STUB1 suppresseses tumorigenesis and chemoresistance through antagonizing YAP1 signaling

Abstract

Yes-associated protein (YAP) is a component of the canonical Hippo signaling pathway that is known to play essential roles in modulating organ size, development, and tumorigenesis. Activation or upregulation of YAP1, which contributes to cancer cell survival and chemoresistance, has been verified in different types of human cancers. However, the molecular mechanism of YAP1 upregulation in cancer is still unclear. Here we report that the E3 ubiquitin ligase STUB1 ubiquitinates and destabilizes YAP1, thereby inhibiting cancer cell survival. Low levels of STUB1 expression were correlated with increased protein levels of YAP1 in human gastric cancer cell lines and patient samples. Moreover, we revealed that STUB1 ubiquitinates YAP1 at the K280 site by K48-linked polyubiquitination, which in turn increases YAP1 turnover and promotes cellular chemosensitivity. Overall, our study establishes YAP1 ubiquitination and degradation mediated by the E3 ligase STUB1 as an important regulatory mechanism in gastric cancer, and provides a rationale for potential therapeutic interventions.

Keywords: STUB1; YAP1; chemoresistance; gastric cancer; ubiquitination.

Figure 1

STUB1 binds and destabilizes Yes‐associated protein 1 (YAP1). A, List of YAP1‐associated ubiquitin ligase proteins identified by mass spectrometric analysis. MGC803 cells stably expressing Flag‐YAP1 were generated and YAP1 complexes were subjected to mass spectrometric analysis. B, MGC803 cells stably expressing control (Ctrl) or the indicated shRNAs and western blot analysis were performed with anti‐YAP1 Ab. C, MGC803 cell lysates were subjected to immunoprecipitation (IP) with control IgG, anti‐STUB1 (left panel), or anti‐YAP1 Ab (right panel). The immunoprecipitates were then blotted with the indicated Abs. D, 293T cells were transfected with myc‐tagged YAP1 and Flag‐tagged STUB1 fragments for 24 h, and lysates were subjected to immunoprecipitation with anti‐Flag M2 beads. Bound proteins were analyzed by western blotting with Myc or Flag Abs. E, The indicated cells were untreated or treated with MG‐132 and western blotting was carried out to examine the indicated protein levels. F, MGC803 cells stably expressing Ctrl or Flag‐STUB1 were subjected to western blotting to examine the indicated protein. G, Cycloheximide (CHX) pulse‐chase assay was carried out in cells as in (F). Right panel, protein levels of YAP1 relative to β‐actin. Results in (B) and (C) are shown as ± SEM of 3 independent experiments. TPR, tetratricopeptide repeat

Figure 2

STUB1 promotes K48‐linked ubiquitination of Yes‐associated protein 1 (YAP1) at the K280 site. A, 293T cells were transfected with Myc‐STUB1, Flag‐YAP, and HA‐tagged ubiquitin plasmid (HA‐ub) as indicated. The polyubiquitylated YAP1 proteins were detected by anti‐HA Ab. B, Cells stably expressing control or STUB1 shRNAs were subjected to ubiquitination assay and the polyubiquitylated YAP1 proteins were detected by anti‐ub Ab. C, Cells transfected with Flag‐YAP1 were treated with or without 17‐AGG. The polyubiquitylated YAP1 proteins were examined as in (B). D, 293T cells were transfected with Myc‐STUB1, Flag‐YAP1 (WT, K102R mutant, K181R mutant, K204R mutant, K280R mutant, and K342R mutant), and HA‐ub. The analysis was undertaken as described for (A). E, 293T cells were transfected with Myc‐STUB1, Flag‐YAP1, and HA‐ub (WT, K6R mutant, K11 mutant, K27 mutant, K29 mutant, K33 mutant, K48 mutant, and K63 mutant). The analysis was undertaken as described for (B). IP, immunoprecipitation

Figure 3

STUB1 regulates cell proliferation and tumor growth through Yes‐associated protein 1 (YAP1). A, B, MGC803 cells stably expressing control (Ctrl) or Flag‐STUB1 plasmids together with or without YAP1 shRNAs were subjected to western blotting to detect the indicated protein levels. YAP1‐regulated target transcription genes were detected by quantitative RT‐PCR. Data were normalized to the β‐actin mRNA (mean ± SD, n = 3). *P < .05; **P < .01. C, MGC803 cells stably expressing Ctrl or Flag‐STUB1 plasmids with or without Flag‐STUB1 plasmids were subjected to western blotting to detect the indicated protein levels. D, Left: colony formation abilities of the cells generated as above were measured after 2 wk. Colony numbers of cellular clones with more than 100 cells was measured (mean ± SEM of 3 independent experiments). Right: statistical analyses were carried out with ANOVA. *P < .05; **P < .01. E, Left: the cells described above and were maintained in soft agar for 3 wk, and colony number per field was determined. Right: statistical analyses were carried out with ANOVA. *P < .05; **P < .01. F‐H, Cells stably expressing Ctrl or shSTUB1 RNAs with or without shYAP1 RNAs were injected into athymic nude mice, as described in the Method 2.8. Tumor growth was measured every 4 d. Images (G) and weight (H) of xenograft tumors are shown (mean ± SD of 6 mice). All of the statistical analyses were carried out with ANOVA. *P < .05; **P < .01

Figure 4

Yes‐associated protein 1 (YAP1) expression negatively correlates with STUB1 expression in clinical gastric cancer (GC) samples. A, Expression of STUB1 and YAP1 in GES‐1 (normal gastric epithelial cell line) and the GC cell lines as indicated. B, A subset of the GC tumor and normal tissues were subjected to western blotting, to examine the STUB1 and YAP1 protein levels. C, Representative staining of STUB1 and YAP1 in GC and normal gastric tissues. D, Quantification of STUB1 and YAP1 protein levels in normal tissue and GC, and the correlation study of STUB1 and YAP1 expression level in GC. Statistical analyses were undertaken with the χ² test, P < .001. R, Pearson's correlation coefficient

Figure 5

STUB1‐Yes‐associated protein 1 (YAP1) axis regulates gastric cancer cells’ response to chemotherapy. A, MGC803 cells stably expressing the indicated constructs were treated with either vehicle or 17‐AGG for 24 h and were then subjected to western blot analysis to examine the indicated protein levels. B, As in (A), cells were treated with mitomycin, cisplatin, and etoposide, and cell survival was determined (mean ± SD, n = 3). C, SGC7901 cells stably expressing control (Ctrl) or STUB1 shRNA with or without YAP1 shRNA were subjected to western blotting to detect the indicated protein levels. D, As in (C), cells were treated with mitomycin, cisplatin, and etoposide, and cell survival was measured (mean ± SD, n = 3). E, Schematic representation of how STUB1 regulates YAP1