The soluble glycoprotein NMB (GPNMB) produced by macrophages induces cancer stemness and metastasis via CD44 and IL-33.

In cancer, myeloid cells have tumor-supporting roles. We reported that the protein GPNMB (glycoprotein nonmetastatic B) was profoundly upregulated in macrophages interacting with tumor cells. Here, using mouse tumor models, we show that macrophage-derived soluble GPNMB increases tumor growth and metastasis in Gpnmb-mutant mice (DBA/2J). GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. Through the CD44 receptor, GPNMB mechanistically activates tumor cells to express the cytokine IL-33 and its receptor IL-1R1L. We also determined that recombinant IL-33 binding to IL-1R1L is sufficient to induce tumor spheroid formation with features of cancer stem cells. Overall, our results reveal a new paracrine axis, GPNMB and IL-33, which is activated during the cross talk of macrophages with tumor cells and eventually promotes cancer cell survival, the expansion of cancer stem cells and the acquisition of a metastatic phenotype.

PO-44 - Tissue factor pathway inhibitor-2 (TFPI-2) is cleaved by PRSS3: implication for tumor endothelial cells migration.

INTRODUCTION: Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor whose expression is up-regulated by VEGF in microvascular and umbilical vein endothelial cells (EC). Despite this, TFPI-2 has been suggested as anti-angiogenic molecule, due to its ability to inhibit the migration/proliferation of EC induced by VEGF. Nothing is known about the precise mechanism of TFPI-2 function tuning in tumor endothelium. AIM: Aim of this study was to investigate the role of TFPI-2 in tumor vasculature, where angiogenesis and vascular remodeling are fundamental for cancer progression. MATERIALS AND METHODS: Tumor-EC were isolated from ovarian carcinomas and cultured in vitro in presence of factors reproducing the tumor microenvironment (VEGF, FGF-2, EGF). TFPI-2 and PRSS3 silencing was achieved by small interfering RNA (siRNA). Tumor-EC migration was assayed by the wound healing assay. Transcript expression was examined by qRT-PCR. Proteolytic reactions were monitored by western blot. RESULTS: We show that tumor-EC express TFPI-2, the majority of which is released and found anchored in the extracellular matrix. Silencing the expression of TFPI-2 enhances tumor-EC migration, confirming TFPI-2 as an anti-angiogenic molecule. We had previously shown that the cancer vasculature express PRSS3, a trypsin family member able to cleave proteins containing the kunitz-type domains; we reasoned that it could potentially inhibit TFPI-2. Herein, we demonstrate in a cell free system that TFPI-2 directly interacts with and is degraded by active PRSS3. In a more complex biological context, active PRSS3 is able to remove TFPI-2 from the extracellular matrix put down by tumor-EC. Accordingly, silencing PRSS3 causes the extracellular accumulation of TFPI-2 that results in the inhibition of tumor-EC migration. CONCLUSIONS: Our results demonstrate for the first time that TFPI-2 is a direct substrate of PRSS3, which hydrolyses TFPI-2 (most likely at the Kunitz-type domains) blocking its anti migratory capability. The proteolytic inactivation of TFPI-2 by PRSS3 might represent a mechanism favoring cancer by increasing angiogenesis and vascular remodeling. ACKNOWLEDGEMENT: Supported by the Italian Association for Cancer Research (AIRC).