The WNK1-ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib

Abstract

Background: The dismal prognosis of advanced ovarian cancer calls for the development of novel therapies to improve disease outcome. In this regard, we set out to discover new molecular entities and to assess the preclinical effectiveness of their targeting.

Methods: Cell lines, mice and human ovarian cancer samples were used. Proteome profiling of human phosphokinases, in silico genomic analyses, genetic (shRNA and CRISPR/Cas9) and pharmacological strategies as well as an ex vivo human preclinical model were performed.

Results: We identified WNK1 as a highly phosphorylated protein in ovarian cancer and found that its activation or high expression had a negative impact on patients' survival. Genomic analyses showed amplification of WNK1 in human ovarian tumours. Mechanistically, we demonstrate that WNK1 exerted its action through the MEK5-ERK5 signalling module in ovarian cancer. Loss of function, genetic or pharmacological experiments, demonstrated anti-proliferative and anti-tumoural effects of the targeting of the WNK1-MEK5-ERK5 route. Additional studies showed that this pathway modulated the anti-tumoural properties of the MEK1/2 inhibitor trametinib. Thus, treatment with trametinib activated the WNK1-MEK5-ERK5 route, raising the possibility that this effect may limit the therapeutic benefit of ERK1/2 targeting in ovarian cancer. Moreover, in different experimental settings, including an ex vivo patient-derived model consisting of ovarian cancer cells cultured with autologous patient sera, we show that inhibition of WNK1 or MEK5 increased the anti-proliferative and anti-tumour efficacy of trametinib.

Conclusions: The present study uncovers the participation of WNK1-MEK5-ERK5 axis in ovarian cancer pathophysiology, opening the possibility of acting on this pathway with therapeutic purposes. Another important finding of the present study was the activation of that signalling axis by trametinib, bypassing the anti-tumoural efficacy of this drug. That fact should be considered in the context of the use of trametinib in ovarian cancer.

Keywords: ERK5; MEK5; WNK1; ovarian cancer; patient biopsies; trametinib.

FIGURE 1

Identification of WNK1 as an active kinase in ovarian cancer. Impact on patient survival. (A) 300 μg of cell lysate from the four ovarian cancer cell lines were hybridised to the phosphokinase array following manufacturer's instructions. The immunodetected signals corresponding to the amount of phosphorylated protein bound were quantified and represented as indicated in Methods section. (B) 70 μg of whole‐cell lysates were used to detect WNK1 and pWNK1 expression in the four cell lines by Western blotting. Calnexin was used as loading control. (C) Box plots representing the levels of pWNK1 in 63 epithelial ovarian cancer samples obtained from 57 patients from the University Hospital of Salamanca, which were quantified from Western blot analysis (shown in Figure S1) using the ImageJ software. The red line represents the threshold value (10 a.u.) selected for sorting the patients into the high or low pWNK1 expression groups. The number of samples from each subtype and the percentage belonging to the high expression cohort are shown. OSC, ovarian serous carcinoma; SBT, serous borderline tumour. (D) Kaplan–Meier curve of the above patients with available clinical data (n = 52), comparing the overall survival of those expressing high levels of pWNK1 (n = 16, red line) with those expressing low levels (n = 36, black line). The median overall survival of each cohort and p value are shown.

FIGURE 2

Genomic analysis of WNK1 and relevance of its dysregulation in ovarian cancer patient's outcome. (A) Firebrowse bioinformatic tool was used for the identification of differentially expressed marker focal events in the ovarian cancer cystadenocarcinoma dataset (n = 579 samples), using Student's t‐test. The top 28 focal level copy number alterations were ranked and represented from lowest to highest frequency. (B) The cBioPortal database was explored to determine gains, high‐level amplifications as well as upregulated mRNA levels of WNK1 gene in ovarian cancer patients. The data were collected, quantified and represented as percentage from the total patients with available data included in the study, as indicated in Methods section. (C) Relationship between WNK1 copy number alterations (CNA) and WNK1 mRNA expression of ovarian cancer samples collected in the TCGA Firehose Legacy dataset. The Pearson correlation coefficient between these variables and the tendency line (red line) are shown. The copy number datasets were generated by the GISTIC algorithm. (D) Overall survival curves of those ovarian serous carcinoma patients from the TCGA Firehose Legacy dataset with available RNAseq mRNA expression and clinical data (n = 303). Patients harbouring WNK1 mRNA upregulated levels (red line) were compared to those without such alteration (blue line). The p value of the study, follow‐up (months) and median overall survival of each cohort are indicated. (E) 120 months follow‐up Kaplan–Meier analysis of the relationship between WNK1 mRNA expression levels and overall survival in ovarian cancer patients (n = 373) collected in the RNA‐seq section of the Kaplan–Meier plotter database. The p value, hazard ratio, median overall survival and number of patients at risk in the low and high expression groups are indicated; FDR = 0.20.

FIGURE 3

Anti‐proliferative effect of WNK1–ERK5 axis knockdown. (A) Cells were infected with a pLKO shControl sequence or two different WNK1 specific shRNA sequences. 70 μg of cell extracts were used to determine the WNK1 knockdown by Western blotting. Calnexin was used as loading control. Dashed line indicates that lanes were cut out from immunoblots. (B) The effect of WNK1 knockdown (sh18 and sh21 or sh86) on cell proliferation was measured at 3 days of culture by the MTT assay. Data are presented as the mean ± SD of an experiment that was repeated three times. *, p ≤ .05; ***, p ≤ .001. (C) 70 μg of whole‐cell lysates were used to detect MEKK2 and MEK5 expression in the four ovarian cancer cell lines by Western blotting with their corresponding antibodies. ERK5 activation was analysed by immunoprecipitating 1 mg of protein followed by Western blotting with the anti‐ERK5 or anti‐pERK5 antibodies. Calnexin was used as loading control. (D) OVCAR8 cells infected with pLKO shControl or sh18 were lysed and protein levels were evaluated on 70 micrograms of cell extracts by Western blotting with their corresponding antibodies. ERK5 and pMEK5 were analysed by immunoprecipitating 1 mg of protein followed by Western blotting with anti‐ERK5 or anti‐pMEK5. Calnexin was used as loading control. (E) Knockdown of MEKK2, (F) MEK5 or (G) ERK5 was carried out by lentiviral infection with specific shRNA sequences. MEKK2, MEK5 (70 μg of cell extracts) and ERK5 (1 mg of immunoprecipitated protein) were analysed by Western blotting with the appropriate antibodies. Calnexin was used as loading control. (H) The effect of MEKK2, MEK5 and ERK5 knockdown on cell proliferation was measured at 3 days of culture by the MTT assay. Data are presented as the mean ± SD of an experiment that was repeated three times. **, p ≤ .01; ***, p ≤ .001.

FIGURE 4

Tumour growth inhibition caused by MEK5 knockout. (A) Western blot showing lack of MEK5 expression in the different OVCAR8 MEK5 CRISPR clones when compared with the control OVCAR8 scramble cells (Sc). ERK5 expression was analysed by immunoprecipitating 1 mg of protein followed by Western blotting with the anti‐ERK5 antibody. MEK1/2 was detected by Western blotting on 70 μg of cell extracts. Calnexin was used as loading control. Note: A lane was cut out from the immunoblot. (B) Effect of MEK5 knockout on the cell proliferation of OVCAR8 cells. Control OVCAR8 Sc cells or OVCAR8 CRISPR MEK5 clones were plated in p6 wells and cultured for 3 days. The proliferation rate was measured by cell counting and represented as percentage from control OVCAR8 Sc cells. Data are presented as the mean ± SD of an experiment that was repeated three times. ***, p ≤ .001. (C) Tumour growth evolution of the mice xenoinjected with the OVCAR8 Sc cells or MEK5 CRISPR cells (#16 and #19 clones) (n = 12 tumours, per group). The mean tumour volume and SEM error bars of each group are represented. The experiment was stopped when mice tumours from the Sc group reached a volume of 420 mm³. The significative p values between groups are indicated (***, p ≤ .001). Data in the inset table indicate the volume percentage of MEK5 CRISPR tumours when compared with scramble tumours. Representative pictures of the tumour size of each group are shown. (D) Western blot showing MEK5 and ERK5 expression in tumours collected at the end of the in vivo experiment. (E) Representative Ki67 immunostaining 20x pictures of tumoural tissue from each condition (scramble and MEK5 CRISPR clones). (F) Quantitation of Ki67 immunostaining from 8 to 10 different images of each condition is represented as percentage of Ki67 positive cells with respect to total. The average percentage (dotted red line), ± SD and p value (***, p ≤ .001) are indicated for each MEK5 CRISPR clone.

FIGURE 5

Impact of pERK1/2 on patient survival. Anti‐tumoural efficacy of double pathway (ERK5 and ERK1/2) blockade. (A) MEK1/2, pMEK1/2, ERK1/2 and pERK1/2 expression were determined by Western blotting. (B) Levels of pERK1/2 in 63 epithelial ovarian cancer samples quantified from immunoblots in Figure S1 using the ImageJ software. The red line represents the threshold value (4 a.u.) selected for sorting the patients into the high or low pERK1/2 expression groups. (C) Kaplan–Meier curve of the above patients with available clinical data (n = 52), comparing the overall survival of those expressing high levels of pERK1/2 (n = 19, red line) with those expressing low levels (n = 33, black line). (D) OVCAR8 cells were treated with trametinib for 4 h and pERK1/2, ERK1/2 and ERK5 expression determined by Western blotting. (E) pERK1/2 and pERK5 levels were quantified from the previous immunoblots (in D) by using the Image Lab software and represented as percentage from control untreated cells. (F) OVCAR8 cells were plated in p6 wells and treated with trametinib and BIX02189, individually and combined, for 3 days. Cell proliferation, measured by cell counting, was represented as percentage from OVCAR8 untreated cells. Data are presented as the mean ± SD of an experiment that was repeated three times. ***, p ≤ .001. (G) OVCAR8 scramble cells and OVCAR8 MEK5 CRISPR clones (#16 and #19) were plated in 24‐well dishes, and 24 h later treated with the indicated doses of trametinib for 48 h. Cell proliferation was measured by an MTT assay, and each condition was represented as percentage from their respective untreated cells. Results are expressed as mean ± SD of an experiment that was repeated twice. ***, p ≤ .001. (H) Mice xenografted with OVCAR8 Sc or MEK5 CRISPR cells were divided into two groups once they reached an initial mean volume of approximately 500 mm³. Each of them received 100 μL of trametinib (0.5 mg/kg) or vehicle, administered i.p. daily for 5 weeks. Tumour progression of Sc and CRISPR groups were measured weekly. At the end point of the experiment, the tumour volume of the trametinib treated mice were relativised to their corresponding untreated (vehicle) groups *, p ≤ .05; **, p ≤ .01; ***, p ≤ .001. (I) Scramble tumours from the vehicle and trametinib treated mice were resected and processed as described in the methods section. pERK1/2 and ERK5 were determined by Western blotting. (J) pERK1/2 (left panel) and pERK5 (right panel) levels from the previous immunoblots (in I) were quantified using the Image Lab software. Comparison of pERK1/2 levels in the scramble tumours treated with vehicle or trametinib was represented as arbitrary units. Comparison of pERK5 expression between groups was represented as percentage of total ERK5 (sum of upper and lower bands). The red lines represent the mean expression values for each group. *, p ≤ .05.

FIGURE 6

Anti‐tumoural effect of MEK5 and MEK1/2 targeting in a three‐dimensional model utilising patient biopsies. (A) Ovarian cancer patient tumour biopsies and plasma samples were obtained from ovarian cancer patients. Tissue biopsies were enzymatically digested and grown in HuP3D cultures made from the matching patient plasma. HuP3D cultures were treated with trametinib and BIX02189 and further evaluated by flow cytometry in terms of mean fluorescence intensity (MFI) expression of pERK1/2 and pERK5 as well as survival. (B) Gating strategy for analysis of patient biopsy material grown in HuP3D cultures. Data acquisition was completed by collecting information for a specified number of events determined by counting beads. Firstly, cellular populations were isolated from beads and then singlets were gated. After that, the live cell population was identified by live/dead viability marker. Following this, CD45− BV510 cells were identified from the live cell population. Finally, EpCAM+ (CD326+) PE‐Cy7 cells and FAP− AF700 cells were identified from the CD45− population. Fluorescence minus one (FMO) controls were used to set the gating for each population BV510 (CD45), PE‐Cy7 (EpCAM) and AF700 (FAP). (C) pERK1/2 expression by EpCAM+ cells from ovarian cancer patients (#1–3) growing within HuP3D cultures after DMSO control (Untreated) and trametinib treatment, quantified as MFI ratio between PE‐anti‐pERK1/2 and FMO PE control. Results are expressed as mean ± SD of an experiment that was repeated three times. An interquartile range (IQR) was used to remove outliers. *, p ≤ .05; **, p ≤ .01. (D) Representative flow cytometry histogram of PE pERK1/2 expression of EpCAM+ cells from patient #1 after DMSO control (untreated) and trametinib treatment, with PE FMO and activated controls, as negative and positive controls, respectively. Vertical line is included to indicate the pERK1/2 positive population defined by FMO control. (E) pERK5 expression by EpCAM+ cells from ovarian cancer patients (#1–3) growing within HuP3D cultures after DMSO control (untreated) and trametinib treatment, quantified as MFI ratio between AF488‐anti‐pERK5 and AF488 FMO control. Results are expressed as in (C). (F) Representative flow cytometry histogram of pERK5 expression (AF488) of EpCAM+ cells from patient #1 after DMSO control (untreated) and trametinib treatment, with AF488 FMO and activated controls, as negative and positive controls, respectively. Vertical line is included to indicate the pERK5 positive population defined by FMO control. (G) Effect of DMSO control (untreated), trametinib alone or trametinib in combination with BIX02189 on primary ovarian cancer EpCAM+ cell survival in HuP3D cultures from patients #1 to 3. Results are expressed as in (C). *, p ≤ .05; **, p ≤ .01. ***, p ≤ .001. (H) Representative flow cytometry histogram of pERK1/2 (PE) and pERK5 expressions (AF488) of EpCAM+ cells from patient #2 after DMSO control (untreated), trametinib alone or trametinib in combination with BIX02189.