Angiopoietin-like 4 (ANGPTL4) drives the progression and metastasis of many solid tumors but has not been described in osteosarcoma tissue. ANGPTL4 also enhances osteoclast activity, which is required for osteosarcoma growth in bone. We, therefore, investigated the expression and function of ANGPTL4 in human osteosarcoma tissue and cell lines

 

Osteosarcoma is the most common primary bone cancer in children and young adults. It is highly aggressive and patients that present with metastasis have a poor prognosis.

Teenagers who are active in sports often complain of pain in the lower femur, or immediately below the knee. If the tumor is large, it can present as overt localized swelling.

Sometimes a sudden fracture is the first symptom because the affected bone is not as strong as normal bone and may fracture abnormally with minor trauma. In cases of more deep-seated tumors that are not as close to the skin, such as those originating in the pelvis, localized swelling may not be apparent.

ANGPTL4 gene is a member of the angiopoietin-like gene family and encodes a glycosylated, secreted protein with a coiled-coil N-terminal domain and a fibrinogen-like C-terminal domain.

Expression of ANGPTL4 in osteosarcoma tissue microarrays was determined by immunohistochemistry. Hypoxic secretion of ANGPTL4 was tested by ELISA and Western blot.

Regulation of ANGPTL4 by hypoxia-inducible factor (HIF) was investigated using isoform-specific HIF siRNA (HIF-1α, HIF-2α). Effects of ANGPTL4 on cell proliferation, migration (scratch wound assay), colony formation and osteoblastogenesis were assessed using exogenous ANGPTL4 or cells stably transfected with ANGPTL4.

Osteoclastogenic differentiation of CD14+ monocytes was assessed by staining for tartrate-resistant acid phosphatase (TRAP), bone resorption was assessed by lacunar resorption of dentine.

ANGPTL4 was immunohistochemically detectable in 76/109 cases. ANGPTL4 was induced by hypoxia in 6 osteosarcoma cell lines, under the control of the HIF-1α transcription factor.

MG-63 cells transfected with an ANGPTL4 over-expression plasmid exhibited increased proliferation and migration capacity and promoted osteoclastogenesis and osteoclast-mediated bone resorption. Individually the full-length form of ANGPTL4 could increase MG-63 cell proliferation, whereas N-terminal ANGPTL4 mediated the other pro-tumourigenic phenotypes.

This study describes a role(s) for ANGPTL4 in osteosarcoma and identifies ANGPTL4 as a treatment target that could potentially reduce tumor progression, inhibit angiogenesis, reduce bone destruction and prevent metastatic events.