TAZ expression as a prognostic indicator in colorectal cancer

Abstract

The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic), and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic target for the treatment of colon cancer.

Figure 1. The correlations among mRNA expression of TAZ (WWTR1), YAP (YAP1), AXL and CTGF in colon cancer specimens.

Scatter plots for (A) TAZ mRNA expression versus AXL mRNA expression, (B) TAZ mRNA expression versus CTGF mRNA expression, (C) YAP mRNA expression versus AXL mRNA expression, and (D) YAP mRNA expression versus CTGF mRNA expression in the GSE14333 colon cancer datasets, and (E) TAZ mRNA expression versus AXL mRNA expression, (F) TAZ mRNA expression versus CTGF mRNA expression, (G) YAP mRNA expression versus AXL mRNA expression, and (H) YAP mRNA expression versus CTGF mRNA expression in the GSE17538 colon cancer datasets.

Figure 2. The associations between TAZ or YAP, and survival in colon cancer patients.

Kaplan-Meier analyses for TAZ mRNA expression in (A) GSE14333 and (B) GSE17538 colon cancer patient datasets. Univariate and Multivariate Cox regression analyses for TAZ mRNA expression, age, tumor stage and tumor grade in (C) GSE14333 and (D) GSE17538 colon cancer patient datasets. Kaplan-Meier analyses for YAP mRNA expression in (E) GSE14333 and (F) GSE17538 colon cancer patient datasets. Univariate and Multivariate Cox regression analyses for TAZ mRNA expression, age, tumor stage and tumor grade in (G) GSE14333 and (H) GSE17538 colon cancer patient datasets.

Figure 3. The associations between AXL or CTGF, and survival in colon cancer patients.

Kaplan-Meier analyses for AXL mRNA expression in (A) GSE14333 and (B) GSE17538 colon cancer patient datasets. Univariate and Multivariate Cox regression analyses for AXL mRNA expression, age, tumor stage and tumor grade in (C) GSE14333 and (D) GSE17538 colon cancer patient datasets. Kaplan-Meier analyses for CTGF mRNA expression in (E) GSE14333 and (F) GSE17538 colon cancer patient datasets. Univariate and Multivariate Cox regression analyses for CTGF mRNA expression, age, tumor stage and tumor grade in (G) GSE14333 and (H) GSE17538 colon cancer patient datasets.

Figure 4. The associations between TAZ-AXL-CTGF expression and survival in colon cancer patients.

Patients were divided into 4 groups according to the number of genes that they overexpressed (expressed at above the Median level) among TAZ, AXL and CTGF. Kaplan-Meier analyses for TAZ-AXL-CTGF mRNA expression in (A) GSE14333 and (B) GSE17538 colon cancer patient datasets. Univariate and Multivariate Cox regression analyses for TAZ-AXL-CTGF mRNA expression, age, tumor stage and tumor grade in (C) GSE14333 and (D) GSE17538 colon cancer patient datasets.

Figure 5. Kaplan-Meier analyses for the mRNA expression of TAZ-AXL-CTGF and its co-regulated genes in the combined colon cancer dataset.

(A) Kaplan-Meier analysis for TAZ-AXL-CTGF mRNA expression in the combined colon cancer datasets. Kaplan-Meier analyses for mRNA expression of (B) MGP, (C), PDLIM3, (D) TAGLN and (E) ZEB2 in colon cancer patients expressing low levels of TAZ-AXL-CTGF (blue line in (A)) in the combined colon cancer datasets.

Figure 6. The prognostic significance of ANO1 and SQLE mRNA expression in colon cancer patients stratified by their TAZ-AXL-CTGF mRNA expression.

Kaplan-Meier analyses for ANO1 mRNA expression in patients overexpressing (A) none, (B) one, (C) two and (D) three of the three genes (TAZ, AXL and CTGF) in the GSE14333 colon cancer patient datasets. Kaplan-Meier analyses for SQLE mRNA expression in patients overexpressing (E) none, (F) one, (G) two and (H) three of the three genes in the GSE14333 colon cancer patient datasets. Kaplan-Meier analyses for ANO1 mRNA expression in patients overexpressing (I) none, (J) one, (K) two and (L) three of the three genes in the GSE17538 colon cancer patient datasets. Kaplan-Meier analyses for SQLE mRNA expression in patients overexpressed in the GSE17538 colon cancer patient datasets.

Figure 7. In vitro and in vivo assays for colon cancer cells expressing scramble shRNA or TAZ shRNA.

(A) Western blot showing that shTAZ specifically knockdown TAZ, but not YAP, and that AXL was down-regulated in shTAZ cells compared to shScr cells. (B) The clonogenicity and non-adherent growth of HCT116 cells expressing shScr or shTAZ were assessed and the number of colonies formed from three repeats was recorded (C) The clonogenicity and non-adherent growth of SW620 cells expressing shScr or shTAZ were assessed and the number of colonies formed from three repeats was recorded. (D) The in vivo tumorigenicity of HCT116 cells expressing shScr or shTAZ was assessed in nude nice, and the tumor formed was excised and weighted (n = 3 in each group). (E) The in vivo tumorigenicity of SW620 cells expressing shScr or shTAZ was assessed in nude nice, and the tumor formed was excised and weighted (n = 3 in each group).