title: 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.
authors: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors: Paul G
authors: PG
authors: Daft
authors: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors: Yang
authors: Y
authors: Yang
authors: Institute of Oral Health Research, Department of Oral and Maxillofacial Surgery, School of Dentistry, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors: Dobrawa
authors: D
authors: Napierala
authors: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors: Majd
authors: M
authors: Zayzafoon
mesh_terms: D000818:Animals; D019898:Autocrine Communication / Q000187:drug effects; D001596:Benzylamines / Q000494:pharmacology; D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016228:Endothelial Growth Factors / Q000494:pharmacology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D009361:Neoplasm Invasiveness; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology; D010456:Peptides, Cyclic / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000187:drug effects; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000096926:Benzenesulfonamides
chemical_list: D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D016228:Endothelial Growth Factors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010456:Peptides, Cyclic; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D013449:Sulfonamides; D018808:Transcription Factor AP-1; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; C478485:cyclo-VEGI; C072105:KN 93; D000096926:Benzenesulfonamides