Yes-associated protein plays oncogenic roles in human sporadic colorectal adenomas

Abstract

The role of Hippo-Yes-associated protein (YAP) in human colorectal cancer (CRC) presents contradictory results. We examined the function of YAP in the early stages of CRC by quantitatively measuring the expression of phospho-YAPS127 (p-YAP) and five APC-related proteins in 145 sporadic adenomas from the Tennessee Colorectal Polyp Study, conducting APC sequencing for 114 adenomas, and analyzing YAP-correlated cancer pathways using gene expression data from 326 adenomas obtained from Gene Expression Omnibus. The p-YAP expression was significantly correlated with YAP expression (r = 0.53, P < .0001) and nuclear β-catenin (r = 0.26, P = .0018) in adenoma tissues. Both p-YAP and nuclear β-catenin were associated with APC mutations (P = .05). A strong association was observed between p-YAP overexpression and advanced adenoma odds (OR = 12.62, 95% CI = 4.57-34.86, P trend < .001), which persisted after adjusting for covariates and biomarkers (OR = 12.31, 95% CI = 3.78-40.10, P trend < .0001). P-YAP exhibited a sensitivity of 77.4% and specificity of 78.2% in defining advanced versus nonadvanced adenomas. Additionally, synergistic interaction was noted between p-YAP positivity and nuclear β-catenin on advanced adenomas (OR = 16.82, 95% CI = 4.41-64.08, P < .0001). YAP-correlated genes were significantly enriched in autophagy, unfolded protein response, and sirtuin pathways showing predominantly pro-tumorigenic alterations. Collectively, YAP plays an oncogenic role in interacting with Wnt as well as other cancer pathways within human sporadic adenomas. P-YAP could be a potential biomarker for human high-risk sporadic adenomas.

Keywords: Hippo-YAP; Wnt pathway; biomarker; colorectal adenoma; oncogene.

Graphical Abstract

Figure 1

Selection and validation of YAP antibodies using TMA control and human adenoma tissues. (a) The YAP expression levels were elevated in CRC, some adenomas, and normal kidney, colon, and placenta tissues. (b) The expression levels of p-YAP were lower than that of YAP and showed negative staining in most normal tissues, including the colon. Scale bars: 100 µm at 10× magnification. (c) In a pilot of 64 adenomas (>4 mm in size), the YAP expression was closely correlated with p-YAP expression (r = 0.525, Pearson correlation coefficients) and YAP nuclear positivity (r = 0.817, Spearman correlation coefficients). Nuclear positively stained cells were scored semiquantitatively as: 0, no staining; 1, 1%–10%; 2, 11%–30%; 3, >31%.

Figure 2

Expression of p-YAP and other biomarkers in adenoma, adjacent nontumor, and normal colorectal tissues. (A) p-YAP expression is weak in the normal rectal mucosa (a) and slightly increased in the small adenoma and adjacent nontumor mucosa (b). The larger adenoma (c) shows an elevated expression of p-YAP in the cytoplasm of tumor cells (the upper-right corner) but may appear in the nuclei in the strong positive area (the lower right corner). (B) The membranous β-catenin can be seen in the normal rectal epithelium (a) and the elevated expression is seen in small adenoma (b) and larger adenoma (c). The nuclear accumulation in the boundary area of the tumor and nontumor in the larger adenoma is shown in the upper-right corner. (C) CtBP is negative in the normal rectal epithelium (a), weakly expressed in small adenoma and nontumor mucosa (b), and strongly expressed in larger adenoma (c) with nuclear staining pattern. (D) Cytoplasmic EB1 signal is strong in the normal rectal epithelium (a) and decreased in small (b) and larger (c) adenomas and adjacent nontumor mucosa. (E) The immunofluorescent dual staining shows that Asef is strongly expressed in the normal rectal mucosa (a) but decreased in small (b) and larger (c) adenomas and adjacent nontumor mucosa (d). Inversely, APCn is weakly expressed in the normal rectal epithelium but increased in small and larger adenoma and adjacent nontumor mucosa. Scale bars: 100 µm at 10X magnification. (F) The quantitative data of biomarkers in adenomas and adjacent nontumor mucosa, using normal rectal tissue as reference.

Figure 3

p-YAP as a biomarker differentiating advanced versus nonadvanced adenomas. (a) Comparison of the ROC curves showed that p-YAP (AUC=0.798, 95% CI: 0.719 - 0.877) was superior to CtBP (AUC=0.648, 95% CI: 0.558 - 0.739) and nuclear β-catenin (AUC=0.570, 95% CI: 0.475 - 0.666) in differentiating advanced adenomas. The best cutoff of p-YAP is 0.735 as a biomarker for advanced adenoma; (b) Example images of p-YAP negative (score < 0.735) and positive (score > 0.735) adenomas. Scale bars, 200 µm; (c) Synergistic relationship of nuclear β-catenin and p-YAP on tumorigenesis.

Figure 4

The frequency and distribution of APC mutations in baseline and metachronous adenoma tissues. (a) Distribution of APC mutations in the 114 baseline adenoma tissues, plotting by the MutPlot (https://bioinformaticstools.shinyapps.io/lollipop/) (b) Distribution of APC mutations in the 32 baseline adenoma tissues. (c) Baseline and metachronous adenomas showed similar APC mutation characteristics in mutation sites, multiplicity, and types (Fisher exact test, P > .05). *Frameshift mutation includes deletion, insertion, and substitution. **One case with missense mutation at exon 3 was excluded (TQ15338), and 7 other cases of missense mutation coexist with stopgain and/or frameshift mutations. ***One case with missense mutation at exon 14 was excluded.

Figure 5

YAP potentially regulated genes and signaling pathways in human adenomas. (a) A total of 2697 genes were correlated with YAP gene expression and significantly enriched in molecular and cellular functions of gene expression, posttranslational modification, protein synthesis, and cell death and survival; (b) YAP-correlated cancer genes identified in this analysis. TSG, tumor suppressor gene; (c) The top six signaling pathways are enriched in the YAP-correlated genes.