Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S.

During progression of rheumatoid arthritis (RA), angiogenesis provides oxygen and nutrients for the cells' increased metabolic demands and number. To turn on angiogenesis, pro-angiogenic factors must outweigh anti-angiogenic factors. We have previously shown that CD147/extracellular matrix metalloproteinase inducer (EMMPRIN) can induce the expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) in a co-culture of the human HT1080 fibrosarcoma and U937 monocytic-like cell lines. However, whether CD147 influences anti-angiogenic factors was not known. We now show that relative to single cultures, the co-culture of these cells not only enhanced pro-angiogenic factors but also decreased the anti-angiogenic factors endostatin and thrombospondin-1 (Tsp-1), generally increasing the angiogenic potential as measured by a wound assay. Using anti-CD147 antibody, CD147 small interfering RNA (siRNA), and recombinant CD147, we demonstrate that CD147 hormetically regulates the generation of endostatin but has no effect on Tsp-1. Since endostatin is cleaved from collagen XVIII (Col18A), we applied different protease inhibitors and established that MMP-9 and proteasome 20S, but not cathepsins, are responsible for endostatin generation. MMP-9 and proteasome 20S collaborate to synergistically enhance endostatin generation, and in a non-cellular system, CD147 enhanced MMP-9 activity and hormetically regulated proteasome 20S activity. Serum samples obtained from RA patients and healthy controls mostly corroborated these findings, indicating clinical relevance. Cumulatively, these findings suggest that secreted CD147 mediates a possibly allosteric effect on MMP-9 and proteasome 20S activities and can serve as a switch that turns angiogenesis on or off, depending on its ambient concentrations in the microenvironment.

Inhibition of tumor growth and metastasis by EMMPRIN multiple antigenic peptide (MAP) vaccination is mediated by immune modulation.

Previously, we have identified a new epitope in EMMPRIN, a multifunctional protein that mediates tumor cell-macrophage interactions and induces both MMP-9 and VEGF. Here, we synthesized this epitope as an octa-branched multiple antigenic peptide (MAP) to vaccinate mice implanted with subcutaneous syngeneic colon (CT26), prostate (TRAMP-C2) or renal (RENCA) cell line carcinomas. Vaccination inhibited, and sometimes regressed, tumor growth in a dose-dependent manner, reaching 94%, 71% and 72% inhibition, respectively, at a 50 µg dose (p < 0.01). Mice with regressed tumors demonstrated immune memory, preventing tumor recurrence upon re-implantation (p < 0.001). When tumor cells were administered through the tail vein to generate lung metastases, vaccination reduced the number of metastatic foci (by 15- and 23-folds, p < 0.001), and increased the median survival time by 25% and 53% in RENCA and CT26 metastases, respectively (p < 0.01) relative to scrambled-MAP controls. No significant adverse responses were observed in all experiments. We show that the tumor microenvironment was immune modulated, as vaccination induced production of EMMPRIN-specific antibodies, increased CD8+ T cells infiltration and cytotoxicity, alleviated immune suppression by decreasing TGFß concentrations, reduced angiogenesis and cell proliferation, and enhanced apoptosis. Thus, our successful active peptide vaccination strategy differs from previous, unsuccessful attempts, both in the selected target (the EMMPRIN epitope) and in the use of a modified, MAP configuration, and demonstrates that this may be an efficient approach for the treatment and prevention of some types of cancer.

Function of miR-146a-5p in Tumor Cells As a Regulatory Switch between Cell Death and Angiogenesis: Macrophage Therapy Revisited.

Tumors survive and progress by evading killing mechanisms of the immune system, and by generating a tumor microenvironment (TME) that reprograms macrophages in situ to produce factors that support tumor growth, angiogenesis, and metastasis. We have previously shown that by blocking the translation of the enzyme inducible nitric oxide synthase (iNOS), miR-146a-5p inhibits nitric oxide (NO) production in a mouse renal carcinoma cell line (RENCA), thereby endowing RENCA cells with resistance to macrophage-induced cell death. Here, we expand these findings to the mouse colon carcinoma CT26 cell line and demonstrate that neutralizing miR-146a-5p's activity by transfecting both RENCA and CT26 cells with its antagomir restored iNOS expression and NO production and enhanced susceptibility to macrophage-induced cell death (by 48 and 25%, respectively, p < 0.001). Moreover, miR-146a-5p suppression simultaneously inhibited the expression of the pro-angiogenic protein EMMPRIN (threefolds, p < 0.001), leading to reduced MMP-9 and vascular endothelial growth factor secretion (twofolds and threefolds, respectively, p < 0.05), and reduced angiogenesis, as estimated by in vitro tube formation and scratch assays. When we injected tumors with pro-inflammatory-stimulated RAW 264.7 macrophages together with i.v. injection of the miR-146a-5p antagomir, we found inhibited tumor growth (sixfolds, p < 0.001) and angiogenesis (twofolds, p < 0.01), and increased apoptosis (twofolds, p < 0.01). This combination therapy increased nitrites and reduced TGFß concentrations in tumor lysates, alleviated immune suppression, and allowed enhanced infiltration of cytotoxic CD8+ T cells. Thus, miR-146a-5p functions as a control switch between angiogenesis and cell death, and its neutralization can manipulate the crosstalk between tumor cells and macrophages and profoundly change the TME. This strategy can be therapeutically utilized in combination with the macrophage therapy approach to induce the immune system to successfully attack the tumor, and should be further explored as a new therapy for the treatment of cancer.

Tumor cell-macrophage interactions increase angiogenesis through secretion of EMMPRIN.

Tumor macrophages are generally considered to be alternatively/M2 activated to induce secretion of pro-angiogenic factors such as VEGF and MMPs. EMMPRIN (CD147, basigin) is overexpressed in many tumor types, and has been shown to induce fibroblasts and endothelial cell expression of MMPs and VEGF. We first show that tumor cell interactions with macrophages resulted in increased expression of EMMPRIN and induction of MMP-9 and VEGF. Human A498 renal carcinoma or MCF-7 breast carcinoma cell lines were co-cultured with the U937 monocytic-like cell line in the presence of TNFα (1 ng/ml). Membranal EMMPRIN expression was increased in the co-cultures (by 3-4-folds, p < 0.01), as was the secretion of MMP-9 and VEGF (by 2-5-folds for both MMP-9 and VEGF, p < 0.01), relative to the single cultures with TNFα. Investigating the regulatory mechanisms, we show that EMMPRIN was post-translationally regulated by miR-146a, as no change was observed in the tumoral expression of EMMPRIN mRNA during co-culture, expression of miR-146a was increased and its neutralization by its antagomir inhibited EMMPRIN expression. The secretion of EMMPRIN was also enhanced (by 2-3-folds, p < 0.05, only in the A498 co-culture) via shedding off of the membranal protein by a serine protease that is yet to be identified, as demonstrated by the use of wide range protease inhibitors. Finally, soluble EMMPRIN enhanced monocytic secretion of MMP-9 and VEGF, as inhibition of its expression levels by neutralizing anti-EMMPRIN or siRNA in the tumor cells lead to subsequent decreased induction of these two pro-angiogenic proteins. These results reveal a mechanism whereby tumor cell-macrophage interactions promote angiogenesis via an EMMPRIN-mediated pathway.