Targeting Mutant (V600E) B-Raf in Melanoma Interrupts Immunoediting of Leukocyte Functions and Melanoma Extravasation

摘要

Polymorphonuclear neutrophils (PMN) facilitate melanoma cell extravasation under dynamic flow conditions by the binding of intercellular adhesion molecule-1 (ICAM-1) on melanoma cells to β2 integrins on PMNs, which is mediated by endogenously produced chemokine interleukin 8 (IL-8) from the tumor microenvironment. However, little is known about the role of B-Raf, the most mutated gene in malignant melanomas, in this process. In this study, we investigated the functional importance of B-Raf in melanoma extravasation by using short interfering RNA to reduce expression/activity of mutant ^V600EB-Raf in melanoma. Results indicated that knockdown of mutant ^V600EB-Raf inhibited melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inhibition of ^V600EB-Raf significantly reduced the constitutive secretion of IL-8 from melanoma cells as well as the capacity of endogenous IL-8 production from the melanoma-PMN microenvironment. Furthermore, a reduction in ICAM-1 expression on melanoma cells was detected following mutant ^V600EB-Raf knockdown. Together, these results suggest that targeting mutant ^V600EB-Raf reduces melanoma cell extravasation by decreasing IL-8 production and interrupting ICAM-1-β2 integrin binding of melanoma cells to the endothelium mediated by PMNs in the microcirculation, which provides a rationale and mechanistic basis for targeting mutant ^V600EB-Raf to inhibit melanoma extravasation and subsequent metastasis development.