MEK5 promotes lung adenocarcinoma

Abstract

MEK5 acts as an oncogenic driver in mice lung cancer and is pivotal for human lung adenocarcinoma http://ow.ly/M9e830mZb8N

FIGURE 1

a) Schematic representation of MEK5 and the sites mutated to create constitutively active MEK5 (MEK5DD). b) The indicated amounts of pCEFL-Flag-MEK5DD were transfected into HeLa cells. ERK5 and MEK5DD were evaluated by Western blotting. c) Expression of MEK5DD in different transgenic mice tissues was evaluated by Western blotting. Note: a lane between lung and kidney was cut out from the Western blots. d) Representative macroscopic image of a MEK5DD transgenic tumoral lung (tumours are indicated by arrows). e) Representative haematoxylin–eosin staining of a lung mass section from a MEK5DD mouse. f) 40× representative immunohistochemical images of napsin-A, cytokeratin (CK)-7 and thyroid transcription factor (TTF)-1 of a lung adenocarcinoma from MEK5DD mouse. An inset image with an isotype control for each antibody was included. g) Western blot analysis of MEK5DD and ERK5 expression in the TG (transgenic) lung tumour compared to NT (nontransgenic) lung from a littermate mouse. h) pERK5 (left) and total ERK5 (right) levels from NT lungs (n=8) versus TG tumoral lungs (n=8) were quantitated from Western blot analysis using ImageJ software and represented in a box plot. The median value for each group is represented as the central line of the box. Black dots represent the outlier values. Statistical comparisons were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) by calculating the p-value according to a two-sided t-test. pERK5 levels were represented as percentage from total ERK5 expression. i) Representative Western blot analyses of MEK5, pMEK5 and ERK5 expression in human lung adenocarcinomas compared to healthy lung tissue (numbers correspond to the tissue bank classification of each patient). N: normal; T: tumour. j) Comparison between MEK5 levels (left panel) or ERK5 levels (right panel) from the total 34 human lung samples. MEK5 and ERK5 expression was quantitated as in figure 1h. k) 120 months follow-up Kaplan–Meier analyses of the relationship between combined MEK5 and ERK5 expression and overall survival in lung adenocarcinoma patients (n=720) collected in the public Kaplan–Meier plotter database. The studies were performed using the multigene classifier tool by selecting the combined mean expression values for both MEK5 (Affymetrix probe id 211370_s_at) and ERK5 (Affymetrix probe id 35617_at) genes on the 2015 version of the database. The cut-off value used to split patients into low or high expression was automatically computed by selecting the “best cut off” tool of the database. l) Representative immunohistochemical analysis of the cellular location of ERK5 in human lung adenocarcinoma. m) NCI-H23 cells were infected with pLKO lentiviral vectors including short hairpin (sh) control (shC), shMEK5 or shERK5 interfering sequences. Protein expression levels were evaluated by Western blotting (top) and the proliferation (bottom) measured by cell counting. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Saliv Gl: salivary gland; CANX: calnexin; au: arbitrary units; HR: hazard ratio. ***: p<0.001.