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Investigation of the role of HACE1 in osteosarcoma Clarkson, Paul William

Abstract

Osteosarcoma is a malignancy of childhood that is characterized by extensive genomic disruption within the tumour cells. It is proposed to have a close relationship with normal osteoblast development. HACE1 is a gene located at 6q21 in humans that has been shown to be a potential tumour suppressor in a wide range of tumours. Disruption or loss of 6q21 is relatively common in osteosarcomas, and mice that are Hace1-/- and p53+/- develop osteosarcomas, amongst other tumour types, while those that are solely p53+/- do not. Immunohistochemistry revealed that a number of osteosarcomas exhibit low expression of HACE1 protein, and where expression is low the protein is restricted to the cytoplasm, while in normal osteoblasts and high-expressing osteosarcomas the expression is nuclear and cytoplasmic. FISH results showed reduced 6q21 copy number in 45% of cases in one series, and in a second series one case out of 16 possessed a disruption in the 6q21 region. To investigate HACE1’s role in osteosarcoma further we developed a novel model for human osteoblasts by harvesting and culturing cells from discarded bone taken as graft during adolescent scoliosis surgery. Comparing the expression of HACE1 in these osteoblastic cells to osteosarcoma cells showed reduced levels of expression in osteosarcoma cells using qRT-PCR, but not by western blot analysis. Re-expression of functionally normal HACE1 in osteosarcoma cells using a lentiviral system significantly altered their behaviour in soft agar assays, Matrigel assays and produced larger subcutaneous tumours in immunodeficient mice. We conclude that HACE1 has a role in osteosarcoma as a growth regulator, and possibly as a tumour suppressor.

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