Kir2.1 Interaction with Stk38 Promotes Invasion and Metastasis of Human Gastric Cancer by Enhancing MEKK2-MEK1/2-ERK1/2 Signaling

Author Manuscript Published OnlineFirst on March 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3776 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Kir2.1 interaction with Stk38 promotes invasion and metastasis of human gastric

cancer by enhancing MEKK2-MEK1/2-ERK1/2 signaling

Cheng-Dong Ji1, Yan-Xia Wang1, Dong-Fang Xiang1, Qiang Liu1, Zhi-Hua Zhou1,

Feng Qian2, Lang Yang1, Yong Ren1, Wei Cui1, Sen-Lin Xu1, Xi-Long Zhao1, Xia

Zhang1, Yan Wang1, Peng Zhang1, Ji-Ming Wang3, You-Hong Cui1,*, Xiu-Wu Bian1,*

1Institute of Pathology and Southwest Cancer Center, and Key Laboratory of Tumor

Immunopathology of Ministry of Education of China, Southwest Hospital, Third

Military Medical University (Army Medical University), Chongqing 400038, China.

2Department of General Surgery, Southwest Hospital, Third Military Medical

University, Chongqing 400038, China.

3Cancer and Inflammation Program, Center for Cancer Research, National Cancer

Institute at Frederick, MD 21702, USA

Running title: Pro-malignancy of Kir2.1 in gastric cancer

Abbreviations: Co-IP: co-immunoprecipitation; EMT: epithelial-mesenchymal

transition; FACS: fluorescence-activated cell sorting; GC: gastric cancer; IHC:

immunohistochemistry; IK+: inwardly rectifying potassium current; IOD: integrated

optical density; K2P: two-pore domain potassium channels; KCa: calcium-activated

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3776 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

potassium channels; Kir2.1: inwardly rectifying potassium channel 2.1; Kv:

voltage-gated channels; MEKK2: mitogen-activated protein kinase kinase kinase 2;

OS: overall survival; PFS: progression-free survival; SDS-PAGE:

SDS-polyacrylamide gel electrophoresis; Smurf1: small mothers against

decapentaplegic-specific E3 ubiquitin protein ligase 1; Stk38:

serine/threonine-protein kinase 38; TNBC: triple-negative breast cancer。

*Correspondence to: Xiu-Wu Bian, M.D., Ph.D. and You-Hong Cui, M.D. Institute

of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military

Medical University (Army Medical University), Chongqing 400038, China.

Telephone: +86 23 68754431; Fax: +86 23 65397004; E-mail: [email protected]

(XB) E-mail:[email protected] (YC)

Conflict of interest statement: The authors disclosed no potential conflicts of

interest.

Precis: Kir2.1 contributes to invasion and metastasis by a non-canonical ion

permeation-independent signaling pathway and may act as a novel prognostic marker

and therapeutic target for gastric cancer.

Main text word count: 4716 words; Number of Figure: 6; Number of table:1.

Abstract

Potassium ion channels are emerging as pro-malignant factors involved in cancer

progression. In this study, we found that invading human gastric cancer (GC) cells

express high level of inwardly rectifying potassium channel 2.1 (Kir2.1). Silencing

Kir2.1 markedly reduced the invasive and metastatic capabilities as well as the

epithelial-mesenchymal transition (EMT) of GC cells. The pro-malignant nature of

Kir2.1 in GC cells was independent of potassium permeation but relied on its

interaction with serine/threonine-protein kinase 38 (Stk38) to inhibit ubiquitination

and degradation of mitogen-activated protein kinase kinase kinase 2 (MEKK2).

Degradation of MEKK2 was mediated by small mothers against

decapentaplegic-specific E3 ubiquitin protein ligase 1 (Smurf1), which resulted in

activation of the MEK1/2-ERK1/2-Snail pathway in GC cells. In human GC tissues,

expression was high and positively correlated with invasion depth and metastatic

status of the tumors as well as poor overall patient survival. Cox regression analysis

identified Kir2.1 as an independent prognostic indicator for GC patients. Our results

suggest that Kir2.1 is an important regulator of GC malignancy and acts as a novel

prognostic marker and a therapeutic target for GC.

Keywords: gastric cancer, invasion, metastasis, Kir2.1, Stk38

Introduction

Gastric cancer (GC) is a major malignant tumor in the digestive system with high

mortality worldwide, especially in East Asia (1,2). Despite the advances in surgical

and other supplemental treatments, the prognosis for patients with advanced GC

remains poor (3). Invasion and metastasis are crucial factors in the progression of GC

(4). Therefore, insight into the mechanisms underlying GC invasion and metastasis

should benefit the development of more effective therapies.

Recently, potassium channels are implicated in promoting the malignancy of tumor

cells. Potassium channels are pore-forming transmembrane proteins that regulate a

multitude of biological processes by selectively transporting potassium ions across the

cell membrane. Based on the structure and activation mechanisms, 78 potassium

channels are divided into four main classes: voltage-gated channels (Kv),

calcium-activated channels (KCa), inward rectifying channels (Kir), and two-pore

domain channels (K2P) (5). Various dysregulated potassium channels that cover all

four classes have been found in different types of human cancer. For example,

voltage-gated potassium channel Kv10.1 was normally expressed in selected brain

areas, but was aberrantly expressed in over 70% human tumors and involved in tumor

cell proliferation, survival, angiogenesis, migration, and invasion (6). In

triple-negative breast cancer (TNBC), KCa3.1 is implicated in the proliferation,

apoptosis, migration and EMT of tumor cells (7). In highly progressive human

astrocytic tumors, the expression of Kir4.1 at both mRNA and protein levels was

markedly increased (8). TREK-1, a two-pore domain (K(2P)) potassium channel, was

highly expressed in prostate cancer associated with abnormal tumor cell proliferation

(9). However, it is unknown whether potassium channels are involved in GC invasion

and metastasis. Hence, we examined the difference in potassium current in invading

and non-invading GC cells by whole-cell patch-clamp. The results showed that

potassium current in invading GC cells was markedly higher than that in non-invading

GC cells. Moreover, the elevated potassium current in invading GC cells exhibited

characteristics of an inwardly rectifying potassium current (IK+), implying that Kir

might be associated with the invasive capability of GC cells. qRT-PCR scanning

revealed that within 15 distinct subunits of Kir family, Kir2.1 was most prominently

expressed in invading GC cells. Whole-cell patch-clamp also showed that Kir2.1 was

the most important contributor of IK+ in invading GC cells. We therefore further

investigated the role of Kir2.1 in the invasion and metastasis of human GC and the

clinical relevance. Our results indicate that Kir2.1 actively promoted GC cell invasion

and metastasis independent of its potassium transport function, but by interaction with

serine/threonine-protein kinase 38 (Stk38) to enhance the signaling of

MEKK2-MEK1/2-ERK1/2-Snail pathway in GC cells. Studies of clinical GC

specimens showed a positive correlation between the levels of Kir2.1 expression and

the invasion depth and metastatic status of human GC in association with poorer

overall patient survival. Thus, Kir2.1 plays an important role in promoting GC

malignancy and acts as an independent indicator of GC prognosis as well as a

potential therapeutic target.

Materials and Methods

Patients and specimens

A total of 349 formalin-fixed and paraffin-embedded GC surgical specimens, in

which 131 specimens contained both tumor and matched adjacent normal tissues,

were collected from patients enrolled in the Southwest Hospital from 2006 to 2007

without prior radiotherapy or chemotherapy. A separate set of samples, which contain

6 fresh surgical tumor and the adjacent normal tissues, was also collected from the

Southwest Hospital. Follow-up information was available for all patients. According

to the WHO standard and American Joint Commission on Cancer TNM staging

system (10), each specimen was histologically examined and the tumor was graded by

two experienced pathologists. Written informed consent was obtained from the

patients or their guardians. This study was performed by the principles of the Helsinki

Declaration and approved by the Ethical Committee of the Southwest Hospital.

Cells and Cell culture

MGC803 human GC cell line was purchased from the Cell Bank of Shanghai

Institute of Cell Biology. Primary GC cell line XN0422 was generated in our

laboratory as described previously (11). All the cells were tested and confirmed

negative for mycoplasma contamination using EZ-PCR Mycoplasma Test Kit (Bioind,

Israel) and authenticated by short tandem repeat profiling and passaged for less than 6

months according to the manufacturer's guideline. The cells were cultured in

RPMI1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA)

o at 37 C in 5% CO2.

Immunohistochemistry and immunoreactivity scores

Immunohistochemical (IHC) staining was performed on 4 μm tissue sections using

an EnVision™ Kit (DAKO, Denmark). After deparaffinized, rehydrated in graded

ethanol, antigen retrieval and blocking, slides were incubated with an anti-Kir2.1

antibody (1:300, Cat.no. ab85492, Abcam) at 4 oC overnight. After washing with PBS,

a horseradish peroxidase (HRP)-conjugated secondary antibody (DAKO, Denmark)

was added and incubated at 37 oC for 30 min. Sections were stained by 3,

39-diaminobenzidine (DAB, DAKO, Denmark) and counterstained with

Hematoxylin.

IHC scoring method was performed as previously described (12). Briefly, five

random IHC images of each slide were captured using an Olympus BX51 microscope.

The images were opened by Image-Pro Plus 5.0 software. The area sum and

integrated optical density (IOD) sum of the positive site (brown) were measured in

pixels using the software. The expression intensity of Kir2.1 was expressed by the

mean value of IOD sum / area sum of 5 photographs for each slide. To ensure data

comparability, the same parameter settings were kept for all photographs. The best

predictive cut-off value of expression intensity was determined to be 0.03438

analyzed with SPSS 20.0. Expression intensity > 0.03438 was defined as Kir2.1high，

low otherwise was defined as Kir2.1 .

Fluorescence-activated cell sorting (FACS)

Single GC cell suspension was prepared by trypsinization of cultured adherent cells

and incubated with anti-human Kir2.1 antibody (Cat.no. orb184788, Biorbyt) for 30

min at room temperature followed by labeled with APC conjugated goat anti-rabbit

secondary antibody. Then the Kir2.1high and Kir2.1low GC cells were sorted by the

FACSAria II cell sorter (BD, USA). Forward side scatter and pulse-width gating were

used for excluding the dead cells, debris, and aggregates. Isotypes matched primary

antibodies were used as controls.

In vitro GC cell invasion assay

Matrigel transwell analyses were performed as previously described (13). To

acquire invading and non-invading GC cells, transwell chambers (8 µm pore size,

Millipore) were coated with 10 µL mixed matrigel (matrigel and RPMI1640, 1:3, v/v).

GC cells were implanted at 5×104 cells/well in 200 µL serum-free RPMI-1640. The

lower chambers were filled with 600 µL RPMI1640 medium supplemented with 10%

FBS. After incubation for 24 h at 37°C, the invading and non-invading cells were

obtained by trypsin enzyme-digesting technique. To examine the affectation of Kir2.1

agonist and antagonist and signaling inhibitors on the invasiveness of GC cells, the

metrigel-transwell system were cultured with or without ML133 (20 µM, TOCRIS) or

Zacopride (10 µM, Sigma-Aldrich) or PD98059 (10 µM, Selleck) or LY3009120 (20

µM, Selleck). After incubation for 24 h at 37°C, the invasive cells were stained with 1%

crystal violet and then counted from five random visual areas under a microscope.

Whole-cell patch-clamp recording

Potassium current was detected by whole-cell patch-clamp as described (14).

Recordings were made at room temperature. Bath (external) solutions were perfused

into the chamber using a gravity-driven perfusion system. The standard bath solution

consisted of (in mM): 100 D-glucose, 5 KCl, 2 MgCl2, 50 NaOH, and 5 glucose.

Recording pipettes were filled with an intracellular solution containing (in mM): 110

KCl, 5 MgATP, 5 NaCl, 2 MgCl2, and 5 glucose. Patch pipettes were made from

thin-walled borosilicate glass and fire polished with a microforge. Data were acquired

using an Axopatch 200B amplifier and filtered at 5 Hz with a Digidata 1322A board.

Acquisition and analysis were performed using an EPC-10 software.

RNA extraction and quantitative PCR

Total RNA was isolated using RNAiso Trizol reagent (Takara, Kyoto, Japan), and

reverse-transcribed with PrimeScript™ RT Master Mix (Takara, Japan) according to

the manufacturer’s instructions. Then a SYBR® Fast qPCR Mix (Takara, Japan) in a

Bio-Rad CFX96 Real-Time PCR Detection System (Bio-rad) was used for qRT-PCR.

qRT-PCR was performed in triplicate and the results were normalized against β-actin.

The sequences of all primers for qRT-PCR are listed in Supplementary Table S1.

Western blot

Immunoblot analyses were performed as previously described (13). GC cells were

washed twice with ice-cold PBS then lysed with protein extraction reagent (Thermo,

USA) containing 1% protease inhibitors (Thermo, USA). The lysate was centrifuged

at 4°C at 16,000×g for 15 min and proteins were separated by 10%

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF

membranes (Millipore, USA). After being blocked with 5 % skim milk for 2 h, the

membranes were incubated at 4 °C overnight with primary antibodies, whose detail

information were descripted in the Supplementary Materials and Method. After

washing with PBST, the membranes were incubated with HRP-conjugated secondary

antibodies (Byotime, China) for 1 h. Then proteins were visualized with SuperSignal

West Femto Maximum Sensitivity Substrate (ECL, Thermo) and detected by a

ChemiDocXRS system (BioRad).

Kir2.1 knockdown, overexpression, and site-directed mutagenesis

To knock down Kir2.1 in GC cells, three targeting shRNAs (named sh-Kir2.1) and

a non-targeting scrambled RNA (named mock) were constructed in lentivirus vectors

(Supplementary Table S2). For Kir2.1 overexpression in GC cells, full-length human

Kir2.1 (named over-Kir2.1) and empty vector (named ctrl) were constructed with a

lentivirus vector. To produce a non-conducting Kir2.1 channel subunit (named

mut-Kir2.1), the 144–146 (Gly-Tyr-Gly) signature sequence that serve as the

selectivity filter of Kir2.1 was mutated to Ala-Ala-Ala (15) and inserted into a

lentivirus vector. All lentivirus particles were packaged by Life Technologies Co. Ltd

(Shanghai, China) and used to infect GC cells with 2 μg/mL polybrene. Stably

transfected cells were selected by 3 μg/mL puromycin in culture.

Peritoneal metastasis in NOD/SCID mice

NOD/SCID mice aging from 4 to 5 weeks and weighing between 17 and 20 grams

were obtained from the Experimental Animal Center of Third Military Medical

University. GC cells suspended in PBS were inoculated intraperitoneally into mice at

1×104 cells per mouse. The mice were sacrificed at the end of 4 weeks after the

implantations. Peritoneal metastases in mesentery were evaluated. Animal

experiments were approved by the Third Military Medical University Animal Ethics

Committee in accordance with the Guide for the Care and Use of Laboratory Animals.

Human phosphokinase array

Cell lysates were diluted and mixed with a cocktail of biotinylated detection

antibodies then added on the Human phosphokinase array (R&D, USA). After

incubated overnight at 4 oC, Streptavidin-horseradish peroxidase and

chemiluminescent detection mix were added, and chemiluminescence was detected

using the ChemiDoc ™ MP System (Bio-Rad, Hercules, California, USA), Image Lab

(Version 5.2 build 14), and automatic exposure settings.

Co-immunoprecipitation

Co-immunoprecipitation (Co-IP) was performed using the Thermo Scientific

Pierce Co-IP kit (Thermo Scientific, Watertown, MA, USA) following the

manufacturer’s protocol. Ten μg each of antibody and IgG were immobilized on

AminoLink Plus coupling resin for 2 h, then washed and incubated with 250 μg GC

cell protein lysate. After incubated overnight at 4 °C, the resin was washed and eluted

using an elution buffer. The eluted proteins were separated by SDS–PAGE and

immunoblotted with indicated antibodies as western blot analysis.

Ubiquitination assay

Ubiquitination assay was performed as previously described (16). Briefly, GC cells

were transfected with plasmids expressing HA-ubiquitin, His-Smurf1, V5-Stk38 and

Flag-Kir2.1 alone or in combination as designed protocol. After 48 h, cells were

treated with proteasome inhibitors MG132 (10 μM, Selleck) for another 6 h. Then the

ubiquitination degradation was detected by immunoprecipitation.

Statistical analysis

All experiments were performed at least three times and results from representative

experiments are presented as the mean ± s.d. by Student’s t-test using SPSS 20.0

software (IBM, USA) and GraphPad Prism. The cut-off value of Kir2.1 IHC staining

scores was analyzed with SPSS. Chi-square analysis was used to evaluate the

relationship between Kir2.1high rate and GC clinicopathological features. The overall

survival (OS) and progression-free survival (PFS) of GC patients were estimated

using Kaplan–Meier method. Cox’s proportional hazard regression model was

established for univariate and multivariate analyses of the combined contribution of

Kir2.1 and clinicopathological features to the OS of patients. P < 0.05 was considered

as statistically significant.

Results

Kir2.1 is highly expressed in invading GC cells

We first measured the potassium current by using a whole-cell patch-clamp

method in both invading and non-invading primary XN0422 GC cells. Compared to

non-invading GC cells, invading cells displayed high level of potassium current,

which was blocked by barium, a nonspecific potassium current blocker (Fig. 1A). As

shown in Fig. 1B, the current density-voltage (pA/pF-V) relation of the potassium

current exhibited typical IK+ characteristics in invading GC cells. The average IK+ at

100 mV in invading cells was markedly higher than that in non-invading cells

(-36.0130 ± 4.9826 vs -3.2529 ± 0.9738, n = 20) (Fig. 1C). Similar results were also

achieved in GC cell line MGC803 cells (Supplementary Fig. S1). These results

suggest presence of functional Kir channels in invading GC cells. We then scanned

gene expression of 15 Kir members by qRT-PCR and found Kir2.1 expression at a

higher level in invading GC cells (Fig. 1D). We subsequently compared Kir2.1

expression between high (> 50 pA) and low (< 5 pA) IK+ -possessed GC cells. As

shown in Fig. 1E, markedly increased expression of Kir2.1 was found in high IK+ GC

cells. Increased Kir2.1 protein in invading GC cells was detected in both GC cell lines

MGC803 and XN0422 (Fig. 1F). These results suggest that aberrant expression of

functional Kir2.1 is associated with the invasive and metastatic capabilities of GC

cells.

Kir2.1high GC cells are highly invasive and metastatic in association with

epithelial-mesenchymal transition (EMT)

By FACS, Kir2.1-high (Kir2.1high) and -low GC cells (Kir2.1low) were isolated, and

the percentage of Kir2.1high GC cells was about 22 % in MGC803 and 14 % in

XN0422 (Supplementary Fig. S2). As shown in Fig. 2A and Supplementary Fig. S3,

Kir2.1high GC cells exhibited more invasive capability compared to Kir2.1low cells in

vitro. The invading cells in Kir2.1high and Kir2.1low subpopulations were 210.60 ±

33.26 vs 96.80 ± 17.02 for MGC803 and 114.40 ± 18.85 vs 65.80 ± 12.40 for

XN0422 cells, respectively. Kir2.1high GC cells also exhibited increased metastatic

capability compared to Kir2.1low cells in a mouse peritoneal metastasis model. The

observed metastatic nodules derived from Kir2.1high and Kir2.1low GC cells were

65.60 ± 5.73 vs 2.60 ± 1.60 for MGC803 and 46.60 ± 4.45 vs 2.40 ± 1.50 for XN0422

cells, respectively (Fig. 2B and C). Knockdown of Kir2.1 (sh-Kir2.1, Supplementary

Fig. S4) significantly inhibited the invasion of GC cells. The invading cells in

sh-Kir2.1 and mock cells were 18.60 ± 4.04 vs 83.20 ± 14.99 for MGC803 and 12.80

± 3.70 vs 46.40 ± 9.96 for XN0422 cells, respectively (Fig. 2D and Supplementary

Fig. S5). Compared to mock cells, sh-Kir2.1 MGC803 and XN0422 cells formed

markedly reduced number of metastatic nodules in mice (Fig. 2E and F). In contrast,

Kir2.1 overexpression (over-Kir2.1, Supplementary Fig. S4) significantly not only

elevated the invasive capability (Fig. 2G and Supplementary Fig. S5) but also

increased the metastatic nodule formation (Fig. 2H and I) by both MGC803 and

XN0422 cells. All metastatic nodules in mice formed by GC cells were confirmed as

human GC by H&E staining (Supplementary Fig. S6). These results suggest that the

presence of Kir2.1 is associated with increased invasion and metastasis of GC cells.

EMT is recognized as a pivotal event for tumor cells to enhance their capacity of

invasion and metastasis (17,18). Kir2.1-overexpressing GC cells contained decreased

epithelial marker E-cadherin but increased mesenchymal marker vimentin and the

transcription factor Snail (Fig. 2J). In contrast, knockdown of Kir2.1 in GC cells

reversed the EMT phenotype, with upregulated E-cadherin and downregulated

vimentin and Snail (Fig. 2K). Although EMT may be regulated by different

transcription factors, examination of major EMT-related transcription factors showed

that overexpressing Kir2.1 only significantly increased Snail expression in GC cells

(Supplementary Fig. S7). These results suggest that Kir2.1 promotes EMT in GC

cells.

The pro-malignant effect of Kir2.1 on GC cells is independent of potassium

permeation

Kir2.1 functions either as a potassium transporter (14,19,20) or by promoting

protein–protein interaction (21-23). We firstly investigated whether the pro-malignant

effect of Kir2.1 was dependent on its channel function in GC cells. Treatment with

ML133, a specific inhibitor of Kir2.1 channel (24), completely interrupted IK+ flux in

both over-Kir2.1 and control GC cells (Supplementary Fig. S8), but did not alter their

invasive capability (Fig. 3A and Supplementary Fig. S9) and the levels of

EMT-related proteins (Fig. 3B). Also, treatment with Zacopride, an IK+ stimulator

(25), potently elevated IK+ (Supplementary Fig. S8), but with little effect on the

invasion capability (Fig. 3C and Supplementary Fig. S9) and EMT phenotype (Fig.

3D) of both over-Kir2.1 and control GC cells. Since extracellular pore-forming region

of Kir2.1 serves as the “ion-selectivity filter”, in which mutation of GYG into AAA

leads to the loss of potassium transport function (15), we introduced a mutant Kir2.1

(GYG to AAA, mut-Kir2.1) into GC cells. The introduction of mutant Kir2.1 did not

increase IK+ (Supplementary Fig. S8), but enhanced the invasive capability (Fig. 3E

and Supplementary Fig. S9), EMT and Snail to the level of over-Kir2.1 in GC cells

(Fig. 3F). These results suggest that the effect of Kir2.1 on increased invasion and

metastasis of GC cells is independent of its ion channel function.

Kir2.1 activates MEK1/2-ERK1/2-Snail-EMT pathway without involvement of

Raf

A phosphokinase array was performed to explore the signaling pathways involving

Kir2.1 in GC cells. As shown in Fig. 4A, Kir2.1 overexpression resulted in elevated

phosphorylation of 11 kinases in XN0422 cells, among which, the phosphorylation

level of ERK1/2 was at the highest level. Markedly enhanced phosphorylation of

ERK1/2 by Kir2.1 overexpression but significantly attenuated phosphorylation of

ERK1/2 by Kir2.1 knockdown were confirmed by immunoblotting. In contrast to

ERK1/2, the phosphorylation of other members of MAPKs, JNK and p38, was

minimally affected in GC cells by Kir2.1 manipulation (Fig. 4B). We further

examined whether Kir2.1-induced ERK1/2 activation involves canonical

Ras-Raf-MEK1/2-ERK1/2 pathway. Treatment over-Kir2.1 GC cells with PD98059

(26), a MEK1/2 inhibitor, significantly attenuated ERK1/2 phosphorylation (Fig. 4C

and Supplementary Fig. S10), accompanied with impaired invasion capability,

down-regulated Snail and vimentin as well as up-regulated E-cadherin expression (Fig.

4D). LY3009120 (27), an inhibitor of pan-Raf (A-Raf, B-Raf and C-Raf) only slightly

attenuated the invasion capability and phosphorylation of MEK1/2 and ERK1/2 in

both control and over-Kir2.1 GC cells (Fig. 4E, 4F and Supplementary Fig. S10).

Thus, the effect of Kir2.1 on the invasion and EMT of GC cells is dependent on

MEK1/2-ERK1/2-Snail but not Raf.

Kir2.1-Stk38 interaction up-regulates MEKK2 to activate MEK1/2-ERK1/2

Since Kir2.1-promoted invasion and metastasis of GC cells were independent of

its potassium channel function, its involvement in protein-protein interaction was

hypothesized. With immunoprecipitation-mass spectrometry, proteins potentially

interacting with Kir2.1 were identified (Supplementary Table S3), with Stk38 and

Rho-associated protein kinase 2 (Rock2), which regulate ERK1/2 pathway

(16,28,29), were selected for further study. Co-IP confirmed that Stk38, but not

Rock2, physically interacted with Kir2.1 in both MGC803 and XN0422 GC cells

(Fig. 5A). Moreover, we further revealed that the interacting site of Kir2.1 with

Stk38 was the C-terminal (aa179-425) region by using truncated mutants

(Supplementary Fig. S11). Since Stk38 regulates MEK1/2-ERK1/2 pathway mainly

by regulating MEKK2, an MAPKKK (30,31). Depletion of MEKK2 with siRNA

significantly decreased the phosphorylation of MEK1/2 and ERK1/2 in both control

and over-Kir2.1GC cells (Fig. 5B).

Stk38 converts MEKK2 from a phosphorylated to a nonphosphorylated form

therefore inhibiting MEKK2 (32). Since specific anti-p-MEKK2 antibody is

unavailable, p-MEKK2 and non-p-MEKK2 may be distinguished by immunoblotting

in which the faster migrating band defines non-phosphorylated or

hypo-phosphorylated MEKK2 (Non-p-MEKK2) while slower migrating band

indicates phosphorylated MEKK2 (p-MEKK2) (32,33). We found that overexpressing

Stk38 markedly reduced the invasiveness (Supplementary Fig. S12) and the levels of

both phosphorylated and non-phosphorylated MEKK2 accompanied by decreased

levels of p-MEK1/2 and p-ERK1/2 (Fig. 5C) in GC cells. However, the relative

density of p-MEKK2 or non-p-MEKK2 remained similar between control and

over-Stk38 GC cells (Fig. 5D), implying that Stk38 regulates

MEKK2-MEK1/2-ERK1/2 mainly by changing the level of MEKK2 protein rather

than affecting its phosphorylation in GC cells. As previously reported (16), Stk38

inhibits MEKK2 by interacting with small mothers against decapentaplegic-specific

E3 ubiquitin protein ligase 1 (Smurf1) to enhance the ubiquitination and degradation

of MEKK2. We thus hypothesized that the interaction between Kir2.1 and Stk38

might attenuate the ubiquitination and degradation of MEKK2 leading to the

activation of the MEK1/2-ERK1/2 pathway. With quantitative Co-IP and

ubiquitination degradation assays, we confirmed that over-expressing Kir2.1 in GC

cells significantly decreased the formation of Smurf1-Stk38 complex (Fig. 5E) and

attenuated the ubiquitination degradation of MEKK2 induced by the complex (Fig.

5F). These results suggest that MEKK2-MEK1/2-ERK1/2 pathway is pivotal to the

invasion and metastasis of GC cells, which is enhanced by the interaction of

Kir2.1with Stk38 to inhibit ubiquitinated degradation of MEKK2.

The expression of Kir2.1 in GC tissues is correlated with clinicopathological

features and the prognosis of GC patients

We further investigated the clinical relevance of Kir2.1 expression in GC. In 349

GC specimens and 131 paired adjacent normal tissues, Kir2.1 protein was mainly

detected in the cytoplasm and membrane of GC cells (Fig. 6A). Kir2.1 was weakly or

not expressed in normal gastric mucosa (Fig. 6Aa). The density of Kir2.1 protein in

GC specimens increased with the invasion depth of tumor (Fig. 6Ab–e). Kir2.1 was

also highly expressed in lymph node metastasis nodules of GC (Fig. 6Af). ROC curve

showed a significant cutoff value of 0.03438 for Kir2.1 as assessed by SPSS 20.0

(Supplementary Fig. S13). The IHC scores of Kir2.1 were significantly higher in 131

cancer tissues than in paired adjacent normal tissues (Fig. 6B). In a separate set of

specimens, quantitative analysis of Kir2.1 in six freshly resected tumor samples and

adjacent normal tissues indicated high levels of Kir2.1 expression in tumor tissues

than in adjacent normal tissues (Fig. 6C, Supplementary Table S4). Higher expression

of Kir2.1 in GC tumors was also found in GEO GSE13911 and GES27342 (Fig. 6D,

Supplementary Table S5). Analysis of clinicopathological features showed that high

expression of Kir2.1 in GC was positively correlated with increased tumor invasion

depth, lymph node metastasis (Fig. 6E), and TNM stage (Table). Kaplan–Meier

analyses showed significant correlation between higher expression of Kir2.1 and

poorer OS and PFS in GC patients (Fig. 6F). KMPLOT database also revealed

negative correlation between increased Kir2.1 expression and poorer prognosis of

patients with GC (Supplementary Fig. S14). Both univariate and multivariate analyses

suggested that Kir2.1 was an independent prognostic indicator for GC patients

(Supplementary Table S6). Thus, elevated Kir2.1 in GC is associated with increased

invasion and metastasis with poorer prognosis of patients.

Discussion

It is well known that aberrant functions of potassium channels result in many types

of channelopathies, such as epilepsy (34), cardiac arrhythmia (34) and neuromuscular

symptoms (35). Emerging evidence demonstrates that potassium channels are

involved in carcinogenesis and progression of many cancers, including GC (36-38).

Liu et al. (39) found that KCNQ1 polymorphisms appear to be independent predictors

of chemotherapeutic response in GC. Ding et al. (40) showed that hERG1 expression

was an independent prognostic factor in GC. In this study, we demonstrated, for the

first time, that Kir2.1 was an important player in the invasion and metastasis capacity

of GC cells. Elevated Kir2.1 expression in GC tissues is associated with cancer

invasion depth, lymph node metastasis, and poor outcome of the patients.

The mechanisms by which potassium channels regulate the malignancy of cancer

vary among different potassium channels and in different cancers. The mechanisms

may be grouped into two types: canonical ion permeation-dependent and

non-canonical ion permeation-independent. Since the basic function of potassium

channels in excitable and non-excitable cells is to conduct potassium across the

membrane to dominate the resting membrane potential, aberrant potassium channel

expression may contribute to the progression of cancer by affecting the resting

membrane potential. It is well known that rapidly proliferating embryonic cells, stem

cells, and cancer cells in general are more depolarized, with resting membrane

potential at -20 to -40 mV, whereas differentiated cell types such as neurons or

cardiomyocytes are hyperpolarized, with resting membrane potential at -60 to -80 mV

(41). A change in resting membrane potential directly or indirectly regulates cancer

cell behaviors, such as proliferation and migration (42-44). Potassium channels also

regulate cancer cell behaviors through non-canonical ion permeation-independent

mechanisms, in which potassium channels utilize their cytoplasmic domains for

protein–protein interactions (17, 39). For example, transfecting either wild-type or

non-conducting Drosophila melanogaster eag channels into NIH3T3 cells induced

comparable levels of cell proliferation (40). Downie et al. (45) found that EAG1

enhanced HIF-1 activity and VEGF secretion, hence tumor vascularization

independently of its ion channel function in NIH3T3 cells. Kir2.2 promoted prostate

cancer cell growth via protein - protein interaction with RelA (42). We found that

Stk38 was critical to mediating a non-canonical ion permeation-independent signaling

for Kir2.1 to enhance GC cell invasion and metastasis.

ERK1/2 signaling pathway is one of the key regulators that drive EMT

switch-dependent metastasis of cancer cells (46-48). We showed that Kir2.1 potently

activated ERK1/2 pathway in GC cells and inhibition of ERK1/2 blocked the effect of

Kir2.1 on the capability of GC cell invasion and metastasis and reversed

Kir2.1-induced EMT in GC cells, indicating that ERK1/2 pathway exerts a pivotal

role in the process of Kir2.1-driven GC cell malignancy.

Classic cascade of ERK1/2 activation includes Ras/Raf/MEK/ERK sequentially,

i.e.: i) Ras recruits and activates Raf (MAP3K); ii) Raf phosphorylates MEK1/2

(MAP2K), then activates ERK1/2 (49,50). The Ras–Raf–MEK1/2–ERK1/2 signaling

(canonical ERK1/2 pathway) is one of the most well-characterized cascades in cancer

cells (51-53). In invading GC cells, however, elevated Kir2.1 activates ERK1/2 in a

MEK1/2-dependent manner, but independent of Raf, implying that other MAP3Ks

may be involved. In fact, there are over 20 MAP3Ks that selectively phosphorylate

and activate different combinations of seven MAP2Ks (54) including MEKK2.

Previous studies have demonstrated that MEKK2 transduces mitogenic signals

emanating from EGFR and FGF2R to JNK and ERK5 cascades (55,56). In recent

years, MEKK2 was also found to exert important activities in ERK pathway

(16,30,57). In this study, we demonstrated that MEKK2 was the necessary MAP3K

for Kir2.1 to activate ERK1/2 pathway. MEKK2 is regulated by Stk38, a binding

partner of Kir2.1 in GC cells. Stk38 is a serine–threonine protein kinase that belongs

to a subfamily of AGC family of kinases (58). Studies have provided compelling

evidence for the important roles of Stk38 in the regulation of MEKK2 signaling.

Stk38 converts MEKK2 from a phosphorylated to a nonphosphorylated form and

inhibits MEKK2 (32). Stk38 also promotes Smurf1-mediated polyubiquitination of

MEKK2 by interacting with Smurf1 (16). In our study, Stk38 was found to inhibit the

function of MEKK2 by Smurf1-mediated polyubiquitination and degradation of

MEKK2. The binding between Kir2.1 and Stk38 interrupted the function of Stk38,

resulting in MEKK2 enrichment and activation of MEKK2-MEK1/2-ERK1/2

signaling. Activated ERK1/2 triggers EMT of GC cells with increased invasion and

metastasis potential. A working model was presented in Supplementary Fig. S15. The

function of Kir2.1 in controlling other malignant behaviors of GC cells, such as

proliferation, drug resistance and apoptosis, requires further investigation.

In conclusion, we report that Kir2.1 control GC cell invasion and metastasis with a

non-canonical ion permeation-independent signaling pathway. Thus, Kir2.1 acts not

only as an additional diagnostic marker, but also as a novel therapeutic target for GC.

Acknowledgments

This research was supported by the Ministry of Science and Technology of the

People's Republic of China (China National Science and Technology Major Project,

2016YFA0101203) to Xiu-Wu Bian; the National Natural Science Foundation of

China (81372555) to You-Hong Cui; the National Natural Science Foundation of

China (81402080) to Yan-Xia Wang and the Chongqing Science and Technology

Commission (cstc2015jcyjA10114) to Dong-Fang Xiang.

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Table. The correlation between Kir2.1 expression in GC tissues and

clinicopathological features of GC patients

Clinicopathological Total No. Kir2.1 p value

parameter Low (%) High(%)

Age, y 0.316

≤60 177 72(40.7) 105(59.3)

>60 172 61(35.5) 111(64.5)

Sex 0.103

Female 89 43(48.3) 46(51.7)

Male 260 100(38.5) 160(61.5)

Differentiation 0.039

Well 31 12(38.7) 19(61.3)

Moderate 115 50(43.5) 65(56.5)

Poor 203 60(29.6) 143(70.4)

T stage 0.000

T1 32 21(65.6) 11(34.4)

T2 66 40(60.6) 26(39.4)

T3 147 61(41.5) 86(58.5)

T4 103 11(10.7) 92(89.3)

Lymph node metastasis 0.000

Absent 129 80(62.0) 49(38.0)

Present 220 63(28.6) 157(71.4)

TNM stage 0.000

Ⅰ 68 48(70.6) 20(29.4)

Ⅱ 117 64(54.7) 53(45.3)

Ⅲ 160 32(20.0) 128(80.0)

Ⅳ 4 0(0) 4(100.0)

Figure legends

Figure 1. Invading GC cells highly express functional Kir2.1. A, whole-cell

patch-clamp measurement showing invading XN0422 GC cells that displayed higher

potassium current compared to non-invading cells. Potassium current was inhibited by

barium (5 mM). B, the current density-voltage (pA/pF-V) curves derived from same

data presented in A with a typical characteristic of IK+. C, higher average IK+ at 100

mV in invading GC cells than in non-invading GC cells. Data are shown as

mean ± S.D., n=20, *, P < 0.05, Student’s t-test. D, the mRNA level of Kir family in

invading and non-invading GC cells. Data are shown as mean ± S.D., n=3. E,

increased Kir2.1 mRNA expressed by GC cells with high current (IK+ > 50 pA)

compared to low current (IK+ < 5 pA) GC cells as detected by qRT-PCR. Data are

shown as mean ± S.D., n=6, ***, P < 0.0001, Student’s t-test. F, higher level of Kir2.1

protein in invading GC cells compared to non-invading GC cells as detected by

Western blotting.

Figure 2. Kir2.1 promotes the invasion and metastasis of GC cells. A,

metrigel-transwell invasion assay showing increased invasion capability of Kir2.1high

GC cells compared to Kir2.1low GC cells. Data are shown as mean ± S.D., n=5, ***, P

< 0.0001, Student’s t-test. B, representative images of intraperitoneal metastasis in

NOD/SCID mice showing significantly higher number of metastatic foci formed by

Kir2.1high GC cells than that formed by Kir2.1low GC cells. C, quantification of

metastatic tumors formed by Kir2.1high and Kir2.1low GC cells. Data are shown as

mean ± S.D., n=5, ***, P < 0.0001, Student’s t-test. D, reduced invasion capability of

sh-Kir2.1 GC cells compared to mock (scrambled shRNA) GC cells in vitro. Data are

shown as mean ± S.D., n=5, NS, not significant, ***, P < 0.0001, ANOVA test. E and

F, reduced metastasis capability of sh-Kir2.1 GC cells in vivo. Data are shown as

mean ± S.D., n=5, **, P < 0.001, Student’s t-test. G, increased invasion capability of

over-Kir2.1 GC cells compared to control (empty vector) GC cells in vitro. Data are

shown as mean ± S.D., n=5, NS, not significant, ***, P < 0.0001, ANOVA test. H and

I, increased metastasis capability of over-Kir2.1 GC cells compared to control (empty

vector) GC cells in vivo. Data are shown as mean ± S.D., n=5, ***, P < 0.0001,

Student’s t-test. J, western blotting showing decreased E-cadherin expression but

enhanced vimemtin and Snail expression in over-Kir2.1 GC cells. K, western blotting

showing up-regulated E-cadherin and down-regulated vimemtin and Snail in

sh-Kir2.1 GC cells.

Figure 3. The pro-malignant effect of Kir2.1 is independent of potassium permeation.

A, similar invasion capability exhibited by GC cells with or without ML133 (20 μM)

treatment. Data are shown as mean ± S.D., n=5, NS, not significant, ***, P < 0.0001,

ANOVA test. B, no changes in the expression of E-cadherin, vimemtin and Snail by

GC cells after treatment with ML133 (20 μM). C, no effect on the invasion capability

of GC cells by Zacopride (10 μM) treatment. Data are shown as mean ± S.D., n=5,

NS, not significant, ***, P < 0.0001, ANOVA test. D, no changes in the expression of

E-cadherin, vimemtin and Snail by GC cells after Zacopride (10 μM) treatment. E,

significantly enhanced invasion capability of GC cells by overexpressing mut-Kir2.1,

with similarly to overexpressing wild type Kir2.1. Data are shown as mean ± S.D.,

n=5, NS, not significant, ***, P < 0.0001, ANOVA test. F, mut-Kir2.1 and

over-Kir2.1 GC cells expressing similar levels of E-cadherin, vimemtin and Snail.

Figure 4. Kir2.1 activates MEK1/2-ERK1/2-Snail-EMT pathway independent of Raf.

A, human phosphokinase antibody array assay showing 11 elevated phosphorylation

kinases (left panel) with ERK1/2 kinases at the highest level (right panel). Data are

shown as mean. B, the over-Kir2.1 GC cells showing enhanced phosphorylation of

ERK1/2 and sh-Kir2.1 GC cells showing attenuated phosphorylation of ERK1/2. JNK

and p38, other two members of MAPKs in GC cells, were unaffected by Kir2.1

expression status. C, decreased invasion capability of GC cells after treatment with

PD98059 (10 μM), a MEK1/2 specific inhibitor. Data are shown as mean ± S.D., n=5,

NS, not significant, ***, P < 0.0001, ANOVA test. D, increased E-cadherin but

decreased vimemtin, Snail and p-ERK1/2 in PD98059 (10 μM) treated GC cells. E,

attenuation of the invasion capability of control GC cells by treatment with

LY3009120 (20 μM), a pan-Raf inhibitor, without effect on over-Kir2.1 GC cells.

Data are shown as mean ± S.D., n=5, ***, P < 0.0001, Student’s t-test. F, attenuation

of the level of p-MEK1/2 and p-ERK1/2 in control GC cells by LY3009120 (20 μM)

treatment, without effect on over-Kir2.1 GC cells.

Figure 5. Kir2.1-Stk38 interaction up-regulates MEKK2 to activate MEK1/2-ERK1/2

in GC cells. A, Co-IP assay showing interaction of Kir2.1 with Stk38 but not with

Rock2. B, decreased expression of MEKK2, p-MEK1/2 and p-ERK1/2 both in

over-Kir2.1 and control GC cells by siMEKK2. C, decreased levels of p-MEKK2 and

non-p-MEKK2 accompanied with decreased p-MEK1/2 and p-ERK1/2 both in

over-Kir2.1 and control GC cells by overexpressing Stk38. D, relative pixel density

showing significantly decreased levels of both p-MEKK2 and non-p-MEKK2 by

overexpressing Stk38, without effect on the ratio between p-MEKK2 and

non-p-MEKK2. Data are shown as mean ± S.D., n=3, *, P < 0.05, Student’s t-test. E,

over-Kir2.1 GC cells showing decreased levels of Smurf1-Stk38 complex in

compared to paired control GC cells. F, overexpressing Kir2.1 blocking ubiquitination

of MEKK2 induced by co-overexpressing Stk38 and Smurf1 in GC cells.

Figure 6. Up-regulation of Kir2.1 in human GC and the correlation with cancer

invasion, lymph node metastasis and outcome of GC patients. A, representative

immunohistochemical images of Kir2.1 in GC specimens; Scale bar =100 μm. Aa,

absence of Kir2.1 staining in normal gastric mucosa. Ab-e, increased Kir2.1 staining

intensity with invasion depth. Af, high Kir2.1 level in both primary tumor and

corresponding metastatic lymph node. B, higher IHC scores of Kir2.1 in 131 GC

tissues compared with paired adjacent normal tissues. Data are shown as mean ± S.D.,

n=131, ***, P < 0.0001, Paired t-test. C, Kir2.1 protein in 6 paired surgically

removed gastric tumor tissues (T) and the adjacent normal tissues (N) showing highly

expressed Kir2.1 in cancerous tissues. D, higher mRNA level of Kir2.1 in cancerous

tissues than in adjacent normal tissues from GEO GES13911 and GES27342. Data are

shown as mean ± S.D., n=31 (GSE13911), n=80 (GSE27342), ***, P < 0.0001, **, P

< 0.001, Paired t-test. E, the relationship between Kir2.1 expression and tumor

invasion depth, and lymph node metastasis of GC. n=349, ***, P < 0.0001,

Chi-square test. F, Kaplan–Meier curves showing the correlation between the levels

of Kir2.1 and the overall survival and progression-free survival of GC patients. n=349

(OS), n= 218 (PFS), Log-rank test.

Kir2.1 interaction with Stk38 promotes invasion and metastasis of human gastric cancer by enhancing MEKK2-MEK1/2-ERK1/2 signaling

Cheng-Dong Ji, Yan-Xia Wang, Dong-Fang Xiang, et al.

Cancer Res Published OnlineFirst March 16, 2018.

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