Stress Response Kinase MK2 Induces Non-canonical Activation of EphA2 in EML4-ALK Lung Cancer Cells

Abstract

The non-canonical phosphorylation of the receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) at Ser-897 plays crucial roles in tumor progression in a tyrosine kinase-independent manner. This phosphorylation is catalyzed by p90 ribosomal S6 kinase (RSK), a kinase downstream of extracellular signal-regulated kinase (ERK). We recently reported that stress-responsive kinase mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), instead of ERK, regulates RSK under cellular stress conditions; however, the function of MK2 in ERK-activated cells is still unknown. We herein clarified that MK2 regulates the RSK-EphA2 axis in ERK-activated echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) lung cancer cells. In addition, an MK2 inhibitor blocked enhancements in cell motility induced by the constitutively activated RSK-EphA2 axis. The present results reveal the importance of MK2 in the ERK-activated non-canonical activation of EphA2.

INTRODUCTION

The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) is highly expressed in various types of cancer cells, including lung cancer.^1,2) Similar to other receptor tyrosine kinases (RTKs), ligand-dependent canonical activation triggers tyrosine autophosphorylation. The canonical activation of RTKs causes the malignant transformation of cancer; however, in the case of EphA2, it exerts a tumor-suppressive effect.^1,3) On the other hand, the non-canonical Ser-897 phosphorylation of EphA2 (pS-EphA2) via intracellular signals independent of ligands and TK activity promotes the malignant progression of cancer, such as cancer cell motility, epithelial-to-mesenchymal transition (EMT), and drug resistance.^1,4,5) We previously identified p90 ribosomal S6 kinase (RSK) as the critical kinase responsible for this phosphorylation. We also reported that ERK signaling induced the RSK-EphA2 axis in cultured lung cancer cells harboring oncogenic mutations in the ALK, EGFR, and KRAS genes. Furthermore, immunohistochemistry revealed that the matched expression of pS-EphA2 and activated RSK correlated with poor survival in lung adenocarcinoma patients.⁵⁾

We recently revealed an alternative upstream signal of the RSK-EphA2 axis, in which the cellular stress-induced p38–MK2 pathway triggered the activation of RSK.⁶⁾ This p38–MK2–RSK–EphA2 pathway promoted glioblastoma cell migration induced by temozolomide, an alkylating chemotherapeutic agent for the treatment of glioblastoma patients. However, the roles of p38 and MK2 in the RSK-EphA2 axis of ERK-activated cancer cells remain unknown. Therefore, we herein attempted to clarify the effects of these stress kinases in EML4-ALK-positive lung cancer cells.

MATERIALS AND METHODS

Antibodies and Reagents

Total antibodies against EphA2 (#6997) and ALK (#3633) and phospho-specific antibodies against EphA2 (Ser-897; #6347), RSK1 (Ser-380; #11989), ALK (Tyr-1604; #3341), and ERK (Thr-202/Tyr-204; #9101) were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.); antibodies against total ERK (C-9), RSK1 (C-21), MK2 (A-11), and β-actin (C-4) were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.); MK2 IN-1 and MK2 IN-3 were from MedChemExpress (Monmouth Junction, NJ, U.S.A.); BI-D1870 was from BioVision (Milpitas, CA, U.S.A.); VX-702, LJH685, ALW-II 41-27, crizotinib, and alectinib were from Selleck Chemicals (Houston, TX, U.S.A.); doramapimod was from Cayman Chemical (Ann Arbor, MI, U.S.A.). All chemical inhibitors were dissolved in dimethyl sulfoxide (Wako Pure Chemical Corporation, Osaka, Japan).

Cell Cultures

H2228, HEK293, and A549 cells were purchased from the ATCC (Manassas, VA, U.S.A.). H3122, A925L, and crizotinib-resistant A925L (CR) cells were previously established.^7,8) H2228, H3122, A925L, CR, and A549 cells were cultured in RPMI medium (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (Merck KGaA, Darmstadt, Germany), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, U.S.A.), 100 U/mL penicillin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and 100 μg/mL streptomycin (Meiji Seika Pharma Co., Ltd.). CR cells were maintained in the presence of 1 μM crizotinib. HEK293 cells were cultured in Dulbecco’s Modified Eagle’s medium (Nissui) supplemented with 10% fetal calf serum (Merck KGaA), 4 mM L-glutamine (Thermo Fisher Scientific), 100 U/mL penicillin (Meiji Seika Pharma Co., Ltd.), and 100 μg/mL streptomycin (Meiji Seika Pharma Co., Ltd.). All cells were cultured at 37 °C in 5% CO₂.

Immunoblotting

Cell lysates were prepared as previously described⁶⁾ and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins in gels were transferred to an Immobilon-P nylon membrane (Merck KGaA). The membrane was treated with BlockAce (KAC Co., Ltd., Kyoto, Japan) and probed with the primary antibody (1 : 1000–1 : 2000) at room temperature. Antibodies were detected using horseradish peroxidase-conjugated anti-rabbit, anti-goat, or anti-mouse immunoglobulin G (1 : 2000, DAKO, Glostrup, Denmark) diluted in Can Get Signal solution (TOYOBO, Osaka, Japan) or phosphate buffered saline containing 0.1% Tween 20 (Wako Pure Chemical Corporation). Bands were detected with an enhanced chemiluminescence system (Thermo Fisher Scientific). Full scans of immunoblotting images are shown in Supplementary Fig. 2.

Transfection of Plasmid DNAs

The expression vectors for EML4-ALK variants were previously generated.⁹⁾ Full-length human MK2 cDNA was synthesized from mRNA isolated from A549 cells. mRNA was extracted using a FastGene Premium kit (NIPPON Genetics, Tokyo, Japan) and cDNA was synthesized using the Prime Script RT reagent kit with gDNA eraser (TaKaRa, Kusatsu, Japan) in accordance with the manufacturer’s instructions. The MK2 cDNA was cloned into pcDNA3.1 (Thermo Fisher Scientific) using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs, Ipswich, MA, U.S.A.). A KOD -Plus- Mutagenesis Kit (TOYOBO) was used to replace the 349th histidine of MK2 with an alanine to generate moderately activated MK2.¹⁰⁾ in accordance with the manufacturer’s instructions HEK293 cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions.

Migration Assay

When a confluent monolayer of CR cells formed on culture dishes, a scratch track was introduced with a pipette tip. The medium was then pumped out and fresh medium was added. After 24 h, cell migration of 5 points was observed using an Olympus microscope (CKX53, Olympus Corporation, Tokyo Japan), and the migration distance was calculated by ImageJ/Fiji software (National Institute of Health, Bethesda, MD, U.S.A.). Analysis was carried out several times and representative results are shown. The significance of differences was analyzed by the Student’s t-test. p-Values < 0.05 were considered to be significant.

RESULTS

EphA2 Phosphorylation at Ser-897 Is Induced in Different Variants of EML4-ALK

Variants of EML4-ALK possess different properties in cancer cells, such as their intracellular localization and response to ALK-tyrosine kinase inhibitors (TKIs)^11–15); therefore, the states of the non-canonical activation of EphA2 may differ among different types of variants. We previously reported that pS-EphA2 was constitutively induced in H2228, a cell line that expresses variant 3a of the EML4-ALK fusion gene.⁵⁾ We herein compared the activities of 3 major EML4-ALK variants, variants 1, 3a, and 5a, expressed in H3122, H2228, and A925L lung cancer cells, respectively. The expression of EphA2 and phosphorylation of EphA2 at Ser-897 were detected in the 3 cell lines, with the highest expression in A925L cells (Figs. 1A and 1B). The ALK-TKIs crizotinib and alectinib inhibited pS-EphA2 (Figs. 1B and 1C). In addition, the overexpression of EML4-ALK variants in HEK293 cells demonstrated that all variants triggered the phosphorylation of ALK at Tyr-1604 as well as the subsequent phosphorylation of EphA2 at Ser-897 (Fig. 1D). These results indicate that the non-canonical activation of EphA2 was elicited regardless of the type of EML4-ALK variant.

Fig. 1. EphA2 Phosphorylation at Ser-897 Is Induced in Different Variants of EML4-ALK Lung Cancer Cells

(A) Whole-cell lysates of H3122 cells, H2228 cells, and A925L cells were immunoblotted with primary antibodies against EphA2 and β-actin. (B–D), H3122 cells, H2228 cells, and A925L cells were treated with 10 μM crizotinib (B) or alectinib (C) for 1 h. (D) HEK293 cells were transfected with the expression vector for EML4-ALK variant 1, variant 3a, variant 5a, or an empty vector. At 24 h post-transfection, cells were stimulated with 10 μM crizotinib for 1 h. Whole-cell lysates were immunoblotted with primary antibodies against phospho-EphA2 at Ser-897 (pS-EphA2), EphA2, phospho-ALK at Tyr-1604 (pY-ALK), ALK, and β-actin.

We previously reported that EML4-ALK induced pS-EphA2 by RSK in H2228 cells.⁵⁾ Similarly, pS-EphA2 was suppressed by the RSK inhibitors BI-D1870 and LJH685 without reductions in ALK TK activity in variant 5a-expressing A925L cells (Fig. 2A). Moreover, variant 5a-mediated pS-EphA2 was clearly suppressed by LJH685 in HEK293 cells (Fig. 2B), indicating that RSK is responsible for pS-EphA2 in EML4-ALK lung cancer cells.

Fig. 2. RSK Regulates pS-EphA2 in EML4-ALK Lung Cancer Cells

A925L cells were treated with 10 μM BI-D1870 or 50 μM LJH685 for 1 h (A). HEK293 cells were transfected with the expression vector for EML4-ALK variant 5a or an empty vector (B). At 24 h post-transfection, cells were stimulated with 50 μM LJH685 for 1 h. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pY-ALK, ALK, and β-actin.

MK2, but Not p38, Regulates the RSK–EphA2 Pathway in EML4-ALK Lung Cancer Cells

We previously indicated that cellular stress induced the p38–MK2 pathway to activate the RSK–EphA2 pathway.⁶⁾ To investigate the roles of p38 and MK2 in EML4-ALK signaling, A925L cells were treated with the p38 inhibitors doramapimod and VX-702 and the MK2 inhibitor MK2 IN-3. Figure 3A shows that p-RSK and pS-EphA2 were not suppressed by either of the p38 inhibitors; however, the MK2 inhibitor blocked the phosphorylation of both without inhibiting ALK-TK or ERK activity (Fig. 3A). Another MK2 inhibitor MK2 IN-1 also suppressed pS-EphA2 (Supplementary Fig. S1). To investigate the involvement of MK2 in the EML4-ALK-mediated RSK–EphA2 pathway, EML4-ALK variant 5a was co-expressed with a moderately active MK2 mutant in HEK293 cells. As shown in Fig. 3B, RSK was more strongly phosphorylated by its co-expression with MK2 than with the single expression of EML-ALK, resulting in the stronger phosphorylation of EphA2 at Ser-897. In addition, MK2 IN-3 suppressed pS-EphA2 in lung cancer cells expressing other variants (Fig. 3C). Collectively, these results show that MK2, but not p38, activated the RSK–EphA2 pathway in EML4-ALK-positive lung cancer cells.

Fig. 3. MK2, but Not p38, Regulates the RSK–EphA2 Pathway in EML4-ALK Lung Cancer Cells

(A) A925L cells were treated with 10 μM doramapimod, VX-702, or MK2 IN-3 (MK2i) for 1 h. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, and phospho-RSK1 at Ser-380 (p-RSK), RSK1, pY-ALK, ALK, and phospho-ERK at Thr-202/Tyr-204 (p-ERK), ERK, and β-actin. (B) HEK293 cells were transfected with the expression vectors for EML4-ALK variant 5a, moderately active MK2, and/or an empty vector. At 24 h post-transfection, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, p-RSK, RSK1, MK2, pY-ALK, ALK, and β-actin. (C) H3122 cells and H2228 cells were treated with 10 μM MK2 IN-3 for 1 h and cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, and β-actin.

The MK2–RSK–EphA2 Pathway Induces the Migration of ALK-TKI-Resistant Cells

Since we previously demonstrated that the p38-MK2-RSK-EphA2 axis plays an important role in cell migration,⁶⁾ we herein investigated whether the MK2-RSK-EphA2 axis also regulates the migration of EML4-ALK cancer cells. Despite the low migratory ability of A925L parent cells, a crizotinib-resistant clone (CR) with a mesenchymal phenotype was found to acquire high motility.⁸⁾ Similar to parent cells, constitutive pS-EphA2 in CR cells was blocked by the RSK and MK2 inhibitors (Figs. 4A and 4B). The scratch assay demonstrated that the EphA2 inhibitor ALW-II 41-27 blocked both pS-EphA2 and cell migration, indicating the regulation of CR cell migration by the non-canonical activation of EphA2 (Figs. 4C and 4D). In addition, the RSK inhibitor BI-D1870 and MK2 inhibitor MK2 IN-3 exerted suppressive effects on cell migration (Figs. 4E and 4F). These results demonstrate that MK2-mediated RSK-EphA2 activation promoted the migration of ALK-TKI-resistant cells.

Fig. 4. The MK2–RSK–EphA2 Pathway Induces the Migration of ALK-TKI-Resistant Cells

(A, B) A925L cells and CR cells were treated with 10 μM BI-D1870, 50 μM LJH685 (A), or 10 μM MK2 IN-3 (MK2i) (B) for 1 h. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, and β-actin. (C) CR cells were treated with 1 μM ALW-II 41-27 for 4 h. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, and β-actin. (D–F) A confluent monolayer of CR cells was scratched and the culture medium was then replaced with fresh medium supplemented with 0.25 μM ALW-II 41-27 (D), 10 μM BI-D1870 (D), or 10 μM MK2 IN-3 (D). After 24 h, cell migration was observed, and the migration distance was calculated. p = 0.00688 (D), 2.43e-05 (D), 0.00199 (D). *p-Values < 0.05 by Student’s t-test were considered to be significant.

DISCUSSION

Differences have been reported in the intracellular localization and TKI response of each EML4-ALK variant.^11–15) However, in our transfection experiments on HEK293 cells, pS-EphA2 was induced in all variants equally and was blocked by ALK-TKI (Fig. 1), indicating that pS-EphA2 was not involved in the localization of EML4-ALK or TKI responses. We also found that the RSK–EphA2 pathway was activated in lung cancer cells harboring variants of EML4-ALK fusion genes, and MK2, instead of p38, induced cell migration by promoting the RSK–EphA2 pathway (Figs. 3A and 3C). The present results revealed that MK2 contributed to the activation of the RSK-EphA2 axis in ERK-activated EML4-ALK lung cancer cells.

Various factors, such as EGFR activation and EMT, have been shown to trigger ALK-TKI resistance.^13,16–20) Upon exposure to crizotinib, epithelial A925L cells changed to a mesenchymal phenotype in resistant CR cells.⁸⁾ pS-EphA2 has been shown to trigger EMT and accelerate mesenchymal cell motility.¹⁾ A previous study reported that EphA2 activated the Wnt/β-catenin pathway by up-regulating the transcription factor c-Myc to induce EMT in gastric cancer cells.²¹⁾ Other groups reported that microRNA-338 regulated the expression of EphA2, which induced EMT through the Wnt/β-catenin/c-Myc pathway.²²⁾ MK2 has also been shown to promote EMT in a c-Myc-dependent manner in nasopharyngeal carcinoma cells.²³⁾ It is important to note that pS-EphA2 and MK2 both induce the expression of c-Myc; therefore, the MK2-RSK-EphA2 axis may regulate the expression of c-Myc to promote EMT in CR cells. Further studies are needed to clarify the relationship between the MK2–RSK–EphA2 pathway and c-Myc in the process of EMT and drug resistance.

A limitation of the present study is that the molecules inducing the activation of MK2 remain unknown. Unexpectedly, inhibitors of p38, a known upstream kinase of MK2,^24–26) did not block the RSK-EphA2 axis (Fig. 3), suggesting a novel mechanism for the regulation of MK2 activation in EML4-ALK lung cancer cells. Therefore, further studies are needed to clarify whether MK2 regulates pS-EphA2 in lung cancers with other typical oncogenic mutations in the EGFR and KRAS genes. In summary, the present results demonstrated that MK2 activated the non-canonical activation of EphA2 in EML4-ALK lung cancer cells, which provides an effective strategy for an intervention of this pathway to prevent the malignant progression of cancer.

Funding

This work was supported by JSPS KAKENHI Grants (Nos. 19K23795 [Y. Z.], 22K06612 [Y. Z.], and 23K24026 [H. S.]), JST Moonshot R&D Grant (No. JPMJMS2021 [H. S.]), the MSD Life Science Foundation, Public Interest Incorporated Foundation (Y. Z.), Pharmacodynamics Research Foundation (Y. Z.), Takeda Science Foundation (Y. Z. and H. S.), and the Extramural Collaborative Research Grant of Cancer Research Institute, Kanazawa University (Y. Z., Seiji Yano, and H. S.).

Author Contributions

F. Z., Y. Z., N. H., and A. T., investigation and analysis; Y. Z., K. M., and H. M., methodology; F. Z. and Y. Z. writing—original draft; F. Z. English proofreading; Satoru Yokoyama and H. S. supervision; Y. Z. and H. S. conceptualization, and project administration; Y. Z., Seiji Yano, and H. S. funding acquisition; N.H., A. T., Satoru Yokoyama, Seiji Yano, K. M., H. M., and H. S. writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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