Vicki L. Rothman

Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin‐1

Thrombospondin‐1 (TSP‐1) is a matrix protein that has been implicated in mechanisms of tumor progression. Our laboratory previously showed that the CSVTCG (cys‐ser‐val‐thr‐cys‐gly) sequence of TSP‐1 functioned as a tumor cell adhesion domain and CSVTCG peptides as well as an anti‐peptide antibody possessed anti‐metastatic activity in a murine model of lung metastasis. In a subsequent study, a putative TSP‐1 binding protein from lung carcinoma was isolated by CSVTCG‐peptide affinity chromatography. In this study, we present the full‐length cDNA of this binding protein isolated from a prostate cancer cell (PC3‐NI) cDNA library. The purified recombinant protein, termed angiocidin, is a potent inhibitor of tumor growth of Lewis Lung carcinoma in vivo and tumor invasion and angiogenesis in vitro. In addition, the recombinant protein inhibits tumor and endothelial cell proliferation and induces apoptosis. The activity of angiocidin both in vivo and in vitro is partially dependent on its TSP‐1 binding activity, since an angiocidin deletion mutant missing a high affinity‐binding site for TSP‐1 failed to inhibit tumor growth in vivo and was less active in its anti‐tumor and anti‐angiogenic activities in vitro. These results suggest that the anti‐tumor activity of TSP‐1 reported in many studies may be mediated in part by binding proteins such as angiocidin. Such proteins may function as tumor‐suppressor proteins, which limit the growth of tumors by inhibiting angiogenesis and cell matrix interaction.

Thrombospondin-1 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human tumor cells: Exploring the functional significance in tumor cell invasion

Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression.

The Interaction of Angiocidin with Tissue Transglutaminase

Angiocidin, a matrix bound and tumor associated protein, has been shown to inhibit tumor progression and angiogenesis. We previously demonstrated that angiocidin binds to thrombospondin-1 and alpha2beta1 integrin. We now show that angiocidin binds and is a preferred substrate for tissue transglutaminase-2 (tTgase). Angiocidin bound tTgase saturably with a Kd of 26 nM, while an angiocidin deletion mutant missing the matrix binding domain of angiocidin failed to bind tTgase. tTgase colocalized with angiocidin on endothelial cells. tTgase bound anti-angiocidin immunoprecipitates of endothelial cell lysates. Breast cancer cells expressing high levels of tTgase attached to angiocidin immobilized on tissue culture plates. Angiocidin was a preferred substrate for tTgase forming high molecular weight cross-linked multimers when treated with tTgase. Cross-linked angiocidin contained iso-peptide bonds as demonstrated by Western blotting and immunohistochemical colocalization studies using endothelial cells treated with angiocidin. Cross-linked angiocidin inhibited cell migration in contrast to monomeric angiocidin and inhibited localization of fibronectin (FN), a pro-tumorigenic matrix protein, into the extracellular matrix (ECM) of tumor and HUVE cells. Our studies provide an additional explanation for the anti-tumor activity of angiocidin suggesting that cross-linked angiocidin disrupts the tumor ECM making it less permissive for tumor growth.

Angiocidin inhibits breast cancer proliferation through activation of epidermal growth factor receptor and nuclear factor kappa (NF-ĸB).

Angiocidin, a tumor-associated peptide, has been previously shown to inhibit tumor progression by blocking angiogenesis. We now show that angiocidin has a direct inhibitory effect on tumor cell proliferation. MDA-MB-231 breast cancer cells were inhibited from proliferating in the presence of epidermal growth factor (EGF) and angiocidin. Angiocidin transfected breast cancer cells also displayed growth inhibition in vitro and failed to develop significant tumors in mice as compared to vector controls. The anti-proliferative effect of angiocidin was reversed by treating the cells with the epidermal growth factor receptor (EGFR) inhibitor 4557W, a potent tyrosine kinase inhibitor. Consistent with these results, we found that treatment of breast cancer cells with angiocidin induced a 2.3 fold increase in EGFR tyrosine 845 phosphorylation while no change in phosphorylation was observed in the remaining 16 phosphorylation sites of EGFR and those of its family members as measured by a human EGFR phosphorylation array. Treatment of breast cancer cells with angiocidin also resulted in the activation of nuclear factor ĸB (Nf-ĸB) and the de novo up-regulation of many down-stream genes transcribed by Nf-ĸB, including cytokines, inflammatory mediators and the cell cycle inhibitor p21(waf1). Therefore, angiocidin is a peptide that not only inhibits tumor angiogenesis but also directly induces inhibition of tumor growth progression through the activation of EGFR and down-stream genes transcribed by Nf-ĸB.

Angiocidin induces differentiation of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a malignant proliferative disorder in which leukemic cells fail to terminally differentiate and accumulate in the blood and bone marrow. Standard AML therapy requires intensive chemotherapy with a low rate of durable remission and is associated with significant treatment-related toxicity, especially in elderly patients. Therefore, new therapeutic options for the treatment of AML are urgently needed. We previously reported that the novel angiogenic inhibitor, angiocidin, induces differentiation of monocytes to macrophages. Here we investigate the effects of angiocidin on AML cells lines and primary AML cells. Differentiation was assessed by flow cytometry measuring the increase in expression of cell surface marker characteristic of normal macrophages. Four AML cell lines (THP-1, Mono-mac-1, HL-60 and MV4-11) and 5 of 10 primary human AML samples showed evidence of differentiation when cultured in vitro for 24 h with 10 μg/mL angiocidin. Additionally, we found that angiocidin promoted secretion of a number of cytokines from the cell lines as well as patient cells. We next evaluated the effect of angiocidin on a xenotransplanted primary human AML sample engrafted in NSG mice. We found angiocidin monotherapy reduced the human AML burden in bone marrow by 63% relative to untreated control. Interestingly, angiocidin+cytosine arabinoside (Ara-C) combination therapy reduced human AML in bone marrow by 79%. We believe the combination of in vitro data supporting the capacity of angiocidin to drive differentiation in multiple AML cell lines and primary human AML samples and its activity in a xenotransplantation model that reproduces the human disease is significant. These observations support the continued evaluation and development of angiocidin as a potential novel, non-toxic therapy for AML.

Abstract 5392: Angiocidin induces differentiation of acute myeloid leukemia (AML) cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Acute myeloid leukemia (AML) is a malignant proliferative disorder where blast stage cells fail to terminally differentiate and accumulate in the blood and bone marrow. AML represents a very significant health risk in the general population with rising incidence in aging adults. From 2004-2008 the median age at diagnosis was 67.8 years and it is estimated 12,950 adults will be diagnosed with 9,050 deaths from AML leukemia in 2011 (http://seer.cancer.gov/statfacts/html/amyl.html, accessed November 2011). Standard AML therapy requires intensive combination chemotherapy with a low rate of durable remissions often requiring stem cell transplantation and associated with significant treatment-related toxicity, especially in elderly patients. Due to this toxicity, the majority of elderly patients are not eligible for therapy. Therefore, new therapeutic options for the treatment of AML are urgently needed. In this study, we present evidence that the human protein angiocidin, discovered in our laboratory, has the potential to be used as a novel, non-toxic therapy for AML. We found that angiocidin can induce both AML cell lines and primary patient cells to increase expression of surface markers characteristic of a more normal differentiated phenotype. We evaluated expression of CD14, a major receptor for the innate immune system in macrophages, CD54 or ICAM-1, the major adhesion receptor used by macrophages for endothelial transmigration, and CD15, a neutrophil phagocytosis receptor. Expression of these receptors was measured by flow cytometry after cells were cultured for 24 hours in media alone or media containing 10 μg/ml angiocidin. Angiocidin up-regulated these receptors by more than 10 fold in 50% of the AML cells tested. The AML cells included the cell line THP-1 and 11 primary AML patient cells. Angiocidin up-regulated CD14 and CD54 in THP-1 cells and in 3 out of 5 M5 phenotype AML patient cells tested and 1 M4 AML phenotype out of 6 M4 samples tested. Angiocidin also up-regulated CD15 in 1 M4 AML patient phenotype out of 6 M4 patient cells tested. These results suggest that angiocidin can differentiate AML cells and therefore has the potential to be developed as differentiation therapy for AML. 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 5392. doi:1538-7445.AM2012-5392

Abstract 3183: The role of G-protein coupled receptor-associated sorting protein 1 (GASP-1) in breast cancer progression

An innovative “2-D High Performance Liquid Electrophoresis” (2-D HPLE) technology was used to identify serum biomarkers associated with the earliest stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 1009s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that G-protein coupled receptor-associated sorting protein 1(GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of stage 2 and stage 3 breast cancer but not in adjacent normal tissue as revealed by Western Blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all 59 cases of archived ductal breast carcinoma tumor samples. When GASP-1 was silenced in MB-231 breast carcinoma, the cells grew more than 5 fold slower than corresponding vector controls. These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for development of breast cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2011-3183