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Role of Protein Kinase D2 (PKD2) in Lymphangiogenesis
Introduction: Protein kinase D2 is a calcium/calmodulin kinase reported to play a critical role in motility, migration and invasion of cancer cells. We previously reported PKD2 to be highly expressed and activated by hypoxia in epithelial tumors including pancreatic, colorectal and gastric cancers, identifying this kinase as a new component of hypoxia signaling. Depletion of PKD2 in tumor cells prevented tumor angiogenesis and subsequent tumor cell proliferation in vivo. In endothelial cells PKDs are activated upon VEGF stimulation by involving tyrosine phosphorylation sites on the VEGF receptor 2 (VEGFR2). Since activation of VEGFR2 has been implicated both in blood and lymph vessels formation, we reasoned PKD2 to play a significant role in tumor lymphangiogenesis as well.
Aim: The aim of this study was to investigate the implication of PKD2 in the formation of lymph vessel during tumorigenesis.
Results: Vascular endothelial growth factor D (VEGF-D) is a secreted glycoprotein shown to regulate angiogenesis and lymphangiogenesis during development and tumor growth. Cultivation of two independent lung cancer cell lines in hypoxic environment revealed that high levels of the O2 sensor protein HIF1a are associated with enhanced VEGF-D expression and augmented VEGF-D promoter activity. Furthermore, RNAi-mediated depletion of PKD2 resulted in impaired hypoxia-induced VEGF-D expression and transcriptional activity.
Our in vivo tumor xenograft experiments conducted on chicken chorionallantoic membrane (CAM) reinforced the role of PKD2 in growth of cancer cells as demonstrated by a significant decrease in the number of Ki-67 positive lung tumor cells after siRNA-mediated ablation of the kinase. The examination of peritumoral lymph vessel formation revealed that decrease in tumor size upon PKD2 abrogation was associated with a marked reduction of the expression of lymphatic endothelium marker Prospero homeobox protein 1 (Prox1), previously reported to be a transcription factor to regulate proliferation, migration and invasion of cancer cells.
Conclusion: Collectively our findings suggest an important role of PKD2 as a critical regulator of tumor growth and tumor angiogenesis and could provide a useful target for regulation of pathological lymph vessel formation.