The Cancer Modeling Section seeks to elucidate the complex molecular/genetic program governing tumor genesis and progression through the development and analysis of genetically engineered mouse models of human cancer. Our efforts in this regard are focused on two tumor types, cutaneous malignant melanoma and the pediatric malignancy rhabdomyosarcoma (RMS). RMS accounts for up to 10% of all pediatric neoplasms and for more than 50% of pediatric soft tissue sarcomas, and is believed to arise from imbalances in skeletal muscle cell proliferation and differentiation (Merlino and Khanna, Genes & Dev. 21: 1275-9, 2007). There are two major subtypes of RMS. We have focused on embryonal RMS, the most common subtype, typically occurs in infants and young children. There is no distinct molecular event that characterizes embryonal RMS, but they tend to demonstrate severe genomic instability. Detailed molecular pathways associated with RMS had been poorly characterized, due in part to the lack of a RMS-prone animal model. We have developed the first relevant model for embryonal RMS, showing that virtually all mice harboring a hepatocyte growth factor/scatter factor transgene (HGF, which regulates its tyrosine kinase receptor MET) and deficiency in the tumor suppressor locus CDKN2A (encoding Ink4a/Arf) rapidly succumbed to highly invasive RMS (Sharp et al., Nature Med. 8: 1276-80, 2002). Highly comparable molecular lesions have also been described for human RMS. These data provide genetic evidence that MET and INK4a/ARF pathways represent critical, synergistic targets in RMS pathogenesis, and suggest a rational therapeutic combination to combat this pediatric sarcoma. Our analysis of the pathways downstream of INK4a/ARF revealed that it was the ARF-MDM2-p53 pathway, and not the INK4a-CDK4-pRb pathway that was the key regulator of this disease (Ha et al., Proc. Natl. Acad. Sci. 104: 10968-73, 2007). A panel of highly and poorly metastatic cell lines was subsequently established from the many RMS tumors arising in our novel mouse model, and used in concert with microarray-based expression profiling to identify a set of genes associated with enhanced metastatic behavior. Multiple functional in vivo studies confirmed that the cytoskeletal linker EZRIN and the homeodomain-containing transcription factor SIX1 both have essential roles in determining the metastatic fate of rhabdomyosarcoma cells (Yu et al., Nature Med. 10: 175-81, 2004). SIX1 was especially intriguing, as it is known to be required for skeletal muscle development. Notably, EZRIN and SIX1 expression levels were also both enhanced in human RMS tissues, significantly correlating with clinical stage. Subsequent molecular analyses showed that the EZRIN gene was a direct transcriptional target of SIX1, and indispensable for SIX1-mediated RMS metastasis (Yu et al., Cancer Res. 66: 1982-9, 2006). We also have found that SIX1 regulates EZRIN expression, at least in part, through epigenetic modification of the chromatin around the EZRIN gene locus, including regulating the states of methylation and acetylation within the histone tails. We are more fully elaborate mechanisms by which the EZRIN gene is regulated, and also determining the efficacy of preclinical drug studies that use histone deacetylase inhibitors or demethylating agents in concert with EZRIN knockdown to treat RMS. EZRIN appears to represent a very promising therapeutic target for patients with advanced stage RMS (Yu et al., PLoS One 5(9):e12710). Our RMS cell lines were employed in the identification and characterization of activating FGFR4 mutations in human RMS tumors, indicating that FGFR4 can function as an oncogene in RMS (Taylor et al., J. Clin. Invest. 119:3395-407, 2009). FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. FGFR4 mutants enhanced tumor proliferation and metastatic potential when expressed in a murine RMScell line. These findings support the potential therapeutic targeting of FGFR4 in RMS. In collaboration with Dr. Crystal Mackall, a new RMS cell line (M3-9-M) was derived from an embryonal RMS occurring in a C57BL/6 mouse transgenic for HGF and heterozygous for mutated p53. Primary tumors and metastases derived from M3-9-M were studied for similarities to human embryonal RMS, and for immunogenicity and immune responsiveness. Primary and metastatic tumors were found to develop after orthotopic injection of M3-9-M into immunocompetent C57BL/6 mice, which mirror human embryonal RMS with regard to histology, gene expression, and metastatic behavior. Whole cell vaccination using irradiated M3-9-M cells or M3-9-M-pulsed dendritic cells (DC)-induced tumor-specific T-cell responses that prevent tumor growth following low-dose tumor injection, and slow tumor growth following higher doses. Administration of anti-CD25 moAbs to deplete CD4(+) CD25(+) FOXP3(+) regulatory T cells prior to tumor vaccination enhanced the potency of the RMS tumor vaccine. Adoptive immunotherapy with M3-9-M primed T cells plus DC-based vaccination resulted in complete eradication of day 10 M3-9-M derived tumors. We concluded that M3-9-M derived murine RMS line was immunogenic and immunoresponsive, regulatory T cells contributed to immune evasion by murine RMS, and adoptive immunotherapy with DC vaccination eradicated low tumor burdens. This work was published (Meadors et al., Pediatr. Blood Cancer 57:921-9, 2011). RMS cell lines are still being used to elaborate metastatic mechanisms in various animal models, and being shared with the general research community. More recently, we have linked expression of Ezrin with that of the MET receptor tyrosine kinase. We have discovered that Ezrin expression is stimulated by HGF and correlates with MET signaling activity. MET stimulation activates Sp1, which in turn induces Ezrin gene transcription. This finding provides a novel molecular mechanism for ways that MET signaling stimulates metastasis. This project has been reduced in priority, but issues associated with RMS still arise in the lab.
| Balakumaran, Arun; Mishra, Prasun J; Pawelczyk, Edyta et al. (2015) Bone marrow skeletal stem/progenitor cell defects in dyskeratosis congenita and telomere biology disorders. Blood 125:793-802 |
| Sworder, Brian J; Yoshizawa, Sayuri; Mishra, Prasun J et al. (2015) Molecular profile of clonal strains of human skeletal stem/progenitor cells with different potencies. Stem Cell Res 14:297-306 |
| Meadors, Joanna L; Cui, Yonghzi; Chen, Qing-Rong et al. (2011) Murine rhabdomyosarcoma is immunogenic and responsive to T-cell-based immunotherapy. Pediatr Blood Cancer 57:921-9 |
| Yu, Yanlin; Zeng, Pingyao; Xiong, Jingbo et al. (2010) Epigenetic drugs can stimulate metastasis through enhanced expression of the pro-metastatic Ezrin gene. PLoS One 5:e12710 |
| Taylor 6th, James G; Cheuk, Adam T; Tsang, Patricia S et al. (2009) Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models. J Clin Invest 119:3395-407 |
