Alpha-CaMKII-induced VEGF expression is critical for the growth of human osteosarcoma.

摘要

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. Despite improvements in OS treatment, more specific molecular targets are needed. One target of interest is alpha-Ca2+/calmodulin-dependent protein kinase II (alpha-CaMKII), a ubiquitous mediator of Ca2+-linked signaling, which has been shown to regulate tumor cell proliferation.;Here, we show that alpha-CaMKII is highly expressed in primary OS tissue, and alpha-CaMKII deletion in human OS cell lines significantly reduces tumor burden in vivo. This inhibition of alpha-CaMKII results in decreased vascular endothelial growth factor (VEGF) protein secretion. Highly aggressive OS cells express VEGF receptor 2 (VEGFR-2), and respond to exogenous VEGF by increasing intracellular calcium. This response is ameliorated by CBO-P11 (VEGFR inhibitor), suggesting that secreted VEGF results in autocrine stimulated alpha-CaMKII activation. Moreover, CBO-P11 and the selective CaMKII inhibitor, KN-93, significantly reduce tumor burden in vivo, suggesting that OS tumor growth is controlled by CaMKII-regulated VEGF.;We expound on these initial studies and show that the FDA approved drugs tamoxifen and bevacizumab inhibit CaMKII, and decrease in vivo tumor growth. Furthermore, we developed a novel preclinical mouse model to examine the metastasis of human OS. After amputation of the tumor containing hind limb, the incidence of pulmonary metastasis in saline treated mice was 100%, with no detectable metastases when mice are subcutaneously injected daily with tamoxifen (10 mg/kg/day) and twice weekly with bevacizumab (2 mg/kg/day). These results suggest that tamoxifen and bevacizumab may be effective at inhibiting primary tumor growth and preventing metastasis.;OS cancer stem cells (CSCs) have been shown to express high levels of c-kit and Stro-1. Using fluorescence-activated cell sorting, we isolated CSCs (CD117+, Stro-1+) and double negative cells (DNCs) (CD117- and Stro-1-) from 143B OS cells. We show that 100% of mice injected with CSCs develop tumors and only 25% of mice injected with DNCs did so. Taken together, our results show a critical role of CaMKII in primary and metastatic tumor growth of human OS.