Abstract
Osteosarcoma is a malignant bone sarcoma characterized by extensive genomic disruption and a propensity for metastatic spread. Osteoid production suggests a close relationship with normal osteoblasts, and the latter are the presumptive cell of origin of this disease. The HACE1 gene, localized to human chromosome 6q21, encodes the HACE1 HECT E3 ligase, a tumor suppressor in diverse tumors that acts in part by targeting the activated form of RAC1 GTPase for proteasomal degradation. Disruption or loss of 6q21 is relatively common in osteosarcomas, and Hace1−/−/Tp53+/− mice frequently develop osteosarcomas, in contrast to Tp53+/− mice, which do not. This suggests an unexplored link between HACE1 loss and osteosarcoma. Here we compared HACE1 expression in normal osteoblasts and osteosarcoma cell lines in vitro by western blotting and quantitative RT-PCR, and in human osteosarcoma specimens by immunohistochemistry. Both HACE1 transcript and protein levels were reduced in osteosarcoma compared to osteoblasts in vitro. Reduced HACE1 expression in osteosarcoma tumors was observed in 76% of cases and associated with high-grade lesions. Further, clonally derived pairs of high and low metastatic osteosarcoma cell lines showed significant downregulation in the high compared to corresponding low metastatic cells. Ectopic expression of HACE1 markedly inhibited anchorage-independent growth and cell motility of HACE1 osteosarcoma cell lines, and was associated with reduced RAC1 activation and decreased reactive oxygen species (ROS). Finally, HACE1 overexpression blocked osteosarcoma xenograft growth and dramatically reduced pulmonary metastases. These findings point to a potential tumor suppressor function for HACE1 in osteosarcoma.
Highlights
Osteosarcoma is a malignant bone tumor commonly arising in areas of rapid bone growth, such as the distal femur and proximal tibia[1,2,3]
The plot shows fractions of copy number loss/gain of a cohort of 113 primary osteosarcoma specimens across chromosome 6. c Diagrammatic representation of the full-length HACE1 protein structure (i) and mutants lacking the HECT domain (ΔHECT; ii) or the ankyrin repeats (ΔANK; iii) used in this study. d OST cells were transfected with the indicated vectors
HEK-293 was used as a positive control while SK-NEP-1 served as a negative control for HACE1 expression. β-Actin was used as a loading control. f Immunofluorescence analysis of HACE1 expression in MG63 osteosarcoma cell line and OB3 osteoblast cell line displaying strong nucleo-cytoplasmic staining in OB3 cells contrary to remarkably weak cytoplasmic staining in MG63
Summary
Osteosarcoma is a malignant bone tumor commonly arising in areas of rapid bone growth, such as the distal femur and proximal tibia[1,2,3]. It represents the most common bone sarcoma, comprising approximately 20% of all bone tumors and about 5% of pediatric tumors[1]. Most cases of osteosarcoma are sporadic, but certain environmental and hereditary factors have been associated with elevated risk osteosarcoma[6,7] The former include exposure to ionizing radiation and Paget disease, with dysregulated bone recycling, both of which wellrecognized risk factors for the development of secondary osteosarcoma[8,9].
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