Amy E. Baek

Abstract Obesity and elevated circulating cholesterol are risk factors for breast cancer recurrence, while the use of statins, cholesterol biosynthesis inhibitors widely used for treating hypercholesterolemia, is associated with improved disease-free survival. Here, we show that cholesterol mediates the metastatic effects of a high-fat diet via its oxysterol metabolite, 27-hydroxycholesterol. Ablation or inhibition of CYP27A1, the enzyme responsible for the rate-limiting step in 27-hydroxycholesterol biosynthesis, significantly reduces metastasis in relevant animal models of cancer. The robust effects of 27-hydroxycholesterol on metastasis requires myeloid immune cell function, and it was found that this oxysterol increases the number of polymorphonuclear-neutrophils and γδ-T cells at distal metastatic sites. The pro-metastatic actions of 27-hydroxycholesterol requires both polymorphonuclear-neutrophils and γδ-T cells, and 27-hydroxycholesterol treatment results in a decreased number of cytotoxic CD8 + T lymphocytes. Therefore, through its actions on γδ-T cells and polymorphonuclear-neutrophils, 27-hydroxycholesterol functions as a biochemical mediator of the metastatic effects of hypercholesterolemia.

Abstract Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8 + T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2 −/− mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2 −/− macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8 + T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.

CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP), an essential second messenger, is particularly important in regulating genes controlling vascular homeostasis. These experiments test the hypothesis that cAMP might positively regulate the expression of CD39 and thereby modulate important vascular homeostatic properties. Cd39 mRNA was induced by 13.8- fold in RAW cells treated with a membrane-permeant cAMP analogue (8-bromo-cyclic AMP; 8-Br-cAMP), stimulation of adenylate cyclase, or prostanoids known to drive cAMP response. Fluorescence-activated cell sorting, immunofluorescence, and TLC assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Br-cAMP but not in cells transfected with short hairpin RNA against CD39. This analogue drove a significant increase in transcriptional activity at the Cd39 promoter although not when the promoter's cAMP-response element sites were mutated. Pretreatment with cAMP-dependent protein kinase (PKA), phosphoinositide 3-kinase (PI3K), or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser133) and ATF2 (Thr71) in a PKA-, PI3K-, and ERK-dependent fashion. Chromatin immunoprecipitation assays demonstrated that binding of phosphorylated CREB1 and ATF2 to cAMP-response element-like sites was significantly increased with 8-Br-cAMP treatment and that binding was reduced with PKA, PI3K, and ERK inhibition, whereas transfection of Creb1 and Atf2 overexpression constructs enhanced cAMP-driven Cd39 mRNA expression. Transfection of RAW cells with mutated Creb1 (S133A) reduced cAMP-driven Cd39 mRNA expression. Furthermore, the cAMP-mediated induction of Cd39 mRNA, protein, and phosphohydrolytic activity was replicated in primary peritoneal macrophages. These data identify cAMP as a crucial regulator of macrophage CD39 expression and demonstrate that cAMP acts through the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways to control a key vascular homeostatic mediator. CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP), an essential second messenger, is particularly important in regulating genes controlling vascular homeostasis. These experiments test the hypothesis that cAMP might positively regulate the expression of CD39 and thereby modulate important vascular homeostatic properties. Cd39 mRNA was induced by 13.8- fold in RAW cells treated with a membrane-permeant cAMP analogue (8-bromo-cyclic AMP; 8-Br-cAMP), stimulation of adenylate cyclase, or prostanoids known to drive cAMP response. Fluorescence-activated cell sorting, immunofluorescence, and TLC assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Br-cAMP but not in cells transfected with short hairpin RNA against CD39. This analogue drove a significant increase in transcriptional activity at the Cd39 promoter although not when the promoter's cAMP-response element sites were mutated. Pretreatment with cAMP-dependent protein kinase (PKA), phosphoinositide 3-kinase (PI3K), or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser133) and ATF2 (Thr71) in a PKA-, PI3K-, and ERK-dependent fashion. Chromatin immunoprecipitation assays demonstrated that binding of phosphorylated CREB1 and ATF2 to cAMP-response element-like sites was significantly increased with 8-Br-cAMP treatment and that binding was reduced with PKA, PI3K, and ERK inhibition, whereas transfection of Creb1 and Atf2 overexpression constructs enhanced cAMP-driven Cd39 mRNA expression. Transfection of RAW cells with mutated Creb1 (S133A) reduced cAMP-driven Cd39 mRNA expression. Furthermore, the cAMP-mediated induction of Cd39 mRNA, protein, and phosphohydrolytic activity was replicated in primary peritoneal macrophages. These data identify cAMP as a crucial regulator of macrophage CD39 expression and demonstrate that cAMP acts through the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways to control a key vascular homeostatic mediator.