Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS) and pleomorphic (pRMS) histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways.

Fig 1. YAP suppression inhibits eRMS cell growth.

(A) YAP suppression by shRNA in RD cells is validated by qRT-PCR (left) and immunoblot (right). Lanes in the immunoblot are from the same membrane but have been rearranged into this order. (B) YAP suppression validation in SMS-CTR cells by qRT-PCR (left) and immunoblot (right). YAP suppression in (C) RD and (D) SMS-CTR cells inhibits cell growth as measured by manual cell counting over four days. **, P<0.01; ***, P<0.001; and ****, P<0.0001.

Fig 2. YAP suppression inhibits proliferation and stimulates apoptosis.

Cell proliferation, as measured by BrdU incorporation, is decreased in (A) RD and (B) SMS-CTR cells stably expressing YAP shRNAs. Immunoblot for cleaved caspase 3 (CC3) shows an increase in apoptosis at days 2 and 4 after shRNA expression in (C) RD and (D) SMS-CTR cells. qRT-PCR for YAP target genes Cyr61 and CTGF show decreased expression with YAP suppression in (E) RD and (F) SMS-CTR cells. Immunoblot analysis of TAZ in (G) RD and (H) SMS-CTR cells expressing YAP shRNAs. *, P< 0.05; **, P<0.01; and ****, P<0.0001.

Fig 3. YAP suppression delays tumor growth in vivo.

(A) In vivo YAP suppression in SMS-CTR xenografts inhibits tumor growth over time. (◊, NT; ■, YAP_sh3; ▲, YAP_sh4). (B) qRT-PCR for YAP, Cyr61, and CTGF show decreased expression in SMS-CTR xenografts expressing YAP shRNAs. Bars represent the average of each treatment group. (C) Quantitation of TUNEL (top) and Ki67 (bottom) staining reflects an increase in apoptosis and decrease in cell proliferation, respectively. (D) Representative images of H&E (left), YAP IHC (middle) and TUNEL staining (right) of the xenograft tumors. *, P<0.05; ***, P<0.001; and ****, P<0.0001. Scale bars: 100μm.

Fig 4. Pharmacologic inhibition of YAP inhibits tumor growth in vivo.

(A) SMS-CTR cells treated with 10μM VP have decreased cell growth, measured by manual cell counting over five days. (B) SMS-CTR subcutaneous xenografts treated with 100mg/kg VP have decreased tumor growth as compared to vehicle control (DMSO). (C) The average tumor weight of VP treated mice is decreased compared to control. (D) Ki67 staining is decreased in VP-treated mice, but TUNEL staining remains the same. **, P<0.01; ***, P<0.001; and ****, P<0.0001. Scale bars: 100μm.

Fig 5. YAP suppression promotes myogenic transcription factor expression.

qRT-PCR for myogenic differentiation genes Mrf4, MyoD, and Myogenin show increased expression with YAP suppression in (A) RD and (B) SMS-CTR cells. *, P< 0.05; ***, P<0.001; and ****, P<0.0001.

Fig 6. eRMS cell lines show increased differentiation with YAP knockdown.

Cells stably expressing YAP shRNAs were cultured in differentiation media for five days, then stained for MF20 expression. Representative images and quantitation of MF20 staining in (A, B) RD and (C, D) SMS-CTR cells. *, P< 0.05; ***, P<0.001. Scale bars: 62.5μm.

Fig 7. YAP expression enables senescence bypass.

(A) Total YAP protein levels in HSMMs ectopically expressing vector, YAPS127A, or YAPWT. (B) Top, population doublings over time of HSMMs expressing vector (●) or YAPS127A (■). Middle, vector-expressing cells show increased β-gal staining as compared to YAPS127A. Bottom, quantitation of β-gal staining. (C) Population doublings of HSMMs expressing YAPWT (top) and β-gal staining (bottom, middle). β-gal staining was performed at PD 16 (Vectors, YAPS127A) and PD 18 (YAPWT). ***, P<0.001; ****, P<0.0001. Scale bars: 125μm.

Fig 8. A genetically defined model of RMS based on serial stable expression of YAP, hTERT and oncogenic Ras.

(A) Schematic of the “YHR” genetic model predicted to form xenograft tumors. (B) Immunoblot validation of YAP, Ras, and actin expression (top) and RT-PCR validation of hTERT and GAPDH expression (bottom) in cell lines. In the pan-Ras blot the top band is the epitope (FLAG)-tagged exogenous oncogenic Ras and the bottom band is the endogenous Ras. (C) Tumor volume as measured over time of YHR (pink), THR (light gray, historical data), SMS-CTR (dark gray) and YHV (black with open circles) xenografts. (D) H&E of two individual YHR tumors (a, b) and representative immunohistochemistry of desmin and MyoD of YHR tumors to confirm skeletal muscle markers (bottom). Scale bars: 100μm.