The Growth and Aggressive Behavior of Human Osteosarcoma Is Regulated by a CaMKII-Controlled Autocrine VEGF Signaling Mechanism

Abstract

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease.

Transcription factors regulate GPR91-mediated expression of VEGF in hypoxia-induced retinopathy

“…Transcription factors control the expression of a given gene by serving as integration centers of different signaling cascades26. Many transcription factors regulate VEGF transcription and expression272829. Daft et al 27.…”

“…Many transcription factors regulate VEGF transcription and expression272829. Daft et al 27. found that the inhibition of CaMKII and VEGFR decreased HIF-1α and AP-1 binding to the VEGF promoter, which is likely responsible for the observed decreases in VEGF transcription.…”

Repetitive Transient Ischemia-Induced Cardiac Angiogenesis is Mediated by Camkii Activation

“…Previous studies have reported that chronic ischemia has a direct association with coronary angiogenesis, but the underlying molecular mechanisms are unclear [23,24]. It has been shown that CaMKII inhibition blunted tumor response to hypoxia [17] and that CaMKII is likely the upstream signal of the hypoxia inducible factor-1 alpha (HIF-1α) in macrophage and some cancer cell lines [14][15][16]. Indeed, oxidative stress and calcium entry caused by repetitive ischemia can both activate CaMKII [25][26][27].…”

“…The primary promoters of VEGF are HIF-1α and activating protein-1 (AP-1). CaMKII was able to up-regulate the expression of HIF-1α in marcrophage [14] and retinal Müller cells [31], and increase AP-1 protein level in osteosarcoma [15]. However, there was study showed that CaMKII inhibitor had no effect on HIF-1α induced VEGF production in rheumatoid synovial fibroblast [32].…”

“…Vascular endothelial growth factor (VEGF) is the vital stimulus for blood-vessel growth. Growing evidence showed that CaMKII activation may have potential connection with the increased expression of VEGF [14][15][16]. It has been demonstrated that CaMKII is capable of regulating the response of tumor to hypoxia via activating VEGF promoters [17].…”