G-protein coupled receptor-associated sorting protein 1 (GASP-1), a ubiquitous tumor marker, promotes proliferation and invasion of triple negative breast cancer.

We have identified the novel protein GASP-1 (G protein coupled receptor-associated sorting protein 1) that appears to be a universal cancer marker and the expression of which in tumor tissue and patient sera is predictive of cancer severity (Tuszynski et al. 2011; Zheng et al. 2012; Zheng 2013; Chang and Tuszynski, 2020). In preliminary results we discovered that a GASP-1 antibody inhibited the growth of the triple negative breast cancer cell line MDA-MB-231 and transient reduction of GASP-1 in these cells decreased their proliferation. To further substantiate these results, we over and under-expressed GASP-1 in stable clones of MDA-MB-231 cells and evaluated their growth and invasive activities. Cells under-expressing GASP-1 failed to grow after 4 days in culture and eventually died. In contrast GASP-1 expressing cells grew exponentially. Similarly, GASP-1 under-expressing cells formed 30% fewer colonies in soft agar as compared to controls and whereas GASP-1 over-expressing cells formed 2-fold more colonies than controls. In tumor cell invasion assays GASP-1 over-expressing cells were over 10-fold more invasive than controls whereas GASP-1 under-expressing cells were over 10-fold less invasive than controls. In IHC staining studies of breast cancer cells, we found that the overexpressed GASP-1 appear in granules of different sizes that are directly correlated with cancer invasiveness. Our results strongly indicate that GASP-1 promotes proliferation and invasion of the triple negative breast cancer cell line MDA-MB-231 and targeting GASP-1 for treatment of breast cancer is indicated.

Long-term efficacy and downstream mechanism of anti-annexinA2 monoclonal antibody (anti-ANX A2 mAb) in a pre-clinical model of aggressive human breast cancer.

There is considerable direct evidence that calcium binding protein ANX A2 is a potential target for treating aggressive breast cancer. The most compelling data are based on the finding of ANX A2 overexpression in aggressive triple negative human breast cancer (TNBC) cell lines and in human breast cancer tissues. Previously, we and others reported a unique role of ANX A2 in cancer invasion, including breast cancer. Moreover, we demonstrated that anti-ANX A2 mAb-mediated immunoneutralization of ANX A2 inhibited invasive human breast cancer growth in a xenograft model. We further evaluated the long-term effects of multiple treatments with anti-ANX A2 mAb and its mechanism of inhibition on human breast tumor growth. We now demonstrate that three treatments with anti-ANX A2 mAb led to significant inhibition of breast tumor growth in immunodeficient mice, and that the anti-tumor response was demonstrable from day 94. After treatment, we followed tumor growth for 172 days and demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated that anti-ANX A2 mAb treatment caused significant inhibition of conversion of tissue plasminogen activator (tPA) in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathways in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for treatment of aggressive breast cancer.

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.

G-protein coupled receptor-associated sorting protein 1 (GASP-1), a ubiquitous tumor marker.

Using an innovative "2-D high performance liquid electrophoresis" (2-D HPLE) technology we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of breast cancer patients and was over-expressed in early and late stage breast tumors (Tuszynski, G.P. et al., 2011). In this study we further investigated the significance of GASP-1 as a tumor marker by investigating the expression GASP-1 in different kinds of tumors as well as in the sera of patients with various cancers. Over expression of GASP-1 was detected in brain, pancreatic, and breast cancers as compared to their respective normal tissues as assessed by immunohistochemical staining of tissue arrays using a "peptide specific" GASP-1 antibody. We found that across these cancers, GASP-1 was expressed approximately 10 fold more in the cancer as compared to normal tissue. The increase in GASP-1 expression was also seen in hyperplastic and inflammatory lesions of breast and pancreatic cancers as compared to normal tissue. GASP-1 was primarily expressed in the tumor epithelium of the epithelial-derived cancers and in the transformed glial cells of the brain tumors. Using a sensitive "competitive ELISA" for GASP-1, we found that sera from patients with brain, liver, breast and lung cancers expressed 4-7 fold more GASP-1 peptide than sera from normal healthy individuals. These studies identify GASP-1 as a potential new serum and tumor biomarker for several cancers and suggest that GASP-1 may be a novel target for development of cancer therapeutics.

G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer.

An innovative "2-D high performance liquid electrophoresis" (2-D HPLE) technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (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 a specific fragment of 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 cancers, 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 of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). 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 the development of breast cancer therapeutics.

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.

Thrombospondin 1, fibronectin, and vitronectin are differentially dependent upon RAS, ERK1/2, and p38 for induction of vascular smooth muscle cell chemotaxis.

BACKGROUND: Thrombospondin 1 (TSP-1), fibronectin (Fn), and vitronectin (Vn) promote vascular smooth muscle cell (VSMC) chemotaxis through a variety of second messenger systems, including Ras, ERK1/2, and p38. HYPOTHESIS: Ras, ERK1/2, and p38 differentially affect TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. METHODS: Bovine VSMCs were transfected with Ras N17 or treated with the following inhibitors: a farnesyl protein transferase (FPT) inhibitor, PD098059 (ERK1/2 inhibitor), or SB202190 (p38 inhibitor). Thrombospondin 1, Fn, and Vn were used as chemoattractants. Results were analyzed by analysis of variance (ANOVA) with post hoc testing (P < .05). RESULTS: Ras N17 transfection or FPT inhibitor treatment inhibited TSP-1-, Fn-, and Vn-induced chemotaxis. PD098059 or SB202190 resulted in more inhibition of VSMC migration to TSP-1 than to Fn or Vn. CONCLUSIONS: Ras appears equally relevant in the signal transduction pathways of TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. Thrombospondin 1-induced migration is more dependent upon ERK1/2 and p38 than Fn- or Vn-included migration.

Thrombospondin-1 (TSP-1) Stimulates Expression of Integrin alpha6 in Human Breast Carcinoma Cells: A Downstream Modulator of TSP-1-Induced Cellular Adhesion.

Thrombospondin-1 (TSP-1) is involved in a variety of different cellular processes including cell adhesion, tumor progression, and angiogenesis. This paper reports the novel finding that TSP-1 upregulates integrin alpha6 subunit in human keratinocytes and human breast cancer cells resulting in increased cell adhesion and tumor cell invasion. The effect of TSP-1 on alpha6 subunit expression was examined in human keratinocytes and breast adenocarcinoma cell lines (MDA-MB-231) treated with TSP-1 and in TSP-1 stably transfected breast cancer cells. TSP-1 upregulated alpha6 message and protein in these cells as revealed by differential display, Northern and Western blot analysis and immunohistochemical localization studies. The increased expression of alpha6 was shown to mediate adhesion and invasion of these cells to laminin, a major component of the basement membrane and extracellular matrix (ECM). These data suggest that TSP-1 plays an integral role in the attachment of cells to the ECM facilitating cell motility and angiogenesis.

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.

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.

Effect of VP12 and viperistatin on inhibition of collagen-receptor-dependent melanoma metastasis.

Viperistatin and VP12 isolated from Vipera paleastinae venom showed a potent inhibitory activity against collagen receptors, alpha1beta1 and alpha2beta1 integrins, respectively. Structurally, viperistatin belongs to the disintegrin family of proteins, whereas VP12 is composed of two subunits VP12A and VP12B displaying amino acid sequence homology with heterodimeric C-lectin type proteins. Viperistatin and VP12 used separately and simultaneously inhibited pro-metastatic activities of melanoma cells lines. The level of inhibition of MV3 and HS.939T human cell lines in cell adhesion and migration assays by both compounds was correlated with expression of alpha1beta1 and alpha2beta1 integrins on the cell surface. MV3 cells express collagen receptors to much higher extent than HS.939T and required the application of higher concentrations of inhibitors to block their adhesion to collagen types I and IV. A melanoma cell transmigration assay through a dHMVEC layer revealed that alpha1beta1 integrin plays a significant role in invasion of HS.939T cells, while alpha2beta1 integrin appears to be more important for MV3 cells. In an animal model of hematogenous metastasis of the mouse B16F10 cell line, the inhibitory effect of viperistatin and VP12 was only partial. These data suggest that collagen receptors may be an interesting target for development of new anti-metastatic therapies.

Regulatory effect of nerve growth factor in alpha9beta1 integrin-dependent progression of glioblastoma.

In the present study we described the role of alpha9beta1 integrin in glioblastoma progression following its interaction with nerve growth factor (NGF). The level of expression of alpha9beta1 on astrocytomas is correlated with increased grade of this brain tumor and is highest on glioblastoma, whereas normal astrocytes do not express this integrin. Two glioblastoma cell lines, LN229 and LN18, that are alpha9beta1 integrin positive and negative, respectively, were used for alpha9beta1 integrin-dependent NGF-induced tumor progression. NGF was a significant promoter of promigratory and pro-proliferative activities of glioblastoma cells through direct interaction with alpha9beta1 integrin and activation of MAPK Erk1/2 pathway. The level of NGF increases approximately threefold in the most malignant glioma tissue when compared with normal brain. This increase is related to secretion of NGF by tumor cells. Specific inhibitors of alpha9beta1 integrin or gene silencing inhibited NGF-induced proliferation of LN229 cell line to the level shown by LN18 cells. VLO5 promoted alpha9beta1-dependent programmed cell death by induction of intrinsic apoptosis pathway in cancer cells. LN229 cells were rescued from proapoptotic effect of VLO5 by the presence of NGF. This disintegrin significantly inhibited tumor growth induced by implantation of LN229 cells to the chorioallantoic membrane (CAM) of quail embryonic model, and this inhibitory effect was significantly abolished by the presence of NGF. alpha9beta1 integrin appears to be an interesting target for blocking the progression of malignant gliomas, especially in light of the stimulatory effect of NGF on the development of these tumors and its ability to transfer proapoptotic signals in cancer cells.

Angiostatic activity of obtustatin as alpha1beta1 integrin inhibitor in experimental melanoma growth.

The presented results show the effect of targeting of collagen receptor, alpha1beta1 integrin expressed on the endothelial cells on the development of experimental melanoma and pathological angiogenesis. Obtustatin, a snake venom KTS-disintegrin, was applied as a specific inhibitor of this integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression in 2 animal systems, mouse and quail. Its oncostatic effect was related to the inhibition of angiogenesis. Obtustatin inhibited the neovascularization ratio on the CAM embryo of quail, which was pathologically induced by the developing tumor. The i.v. administration of obtustatin completely blocked cancer growth of MV3 human melanoma in nude mice. In B16F10 syngeneic mouse model treatment with the disintegrin revealed a lower effect, although the development of the tumor was significantly reduced for both dosages. The mechanism of obtustatin action is related to the blocking of microvascular endothelial cell proliferation, which undergoes apoptosis in caspase-dependent manner. Summarizing, we present studies of low molecular weight disintegrin, obtustatin as a potential therapeutic compound for treatment of melanoma that contain a high level of vascularization.

The novel angiogenic inhibitor, angiocidin, induces differentiation of monocytes to macrophages.

We previously showed that angiocidin, a tumor and vascular associated protein, is a potent inhibitor of angiogenesis and tumor growth. Angiocidin is a multidomain protein that exerts its antiangiogenic activity through multiple mechanisms, including effects on cell matrix interaction. Here, we describe another activity of angiocidin that may contribute to its antitumor activity. We show that angiocidin activates monocytes to secrete a mixture of proinflammatory cytokines and induces them to differentiate into macrophage-like cells. Using the monocytic cell line THP-1, we show that angiocidin induces the cells to become adherent and phagocytic, express macrophage markers, and secrete matrix metalloproteinase-9. Microarray analysis of control and angiocidin-treated THP-1 cells revealed that angiocidin up-regulated p105/p50, p100/p52, and rel B, components of the nuclear factor-kappaB (NF-kappaB) pathway. We confirmed the microarray data and showed that angiocidin induced phosphorylation of I kappa beta, p50, and p65 and translocation of p50 and p65 to the nucleus. We also showed that angiocidin activated up-stream mediators of NF-kappaB, such as the mitogen-activated protein kinase (MAPK) pathway and phosphoinositide-3 kinase (PI3K). Blockage of NF-kappaB and MAPK activation with small molecule inhibitors completely prevented angiocidin-mediated secretion of cytokines from THP-1 cells, but did not inhibit their adhesive phenotype. Blocking PI3K inhibited both secretion of cytokines, as well as the adhesive phenotype. These data suggest that angiocidin activates monocytes to secrete cytokines and differentiates them to a macrophage-like phenotype through at least two pathways mediated by MAPK and NF-kappaB, as well as PI3K.

Integrin alpha9 beta1 is a receptor for nerve growth factor and other neurotrophins.

The integrin alpha9beta1 is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin C and osteopontin. We found that this integrin is a receptor for nerve growth factor (NGF) and two other neurotrophins, brain-derived neurotrophic factor and NT3, using a cell adhesion assay with the alpha9SW480 cell line. Interaction of alpha9beta1 with NGF was confirmed in an ELISA assay by direct binding to purified integrin. alpha9beta1 integrin binds to neurotrophins in a manner similar to another common neurotrophin receptor, p75(NTR) (NGFR), although alpha9beta1 activity is correlated with induction of pro-survival and pro-proliferative signaling cascades. This property of alpha9beta1 resembles the interaction of NGF with a high affinity receptor, TrkA, however, this integrin shows a low affinity for NGF. NGF induces chemotaxis of cells expressing alpha9beta1 and their proliferation. Moreover, alpha9beta1 integrin is a signaling receptor for NGF, which activates the MAPK (Erk1/2) pathway. The alpha9beta1-dependent chemotactic ability of NGF appears to result from the activation of paxillin.

Angiocidin inhibitory peptides decrease tumor burden in a murine colon cancer model.

INTRODUCTION: We have recently developed two inhibitory peptides that target angiocidin, a key mediator of tumor progression and angiogenesis. In this study, we investigate the expression of angiocidin in human colon cancer specimens and evaluate the therapeutic efficacy of our angiocidin inhibitory peptides. METHODS: We created a colon cancer tissue array containing primary tumor, normal colon, negative and positive lymph nodes, and liver metastases (when available) from 159 consecutive colon cancer specimens. Angiocidin expression was determined by immunohistochemistry. The efficacy of 6-mer and 25-mer angiocidin inhibitory peptides was determined in a murine model of human colon cancer. Treatment efficacy was based on primary tumor volume and measures of tumor burden, including internal disease score and health score. Western blots were used to determine angiocidin expression in xenografts. RESULTS: Eighty-nine percent of primary tumors and 91% of positive lymph nodes expressed angiocidin. Normal colon was negative in 94% of specimens, and normal lymph nodes were negative or weakly positive in 79% of specimens. All liver metastases were positive for angiocidin. Animals in both peptide treatment groups showed improvement in health score and internal disease score compared with control animals (P = 0.001). Treatment with 6-mer and 25-mer peptide resulted in 3-fold and 16-fold reductions, respectively, in primary tumor volume (P = 0.001). Angiocidin expression in primary tumors of peptide-treated mice correlated with tumor burden (P < 0.05). CONCLUSIONS: Angiocidin is overexpressed in human colon cancer specimens. Angiocidin-inhibitory peptides are well tolerated in vivo and effectively reduce primary tumor volume and tumor burden in human colon cancer xenografts.

Interaction of alpha9beta1 integrin with thrombospondin-1 promotes angiogenesis.

Thrombospondin-1 is a multifunctional protein interacting with several cell surface receptors including integrins. We found that it is a ligand for alpha9beta1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with alpha9beta1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing alpha9beta1 integrin activated signaling proteins such as Erk1/2 and paxillin. Blocking of this integrin by monoclonal antibody and the met-leu-asp-disintegrin inhibited dermal human microvascular endothelial cell proliferation and NoC1-induced migration of these cells. Immunohistochemical studies revealed that alpha9beta1 is expressed on microvascular endothelium in several organs including skin, lung, heart and brain. NoC1 induced neovascularization in an experimental quail chorioallantoic membrane system and Matrigel plug formation assay in mice. This proangiogenic activity of NoC1 in vivo was inhibited by alpha9beta1 inhibitors. In summary, our results revealed that alpha9beta1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.

Clinical significance of serum angiocidin levels in hepatocellular carcinoma.

Angiocidin, a tumor-secreted protein, was measured in serum of 27 healthy volunteers and 33 hepatocellular carcinoma (HCC) patients. Healthy controls either hepatitis B surface antigen (HBsAg) positive or negative showed undetectable levels. Patients had levels of angiocidin ranging from 15.09 to 195.73 pg/ml. Patients with stages III-IV had higher levels of angiocidin (97+/-13 pg/ml, n=17) compared to those with stages I-II (63+/-37 pg/ml, n=16), p<0.043. Patients with microsatellite tumor nodules had higher average levels (98+/-55 pg/ml, n=17) compared to those without microsatellite nodules (51+/-27 pg/ml, n=20), p<0.032. Our studies suggest that angiocidin predicts advanced stage and intra-hepatic metastasis.

Integrin alpha2beta1 mediates the anti-angiogenic and anti-tumor activities of angiocidin, a novel tumor-associated protein.

We recently characterized an anti-tumor protein termed angiocidin. Here, we report that angiocidin may inhibit angiogenesis by binding collagen and its receptors. Angiocidin bound purified type I collagen and alpha2beta1 with high affinity. K562 cells expressing alpha2beta1 bound and adhered to angiocidin while K562 cells which only expressed alpha5beta1 integrin showed no binding and adhesion. Binding was specific since a neutralizing antibody against alpha2beta1 inhibited binding but antibodies against alpha5beta1 had no effect. Additionally, angiocidin co-localized with alpha2beta1 on K562 alpha2beta1 transfected cells, pancreatic cancer colo 357 cells, breast cancer MB-231 cells and human umbilical endothelial vein (HUVE) cells. In an alpha2beta1-dependent collagen gel angiogenesis assay, angiocidin showed potent inhibitory activity. We identified a 20-amino-acid amino terminal peptide of angiocidin that bound both alpha2beta1 and type I collagen. This peptide promoted alpha2beta1-dependent cell adhesion and inhibited tumor growth and angiogenesis. Taken together, these results are consistent with the conclusion that the anti-tumor activity of angiocidin arises from its ability to ligate collagen and alpha2beta1 on endothelial cells and tumor cells. Our results provide support for the concept that targeting matrix-cell interactions is a viable strategy for the development of anti-cancer therapeutics.

Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis Lung Carcinoma tumor growth via blocking of plasminogen activation: possible biochemical mechanism of angiostatin's action.

Angiostatin, the N-terminal four kringles (K1-4) of parent molecule plasminogen, is reported to block Lewis Lung Carcinoma (LLC) tumor growth and metastasis. However, angiostatin's mechanism of action is unclear. We earlier reported that angiostatin binds to cell surface annexin II through the lysine-binding domain (kringles 1-4) [Tuszynski, G.P., Sharma, M., Rothman, V.L., Sharma, M.C., 2002. Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc. Res. 64:448-462.]). We now show that annexin II on the cell surface of LLC cells regulates conversion of plasminogen to plasmin. Activation of plasminogen to plasmin is time-dependent, with the linear activation lasting up to 120 min. Monoclonal antibodies to annexin II reduced plasminogen activation by 92.6%, suggesting a specific role of annexin II in plasmin generation. Angiostatin also reduced plasmin generation by 81.6%, suggesting that angiostatin may be competing with plasminogen through lysine-binding domain. epsilon-Aminocaproic acid, a lysine analogue, effectively blocked plasminogen activation indicating that, indeed, the lysine-binding site of the kringles domain is required for activation. These data suggest that annexin II may be a receptor target for angiostatin's action. Therefore, we tested the effect of high affinity monoclonal antibody to annexin II in mouse model of LLC. A single dose of antibody treatment inhibited LLC tumor growth almost 70% with concomitant inhibition of circulating plasmin generation and its proteolytic activity. Taken together, it is possible that inhibition of LLC tumor growth and metastasis reported by angiostatin therapy may be due to blocking of annexin-II-dependent plasmin generation. Plasmin is known to influence angiogenic, invasive and metastatic capability of tumors.