Legumain-deficient macrophages promote senescence of tumor cells by sustaining JAK1/STAT1 activation

Highlights

• Depletion of legumain in macrophages suppresses tumor growth and activates senescence in tumor cells.

• Downregulation of legumain in macrophage leads to sustained activation of JAK1/STAT1 and pro-inflammatory phenotype.

• Macrophage-derived IL-1β mediates the pro-senescence effect of Lgmn^-/- macrophages on tumor cells.

• Transplantation of Lgmn^-/- bone marrow suppresses tumor growth through activating tumor senescence.

Abstract

Macrophages serve as the first line of communication between tumors and the rest of the immune system, and understanding the interplay between macrophage and tumor cells is essential for developing novel macrophage-based strategy against tumor. Here, we show that deletion of legumain in macrophages activates senescence of tumor cells. Macrophage derived IL-1β mediates the pro-senescent effect of Lgmn^-/- macrophages since blockage of IL-1β reverses the senescence phenotype in both a coculture model of macrophage and tumor cells and an orthotopic mouse model of breast cancer. Sustained activation of JAK1/STAT1 signaling and increased iNOS were found in the tumor cell-cocultured Lgmn^-/- macrophages, which were necessary for IL-1β expression and secretion. Applying a specific STAT1 agonist mimics the inductive effect of legumain deletion on IL-1β expression in macrophages, and the effect can be blocked via inhibition of iNOS. Legumain and integrin αvβ3 interact to prevent STAT1 signaling in macrophages, and blockage of integrin αvβ3 stimulates STAT1 activation. Therapeutically, transplantation of bone marrow from Lgmn^-/- mice suppresses the malignant growth of tumor by upregulating tumor cell senescence. Therefore, our finding highlights legumain in macrophages as a potential therapeutic target for tumors.

Introduction

Macrophages (MΦ) account for one of the largest cell populations in the tumor microenvironment [1,2]. There exist at least two MΦ polarized directions, pro-inflammatory ‘M1’ or pro-tumor ‘M2’ [3,4]. Although it has been accepted that tumor-associated MΦs (TAMs) are largely involved in many aspects of malignant tumor growth, the regulatory processes of distinct MΦ polarization, as well the mechanism underlying the effect of different activated macrophages on the tumor progress is still obscure [5,6].

Recent studies have shown that legumain, a highly conserved asparaginyl endopeptidase, correlates closely with the poor prognosis of human tumors [[7], [8], [9]]. Notably, legumain is overexpressed in both tumor cells and TAMs in the tumor microenvironment [10]. Depletion of TAMs via legumain-based DNA vaccine led to significant suppression of malignant tumor growth [11]. Lin et al. reported the pro-metastatic effect of tumor cell-derived legumain and circulatory legumain as the prognostic marker in breast cancer patients [12]. Considering the entirely different properties of MΦs and tumor cells, along with the critical role of TAMs in determining the nature of the tumor, we sought to understand the particular mechanism underlying the effect of MΦ-derived legumain in the tumor microenvironment.

Previous reports have indicated that legumain works mainly as a lysosomal protease after maturation upon sequential removal of C- and N- terminals [13]. However, protease-independent functions of legumain have been increasingly reported outside the lysosome, including in the nucleus, at the cell surface, and extracellularly [14,15]. In diverse pathological settings including cancer or Alzheimer's disease, the molecular properties of legumain has been associated with the corresponding cellular compartments and milieus [16,17]. Structure determination of legumain provides the theoretical support for its activation, conformational stability and enzymatic function in the zymogenic and fully activated forms [18].

Here, we investigate the effect of MΦ-derived legumain on the polarization of TAMs and the consequential influence on the tumor growth through interaction with integrin αvβ3. We find that deletion of legumain in TAMs leads to sustained activation of STAT1, which increases iNOS and secretion of IL-1β, therefore induces senescence of tumor cells. Through transplantation of bone marrow from Lgmn^-/-mice, we demonstrate that Lgmn^-/- MΦs activates the senescence program, thereby suppressing malignant growth of the tumor.