MST1/Hippo promoter gene methylation predicts poor survival in patients with malignant pleural mesothelioma in the IFCT-GFPC-0701 MAPS Phase 3 trial

Abstract

Background: The Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS/NCT00651456) phase 3 trial demonstrated the superiority of bevacizumab plus pemetrexed-cisplatin triplet over chemotherapy alone in 448 malignant pleural mesothelioma (MPM) patients. Here, we evaluated the prognostic role of Hippo pathway gene promoter methylation.

Methods: Promoter methylations were assayed using methylation-specific polymerase chain reaction in samples from 223 MAPS patients, evaluating their prognostic value for overall survival (OS) and disease-free survival in univariate and multivariate analyses. MST1 inactivation effects on invasion, soft agar growth, apoptosis, proliferation, and YAP/TAZ activation were investigated in human mesothelial cell lines.

Results: STK4 (MST1) gene promoter methylation was detected in 19/223 patients tested (8.5%), predicting poorer OS in univariate and multivariate analyses (adjusted HR: 1.78, 95% CI (1.09-2.93), p = 0.022). Internal validation by bootstrap resampling supported this prognostic impact. MST1 inactivation reduced cellular basal apoptotic activity while increasing proliferation, invasion, and soft agar or in suspension growth, resulting in nuclear YAP accumulation, yet TAZ cytoplasmic retention in mesothelial cell lines. YAP silencing decreased invasion of MST1-depleted mesothelial cell lines.

Conclusions: MST1/hippo kinase expression loss is predictive of poor prognosis in MPM patients, leading to nuclear YAP accumulation and electing YAP as a putative target for therapeutic intervention in human MPM.

Fig. 1

a Flow chart. b Kaplan–Meier curve of overall survival according to the MST1 promoter status (methylated or unmethylated)

Fig. 2

MST1 depletion causes morphological changes. a–d MST0-211H and H2452 cells were transfected with siNeg, siMST1, or siMST1+pcDNAMST1, analysed 48 h after transfection. Expression of MST1 was analysed by reverse transcription-quantitative real-time PCR (RT-qPCR) (a) and western blot (b), using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Quantification of number of cytoplasmic extensions and their size (µm) after α-Tubulin staining (c) and quantification of Fascin expression (d) by immunofluorescence and confocal microscopy in almost 200 cells using ImageJ software. Representative confocal pictures (c, d) are presented for cells stained for α-Tubulin (red), Fascin (green), and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). For all histograms, error bars indicate the SEM of at least three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, using an analysis of variance (ANOVA) test followed by Dunnett’s test

Fig. 3

MST1 depletion increases cell invasion and anchorage-independent growth and decreases spheroid diameter. a–c MST0-211H and H2452 cells were transfected with siNeg, siMST1, or siMST1+pcDNAMST1, experiments were performed 48 h after transfection. a Invasion capacity of transfected cells on BioCoat Matrigel Invasion Chamber for 48 h. b Quantification of colonies in soft agar after 21 days. c Quantification of spheroid after 6 days. Representative picture is provided under histograms. For all histograms, error bars indicate the SEM of at least four independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, using an analysis of variance (ANOVA) test followed by Dunnett’s test

Fig. 4

MST1 depletion decreases apoptosis and increases cell proliferation. a–d MST0-211H and H2452 cells were transfected with siNeg, siMST1, or siMST1+pcDNAMST1. Experiments performed 48 h after transfection. a Caspase 3/7 activity. b DNA fragmentation. c Quantification of cytochrome c (red) and representative immunofluorescent confocal picture, nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). d Cell proliferation assays. For all histograms, error bars indicate the SEM of at least three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, using an analysis of variance (ANOVA) test followed by Dunnett’s test

Fig. 5

MST1 depletion increases nuclear Yes-associated protein (YAP) but decreases nuclear transcriptional co-activator with PDZ-binding motif (TAZ). a–d MST0-211H, H2452, and H2052 cells were transfected with siNeg, siMST1, or siMST1+pcDNAMST1. Experiments performed 48 h after transfection. a YAP nuclear and cytoplasmic localisation assayed by immunofluorescence. YAP/TAZ activity by quantifying ANKDR1 expression in b H2452 or c H2052 cells. d Quantification of TAZ, YAP, and PSer127-YAP protein levels using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. For all histograms, error bars indicate the SEM of at least three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, using an analysis of variance (ANOVA) test followed by Dunnett’s test

Fig. 6

Yes-associated protein (YAP) or transcriptional co-activator with PDZ-binding motif (TAZ) depletion decreases cell invasion and anchorage-independent growth while increasing apoptosis. a–d MSTO-211H and H2452 cells were transfected with siNeg, siYAP, siTAZ, and siMST1 in combination or not with pcDNA YAP or TAZ. Experiments performed 48 h after transfection. a Invasion capacity of transfected cells on BioCoat Matrigel Invasion Chamber; representative image of YAP and TAZ extinction shown under the histograms. b Quantification of colonies in soft agar after 21 days. Representative picture is provided above (a) or under (b) histograms. c Caspase 3/7 activity. d Invasion capacity of transfected cells on BioCoat Matrigel Invasion Chamber for 48 h. For all histograms, error bars indicate the SEM of at least three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, using an analysis of variance (ANOVA) test followed by Dunnett’s test