The Hippo effector TAZ (WWTR1) transforms myoblasts and TAZ abundance is associated with reduced survival in embryonal rhabdomyosarcoma

Abstract

The Hippo effector YAP has recently been identified as a potent driver of embryonal rhabdomyosarcoma (ERMS). Most reports suggest that the YAP paralogue TAZ (gene symbol WWTR1) functions as YAP but, in skeletal muscle, TAZ has been reported to promote myogenic differentiation, whereas YAP inhibits it. Here, we investigated whether TAZ is also a rhabdomyosarcoma oncogene or whether TAZ acts as a YAP antagonist. Immunostaining of rhabdomyosarcoma tissue microarrays revealed that TAZ is significantly associated with poor survival in ERMS. In 12% of fusion gene-negative rhabdomyosarcomas, the TAZ locus is gained, which is correlated with increased expression. Constitutively active TAZ S89A significantly increased proliferation of C2C12 myoblasts and, importantly, colony formation on soft agar, suggesting transformation. However, TAZ then switches to enhance myogenic differentiation in C2C12 myoblasts, unlike YAP. Conversely, lentiviral shRNA-mediated TAZ knockdown in human ERMS cells reduced proliferation and anchorage-independent growth. While TAZ S89A or YAP1 S127A similarly activated the 8XGTIIC-Luc Hippo reporter, only YAP1 S127A activated the Brachyury (T-box) reporter. Consistent with its oncogene function, TAZ S89A induced expression of the ERMS cancer stem cell gene Myf5 and the serine biosynthesis pathway (Phgdh, Psat1, Psph) in C2C12 myoblasts. Thus, TAZ is associated with poor survival in ERMS and could act as an oncogene in rhabdomyosarcoma. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Keywords: Hippo pathway; TAZ; WWTR1; embryonal rhabdomyosarcoma; myoblasts.

Figure 1

TAZ is associated with reduced survival in ERMS. (A) Examples of rhabdomyosarcoma, showing either negative or positive TAZ immunostaining; positive TAZ staining shows both strong nuclear and cytoplasmic TAZ staining, shown at higher magnification in Figure S1A (see supplementary material); scale bar = 50 µm. (B) Percentage survival of ERMS and ARMS patients who had sufficient follow‐up to be included in the survival analysis scored positive and negative for TAZ; the table shows patients at each given time point, ie those who had not died or had been lost to follow‐up. (C) Abundance and localization of TAZ in ERMS and ARMS. (D) (Left) Expression of WWTR1 and protein abundance in cultured human skeletal myoblasts, RD and RH30 cells; data are presented as mean ± SD, where n = 3 and * denotes a significant difference (p < 0.05), as assessed using one‐way ANOVA when compared to human myoblasts. (E) Genomic regions on chromosome 3 that include the WWTR1 locus were gained in five of 43 (12%) fusion‐negative rhabdomyosarcoma samples 33.

Figure 2

TAZ and TAZ S89A enhance proliferation in C2C12 myoblasts. (A) pMSCV map and schematic drawing of the control vector, TAZ and TAZ S89A‐encoding inserts. (B) Validation of human WWTR1 expression and TAZ protein levels in cells transduced with TAZ or TAZ S89A‐encoding retroviruses. (C) Example images of C2C12 myoblasts transduced with control vector, TAZ or TAZ S89A‐encoding retroviruses and immunostained 2 days later for the proliferation marker Ki67, and quantification of the proportion of Ki67‐expressing myoblasts; scale bar = 50 µm; data are expressed as mean ± SD, where n = 3 and * denotes a significant difference (p < 0.05), as assessed using one‐way ANOVA when compared to control.

Figure 3

TAZ or mutant TAZ S89A promote transformation of C2C12 myoblasts. (A) Example images showing colony formation in C2C12 myoblasts transduced with empty vector, TAZ or TAZ S89A‐encoding retroviruses; TAZ or TAZ S89A significantly increase colony number and size when compared to control vector. (B) Expression of TAZ or TAZ S89A did not prevent myogenic differentiation of C2C12 myoblasts when compared to control (empty vector). TAZ or TAZ S89A, actually significantly increased the fusion index after 72 h. Data are expressed as mean ± SD, where n = 3 and * indicates a significant difference (p < 0.05) using one‐way ANOVA when compared to control; scale bar = (A) 500 µm; (B) 50 µm

Figure 4

TAZ S89A activates genes associated with human cancer. (A) Effects of TAZ, mutant TAZ S89A and YAP S127A expression on Hippo pathway reporter constructs 8XGTIIC–Luc, CTGF–Luc and Brachyury–Luc in C2C12 myoblasts. (B) Validation of the expression of human WWTR1 and effects of TAZ or TAZ S89A expression in C2C12 myoblasts on expression of Ctgf, Myf5, Phgdh, Psat1 and Psph. Data are expressed as mean ± SD, where n = 3 and * indicates a significant difference (p < 0.05) when compared to control using one‐way ANOVA

Figure 5

TAZ knockdown reduces proliferation and transformation behaviour in RD cells. (A) Transduction of RD cells with scrambled or TAZ shRNA‐encoding lentiviruses for 24 h followed by 72 h puromycin selection reduced TAZ protein and mRNA but did not affect YAP expression, as assessed by western blot and qPCR. (B) Example images showing number of colonies and their size with RD cells transduced with either scrambled or TAZ shRNA; TAZ knockdown significantly reduced colony number when compared to control. (C) Example images of RD cells transduced with scrambled or TAZ shRNA lentiviruses and incorporation of EdU visualized and quantified. Data are expressed as mean ± SD, where n = 3 and * denotes a significant difference (p < 0.05) when compared to control using unpaired t‐test; scale bar = (B) 500 µm; (C) 50 µm

Figure 6

TAZ knockdown reduces the levels of known oncogenes in RD cells. (A) shRNA‐mediated knockdown of TAZ in RD cells had no effect on myogenic differentiation, as shown by immunostaining with MyHC. (B) Expression of CYR61, E2F1, BIRC5 and activity of the 8XGTIIC–Luc reporter was assessed in RD cells after TAZ knockdown. Data are expressed as mean ± SD, where n = 3 and * indicates a significant difference (p < 0.05) when compared to control using unpaired t‐test; scale bar = 50 µm