Upstream regulation of TAZ and YAP in sarcomas: Towards combinatorial therapytargeting the Hippo pathway

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Sarcomas are cancers arising within bone and soft tissue which rank among the most difficult cancers to treat. Limb-salvage therapy has reduced the incidence of disfiguring amputations, however many sarcomas involve crucial anatomical structures precluding this approach. Additionally, five year survival for metastatic sarcomas is only 16%, underscoring the need for new therapeutic targets. The Hippo pathway is a highly conserved serine/threonine kinase cascade which negatively regulates the TAZ and YAP transcriptional coactivators. TAZ and YAP have emerged as important oncogenes in a number of cancers including breast, colon, liver, lung, pancreas, and thyroid cancers. We have shown that TAZ/YAP are activated in the majority of sarcomas (66%), however upstream mechanisms regulating TAZ and YAP are poorly understood, representing a significant gap in knowledge. Our objective in this proposal is to dissect the signal transduction cascades that activate TAZ and YAP in sarcomas. Our central hypothesis is that histone deacetylation of the regulatory regions of the core Hippo kinases and MAP4 kinases works in tandem with PI3 kinase signaling to activate TAZ/YAP. This hypothesis is supported by studies showing that loss of expression of the Hippo kinases is common in both sarcoma clinical samples and cell lines as well as pharmacological studies showing that inhibition of histone deacetylases (HDACs) lead to reconstitution of the Hippo kinases thus inhibiting TAZ/YAP. A parallel set of studies utilizing genetically engineered and xenograft mouse models and sarcoma cell lines have shown that TAZ and YAP differentially mediate PI3 kinase signaling in undifferentiated pleomorphic sarcoma and pediatric rhabdomyosarcomas. We plan to test our central hypothesis with the following specific aims: Aim 1) Identify how TAZ and YAP differentially contribute to PI3 kinase-mediated sarcomagenesis. We will stimulate PI3K signaling in rhabdomyosarcoma cell lines that are wild-type, TAZ knockout, YAP knockout, and TAZ/YAP knockout and determine how TAZ and YAP differentially contribute to the PI3 kinase transcriptome by RNA-Seq gene expression analysis. We will also stimulate PI3K signaling in the muscle of sarcoma-prone mice that are Trp53fl/flPtenfl/fl and that are Tazfl/flYapfl/fl, Tazfl/fl, Yapfl/fl, or Taz/Yap wild type and compare tumor latency and gene expression profiles. Aim 2) Determine if histone deacetylation of MST1/2 and the MAP4 kinases activates TAZ and YAP. We will inhibit HDACs and assess occupancy of H3K27ac at promoter/enhancer regions of MST1/2 and the MAP4 kinases (MM kinases) in rhabdomyosarcoma cell lines. To test the importance of the MM kinases in tumor progression in vivo, we will delete genes encoding the LATS1/2 kinases (that are downstream of the MM kinases) in sarcoma-prone mice. To achieve this we will inject Trp53fl/fl mice that are Lats1fl/flLats2fl/fl or are Lats1/Lats2 wild-type with intramuscular adeno-Cre virus. Aim 3) Determine if inhibition of HDACs and the PI3K-Akt axis diminish proliferation and metastasis. We will determine if combinatorial inhibition of histone deacetylases and the PI3 kinase pathway will inactivate TAZ/YAP. This will be tested in vitro with proliferation and cell cycle analysis assays. The effect of combination therapy on spontaneous metastases will be tested in a sarcoma xenograft mouse model. The proposal is innovative because it targets two incompletely understood parallel pathways upstream of TAZ/YAP, expanding the focus of the field beyond targeting TAZ/YAP themselves, and thus addressing a significant gap in knowledge. The project is significant because it identifies upstream pathways regulating TAZ/YAP that can be inhibited pharmacologically, providing a mechanism to make therapeutic targeting of TAZ and YAP clinically feasible for sarcomas and potentially other cancers.