Alveolar rhabdomyosarcoma (aRMS) is a childhood soft tissue sarcoma driven by the signature (P3F) fusion gene. Five-year survival for aRMS is<50%, with no improvement in over 4 decades. Although the transcriptional coactivator TAZ is oncogenic in carcinomas, the role of TAZ in sarcomas is poorly understood. The aim of this study was to investigate the role of TAZ in P3F-aRMS tumorigenesis. After determining from publicly available datasets that TAZ is upregulated in human aRMS transcriptomes, we evaluated whether TAZ is also upregulated in our myoblast-based model of P3F-initiated tumorigenesis, and performed IHC staining of 63 human aRMS samples from tissue microarrays. Using constitutive and inducible RNAi, we examined the impact of TAZ loss of function on aRMS oncogenic phenotypes and tumorigenesis Finally, we performed pharmacologic studies in aRMS cell lines using porphyrin compounds, which interfere with TAZ-TEAD transcriptional activity. TAZ is upregulated in our P3F-initiated aRMS model, and aRMS cells and tumors have high nuclear TAZ expression. , TAZ suppression inhibits aRMS cell proliferation, induces apoptosis, supports myogenic differentiation, and reduces aRMS cell stemness. TAZ-deficient aRMS cells are enriched in G-M phase of the cell cycle. , TAZ suppression attenuates aRMS xenograft tumor growth. Preclinical studies show decreased aRMS xenograft tumor growth with porphyrin compounds alone and in combination with vincristine. TAZ is oncogenic in aRMS sarcomagenesis. While P3F is currently not therapeutically tractable, targeting TAZ could be a promising novel approach in aRMS. .

SUMMARYCellular heterogeneity is frequently observed in cancer, but the biological significance of heterogeneous tumor clones is not well defined. Using multicolor reporters and CRISPR-Cas9 barcoding, we trace clonal dynamics in a mouse model of sarcoma. We show that primary tumor growth is associated with a reduction in clonal heterogeneity. Local recurrence of tumors following surgery or radiation therapy is driven by multiple clones. In contrast, advanced metastasis to the lungs is driven by clonal selection of a single metastatic clone (MC). Using RNA sequencing (RNA-seq) and in vivo assays, we identify candidate suppressors of metastasis, namely, Rasd1, Reck, and Aldh1a2. These genes are downregulated in MCs of the primary tumors prior to the formation of metastases. Overexpression of these suppressors of metastasis impair the ability of sarcoma cells to colonize the lungs. Overall, this study reveals clonal dynamics during each step of tumor progression, from initiation to growth, recurrence, and distant metastasis.

A hallmark of fusion-positive alveolar rhabdomyosarcoma (aRMS) is the presence of a chromosomal translocation encoding the fusion oncogene. Primary cell-based modeling experiments have shown that is necessary, but not sufficient for aRMS tumorigenesis, indicating additional molecular alterations are required to initiate and sustain tumor growth. Previously, we showed that -positive aRMS is promoted by dysregulated Hippo pathway signaling, as demonstrated by increased YAP1 expression and decreased MST activity. We hypothesized that ablating MST/Hippo signaling in a genetically engineered mouse model (GEMM) of aRMS would accelerate tumorigenesis. To this end, MST1/2-floxed ( ) mice were crossed with a previously established aRMS GEMM driven by conditional expression of from the endogenous locus and conditional loss of in (myogenic factor 6)-expressing cells. Compared with controls, animals displayed accelerated tumorigenesis ( < 0.0001) and increased tumor penetrance (88% vs. 27%). GEMM tumors were histologically consistent with aRMS. GEMM tumor-derived cell lines showed increased proliferation and invasion and decreased senescence and myogenic differentiation. These data suggest that loss of MST/Hippo signaling acts with expression and loss to promote tumorigenesis. The rapid onset and increased penetrance of tumorigenesis in this model provide a powerful tool for interrogating aRMS biology and screening novel therapeutics. A novel mouse model sheds light on the critical role of Hippo/MST downregulation in PAX3-FOXO1-positive rhabdomyosarcoma tumorigenesis. .

Prior research has implicated the involvement of cell adhesion molecule N-cadherin in tissue fibrosis and remodeling. We hypothesize that anomalies in N-cadherin protein processing are involved in pathological fibrosis. Diseased tissues associated with fibrosis of the heart, lung, and liver were probed for the precursor form of N-cadherin, pro-N-cadherin (PNC), by immunohistochemistry and compared to healthy tissues. Myofibroblast cell lines were analyzed for cell surface pro-N-cadherin by flow cytometry and immunofluorescent microscopy. Soluble PNC products were immunoprecipitated from patient plasmas and an enzyme-linked immunoassay was developed for quantification. All fibrotic tissues examined show aberrant PNC localization. Cell surface PNC is expressed in myofibroblast cell lines isolated from cardiomyopathy and idiopathic pulmonary fibrosis but not on myofibroblasts isolated from healthy tissues. PNC is elevated in the plasma of patients with cardiomyopathy (p ≤ 0.0001), idiopathic pulmonary fibrosis (p ≤ 0.05), and nonalcoholic fatty liver disease with cirrhosis (p ≤ 0.05). Finally, we have humanized a murine antibody and demonstrate that it significantly inhibits migration of PNC expressing myofibroblasts. Collectively, the aberrant localization of PNC is observed in all fibrotic tissues examined in our study and our data suggest a role for cell surface PNC in the pathogenesis of fibrosis.