MAP4K3/GLK promotes lung cancer metastasis by ... · 8/20/2019 · 3 INTRODUCTION. M. ore than 90%...

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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

Research. on January 26, 2021. © 2019 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 20, 2019; DOI: 10.1158/0008-5472.CAN-19-1402

mailto:[email protected]

http://cancerres.aacrjournals.org/

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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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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




19

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




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




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




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




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




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




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




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




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




28

Bio-Development Foundation (TBF) Chair in Biotechnology.

COMPETING FINANTIAL INTERESTS

The authors declare no competing financial interests.

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their functional classification. J Biol Chem 2004;279:31833-41




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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




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.




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




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




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




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




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.




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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