Abstract
Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 co-expression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS.
Highlights
Osteosarcoma (OS) is the most common primary malignant tumor of the bone
Two weeks after Dox removal, at 5 weeks of age, quantitative real-time PCR analyses indicated that Wls expression was significantly decreased in tumor-bearing bones from WlsΔOB-OS mice compared to Wls floxed, H2k-c-fosLTR littermates that do not carry the Osx-tetO-cre allele (WlsWT-OS), consistent with activation of Cre expression (Fig. 1b)
When Wls was inactivated at birth before the onset of c-Fos transgene expression (Supplementary information, Fig. S1b), very few tumors were observed in 5 week-old WlsΔOB-OS mice and tumor volume and burden were decreased (Supplementary information, Fig. S1c–e)
Summary
Osteosarcoma (OS) is the most common primary malignant tumor of the bone. With a worldwide yearly incidence of ~1–3 cases per million, OS arises mostly in children and adolescents during bone growth,[1] with a second incidence peak after 50 years.[2]. Osteomas are part of the extra-colonic manifestations of FAP,[38] while APC somatic mutations are found in several tumor types including OS.[39,40] Wnt target genes, such as MYC and TWIST, are amplified in human OS, while Wnt inhibitory factor 1 (WIF1) is epigenetically silenced.[41,42,43] WLS/GRP177, encoding the exclusive cargo protein for Wnt ligands and essential to the secretion of all mammalian Wnts,[44,45] was recently identified as a likely oncogene in human OS.[46] These observations and the aberrant expression of numerous Wnt ligands, FZD/LRP receptors and Wnt inhibitors collectively support an implication of Wnt signaling in OS. LOXL2 is co-expressed with WNT7B/WNT9A and with FOS in a substantial fraction of human OS specimens and therapeutic targeting of Loxl[2] with a small molecule inhibitor, with shRNA or with blocking antibodies delays OS formation in experimental OS models
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