Adonia E. Papathanassiu

Tissue factor pathway inhibitor inhibits endothelial cell proliferation via association with the very low density lipoprotein receptor.

Tissue factor pathway inhibitor (TFPI) contains three Kunitz-type proteinase inhibitor domains and is a potent inhibitor of tissue factor-mediated coagulation. Here, we report that TFPI inhibits the proliferation of basic fibroblast growth factor-stimulated endothelial cells. A truncated form of TFPI, containing only the first two Kunitz-type proteinase inhibitor domains, has very little antiproliferative activity, suggesting that the carboxyl-terminal region of TFPI is responsible for this activity. Binding studies revealed that full-length TFPI, but not the truncated TFPI molecule, is recognized by the very low density lipoprotein receptor (VLDL receptor) indicating that this receptor is a novel high affinity endothelial cell receptor for TFPI. The antiproliferative activity of TFPI on endothelial cells is inhibited by the receptor-associated protein, a known antagonist of ligand binding by the VLDL receptor, and by anti-VLDL receptor antibodies. These results confirm that the antiproliferative activity of TFPI is mediated by the VLDL receptor and suggest that this receptor-ligand system may be a useful target for the development of new anti-angiogenic and antitumor agents.

Abstract 2683: Inhibition of BCAT1 suppresses the expression of pro-metastatic proteins and reduces cancer metastasis

Recent evidence suggests that the cytosolic presence of branched chain amino acid aminotransferase (BCAT1) in adult tissues is associated with diverse pathological conditions including cancer and inflammatory diseases such as rheumatoid arthritis; in these diseases, BCAT1 appears to modulate the expression of many key proteins and to be a new druggable target. In cancer, BCAT1 physically associates with CD147 and regulates the levels and the cellular localization of the protein in a number of different cell lines in vitro and in vivo. Silencing of Bcat1 gene downregulates the total expression of CD147 in MDA-MB-435 cells, while inhibition of BCAT1 by sodium 4-methyl-5-oxo-hexanoate (ERG240) leads to suppression of the extracellular CD147 in the same cells. In turn, suppression of extracellular CD147 results in downregulation of CD147-associated proteins such as MCT4, matrix metalloproteinases (MMPs), cyclophilin A, and GAPDH. Furthermore, inhibition of BCAT1 is associated with prevention of cancer cell migration and reduction in secretion of metabolic by-products such as lactate. Surprisingly, treatment of MDA-MB-231 tumor-bearing animals with 2-deoxyglucose (2DG), a well-known inhibitor of glycolysis, leads to a decrease in the levels of c-MYC and BCAT1, an increase in the total expression of CD147, and the increased shedding of the extracellular CD147 into the circulation. This observation suggests that BCAT1-driven regulation of CD147 in cancer cells involves a complex mechanism. Inhibition of tumoral BCAT1 is expected to adversely affect the metastatic potential of various tumors not only through a reduction in cell motility but also through alterations in the tumor microenvironment, driven by inhibition of extracellular CD147. Citation Format: Adonia E. Papathanassiu, Hong A. Vu. Inhibition of BCAT1 suppresses the expression of pro-metastatic proteins and reduces cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2014-2683

Abstract 5416: Metabolic control of the prometastatic protein CD147.

Apart from a recent report suggesting that hypoxia upregulates CD147, little is known about the mechanisms responsible of the tumoral overexpression of CD147, a transmembrane and secretory glycoprotein known to promote metastasis through multiple mechanisms. Here, we report that the total and surface concentration of CD147 appear to be under metabolic control. Glucose (Glc) deprivation in MDA-MB-231 and MDA-MB-435 tumor cells leads to a decrease in the levels of total CD147 and a dramatic increase in the expression of cell surface CD147. A similar mobilization of CD147 to the plasma membrane was observed with glutamine (Gln) deprivation in MDA-MB-435 cells; when the cells were deprived of both nutrients (Glc and Gln) a dramatic increase (∼6-fold) in the cell surface expression of CD147 was observed. Plasma membrane associated CD147 is known to promote metastasis through homotypic and heterotypic interactions between cancer and stromal cells. An 80% reduction in the membrane found CD147 was observed when Gln-deprived cells were treated with ERG-240, a small molecule that inhibits the cytosolic form of BCAT, the enzyme responsible for the reversible transamination of leucine (Leu). On the other hand, Leu deprivation was shown to decrease CD147 by 6-7 fold in both cancer cell lines tested here. Treatment of the cells with ERG-240 partially reversed the increase in the levels of CD147 seen after supplementation of the Leu-deprived cells with 105 mg/L Leu. CD147 is known to possess 3 N-glycosylation sites and to exist in forms with various degrees of glycosylation; the fully glycosylated protein is associated with an increased prometastatic potential. Inhibitors of N-glycosylation, such as 2-deoxyglucose (2DG), are known to possess anticancer properties. In the present study, treatment of MDA-MB-231 cells in vitro with 10 mM 2DG led to 80% reduction of the cell surface expression of highly glycosylated CD147 without the concomitant reduction in the total concentration of CD147. In contrast, treatment of athymic female nude mice, bearing orthotopically implanted MDA-MB-231 tumors, with 125 mg/Kg 2DG twice a week led to tumors that expressed markedly increased levels of the highly glycosylated CD147 variant (Control/Treated or T/C=2.3, p=0.01). The increased expression of the protein in the 2DG-treated tumors was accompanied with an increase in the levels of circulating human CD147 (T/C=1.7, p=0.01) normalized per tumor volume. Collectively, the data point towards a metabolic control of CD147 and suggest that inhibition of tumor growth due to nutrient deprivation may initiate pathways that promote metastasis. Citation Format: Adonia E. Papathanassiu, Hong A. Vu. Metabolic control of the prometastatic protein CD147. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2013-5416

Abstract 3216: Cytosolic leucine metabolism regulates the expression of prometastatic proteins: The genesis of a new druggable target

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Branched chain amino acid metabolism begins with the reversible transamination of the amino acids to the corresponding α-keto acids. The reaction is catalyzed by the enzyme Bcat. The cytosolic form of this enzyme, also known as Bcat1, is thought to be expressed only in embryonic tissues, in select adult organs (brain, ovary), and in c-myc induced brain tumors. In the current study, Bcat1 is shown to be expressed by a variety of tumor cell lines including MCF-7, MDA-MB-231, MDA-MB-435, and HT-29. Its expression appears to be associated with the ability of the cells to uptake glucose since treatment of MDA-MB-231 with 2-deoxyglucose leads to the suppression of the cellular Bcat1 levels in vitro and in vivo. In the cytosol, Bcat1 appears to complex with CD147, a protein known to promote cancer growth and metastasis through multiple pathways. Silencing of Bcat1 in MDA-MB-231 and MDA-MB-435 cells results in the downregulation of CD147, while inhibition of Bcat1 by ERG-240, a small molecule structurally related to leucine, leads to downregulation of the cell surface expressed CD147 and its transmembrane partner monocarboxylate transporter-4 (MCT-4) in MDA-MB-231 breast cancer cells. MCT-4 is known to mediate the expedient removal of lactate, a cytotoxic by-product of cancer glycolysis, from the cytosol. Furthermore, treatment of MDA-MB-435 cells with 10 mM ERG-240 results in downregulation of matrix metalloproteinase 9 (MMP-9) production, while treatment of normal adult fibroblasts with either ERG-240 or conditioned media of MDA-MB-435 cells previously exposed to ERG-240 results in downregulation of MMP-2 production. The later is attributed to the ability of ERG240 to suppress the presence of soluble CD147 in the conditioned media. Exocytosis of CD147 has been implicated in the formation of the pre-metastatic niche through inductions of MMPs and VEGF. Inhibition of Bcat1 by ERG-240 suppresses cell proliferation in various tumor cell lines with an IC50 value of 5 to 10 mM. ERG240-induced inhibition of MDA-MB-231 cell migration is also observed. The results from an on-going in vivo study are presented. In summary, our experiments suggest that: a) metabolism of branched chain amino acids is intimately linked with the expression of proteins controlling metastatic activities of tumor cells and b) Bcat1, which is not expressed by normal adult tissues, is an attractive target for the development of new, non-toxic anticancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3216. doi:1538-7445.AM2012-3216