Associate Research Specialist Children's Hospital Los Angeles Los Angeles, California, United States
Introduction:: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue cancer categorized by two subtypes, fusion protein-positive (FPRMS) and negative (FNRMS), based on the existence of PAX3-FOXO1 fusion gene. Worse clinical outcomes of FPRMS than FNRMS require understanding the effect of the fusion gene and corresponding fusion protein on a worse clinical outcome. However, how the expression levels are determined and regulate related outcomes still need to be fully understood. FPRMS shows the lower expression of the fusion gene in the leading edge of collective tumor tissue where cells directly contact extracellular matrix (ECM). Thus, we suggest that ECM suppresses fusion gene expression, driving cell migration, reflective of metastatic invasion.
Materials and Methods:: We seeded RH30 and RH41 FPRMS cell lines on the plate coated with varying concentrations of collagen type I (0, 1, 3, and 10 ug) and assessed the expression level of FOXO1-PAX3 via immunoblotting and corresponding cell phenotypes such as proliferation and migration. Additionally, we created cell lines that can induce knockdown of fusion gene expression. Using these, we investigated molecular and phenotypic changes induced by knockdown of the fusion gene, modeling cells with lower expression of the fusion gene.
Results, Conclusions, and Discussions:: We discovered that FPRMS cells showed an inversely proportional of fusion protein levels to collagen concentration. Additionally, the cells showed faster migration and slower proliferation on the highest collagen-coated plates and vice versa. These results suggest that lower fusion protein may promote cell migration and suppress cell proliferation. Indeed, the fusion gene knockdown cells showed higher expression of genes related to migration (cell-ECM interaction) and lower expression of genes related to proliferation (CDK6-cyclin D1). Consistently, the knockdown cells exhibited faster cell migration.
The heterogeneity of the FPRMS indicates discrete expressions of PAX3-FOXO1, regulated by ECM in the tumor microenvironment, determining invasion vs. proliferation.
