EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

Abstract

Background: The echinoderm microtubule-associated protein-like-4 anaplastic lymphoma kinase (EML4-ALK) fusion gene was identified in a subset of non-small cell lung cancer (NSCLC) patients. They responded positively to ALK inhibitors. This study aimed to characterize the mechanisms triggered by EML4-ALK to induce NSCLC transformation.

Methods: HEK293 and NIH3T3 cells were transfected with EML4-ALK variant 3 or pcDNA3.1-NC. H2228 cells were transfected with siRNA-EML4-ALK or siRNA-NC. Cell viability and proliferation were measured by the CCK-8 and EdU methods, respectively. Flow cytometry revealed apoptosis. Gene expression profiles were generated from a signaling pathway screen in EML4-ALK-regulated lung cancer cells and verified by qPCR and Western blotting. The co-immunoprecipitation and immunohistochemistry/ immunofluorescence determined the interaction and colocalization of JAK2-STAT pathway components with EML4-ALK.

Results: Microarray identified several genes involved in the JAK2-STAT pathway. JAK2 and STAT6 were constitutively phosphorylated in H2228 cells. EML4-ALK silencing downregulated phosphorylation of STAT6. Expression of EML4-ALK in HEK293 and NIH3T3 cells activated JAK2, STAT1, STAT3, STAT5, and STAT6. In EML4-ALK-transfected HEK293 cells and EML4-ALK-positive H2228 cells, activated STAT6 and JAK2 colocalized with ALK. STAT3 and STAT6 were phosphorylated and translocated to the nucleus of H2228 cells following IL4 or IL6 treatment. Apoptosis increased, while cell proliferation and DNA replication decreased in H2228 cells following EML4-ALK knockdown. In contrast, HEK293 cell viability increased following EML4-ALK overexpression, while H2228 cell viability significantly decreased after treatment with ALK or JAK-STAT pathway inhibitors.

Conclusions: Our data suggest that the aberrant expression of EML4-ALK leads to JAK2-STAT signaling pathway activation, which is essential for the development of non-small cell lung cancer.

Keywords: EML4-ALK; JAK2-STAT pathway; Non-small cell lung cancer; Transformation.

Fig. 1

Microarray analysis of H2228 cells without or with EML4-ALK knockdown and of HEK293 cells expressing or not the EML4-ALK variant 3. a The heat map shows the regulation of a number of JAK2-STAT pathway genes. b Expression of JAK2-STAT pathway-related genes measured by real-time PCR

Fig. 2

Activation status of STAT proteins in EML4-ALK-positive lung cancer. a STAT6 and JAK2 were constitutively phosphorylated in H2228 cells. EML4-ALK knockdown downregulated phosphorylation of JAK2 and STAT6 in H2228 cells. b colocalization of ALK, p-STAT6, and p-JAK2 staining in primary tumor tissue. c Representative images of ALK,p-JAK2 and p-STAT6 expression by immunohistochemical analysis in the primary tumors and metastatic tumors of EML4-ALK-positive lung cancer patients

Fig. 3

EML4-ALK transfection activated JAK2, STAT1, STAT3, STAT5, and STAT6 in HEK293 and NIH3T3 cells

Fig. 4

Interactions between components of the JAK2-STAT pathway and EML4-ALK. a Active STAT6 and JAK2 colocalized with ALK in EML4-ALK-transfected HEK293 cells and in H2228 cells. b: ALK and phosphorylated STAT6 protein were co-immunoprecipitated with JAK2 in EML4-ALK-transfected HEK293 cells

Fig. 5

IL4 and IL6 affected the JAK2-STAT pathway in EML4-ALK-positive cells. a STAT3 and STAT6 were phosphorylated and translocated to the nuclei of H2228 cells following IL4 or IL6 treatment. b Microarray analysis of H2228 cells stimulated or not with IL4. The heat map identifies modified proteins associated with the JAK2-STAT pathway, the regulation of the actin cytoskeleton, the cell cycle, cell adhesion, and the positive regulation of cell proliferation

Fig. 6

Effect of oncogenic EML4-ALK tyrosine kinase on the biological behaviors of lung cancer cells. a EML4-ALK levels in transfected H2228 and HEK293 cells. b Increased apoptosis in H2228 cells following EML4-ALK knockdown measured by flow cytometry. c Cell viability, measured by the CCK8 assay, was increased by EML4-ALK expression in HEK293 cells. d–e Viability and proliferation of H2228 cells decreased after knockdown of EML4-ALK or STAT6. f H2228 cell viability following treatment with ALK or JAK-STAT pathway inhibitors as measured by the CCK-8 assay. *P < 0.05