Analysis of the role of Wiskott-Aldrich Symdrome protein in macrophage functions in inflammation and tumor metastasis

Abstract

Wiskott-Aldrich syndrome protein (WASp) is a nucleation promoting factor that integrates receptor signaling with actin cytoskeleton rearrangement. WASp is exclusively expressed by hematopoietic cells and WASp-deficiency of macrophages results in defective chemotaxis towards chemoattractant CSF-1. Using a mouse derived monocyte/macrophage cell line, RAW/LR5, in which endogenous WASp expression was reduced by short hairpin RNA (shWASp cells), it was determined that CSF-1 induced protrusions from shWASp cells were directional but persisted less compared to control cells. However, WASp-deficiency did not affect the recruitment of murine WASp-/-peritoneal macrophages in a mouse model of thioglycolate-induced peritonitis. Interestingly, activated WASp-/-peritoneal macrophages exhibited an increase in expression of a homologue known as neural-WASP (N-WASP) compared to unstimulated WASp-/-peritoneal macrophages. Since N-WASP is a homologue of WASp that is co-expressed by hematopoietic cells, whether N-WASP was compensating for the loss of WASp in activated macrophages was examined. Activated WASp-deficient peritoneal macrophages showed increased matrix degradation, suggesting that the expression levels of WASp and N-WASP influenced their roles in actin cytoskeleton rearrangement in macrophages.;Previous studies demonstrated the existence of a paracrine interaction between EGF-secreting macrophages and CSF-1-secreting breast carcinoma cells, which promotes mutual co-migration and tumor progression. Unlike control RAW/LR5 or wildtype (WASp+/+) bone marrow macrophages, WASp-deficient macrophages failed to promote the elongation and invasion of either rat (MTLn3) or human (MDA-MB 231 and 231-4173) metastatic breast carcinoma cell lines in vitro. The defect in carcinoma cell invasion could be rescued by addition of conditioned media from control macrophages, in an EGF-dependent manner, suggesting WASp may be required for processing of surface-bound EGF. Xenograft competent Rag2-deficient mice were crossed with WASp-deficient mice. While primary tumor growth of either rat (MTLn3-Cerulean) or human (MDA-MB 231-4173-Cerulean) cells was unaffected, carcinoma cell motility, invasion and lung metastasis were all significantly reduced in WASp-/-mice. Furthermore, results from a transgenic mouse model of breast cancer (PyMT) supported the data from the orthotopic tumor models. These results suggest that WASp is required for macrophage dependent breast carcinoma cell motility and invasion in the tumor microenvironment leading to metastasis, suggesting inhibition of WASp may have therapeutic benefit in the treatment of breast cancer.