Src, PKCalpha, and PKCdelta are required for alphavbeta integrin-mediated metastatic melanoma invasion

Background: Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor alpha v beta 3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis. Results: Two melanoma cell lines C8161.9 and M14 both express high levels of alpha v beta 3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an alpha v beta 3-depenent manner. Elevated levels of PKC alpha and PKC delta, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed alpha v beta 3-dependent invasion. Furthermore, over expression of Src or PKCa and PKCd was sufficient to confer alpha v beta 3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKC alpha expression, but not PKC delta, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKC alpha restored focal adhesion formation and partially restored stress fiber formation, while loss of PKC delta primarily restored stress fibers. Conclusion: The misregulated expression of PKC alpha and PKC beta and elevated Src activity in metastatic melanoma cells is required for efficient alpha v beta 3-mediated invasion. PKC alpha and Src enhance alpha v beta 3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKC alpha influences focal adhesion formation, while PKCd controls stress fibers