Targeting mutant (V600E) B-Raf in melanoma interrupts immunoediting of leukocyte functions and melanoma extravasation

Polymorphonuclear neutrophils (PAIN) facilitate melanoma cell extravasation under dynamic flow conditions by the binding of intercellular adhesion molecule-1 (ICAM-1) on melanoma cells to beta(2) 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 E-V600 B-Raf in melanoma. Results indicated that knockdown of mutant (EB)-E-V600-Raf inhibited melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inhibition of (EB)-E-V600-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 (EB)-E-V600-Raf knockdown. Together, these results suggest that targeting mutant (EB)-E-V600-Raf reduces melanoma cell extravasation by decreasing IL-8 production and interrupting ICAM1-beta(2) integrin binding of melanoma cells to the endothelium mediated by PMNs in the microcirculation, which provides a rationale and mechanistic basis for targeting mutant E-V600 BRaf to inhibit melanoma extravasation and subsequent metastasis development.