Red kit
(Sigma) according to the manufacturer’s instructions. Briefly, cells were incubated
with anti-Flag and anti-Myc antibodies, followed by species-specific secondary
antibodies conjugated with oligonucleotides (PLA probes). After ligation and
amplification reactions, the signal from each pair of PLA probes in close proximity (<
40 nm; GLK-IQGAP1 interaction) was visualized as an individual red spot and
analyzed by Leica DM2500 upright fluorescence microscope. For cell line
experiments, at least 5 different fields were randomly selected, and the number of red
spots per cell was counted. Each experiment was repeated at least three times.
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For experiments using human pulmonary tissues, tissue sections were
deparafinized, antigen retrieved, and nonspecific-binding blocked, followed by in situ
PLA assays using first antibodies for IQGAP1 (1:4,000, CUSABIO) plus either GLK
(1:3,000, mAb clone C3) or phospho-IQGAP1 Ser-480 (1:2,000, Allbio Science). The
monoclonal antibody for phosphorylated IQGAP1 Ser-480 was generated by
immunization of a mouse with phospho-peptides (human IQGAP1 epitope:
473NTVWKQL[pS] SSVTGLT
487). The tissue sections were then incubated with
species-specific secondary antibodies conjugated with oligonucleotides (PLA probes),
followed by ligation and amplification reactions. The number of PLA signals per
tissue (3.14 mm2) was counted.
Statistical analysis.
All experiments were repeated at least three times. The associations between
metastasis and GLK transgene were evaluated using the Fisher’s exact test. To
evaluate normality of each column data, Kolmogorov-Smirnov and Shapiro-Wilk tests
were performed. The statistical significance between two unpaired groups was
analyzed using the two-tailed Student’s t-test (for normally distributed data) or using
the two-tailed Mann-Whitney U-test (for non-normally distributed data). Cluster
analyses (hierarchical clustering and subsequent k-means clustering) were used to
divide patients into subgroups. Kaplan-Meier survival analyses were performed to
show the difference in the survival between subgroups (e.g., PLA signal-High versus
PLA signal-Low). The log-rank test was used to calculate the significance of the
survival distributions between two groups. Data were calculated using SPSS 19
software. A P value of<0.05 was considered statistically significant (*, P value<
0.05; **, P value<0.01; ***, P value<0.001). All statistical analyses of clinical data
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were further independently verified by two biostatisticians at Institute of Population
Sciences of National Health Research Institutes.
RESULTS
GLK induces distant metastasis of lung cancer
In order to study the role of GLK in cancer progression, we generated the whole-body
GLK transgenic (PolⅡ-GLK Tg) mice using RNA polymeraseⅡ(PolⅡ)
promoter-driven human GLK cDNA (Figure 1a). GLK overexpression was confirmed
by real-time PCR (Figure 1b). Because GLK overexpression is correlated with cancer
recurrence of human non-small cell lung cancer (NSCLC) and hepatoma (34,35), we
characterized whether PolⅡ-GLK Tg mice spontaneously develop lung cancer or
liver cancer using immunohistochemistry analysis. The data showed that 1-year-old
PolⅡ-GLK Tg mice did not develop any lung cancer or liver cancer (Supplementary
Fig. S1a). To study the effect of GLK on cancer progression, PolⅡ-GLK Tg mice
were bred with a genetically modified lung cancer mouse line, the lung-specific
pulmonary surfactant protein A (SPA) promoter-driven EGFR-deletion mutant
transgenic (SPA-EGFRdel
Tg) mouse line (43). Consistent with the published results
(43), all 1-year-old SPA-EGFRdel
Tg mice (9/9) indeed developed lung cancer (PCNA
positive) (44) (Figure 1c), and so did SPA-EGFRdel
;PolⅡ-GLK Tg mice (15/15)
(Figure 1c). Immunohistochemistry analysis using anti-EGFR-deletion mutant
antibodies showed that EGFRdel
-expressing cells are detected in the lung cancer of
both SPA-EGFRdel
Tg mice and SPA-EGFRdel
;PolⅡ-GLK Tg mice (Figure 1d). We
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next studied whether GLK transgene induces lung cancer (EGFRdel
-positive)
metastasis to other organs in SPA-EGFRdel
;PolⅡ-GLK Tg mice. We performed
immunohistochemistry using anti-EGFR-deletion mutant antibodies on the tissues of
the cervical lymph nodes, the liver, and the brain from wild-type and three different
transgenic mice. For regional metastasis to cervical lymph nodes, all but one (14/15)
SPA-EGFRdel
;PolⅡ-GLK Tg mice displayed numerous metastatic
EGFRdel
-expressing lung cancer cells in cervical lymph nodes. In contrast, only three
of nine SPA-EGFRdel
Tg mice showed a few metastatic EGFRdel
-expressing lung
cancer cells in cervical lymph nodes (Figure 1e and Supplementary Fig. S1b). For
distant metastasis, all SPA-EGFRdel
;PolⅡ-GLK Tg mice displayed metastasis of
EGFRdel
-expressing lung cancer cells to the brain (14/15) or liver (15/15) (Figure 1e).
In the 9 control SPA-EGFRdel
Tg mice, only one SPA-EGFRdel
Tg mouse (1/9)
developed both brain metastasis and liver metastasis, only one (1/9) developed liver
metastasis, and remaining 7 mice did not develop any detectable distant metastasis
(Figure 1e). These results suggest that GLK induces distant metastasis of lung cancer
to the brain and liver.
GLK promotes migration of lung epithelial cells
We next studied the underlying mechanism of GLK-induced lung cancer metastasis.
Knockdown of microRNA let-7c or microRNA-199a-5p promotes cell migration of
cancer cell lines and also upregulates GLK levels (36,37), suggesting that GLK may
induce lung cancer metastasis by promoting cancer cell migration. We first evaluated
whether GLK indeed promotes lung cell migration by transwell migration assays. We
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used GLK overexpression and shRNA knockdown approaches to study the role of
GLK in regulating migration of two human lung cancer cell lines. HCC827 lung
cancer cells expressed lower levels, while H661 lung cancer cells expressed higher
levels, of endogenous GLK proteins than those of the normal lung cell line NL20
(Supplementary Fig. S2a). The HCC827 and H661 cells were transfected with a
GLK-expressing plasmid and GLK-specific shRNA constructs, respectively. The
migrated cell number was increased by GLK overexpression (Supplementary Fig. S2b)
but attenuated by GLK shRNA knockdown (Supplementary Fig. S2c).
Overexpression or knockdown of GLK was confirmed by immunoblotting analysis
(Supplementary Fig. S2d, S2e). MTT assays showed that neither overexpressing nor
downregulating GLK affected cell proliferation during the incubation time period (16
hours) of cell migration assays (Supplementary Fig. S2f, S2g), excluding the
possibility that the observed phenotypes were due to changes in cell proliferation. The
involvement of GLK in cell migration was further evaluated using primary lung
epithelial cells from previously generated GLK-deficient mice (28) and newly
generated GLK transgenic (PolⅡ-GLK) mice. The expected GLK protein levels in
GLK-deficient and transgenic mice were confirmed by immunoblotting analysis
(Supplementary Fig. S3a). The primary lung epithelial cells were isolated from mice
and subjected to transwell migration assays. The number of primary lung epithelial
cells that migrated through transwells in a uniform field was increased for cells from
GLK transgenic mice (Supplementary Fig. S3b, S3c), whereas the migrated cell
number was decreased for cells from GLK-deficient mice, compared to that of
wild-type mice (Supplementary Fig. S3b, S3c).
The regulation of cell migration by GLK was further verified by examining cell
motility of primary lung epithelial cells by time-lapse live cell imaging using Nikon’s
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Structured Illumination Microscopy. The migrating cells were defined by their
displacement lengths that are more than 15 μm during a 3-hour period. The lung
epithelial cells isolated from GLK transgenic mice showed a marked increase in the
percentage of migrating cells compared to that of wild-type mice (Supplementary Fig.
S3d, S3e; Movie S1). Conversely, GLK-deficient lung epithelial cells showed a
decrease in the percentage of migrating cells compared to that of wild-type mice
(Supplementary Fig. S3d, S3e; Movie S1). Moreover, the average migrating length of
GLK transgenic lung epithelial cells was significantly increased, whereas that of
GLK-deficient lung epithelial cells was significantly decreased (Supplementary Fig.
S3f). These results indicate that GLK plays an important role in regulating cell
migration.
GLK interacts directly with IQGAP1
To identify the GLK-targeted molecule involved in GLK-regulated cell migration, we
characterized GLK-interacting proteins in anti-Flag immunocomplexes isolated from
Flag-GLK-transfected HEK293T cells. Following immunoprecipitation of GLK, the
GLK-interacting proteins were resolved by SDS-PAGE and visualized by silver
staining (Figure 2a). The seven most prominent protein bands enhanced in
GLK-transfected cells were sliced and then digested by trypsin, and the resulting
protein peptides were subjected to mass spectrometry analysis. Moreover, four
pervanadate-induced tyrosine phosphorylation sites (Tyr-366, Tyr-379, Tyr-574, and
Tyr-735) on GLK proteins were identified by mass spectrometry analysis (Figure 2a,
right panel). We identified several putative GLK-interacting proteins, including
myosin, IQGAP1 (Figure 2b), vimentin, drebrin, and heat shock protein 70 (HSP70)
(ordered by database search scores from highest to lowest). Among these proteins,
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IQGAP1, a positive regulator of cell migration, was selected for further study,
whereas myosins, heat-shock proteins, and cytoskeletal proteins are common
contaminant proteins detectable by mass spectrometry. Next, we confirmed the
interaction between GLK and IQGAP1 using reciprocal co-immunoprecipitation
assays (Figure 2c, 2d). GLK was co-immunoprecipitated with Flag-tagged IQGAP1
proteins with an anti-Flag antibody (Figure 2c, 2d). This co-immunoprecipitation
between GLK and IQGAP1 was abolished by GLK (Y735F) mutation (Figure 2e),
suggesting that Tyr-735 phosphorylation of GLK protein is important for the
interaction between GLK with IQGAP1. In situ proximity ligation assay (PLA) with a
combination of PLA probes corresponding to Flag (Flag-tagged GLK) and Myc
(Myc-tagged IQGAP1) showed strong PLA signals in cells overexpressing both
proteins than those overexpressing each alone (Figure 2f, 2g). The PLA signals
suggest a direct interaction (< 40 nm) between GLK and IQGAP1. Moreover, the
fluorescence resonance energy transfer (FRET) assay using CFP-tagged GLK and
YFP-tagged IQGAP1 fusion proteins showed a direct interaction (1-10 nm) between
these two molecules (Figure 2h). To further confirm the direct interaction,
co-immunoprecipitation experiments were performed using purified proteins.
Flag-tagged GLK and Myc-tagged IQGAP1 proteins from HEK293T cell lysates
were purified by eluting immunocomplexes with Flag and Myc peptides, respectively.
The co-immunoprecipitation assays showed an interaction between purified GLK and
IQGAP1 proteins (Figure 2i). The data from three different approaches (PLA, FRET,
and purified proteins) suggest that GLK interacts directly with IQGAP1.
GLK promotes cell migration through IQGAP1
To demonstrate the role of IQGAP1 in GLK-induced cell migration, we studied
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whether IQGAP1 knockout attenuates GLK-promoted cell migration of primary lung
epithelial cells. We first generated IQGAP1 knockout mice using TALEN technology
(Figure 3a); IQGAP1 knockout was characterized by immunoblotting analyses
(Figure 3b). IQGAP1 knockout mice were then bred with GLK transgenic mice. The
primary lung epithelial cells were isolated from the offspring or the parental GLK
transgenic (PolⅡ-GLK) mice and subjected to transwell migration assays. The
migrated cell number of primary lung epithelial cells from GLK transgenic mice was
drastically decreased by IQGAP1 homozygote knockout (Figure 3c, 3d), and was
modestly decreased by IQGAP1 heterozygote knockout (Figure 3c, 3d). The
migration dynamics of primary lung epithelial cells from the offspring were further
examined by time-lapse live cell imaging using Nikon’s Structured Illumination
Microscopy. The percentage of migrating epithelial cells of GLK transgenic mice was
increased compared to that of wild-type mice (Figure 3e, 3f). The GLK-induced
migration of GLK transgenic epithelial cell was significantly reduced by IQGAP1
homozygote knockout (Figure 3e, 3f; Movie S2), and modestly reduced by IQGAP1
heterozygote knockout (Figure 3e, 3f; Movie S2). Moreover, the migration lengths of
primary lung epithelial cells were increased by GLK transgene compared to those of
wild-type cells (Figure 3g). Whereas the GLK-induced migration lengths were
decreased by IQGAP1 knockout (Figure 3g). Next, we studied whether similar results
can be obtained using the HCC827 lung cancer cell line (Supplementary Fig. S4). The
HCC827 cells were transfected with GLK plasmid alone or together with each of two
different IQGAP1 shRNA constructs. As compared to control cells, the migration
ability of HCC827 cells was enhanced by GLK transfection alone but reduced by
IQGAP1 knockdown with individual IQGAP1 shRNAs even in the presence of GLK
overexpression (Supplementary Fig. S4a). GLK overexpression and IQGAP1
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17
knockdown were confirmed by immunoblotting analysis (Supplementary Fig. S4b).
No major difference was seen in cell growth between each group during the time
period of cell migration assays (Supplementary Fig. 4c). Overall, these data suggest
that GLK promotes cell migration through IQGAP1.
GLK co-localizes with IQGAP1 predominantly at the leading edge of migrating
cells
We then investigated how GLK promotes IQGAP1-mediated cell migration. First, we
studied whether GLK co-localizes with IQGAP1 in migrating cells. The GLK-mGFP
and IQGAP1-Tomato proteins were co-expressed in HCC827 lung cancer cells and
monitored by time-lapse live cell imaging using either confocal microscopy (Figure
4a) or super-resolution N-SIM microscopy (Figure 4b-d). Data showed that GLK was
localized diffusely throughout the cell, including the plasma membrane (Figure 4a).
The IQGAP1 distribution pattern was similar to that of GLK (Figure 4a). Notably,
super-resolution N-SIM imaging showed that GLK and IQGAP1 were co-localized
mainly at the cell membrane (Figure 4b). During cell migration, the cell formed
lamellipodia and spike-like filopodia at the migratory front of the cell (45). The
super-resolution N-SIM microscopy showed a striking co-localization of GLK and
IQGAP1 predominantly at filopodia and lamellipodia of the cell during the extension
step prior to cell body movement. (Figure 4c and Movie S3). Moreover, time-lapse
super-resolution live cell imaging also showed co-localization of GLK and IQGAP1
predominantly at the leading edge of the migrating cells (Figure 4d, Movie S4, S5).
These data indicate that GLK and IQGAP1 co-localize predominantly at the leading
edge and may cooperate to promote cell migration.
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GLK proline-rich regions mediate the binding to the IQGAP1 WW domain
WW domains of proteins recognize proline-rich motifs and trigger downstream
signaling pathways (46-48). GLK and other MAP4K members contain an N-terminal
kinase domain, a C-terminal Citron homology (CNH) domain, and several
proline-rich motifs in the middle (11,12). MAP4K1 (also named HPK1) interacts with
its interacting proteins via the proline-rich motifs (23). In addition, IQGAP1 binds to
the MAP kinase ERK2 via its WW domain (49), which preferentially recognizes
ligands containing proline-rich sequence (47). We thus asked whether GLK and
IQGAP1 interact with each other through the proline-rich domain and the WW
domain, respectively.
We tested whether IQGAP1 interacts with one or both of the two potential WW
domain-recognized proline-rich regions of GLK. We generated three GLK mutants
(P436/437A, P478/479A, and P436/437A;P478/479A) by substitution of the
Pro-436/437 and/or Pro-478/479 residues to alanine within the two proline-rich
regions (Supplementary Fig. S5a-d). The IQGAP1 WW domain mutant (∆WW) was
also generated (Supplementary Fig. S5a-d). Different pairs of these mutants and their
wild-type constructs were then co-transfected into HEK293T cells. Overexpression of
wild-type or mutant GLK and IQGAP1 proteins were confirmed by immunoblotting
analysis (Supplementary Fig. S5d). In situ proximity ligation assay (PLA) assay with
a combination of PLA probes corresponding to Flag (Flag-tagged GLK) and Myc
(Myc-tagged IQGAP1) showed strong PLA signals in cells overexpressing both
proteins. The PLA signals were reduced by overexpression of either GLK
(P436/437A) or GLK (P478/479A) mutant (Supplementary Fig. S5b and c). Moreover,
the interaction between GLK and IQGAP1 was completely abolished by double
mutations (P436/437A;P478/479A) of both GLK proline-rich regions (Supplementary
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Fig. S5b and c). In addition, the PLA signals were reduced by overexpression of
IQGAP1 (∆WW) mutant (Supplementary Fig. S5b and c). Collectively, these data
indicate that Pro-478/479 and Pro-436/437 regions of GLK mediate its binding to the
WW domain of IQGAP1.
GLK promotes cell migration by phosphorylating IQGAP1 at Ser-480
Because GLK directly binds to IQGAP1, we speculated that GLK may be a kinase
that regulates IQGAP1-mediated cell migration. To determine whether GLK
phosphorylates IQGAP1, in vitro kinase assay was conducted using purified proteins
of GLK, GLK kinase-dead (K45E) mutant, and IQGAP1. IQGAP1 phosphorylation
was induced by GLK but not GLK kinase-dead (K45E) mutant (Figure 5a). Following
SDS-PAGE fractionation and mass spectrometry analysis, Ser-480 was identified as
the GLK-phosphorylated residue on IQGAP1 (Figure 5b). Next, we tested whether
the GLK-induced IQGAP1 Ser-480 phosphorylation regulates the activation of Cdc42
or Rac1, as well as the interaction of IQGAP1 with Cdc42 or Rac1.
Immunoprecipitation data showed that active (GTP-binding) Cdc42 proteins were
increased in GLK plus IQGAP1-overexpressing cells; conversely, active Cdc42
protein levels were attenuated by overexpression of GLK plus IQGAP1 (S480A)
mutant (Figure 5c, lower panel). In contrast, active (GTP-binding) Rac1 protein levels
were not increased by GLK plus IQGAP1 overexpression (Figure 5d, lower panel).
These results were further supported by ELISA results of Cdc42 and Rac1 activation
(Figure 5e). In addition, co-immunoprecipitation data showed that the interaction of
IQGAP1 with either Cdc42 or Rac1 was not affected by the IQGAP1 (S480A)
mutation (upper panel of Figure 5c and 5d). To evaluate the role of IQGAP1 Ser-480
phosphorylation in IQGAP1-mediated cell migration, HCC827 cells were
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20
co-transfected with IQGAP1 (S480A) phosphorylation-defective mutant and GLK
plasmids. The transwell migration assays showed that the migrated cell number of
GLK-overexpressing cell was decreased by overexpression of IQGAP1 (S480A)
mutant (Figure 5f, top-right panel; Figure 5g and 5h). Conversely, overexpression of
two IQGAP1 Ser-480 phosphomimetic (S480D and S480E) mutants induced a higher
cell migration ability than that of overexpression of IQGAP1 or IQGAP1 (S480A)
mutant in HCC827 lung cancer cells (Supplementary Fig. S6a-c). These results
suggest that IQGAP1 Ser-480 phosphorylation is responsible for IQGAP1 activation
and IQGAP1/Cdc42-mediated cell migration.
Our results suggest that GLK interacts with and phosphorylates IQGAP1. We
next studied the interaction between GLK proline regions and IQGAP1 WW domain
indeed controls the GLK-IQGAP1-induced cell migration. HCC827 lung cancer cells
co-transfected with GLK and IQGAP1 displayed enhancement of migration than that
of vector control cells, whereas co-transfection of GLK plus IQGAP1 (∆WW) mutant
abrogated the enhanced cell migration (Figure 5f, 5g). Overexpression of GLK
(P436/437A), (P478/479A), or (P436/437A;P478/479A) mutant attenuated
GLK-induced cell migration (Figure 5f, 5g). Overexpression of GLK and IQGAP1
was confirmed by immunoblotting analysis (Figure 5h).
IQGAP1 mediates GLK-induced cancer metastasis
We next studied whether GLK promotes metastasis of lung cancer through
IQGAP1-mediated cancer cell migration. To shorten the time (12 months) required
for the development of lung cancer metastasis in SPA-EGFRdel
;PolⅡ-GLK Tg mice,
we generated a GLK mutant transgenic mouse line (PolⅡ-GLKE351K
Tg), which
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21
expressed a constitutively activated GLK (E351K) mutant (Figure 6a-c). Notably, the
GLK (E351K) mutation was reported in the supplementary information of a previous
publication (27); however, the functional consequence of GLK (E351K) mutation has
not been demonstrated until this study (Figure 6a, 6b). Overexpression of GLK
(E351K) mutant was confirmed by real-time PCR (Figure 6d). Next, we bred PolⅡ
-GLKE351K
Tg mice with SPA-EGFRdel
Tg mice to generate SPA-EGFRdel
;PolⅡ
-GLKE351K
Tg mice, which displayed enhanced GLK protein levels in the lung
compared to those of SPA-EGFRdel
Tg mice (Figure 6e). SPA-EGFRdel
;PolⅡ
-GLKE351K
Tg mice (8/8) indeed developed lung cancer (Figure 6f) and
regional/distant metastasis at a younger age (7-month-old) than that of
SPA-EGFRdel
;PolⅡ-GLK Tg mice. All 7-month-old SPA-EGFRdel
;PolⅡ-GLKE351K
Tg mice displayed distant metastasis of EGFRdel
-expressing lung cancer cells to the
brain and/or liver (both brain and liver [6/8], brain only [1/8], and liver only [1/8]
(Figure 6g). In contrast, all SPA-EGFRdel
;PolⅡ-GLKE351K
;IQGAP1-/-
mice did not
develop any distant metastasis (3/3) at 7-month-old age (Figure 6g). These data
suggest that GLK induces distant metastasis through IQGAP1 in SPA-EGFRdel
lung
cancer model. To verify this notion, we studied the interaction between GLK and
IQGAP1, as well as IQGAP1 Ser-480 phosphorylation in tissues of human non-small
cell lung cancer (NSCLC).
GLK-IQGAP1 complex is correlated with poor survival of human NSCLC
To study the interaction of GLK with IQGAP1 in NSCLC tissues, we collected
clinical lung tissues from 7 human NSCLC patients who underwent pulmonary
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22
resection. We also employed a commercially available pulmonary tissue array
containing 85 NSCLC tissues (including 49 squamous cell carcinoma, 17
adenocarcinoma, 11 bronchioloalveolar carcinoma, and 8 large cell carcinoma) and
68 normal adjacent tissues, as well as 3 small cell lung carcinoma tissues. These
tissues were subjected to in situ proximity ligation assay (PLA) with a combination of
paired PLA probes corresponding to GLK and IQGAP1. The data showed multiple
strong PLA signals in most (81/92) of NSCLC tissues but not in any small cell
carcinoma tissues (Figure 7a and 7b). Most (61/68) of normal adjacent tissues from
NSCLC patients did not display any PLA signals, while 7 of 68 normal adjacent
tissues showed much less PLA signals. The few PLA signals in the normal adjacent
tissues of NSCLC patients may be metastatic cancer cells migrating from original
lesion. These results suggest that the GLK-IQGAP1 complex in pulmonary tissue
may be a diagnostic biomarker for NSCLC. Next, we studied whether the
GLK-IQGAP1 complex could act as a potential prognostic biomarker for NSCLCs.
NSCLC (squamous cell carcinoma and adenocarcinoma) patients, whose survival data
were available, were divided into four PLA-signal subgroups after cluster analyses.
The two subgroups with highest PLA signals contained only one and two patients,
respectively, thus, these two subgroups were excluded from further analysis. The
remaining two subgroups (n = 63) were subjected to Kaplan-Meier survival analysis
using the survival data (available from the provider). NSCLC patients with high PLA
signals showed poor survival during follow-up periods (n = 63, PLA signal-High
versus PLA signal-Low, P = 0.069) (Figure 7c). The higher P value (P = 0.069) may
be due to the exclusion of three data with highest PLA signals. Nevertheless, NSCLC
patients with more GLK-IQGAP1 complexes have a lower survival rate than that of
the patients with less GLK-IQGAP1 complexes. Because 40% to 60% of NSCLC
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23
patients die of cancer recurrence after cancer resection (50), we studied whether the
GLK-IQGAP1 complex is associated with NSCLC metastasis. The cancer cells with
the GLK-IQGAP1 complex particularly accumulated on/near the vascular wall in the
lung (Figure 7d); GLK-IQGAP1 complex-positive cells also existed in lumen of the
blood vessel (Figure 7d). Moreover, the bone, lymph node, or soft tissue section with
metastatic carcinoma displayed GLK-IQGAP1 complex-positive cells (Figure 7d), the
cancer cells was verified using (proliferating cell nuclear antigen) PCNA staining
(Figure 7d). Merged images show that the GLK-IQGAP1 complex-positive cells in
these tissues were indeed cancer cells (Figure 7d). The data suggest that lung cancer
cells with the GLK-IQGAP1 complex tend to be metastatic. Next, we examined the
GLK-induced IQGAP1 Ser-480 phosphorylation in human NSCLC tissues. After
several failed attempts, we finally obtained a monoclonal antibody (mAb) against
IQGAP1 Ser-480 phosphorylation. However, the immunostaining signal using
phospho-IQGAP1 mAb was not strong enough to provide a discernible signal. To
enhance the specificity and staining signal of anti-phospho-IQGAP1 mAb, we
performed PLA that amplifies phosphorylation signals (51) like a polymerase chain
reaction with a combination of paired PLA probes corresponding to IQGAP1 and
phospho-IQGAP1 Ser-480. The antibody specificity was demonstrated using IQGAP1
S480A mutant-expressing cells and IQGAP1-knockout lung cancer tissues
(SPA-EGFRdel
;PolⅡ-GLKE351K
;IQGAP1-/-
) (Supplementary Fig. S7a and b). Using
human NSCLC tissues, we found multiple PLA signals of IQGAP1 Ser-480
phosphorylation in 82.7% (72/87) of tumor tissues tested (Figure 7e and f). The
phospho-IQGAP1 PLA signals coexisted with PCNA staining in the same cells
(Figure 7e). Moreover, NSCLC (squamous cell carcinoma and adenocarcinoma)
patients were divided into PLA signal-High and PLA signal-Low subgroups after
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24
cluster analyses. Kaplan-Meier survival analysis showed that NSCLC patients with
high phospho-IQGAP1 PLA signals had poor survival during follow-up periods (n =
63, PLA signal-High versus PLA signal-Low, P = 0.037) (Figure 7g). Collectively,
our findings suggest that GLK promotes cell migration and cancer metastasis by
direct binding to and phosphorylating IQGAP1 (Graphic Abstract).
DISCUSSION
Cell migration plays a critical role in cancer progression and cancer metastasis.
Identification of signaling molecules that regulate cell migration should help
development of novel therapeutic approaches for cancer metastasis. GLK
overexpression is correlated with cancer recurrence of human lung cancer or
hepatoma (34,35). Here we report that GLK is a key kinase controlling
IQGAP1-mediated cell migration and cancer metastasis. Our data showed that cell
migration of primary lung epithelial cells was enhanced by GLK transgene but
inhibited by GLK deficiency or IQGAP1 knockout. Moreover, distant metastasis of
the lung cancer mouse model was significantly enhanced by GLK transgene, whereas
GLK-induced distant metastasis of lung cancer was abolished by IQGAP1 knockout.
Most importantly, the GLK-IQGAP1 complex was induced in tumor tissues of
NSCLC patients, and the number of the protein complex was correlated with poor
survival of NSCLC patients. These findings suggest that the GLK-IQGAP1 pathway
is a therapeutic target for cancer metastasis or cancer recurrence.
A key finding of this study is that GLK induces IQGAP1-mediated cell
migration by directly phosphorylating IQGAP1 at Ser-480 residue. It has been
proposed that IQGAP1 activity may be regulated by autoinhibition through a
C-terminal intramolecular interaction; PKC-inducedphosphorylation of IQGAP1 at
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25
Ser-1443 inhibits its intramolecular interaction to facilitate IQGAP1 function in
cytoskeletal regulation (8). Whereas GLK phosphorylated IQGAP1 at Ser-480 located
in the N-terminal region. A previous report indicates that IQGAP1 promotes cell
motility by activating Rac1 and Cdc42 (52). The GLK-induced S480 phosphorylation
of IQGAP1 promoted Cdc42 activation and cell migration without affecting the
interaction of IQGAP1 with Cdc42. Notably, the GLK-induced IQGAP1 Ser-480
phosphorylation did not regulate the activation of Rac1, which is responsible for
directional/persistent migration instead of Cdc42-regulated random migration (53).
Consistent with the selective regulation of Cdc42 by the GLK-IQGAP1 pathway,
GLK-promoted cell migration of primary lung epithelial cells was non-directional cell
migration. Furthermore, IQGAP1 Ser-480 phosphorylation was indeed detectable in
tumor tissues of human NSCLC patients. These results indicate that IQGAP1 activity
can be regulated by multiple phosphorylation events. Thus, IQGAP1 may interact
with different sets of effector proteins in response to activation by distinct
phosphorylation sites of IQGAP1.
Another important finding of this study is the direct binding of GLK to IQGAP1.
Our data showed that two proline regions (Pro-436/437 and Pro-478/479) of GLK and
the WW domain of IQGAP1 were required for the interaction between GLK and
IQGAP1. Consistently, the transwell cell migration assays showed that
overexpression of either GLK (P436/437;478/479A) mutant or IQGAP1 (∆WW)
mutant reduced the cell migration of lung cancer cells (Figure 5f and g). Interestingly,
using primary epithelial cells, we found that IQGAP1 KO abolished GLK
transgene-induced cell migration in transwell migration assays (Figure 3c and d);
however, co-transfection of GLK (P436/437;478/479A) and IQGAP1 (∆WW) did not
efficiently abolish lung cancer cell migration. This phenomenon may be due to the
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26
homodimer formation (25,54) between exogenous mutant GLK proteins, and
endogenous wild-type GLK proteins or between exogenous mutant IQGAP1 proteins
and endogenous wild-type IQGAP1 proteins. WW domains are divided into four
groups (Group I to Group IV) with different binding preferences for proline-rich
motifs (55): the Group I binds Pro-Pro-X-Tyr motifs (where X is any amino acid); the
Group II binds Pro-Pro-Leu-Pro motifs; the Group III binds polyproline motifs
flanked by Arg or Lys; and the Group IV binds phospho-Ser/Thr-Pro containing
motifs. Due to the ability of binding to each other’s cognate ligands, the Group II and
III WW domains have been redefined as one single Group II/III WW domain (56).
Our data showed that GLK binding to the WW domain of IQGAP1 was mediated by
two proline-rich regions Pro-436/437 (432
PPPLPP437
) and Pro-478/479 (477
RPPPPR482
)
of GLK, which match to the recognition sequence of the Group II and Group III WW
domains, respectively. This finding indicates that IQGAP1 contains the Group II/III
WW domain.
Our results of N-SIM time-lapse live cell imaging showed that IQGAP1
displayed a polarized distribution in migratory lung cancer cells, and that GLK was
highly co-localized with IQGAP1 at filopodia and lamellipodia of the polarized
membrane during cell migration. The data support that GLK cooperates with IQGAP1
to promote cell migration. Previous reports indicate that IQGAP1 localizes at the
leading lamellipodia of migrating cells and promotes cell motility by activating Rac1
and Cdc42 (52). Our results showed that GLK-induced IQGAP1 Ser-480
phosphorylation enhances Cdc42 activation and subsequent cell migration. Cancer
cell migration contributes to cancer progression and cancer metastasis. Consistently,
the extent of interaction between GLK and IQGAP1 was correlated with poor survival
of NSCLC patients. The lung cancer cells containing the GLK-IQGAP1 complex
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27
accumulated around tumor blood vessel, suggesting their tendency to extravasation
into the tumor blood vessel. The distal metastatic carcinoma tissues also showed
GLK-IQGAP1 complex-containing cells. Collectively, GLK may bind to and activate
IQGAP1 at the leading edge of migrating cells, leading to Cdc42-mediated cell
migration and cancer metastasis.
In conclusion, GLK plays a crucial role in promoting cell migration and cancer
metastasis by directly binding to and phosphorylating IQGAP1. These findings
suggest that the GLK-IQGAP1 complex is a potential therapeutic target for cancer
recurrence.
ACKNOWLEDGEMENTS
We thank members of Tan Lab for technical assistance, including Ms. Ching-Yi Tsai
for IHC staining, PLA assay, and G-LISA activation assay, as well as Ms. Ting-Shuan
Kung for mouse breeding and IHC staining. We thank the Transgenic Mouse Core
(NHRI, Taiwan) for generation of transgenic and knockout mice. We thank the Core
Facilities of National Health Research Institutes (NHRI, Taiwan) for tissue
sectioning/H&E staining, confocal microscopy, live cell imaging, and
super-resolution N-SIM microscopy. We thank the Laboratory Animal Center
(AAALAC accredited) of NHRI for mouse housing. We thank Institute of Biological
Chemistry of Academia Sinica for mass spectrometry. We also thank Dr. Shao-Chun
Hsu and the Imaging Core Facility of the Institute of Cellular and Organismic Biology,
Academia Sinica for using the software Imaris (Version 9.1.2). This work was
supported by grants from the National Health Research Institutes, Taiwan
(IM-105-PP-01 and IM-105-SP-01, to T.-H.T.) and Ministry of Science and
Technology, Taiwan (MOST-106-2321-B-400-013 to T.-H.T.). T.-H.T. is a Taiwan
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28
Bio-Development Foundation (TBF) Chair in Biotechnology.
COMPETING FINANTIAL INTERESTS
The authors declare no competing financial interests.
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33
FIGURE LEGENDS
Figure 1. GLK induces distant metastasis of lung cancer.
(a) Schematic diagram of the PolⅡ-GLK transgenic construct. In GLK transgenic
mice, human GLK cDNA was driven by the mouse RNA polymerase II (PolⅡ)
promoter. (b) Real-time PCR analyses of transgenic human GLK (hGLK) mRNA
levels in murine peripheral blood cells from mice. The human GLK mRNA levels
were normalized to mouse Srp72 mRNA levels. WT, n = 6; PolⅡ-GLK, n = 12.
Means ± SEM are shown. WT, wild-type littermate controls; PolⅡ-GLK, PolⅡ
-GLK transgenic mice. (c) Representative immunohistochemistry of a lung cancer
maker proliferating cell nuclear antigen (PCNA) or H&E staining in lung tissues from
8-month-old wild-type (WT), SPA-EGFRdel
transgenic, or SPA-EGFRdel
;PolⅡ-GLK
transgenic mice. Scale bar, 100 μm. (d and e) Representative immunohistochemistry
of EGFR-deletion mutant expression or H&E staining in the lung (d), cervical lymph
nodes (e), brain (e), or liver (e) from 1-year-old wild-type (WT), SPA-EGFRdel
transgenic, PolⅡ-GLK transgenic, and SPA-EGFRdel
;PolⅡ-GLK transgenic mice.
LN, cervical lymph nodes. Scale bar, 100 μm. Comparison of EGFR-deletion mutant
expression in indicated tissues from individual groups (lower panel). § denotes that
only three of nine SPA-EGFRdel
Tg mice showed a few metastatic
EGFRdel
-expressing lung cancer cells in cervical lymph nodes.
Figure 2. GLK interacts directly with IQGAP1.
(a) Silver-stained gel of anti-Flag immunoprecipitates from HEK293T cells
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34
transfected with empty vector or Flag-tagged GLK in the presence or absence of the
tyrosine phosphatase inhibitor pervanadate (25 μM). Arrows indicate the positions of
GLK or GLK-interacting proteins. Four pervanadate-induced tyrosine
phosphorylation residues of GLK proteins were listed at right panel. (b) Identification
of IQGAP1 by mass spectrometric sequencing of proteins from the silver-stained gel.
Sequence coverage, 28.79%. (c and d) Co-immunoprecipitation of anti-Flag (c) or
anti-Myc (d) immunocomplexes from lysates of HEK293T cells transfected with
Flag-tagged GLK, Myc-tagged IQGAP1, or both plasmids. The whole-cell lysate
immunoblots before immunoprecipitation (Pre-IP) are shown at the bottom of each
panel. β-Tubulin was used as the loading control. (e) Co-immunoprecipitation of
anti-Flag immunocomplexes from lysates of HEK293T cells transfected with either
Flag-tagged GLK or Flag-tagged GLK mutant (Y366F, Y379F, Y574F, or Y735F).
HEK293T cells were treated with 25 μM pervanadate for 2 h. (f) In situ PLA assays
of HEK293T cells transfected with empty vector, 3xFlag-tagged GLK, Myc-tagged
IQGAP1, or GLK together with IQGAP1 plasmids. Arrows indicate the PLA signals
(red spots). For PLA, each red dot represents for a direct interaction. Original
magnification, x40. Scale bar, 10 μm. (g) The relative PLA signal of each group per
field is shown on the plot. (h) FRET assays of HEK293T cells transfected with the
indicated plasmids encoding CFP- and YFP-fused proteins. (i)
Co-immunoprecipitation (Co-IP) assays of purified Flag-tagged GLK and
Myc-tagged IQGAP1 proteins. Flag-tagged GLK and Myc-tagged IQGAP1 proteins
from HEK293T cell lysates were eluted with Flag and Myc peptides, respectively.
Data shown (a-i) are representative results of three independent experiments. **, P
value<0.01; ***, P value<0.001.
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35
Figure 3. GLK promotes cell migration through IQGAP1.
(a) Schematic diagram of the mouse IQGAP1 wild-type allele and the targeted
IQGAP1 mutant allele. IQGAP1 knockout mice were generated by TALEN-mediated
gene targeting. The deletion of one bp on exon 1 results in a 57 amino acid (a.a.)
frame-shift mutant. (b) Immunoblotting of IQGAP1 and β-Tubulin proteins from cells
of wild-type (WT), IQGAP1+/-
heterozygote, and IQGAP1-/-
homozygote mice. (c)
Transwell migration assays of lung epithelial cells from wild-type (WT), GLK
transgenic (PolⅡ-GLK), GLK transgenic/IQGAP1-heterozygous (PolⅡ
-GLK;IQGAP1+/-
), and GLK transgenic/IQGAP1 knockout (PolⅡ-GLK;IQGAP1-/-
)
mice. Nikon’s Structured Illumination microscope was used. Original magnification,
x10. Scale bar, 200 μm. (d) The number of migrated cells in (c) was shown on the plot.
(e) Cell tracking in time-lapse microscopy images from of lung epithelial cells from
littermate wild-type (WT), GLK transgenic (PolⅡ-GLK), GLK
transgenic/IQGAP1-heterozygous (PolⅡ-GLK;IQGAP1+/-
), and GLK
transgenic/IQGAP1 knockout (PolⅡ-GLK;IQGAP1-/-
) mice. The tracked path of the
migration cell is shown as the line. Original magnification, x20. Scale bar, 50 μm. (f)
The percentage of migrated cells in (e) was shown on the plot. (g) The average
migration length of cells in (e) was shown on the plot. Data shown (a-f) are
representative results of three independent experiments. *, P value<0.05; **, P value
<0.01.
Figure 4. GLK co-localizes with IQGAP1 in the protrusive region and
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36
accumulate at the leading edge of migrating lung cancer cells.
(a) The confocal fluorescence imaging of HCC827 cells co-expressing GLK-mGFP
and IQGAP1-Tomato fusion proteins. The left and middle columns show the
GLK-mGFP (green) and IQGAP1-Tomato (red). Nuclei were stained with Hoechst
33342 (blue). Leica TCS SP5 microscope was used. Original magnification, x60.
Scale bar, 10 μm. (b) N-SIM imaging of HCC827 cells co-transfected with plasmids
encoding GLK-mGFP and IQGAP1-Tomato fusion proteins. The left column shows
the GLK-mGFP (green). The middle and right columns show the IQGAP1-Tomato
(red) and merged images. Merging of GLK (green) and IQGAP1 (red) appeared
yellow. Original magnification, x60. Scale bar, 5 μm. (c) N-SIM time-lapse live cell
imaging of an HCC827 cell co-expressing GLK-mGFP and IQGAP1-Tomato. Time
lapse is shown in h:min. Original magnification, x60. Scale bar, 5 μm. (d) N-SIM
time-lapse live cell imaging of HCC827 cells co-expressing GLK-mGFP and
IQGAP1-Tomato. Time lapse is shown in min:sec. Original magnification, x60. Scale
bar, 5 μm. Arrows denote the direction of the migrating cell. See also Movie S3-S5.
Figure 5. Phosphorylation of IQGAP1 at Ser-480 by GLK controls lung cancer
cell migration.
(a) In vitro kinase assay and immunoblotting of purified wild-type GLK, kinase-dead
GLK, and IQGAP1. Phosphorylation of IQGAP1 was then quantified by a Typhoon
scanner (GE). (b) Mass spectrometry analysis of the tryptic peptides of IQGAP1 to
identify the peptide containing phosphorylated Ser-480. (c) Active (GTP-binding)
Cdc42 proteins were immunoprecipitated from lysates of HEK293T cells
co-transfected with Cdc42 and GLK plus either IQGAP1 or IQGAP1 (S480A) mutant,
followed by immunoblotting analyses. Lower panel showed the
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37
co-immunoprecipitation (interaction) between IQGAP1 and Cdc42. (d) Active
(GTP-binding) Rac1 proteins were immunoprecipitated from lysates of HEK293T
cells co-transfected with Rac1 and GLK plus either IQGAP1 or IQGAP1 (S480A)
mutant, followed by immunoblotting analyses. Lower panel showed the
co-immunoprecipitation (interaction) between IQGAP1 and Rac1. (e) Cdc42 or Rac1
enzymatic activity the cell lysates as in (c) or (d) was determined using G-LISA
Activation Assay Biochem Kit. Positive, positive controls from the assay kit. SA,
IQGAP1 (S480A) mutant. (f) Migration assays of HCC827 cells transfected with
Flag-tagged GLK or GLK (P436/437A;P478/479A) plasmid plus the plasmid
expressing Myc-tagged IQGAP1, IQGAP1 (S480A), or IQGAP1 (∆WW). Original
magnification, x10. Scale bar, 20 m. (g) The relative number of migrated cells per
field is shown on the plot. (h) Immunoblotting of GLK and IQGAP1 proteins from
HCC827 cells transfected with the indicated plasmids. Data shown are representative
results of three (a-d, f) or two (e) independent experiments. *, P value<0.05.
Figure 6. GLK induces distant metastasis of lung cancer.
(a) Constitutively activated GLK (E351K) mutant induces higher phosphorylation
levels of IKK than wild-type GLK. Immunoblotting of phospho-IKK and GLK
proteins from Jurkat T cells transfected with the indicated plasmids. Tubulin was used
as the loading control. (b) ADP-based kinase assays of GLK or GLK (E351K) mutant
proteins. Flag-tagged GLK or GLK (E351K) proteins were immunoprecipitated from
transfected HEK293T cell lysates. (c) Schematic diagram of the PolⅡ-GLK-E351K
transgenic construct. Constitutively activated human GLK (E351K) mutant cDNA
was driven by the mouse RNA polymerase II (PolⅡ) promoter. (d) Real-time PCR
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38
analyses of transgenic human GLK-E351K (hGLKE351K
) mRNA levels in murine
peripheral blood cells from mice. The human GLK mRNA levels were normalized to
mouse Srp72 mRNA levels. WT, n = 4; PolⅡ-GLKE351K
, n = 4. Means ± SEM are
shown. WT, wild-type littermate controls; PolⅡ-GLKE351K
, GLKE351K
transgenic
mice. (e) Immunohistochemistry of GLK expression in the lung cancer from
SPA-EGFRdel
transgenic mice and SPA-EGFRdel
;PolⅡ-GLKE351K
transgenic mice.
Scale bar, 100 μm. (f) Representative immunohistochemistry of EGFR-deletion
mutant expression and H&E staining in the lung cancer from 7-month-old
SPA-EGFRdel
;PolⅡ-GLKE351K
transgenic mice and SPA-EGFRdel
;PolⅡ
-GLKE351K
;IQGAP1-/-
mice. Scale bar, 100 μm. (g) Representative
immunohistochemistry of EGFR-deletion mutant expression or H&E staining in the
brain and liver from 7-month-old SPA-EGFRdel
;PolⅡ-GLKE351K
transgenic mice and
SPA-EGFRdel
;PolⅡ-GLKE351K
;IQGAP1-/-
mice. Scale bar, 100 μm. Comparison of
EGFR-deletion mutant expression in tissues from individual groups (lower panel).
Figure 7. GLK-IQGAP1 complex is correlated with poor survival of human
NSCLC.
(a) In situ PLA assays of the interaction between GLK and IQGAP1 in normal
adjacent tissues, squamous cell carcinoma (one type of NSCLC) tissues,
adenocarcinoma (one type of NSCLC) tissues, and small cell lung carcinoma (SCLC)
tissues from representative patients. Original magnification, x40. (b) The PLA signals
of the interaction between GLK and IQGAP1 each group per tissue (3.14 mm2) are
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39
shown on the plot. NSCLCs included squamous cell carcinoma (SCC, n = 56),
adenocarcinoma (ADC, n = 17), bronchioloalveolar carcinoma (BC, n = 11), and large
cell carcinoma (LCC, n = 8). Normal adjacent (NA) tissues, n = 68. Small cell lung
carcinoma (SCLC), n = 3. (c) Kaplan-Meier estimates of survival according to
GLK-IQGAP1 PLA signals (PLA signal-High versus PLA signal-Low) of NSCLCs (n
= 66). P values were calculated using the log-rank test. (d) In situ PLA assays of the
interaction between GLK and IQGAP1 in metastatic NSCLC cells of the lung, the
lymph node, and the bone tissues. PCNA staining (in green, FITC) was used to label
lung cancer cells. Dotted lines indicate the vascular wall of blood vessels in the lung
tissue. (e) In situ PLA assays of phosphorylated IQGAP1 Ser-480 in the tumor tissues
from a representative squamous cell carcinoma patient and a representative
adenocarcinoma patient using a combination of paired PLA probes corresponding to
IQGAP1 and phospho-IQGAP1 (Ser-480). PCNA staining (in green, FITC) was used
to label lung cancer cells. Original magnification, x40. (f) The PLA signals of the
interaction between GLK and IQGAP1 each group per tissue (3.14 mm2) are shown
on the plot. NSCLCs included squamous cell carcinoma (SCC, n = 53),
adenocarcinoma (ADC, n = 17), bronchioloalveolar carcinoma (BC, n = 11), and large
cell carcinoma (LCC, n = 8). Normal adjacent (NA) tissues, n = 68. Small cell lung
carcinoma (SCLC), n = 3. (g) Kaplan-Meier estimates of survival according to
p-IQGAP1 (Ser-480) PLA signals (PLA signal-High versus PLA signal-Low) of
NSCLCs (n = 63). Among the tissue arrays, three SCC tissues on the tissue array slide
were damaged; therefore, only 63 of 66 tissues were analyzed. P values were
calculated using the log-rank test.
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Published OnlineFirst August 20, 2019.Cancer Res Huai-Chia Chuang, Chih-Chi Chang, Chiao-Fang Teng, et al. phosphorylating and activating IQGAP1MAP4K3/GLK promotes lung cancer metastasis by
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