Inflammation, NLRP3 Inflammasomes, Macrophages and Breast Cancer Progression
One in 8 women will be diagnosed with breast cancer in their lifetime and while breast cancer mortality has declined since the 1980s, it remains the 2nd leading cause of cancer-related deaths among women. Standard of care is determined by anatomical staging and molecular biomarkers, but generally includes surgery, systemic or targeted chemotherapy or immunotherapy. Therapy resistance remains challenging and is exacerbated by the presence of cancer-stem cells and inflammation. Tumor microenvironment heterogeneity – especially high macrophage infiltration - in breast cancer is associated with larger tumor size, increased vascularization, lower hormone and growth factor expression and lower overall survival. Macrophage phenotype is highly plastic and on a continuum between M1 and M2 macrophages. M1 or classically-activated macrophages are phagocytic, pro-inflammatory and express high levels of inducible nitric oxide synthase (iNOS). In contrast, M2 or alternatively activated macrophages are endocytic and only partially phagocytic, immunosuppressive, and associated with poor prognostic outcomes Additionally, M2 macrophages express arginase 1 and surface marker CD206 and secrete VEGF and matrix metalloproteinases that promote angiogenesis and matrix remodeling, respectively. Macrophage activation occurs, in part, through cell membrane bound pattern recognition receptors (PRRs) interacting with external pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) including inflammasomes. In particular, the NLRP3 inflammasome is a multiprotein complex which consists of the intracellular receptor, NLRP3, the adaptor, ASC1, and effector caspase, caspase 1. Upon activation, these proteins oligomerize and, through caspase activity, cleave pro-inflammatory IL-1β and IL-18 cytokines into their mature active forms. Furthermore, pyroptosis, i.e., an inflammatory form of cell death, that can supersede inflammasome activation is mediated by cleavage of gasdermin D and membrane pore formation. First, our results demonstrate through in vitro treatment of macrophages with known inflammasome activators and secretions from tumor cells that macrophages differentially express NLRP3 inflammasomes and NLRP3 inflammasome activation was associated with the development of a pro-tumorigenic macrophage phenotype. Second, our in vivo data in immunocompetent, orthotopic, pre-clinical, murine model injected with 4T1 tumors alone or combined with J774 macrophages demonstrate that NLRP3 inflammasome activation in macrophages support tumor proliferation by increased cancer-stem cells and metastasis. Our findings also determined the efficacy of NLRP3 inflammasome inhibition and chemotherapy in in vitro co-cultures and in vivo immunocompetent, orthotopic, preclinical, murine models demonstrating that the MCC950+5-FU treatment combination decreased inflammasome complex expression and tumor proliferation in vitro and reduced in vivo tumor growth and leukocyte infiltration in mice co-implanted with 4T1 tumor cells and J774 macrophages. Lastly, our mechanism investigations assessed whether NLRP3 inflammasome activation or pore-mediated cytokine secretion and subsequent autocrine signaling were involved in the generation of the pro-tumorigenic shift in macrophage phenotype associated with NLRP3 inflammasome activation. While following incubation with specific P2RX7 antagonist and gasdermin D inhibitor, inflammasome protein expression remained unchanged, the gasdermin D inhibition using disulfiram effectively limited pro-inflammatory IL-1β cytokine secretion. Moreover, blocking either P2RX7 signaling or gasdermin D pore formation reversed macrophage polarization toward an M2-like pro-tumorigenic phenotype. Taken together, our work highlights the role of the NLRP3 inflammasome activation in the pro-tumorigenic and immunosuppressive phenotype shift in macrophages. Ongoing and future studies will further explore the potential of targeting the NLRP3 inflammasome pathway to alter to modulate pro-tumorigenic and immunosuppressive macrophage within the tumor microenvironment to more effectively prevent breast cancer progression.