STUB1 suppresseses tumorigenesis and chemoresistance through antagonizing YAP1 signaling.

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.

cycloheximide

gastric cancer

heat shock protein

carboxy terminus of Hsc70 interacting protein

tetratricopeptide repeat

Yes‐associated protein

INTRODUCTION

Although the incidence of malignant GC declined in many developed countries from the 1940s to the 1980s, this cancer remains a major public health problem throughout the world, as it is the most common malignant gastrointestinal cancer, especially in East Asia,1 and causes 12% of all cancer‐related deaths each year.2 Over 95% of gastric tumors are adenocarcinomas histologically classified as either intestinal or diffuse type.3 Gastric cancer is a multifactorial and multistep disease that involves activation of oncogenes and inactivation of tumor suppressor genes during GC progression.4 Studies have found that inactivation of Hippo signaling leads to proproliferative and antiapoptotic signaling associated with increased cancer risk.5, 6, 7, 8, 9

As a key downstream effector, YAP1 plays a key role in the Hippo pathway to control cell proliferation and growth. Dysregulation of the Hippo/YAP1 pathway is involved in cancer development.10 Elevated YAP1 activity and/or YAP1 overexpression has been observed in a subset of primary human cancers,11 and elevated YAP1 protein expression and nuclear localization are correlated with poor prognosis.12, 13, 14, 15 The activity of YAP1 is tightly governed by posttranslational modification. Several studies have elucidated that a deubiquitinase, DUB3, regulates YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins.16 Recently, it was reported that the deubiquitination enzyme USP9X deubiquitinates and stabilizes YAP1, thereby promoting cancer cell survival.10 In addition, YAP1 can be regulated by other posttranslational modification. Lats and CK1 coordinately phosphorylate YAP1 and subsequently recruit the SCF (beta‐TRCP) E3 ubiquitin ligase, which catalyzes YAP1 ubiquitination, ultimately leading to YAP1 degradation.17 A recent study found that Fbxw7 regulated YAP1 protein abundance by targeting YAP1 for ubiquitination and proteasomal degradation in hepatocellular carcinoma.18 Thus, the role of YAP1 as a promising and important therapeutic target has been increasingly recognized. However, research regarding specific YAP1 inhibitors and their potential therapeutic use in cancers remains very limited, with only a few reports to date, limited to small‐molecule inhibitors.19 Therefore, there is a great need to identify new prognostic markers as well as to develop novel therapeutic strategies in GC treatment. In this work, we aimed to identify the signaling pathway controlling YAP1 stabilization and the regulatory function and mechanism of YAP1 in the Hippo pathway, which can be exploited for potential therapeutic interventions.

Here, we report that STUB1 regulates GC cell proliferation and response to therapeutic drugs through the YAP1 protein. Mechanistically, we found that STUB1 is the E3 ligase responsible for YAP1 ubiquitination at K280 and degradation. Downregulation of STUB1 promoted GC proliferation, tumorigenesis, and chemoresistance in a YAP1‐dependent manner. Furthermore, YAP1 overexpression was observed in gastric cancers, and was correlated with low expression of STUB1, suggesting that the STUB1‐YAP1 axis might have a role in the pathogenesis of GCs.

MATERIALS AND METHODS

Cell culture, constructs, and Abs

HEK293T cells and the human GC cell lines SGC7901, MGC803, MKN45, 9811P, HGC27, BSG823, MKN28, AGS, and BGC803 were purchased from the National Infrastructure of Cell Line Resources of China. All cell lines were tested and authenticated by karyotyping analysis on 1 January 2018, and confirmed by the National Infrastructure of Cell Line Resources of China. Expression plasmids containing pCMV‐Flag‐STUB1, pCMV‐Myc‐STUB1, pCMV‐Flag‐YAP1, and pCMV‐Myc‐YAP1 were constructed as previously described,20 and the different Flag‐tagged STUB1 fragments and mutations were generated as previously described.21 The HA‐tagged ubiquitin (HA‐ub) plasmid and mutation constructs were kindly provided by Professor Jianfei Qi from the University of Maryland Cancer Center. Mutations were produced using a QuikChange Site‐Directed Mutagenesis Kit (Stratagene) and validated by DNA sequencing.

The anti‐STUB1 (C3B6) rabbit mAb #2080, the anti‐YAP1 (D24E4) rabbit mAb #8418, and anti‐HSP90 (C45G5) rabbit mAb #4877 were purchased from Cell Signaling Technology. Anti‐ub (sc‐8017) Abs were purchased from Santa Cruz Biotechnology. Antibodies against HA (H9658), FLAG (F1804), and β‐actin (A1978) were purchased from Sigma. The HSP90 inhibitor 17‐AAG was purchased from Cell Signaling Technology (8132S) and used at 10 μmol/L.

Cell survival assay

Gastric cancer cell lines stably expressing the indicated constructs were incubated for 24 hours and were then treated with mitomycin, cisplatin, or etoposide at the indicated doses. After 36 hours, the 96‐well plates were read in an Epoch2 microplate reader (BioTek Instruments). The cell survival ratio calculated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐ 2H‐tetrazolium assay (Promega).

Soft agar colony formation assays

The indicated GC cells were plated in 0.2% (w/v) agarose with a base layer of 0.5% (w/v) agarose. Both layers contained complete medium. After 2 weeks, colonies were counted by using a light microscope at 4× magnification with a numerical aperture 0.10 objective lens (ECLIPSE 80i; Nikon).

Coimmunoprecipitation

Cells were harvested and washed with PBS. Cells were then lysed with NETN buffer (20 mmol/L Tris‐HCl, pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% Nonidet P‐40) containing 50 mmol/L b‐glycerophosphate, 10 mmol/L NaF, and 1 mg/mL each of pepstatin A and aprotinin. Whole cell lysates obtained by centrifugation were incubated with 2 μg of the indicated Ab and protein A or protein G Sepharose beads (Amersham Biosciences) for 4 hours at 4°C. After washing with NETN buffer 3 times, immunocomplexes were separated by SDS‐PAGE. Immunoblotting was carried out following standard procedures.

Protein identification by mass spectrometry

Flag‐tagged YAP1 or empty control lentiviral vector was transduced into MGC803 cells in five 15‐cm dishes. Immunoprecipitation of Flag‐YAP1 was carried out as described above. The precipitated proteins were eluted with 3× flag peptides. The eluted samples were subjected to in‐solution trypsin digestion, followed by liquid chromatography‐MS analysis and protein identification was undertaken using the Mascot (version 2.3.02) program and compared against the UniProt human protein database (released December 2014). The following search parameters were used: proteins were digested by trypsin; 2 missed cleavages were allowed; carbamidomethylation was set as the fixed modification, whereas oxidation (M) was considered the variable modification; an initial mass deviation of the precursor ion and fragment ions of up to 30 ppm and 0.1 Da, respectively, were allowed; the false discovery rate was set at 1%; the protein score was set at R ≥ 40 and the number of unique peptides was set at R ≥ 2.

In vivo ubiquitination assay

This procedure was carried out as previously described.21 Briefly, cells were cotransfected with the indicated plasmids for 24 hours, and were treated with 10 μmol/L MG132 for the indicated number of hours prior to harvesting. Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche). Flag‐YAP1 was immunoprecipitated using anti‐Flag Abs and protein A/G agarose beads. Polyubiquitinated YAP1 was detected using anti‐HA or anti‐ub Abs.

Immunohistochemistry

Normal GC tissue samples and gastric tumor tissue samples were collected at Clinical Medical College of Jinan University. Tissue sample collection was approved by the Internal Review and Ethics Boards of Jinan University. Tissue microarray chips containing normal gastric tissue samples and GC tumor tissue samples were obtained from Shanghai OUTDO Biotech. Immunohistochemical staining and quantification were undertaken as described previously.21 The immunostaining was blindly scored by pathologists. The immunohistochemical score was calculated as described previously.21 The χ2 test and Pearson's correlation coefficient were used for statistical analysis of the correlation between STUB1 and YAP1 expression.

Athymic nude mouse tumor formation assay

Six‐week‐old female BALB/c nude mice were obtained from the Model Animal Research Center of Jinan University and housed under pathogen‐free conditions in the animal experiment center of Jinan University. A total of 1 × 106 MGC803 cells stably expressing control shRNA, shSTUB1, or shSTUB1 with shYAP1 were injected s.c. into female BALB/c nude mice. Every 4 days, the tumor volumes were measured following a standard protocol. Data were analyzed using ANOVA. Following the blinding procedures, 2 persons undertook all the mouse experiments as a study group. Dr. Song‐Hui Xu injected the cells into the mice and Dr. Dong‐e Tang measured the tumors and analyzed the data. All protocols involving live mice were approved by the Animal Care and Use Committee of Jinan University. Mice were killed when the standard situations occurred.

Statistical analysis

Data for the cell proliferation and colony formation assays are presented as the mean ± SEM of 3 independent experiments. Data for the xenograft tumor growth study are presented as the mean ± SD of 6 mice. A 2‐tailed, unpaired Student's t test, ANOVA, and χ2 test were utilized for statistical analyses (*P < .05; **P < .01).

Supplementary materials and methods in Appendix S1.

RESULTS

STUB1 is a YAP1 binding protein and destabilizes YAP1

The Hippo pathway has been implicated in suppressing tissue overgrowth and tumor formation by inhibiting the oncogenic activity of YAP1.22 The dysregulation of the Hippo/YAP1 pathway is involved in cancer development.11, 23, 24 However, the ubiquitin ligase that regulates YAP1 protein stability in human cancers remains largely unknown. To identify YAP1‐interacting ubiquitinases, we used cells stably expressing Flag‐YAP1 to undertake tandem affinity purification and mass spectrometry analysis; several proteins were identified, including 4 ubiquitin ligases (RNF4, WWP1, STUB1, and CBX4), as YAP1 interactors (Figure 1A). To confirm which ubiquitin ligase is responsible for YAP1 degradation, we first examined the effects of these 4 ubiquitin ligases on YAP1 expression. We stably expressed shRNAs targeting these proteins individually in the MGC803 human GC cell line (Figure S1A). Only one, STUB1, significantly increased endogenous YAP1 protein expression (Figure 1B). Then we investigated the interaction between STUB1 and YAP1. Immunoblotting assays in the coimmunoprecipitation experiment showed that endogenous YAP1 coimmunoprecipitated with endogenous STUB1 (Figure 1C). STUB1 is a cochaperone protein and E3 ubiquitin ligase that regularly interacts with the molecular chaperones Hsc70‐Hsp70 and Hsp90 through its TPR domain, whereas its E3 ubiquitin ligase activity is restricted to the U‐box domain.25, 26 To determine which domain of STUB1 is responsible for the STUB1‐YAP1 interaction, we coexpressed full‐length STUB1 with TPR domain, U‐box domain, and the middle region between TPR domain and U‐box domain fragments of STUB1 in 293T cells. Coimmunoprecipitation and western blot analysis revealed that the TPR domain of STUB1 interacted with YAP1 (Figures 1D and S1B). Next, we investigated whether the binding of STUB1 to YAP1 requires the molecular chaperone Hsp90. As shown in Figure S1D,E, the decrease in the YAP1 level was significantly reversed by knockdown or inhibition of Hsp90, suggesting that STUB1 might need the molecular chaperone Hsp90 to facilitate TPR domain‐dependent ubiquitination of YAP1. In addition, we examined whether the Hippo‐resistant YAP mutant (YAP‐5SA) could be regulated by STUB1. As shown in Figure S1F, we found that overexpression of STUB1 reduced levels of both the WT YAP1 and mutant YAP1 (YAP1‐5SA). As STUB1 is a ubiquitination enzyme, we hypothesized that STUB1 might regulate the protein level of YAP1. First, we found the decrease in the YAP1 level was reversed by the addition of the proteasome inhibitor MG132, suggesting that STUB1 regulates the YAP1 level in a proteasome‐dependent manner (Figure 1E). Next, when we overexpressed STUB1 in 2 GC cell lines, we found that STUB1 upregulation decreased the YAP1 protein level (Figure 1F), with no effect on the YAP1 mRNA level (Figure S1C). We then hypothesized that STUB1 might regulate YAP1 stability, and we treated cells with CHX and determined the half‐life of YAP1. As shown in Figure 1G, YAP1 stability was dramatically decreased in STUB1‐overexpressing cells. In addition, we found that the half‐life of STUB1 was approximately 14 hours (Figure S1G). Taken together, these results indicate that STUB1 binds and destabilizes YAP1 through its TPR domain, which needs the molecular chaperone Hsp90 to facilitate.

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

STUB1 ubiquitinates YAP1 at K280 through K48‐linked polyubiquitination

We next examined whether STUB1 regulates the level of YAP1 ubiquitination in cells. As shown in Figure 2A, STUB1 overexpression resulted in a significant increase in YAP1 polyubiquitination. Conversely, knocking down STUB1 decreased the polyubiquitination of YAP1 (Figure 2B). Several studies have reported that the T246M mutation of STUB1 abolishes its ubiquitin ligase activity.27, 28, 29 To test whether STUB E3 ligase activity is required for YAP1 ubiquitination, we transfected 293T cells with STUB1 (WT or T246M mutant). We found that the T246M mutation of STUB1 did not increase YAP1 ubiquitination (Figure S2A), suggesting that STUB E3 ligase activity is indispensable for YAP1 ubiquitination. To test whether STUB1 can directly ubiquitinate YAP1, we carried out the in vitro ubiquitination assay using the purified His‐YAP1, GST‐STUB1 (WT or T246M mutant) and E1/E2/ubiquitin. His‐YAP1 was precipitated and analyzed by western blotting with ubiquitin Abs. As shown in Figure S2B, the WT GST‐STUB1 could directly ubiquitinate His‐YAP1 in the in vitro reaction, but not the T246M mutant. 17‐AAG is a heat shock protein inhibitor that shows antitumorigenic and antiangiogenic properties in vitro and in in vivo animal models.30, 31 Heat shock proteins can promote the ubiquitination and degradation of proteins through cooperative interaction with the E3 ubiquitin ligase STUB1.32 Here, we used 17‐AAG to treat cells and found significantly reduced YAP1 ubiquitination (Figure 2C). To identify the specific lysine sites in the YAP1 protein with ubiquitination modification, we used UbPred software (http://www.ubpred.org/). Five potential ubiquitination sites at lysine residues were found in the YAP1 protein (Table S1). We subsequently generated YAP1 mutants in which these lysine residues were replaced with arginines. First, we found that STUB1 overexpression increased the polyubiquitination levels in the K102R, K181R, K204R, and K342R YAP1 mutants but not in the K280R YAP1 mutant (Figure 2D). Second, STUB1 overexpression did not decrease the protein levels of the K280R YAP1 mutant compared with those of the other four YAP1 mutants (Figure S2C). To further confirm that K280 is a critical residue that regulates STUB1 mediated YAP1 degradation, we treated cells with CHX and determined the half‐life of WT YAP1 and K280R YAP1 mutant after transfection with STUB1. As shown in Figure S2D, the K280R YAP1 mutant was more stable than the WT YAP1. Furthermore, we found that STUB1 overexpression increased the polyubiquitination levels in the WT, K6R, K11R, K27R, K29R, K33R, and K63R HA‐ubiquitin mutants but not in the K48R mutant (Figure 2E) and the cells transfected the plasmid of K48R HA‐ubiquitin could partially increase the YAP1 expression caused by transfection of WT HA‐ubiquitin (Figure S2E), suggesting that YAP1 as a novel target of STUB1‐mediated K48‐linked ubiquitin. Taken together, these results suggest that STUB1 ubiquitinates YAP1 at the K280 site by K48‐linked polyubiquitination.

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

STUB1 regulates cell proliferation and tumor growth through the YAP1 pathway

We asked whether STUB1 functions as a tumor‐suppressing protein by regulating YAP1. First, we assessed the effect of STUB1 on YAP1 transactivation. We overexpressed STUB1 in MGC803 cells with YAP1 silencing (Figure 3A) and examined the transcription of YAP1‐regulated target genes (ANKRD1, Cyr61, and CTGF). As shown in Figure 3B, silencing YAP1 dramatically decreased the transcription of ANKRD1, Cyr61, and CTGF (column 1 vs column 3) and STUB1 overexpression also reduced the transcription of YAP1‐regulated target genes (column 1 vs column 2), whereas STUB1 overexpression did not show any additional effect in YAP1‐depleted cells (column 3 vs column 4). These results suggest that STUB1 regulates YAP1‐dependent transcription. To investigate the biological function of STUB1 in YAP1‐dependent cells, we evaluated proliferation and anchorage‐independent growth of STUB1‐overexpressing cells following the upregulation of YAP1 expression in these cells (Figure 3C). We observed that YAP1 overexpression markedly increased both the proliferation (Figure 3D, column 1 vs column 2) and anchorage‐independent growth (Figure 3E, column 1 vs column 2) of MGC803 GC cells. Conversely, STUB1 overexpression decreased both the proliferation (Figure 3D, column 1 vs column 3) and anchorage‐independent growth (Figure 3E, column 1 vs column 3) of MGC803 GC cells, whereas restoration of YAP1 in cells with STUB1 overexpression significantly reversed the effect of STUB1 overexpression (Figure 3C‐E, column 3 vs column 4). Conversely, we reduced YAP1 expression in MGC803 cells with STUB1 silencing (Figure S3A) and examined cell proliferation and anchorage‐independent growth. We found that STUB1 knockdown markedly increased the proliferation (Figure S3B) and anchorage‐independent growth (Figure S3C) of MGC803 GC cells, whereas downregulation of YAP1 could significantly reverse the effect of STUB1 knockdown.

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

To investigate the biological function of the STUB1‐YAP1 interaction in GC cells in vivo, we used a xenograft gastric tumor model in which the indicated numbers of MGC803 cells were injected into athymic nude mice and tumor growth was monitored. Mice implanted with STUB1 shRNA‐expressing MGC803 cells showed increased tumor growth throughout the experiment compared with that in mice implanted with control shRNA‐expressing cells (Figure 3F). At 23 days after tumor cell implantation, we observed a more than 2.5‐fold increase in the volume (Figure 3F) and a 2‐fold increase in the weight of the tumors formed by STUB1‐depleted MGC803 cells (Figure 3G,H). Notably, silencing of YAP1 in MGC803 cells expressing STUB1 shRNA fully reversed the tumor‐promoting effect of STUB1 shRNA (Figure 3F‐H). Taken together, these findings indicate that the loss of STUB1 promotes tumorigenesis through the upregulation of YAP1.

STUB1 is downregulated in human gastric tumors and correlates with the YAP1 protein level

As a downstream effector, YAP1 plays a key role in the Hippo pathway to control tissue overgrowth and tumor formation. YAP1 has primarily been reported as an oncoprotein; elevated expression and nuclear localization of YAP1 have been frequently observed in human cancers.7, 33, 34, 35, 36, 37, 38, 39 Posttranscriptional and posttranslational regulation of YAP1 have been reported to contribute substantially to the development of human cancer.10, 40, 41, 42 As YAP1 plays a key role in human cancer development, it is possible that in human cancers STUB1 promotes the ubiquitination and destabilization of YAP1. First, we measured the expression of STUB1 and YAP1 in GC cell lines and cancer tissue samples. As shown in Figure 4A, low STUB1 protein levels correlated with high YAP1 expression in these GC cells compared with normal gastric epithelial GES‐1 cells. Furthermore, low STUB1 protein levels correlated with increased YAP1 expression in most GC samples (Figure 4B). To determine the relevance of YAP1 regulation by STUB1 in patients, we undertook immunohistochemical staining of YAP1 and STUB1 (Figure 4C) in GC tissue microarrays. Notably, downregulation of STUB1 expression and high YAP1 expression were observed in 67.9% (72/106) and 74.5% (79/106) of gastric tumors, whereas only 27.3% (6/22) and 31.8% (7/22) of normal mammary tissues showed low STUB1 expression and high YAP1 expression (Figure 4D), respectively, suggesting that STUB1 was downregulated but YAP1 was upregulated in human gastric tumors. Moreover, a significant negative correlation (R = −0.305, P < .001) between the STUB1 and YAP1 protein levels was observed in these gastric carcinomas: 84.7% (61/72) of tumors with low STUB1 expression also displayed high YAP1 expression (Figure 4D). However, it should be noted that 16.98% (18/106) of all tumor specimens had high YAP1 expression but high STUB1 expression (Figure 4D). Collectively, these data suggest that loss of STUB1 might contribute to upregulation of YAP1 in a substantial fraction of human gastric tumors, whereas in other gastric tumors, YAP1 can be activated by different mechanisms, including genetic alterations and upregulation of YAP1 deubiquitinases such as USP743 and USP9X.10

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

STUB1 regulates the response of GC cells to chemotherapy through YAP1

We found that STUB1 negatively regulates the expression of YAP1, which plays a key role in chemoresistance in different malignancies.10, 44, 45, 46 We next examined whether STUB1 plays a role in the response of GC to chemotherapy. To investigate the role of STUB1 in the response of GC to chemotherapy, MGC803 cells stably expressing YAP1 shRNAs, which responded to mitomycin C, cisplatin, and etoposide, were subsequently treated with either vehicle or 17‐AAG. As shown in Figure 5A, we found that 17‐AGG inhibited YAP1 degradation. In addition, cells treated with 17‐AAG were significantly resistant to chemotherapy, whereas YAP1 knockdown promoted cellular chemosensitivity (Figure 5B). However, 17‐AAG treatment of cells with stable expression of YAP1 shRNAs reversed the sensitivity to chemotherapy (Figure 5B). Similarly, silencing of STUB1 in SGC7901 cells using 2 specific shRNAs significantly increased the YAP1 protein levels (Figure 5C) and increased cell resistance to mitomycin C, cisplatin, and etoposide (Figure 5D), whereas YAP1 knockdown in STUB1‐depleted cells reversed the sensitivity to chemotherapy (Figure 5D). These results establish an important role for STUB1 in regulating the chemotherapeutic response in GC through YAP1 signaling.

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

In summary, we showed that STUB1 interacts with YAP1 and promotes its ubiquitination, ultimately leading to YAP1 degradation (Figure 5E). We revealed STUB1 as a negative regulator of YAP1 in GC cell proliferation and as a potential biomarker for predicting chemoresistance.

DISCUSSION

Our study found that YAP1 promoted GC proliferation in vitro and in vivo, and we elucidated a novel mechanism underlying the effect of STUB1 on cancer progression. Specifically, we revealed the interaction of STUB1 with YAP1 and the subsequent ubiquitination and degradation of YAP1. STUB1 is an E3 ubiquitin ligase that mediates K48‐linked polyubiquitination of YAP1 at K280.

YAP1, a major factor in the Hippo pathway controls multiple cellular processes related to proliferation and apoptosis, and its dysregulation has been linked to various cancers.10 Here, we found that the human YAP1 protein levels were significantly increased in GC tissues compared with those in normal gastric tissues. Our data consistently supported the reports that YAP1 functions as a potential oncogene and is associated with the prognosis of many human cancers including prostate, breast, ovarian, and hepatocellular cancers.11, 47, 48, 49 Silencing YAP1 significantly suppressed GC proliferation and tumorigenesis, indicating that YAP1 could be a novel therapeutic target in GC. Our findings suggest that YAP1 could be a novel independent prognostic factor in GC patients.

The process of ubiquitination is triggered by the coordinated action of 3 classes of enzymes, including E1 ubiquitin activating enzymes, E2 ubiquitin‐conjugating enzymes, and E3 ubiquitin ligases. Among these enzymes, the E3 ligase determines the substrate specificity of the process. The STUB1 protein (the carboxy terminus of the Hsc70‐interacting protein), composed of a TPR domain at its amino terminus that interacts with chaperone proteins (Hsc70, Hsp70, and Hsp90) and a U‐box domain at its carboxy terminus with E3 ubiquitin ligase activity, functions as a link between the chaperone and proteasome systems.50, 51 Numerous reports have indicated that STUB1 acts as a tumor suppressor because it induces the ubiquitination and degradation of several oncogenic proteins, such as mutant p53,51 SRC‐3,52 Smad3,53 c‐ErbB2/neu,54 Dbl,55 Runx1,56 hypoxia‐inducible factor‐1a,57 the estrogen receptor,58 and the Met receptor.59 A recent study showed that STUB1 expression is significantly decreased in GC lesions compared with that in paired noncancerous tissues, and that this decrease might be associated with STUB1 promoter methylation in GC cells.60 Thus, identifying novel substrates is essential for understanding STUB1 biology and its implications in GC progression and drug resistance. Here, we found that the TPR domain of STUB1 interacts with YAP1 and promotes K48‐linked polyubiquitination of YAP1 at K280. This finding explains why this E3 ligase accounts for YAP1 ubiquitination; several studies have reported that YAP1 undergoes deubiquitinase‐mediated stabilization10, 43, 61 but have not identified which E3 ligase mediates YAP1 ubiquitination. The mechanisms through which E3 ubiquitin ligases and deubiquitinases balance YAP1 expression could explain why the activity of YAP1 is tightly regulated under physiological conditions, whereas elevated YAP1 activity and/or overexpression has been observed in different cancer types.11

STUB1 reportedly induces the degradation of MST1, an upstream inhibitor of YAP, in an Hsp70‐interacting protein (CHIP)‐dependent manner under different stresses and in different kinds of cancer.62, 63 This regulation seems to be the opposite of YAP1 activity, because we found that the interaction of STUB1 with YAP1 and subsequent ubiquitin and degradation of YAP1 proceeds in an Hsp90‐dependent manner. Future studies are needed to determine whether STUB1 regulates MST1 degradation in GC cancer.

In summary, we revealed a connection between STUB1 and the Hippo pathway in GC. In this study, we demonstrated that STUB1 may target YAP1 for ubiquitin and destabilization, thereby inhibiting GC growth and tumor progression. The tumor suppressor role of STUB1 was partially reversed by inhibition of YAP1 activity in vitro and in vivo. Interestingly, STUB1 expression was negatively correlated with YAP1 protein expression in GCs. Our study provides evidence that inhibition of YAP1 activity could be used to sensitize cancer cells to radiotherapy and chemotherapy, although the clinical effect of YAP1 inhibitors needs further testing. Furthermore, our findings indicate that YAP1 ubiquitination and degradation mediated by the E3 ligase STUB1 ubiquitin and degradation affects the response to chemotherapeutic agents, which has broader implications for the treatment of other cancers and should be investigated in the future.

DISCLOSURE

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.

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