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Accumulating evidence indicates that serum and tissue levels of lectin, galactoside-binding soluble 3 binding protein (LGALS3BP), a secreted glycoprotein, are elevated in human cancers. Recently, we have identified LGALS3BP as a factor capable of stimulating angiogenesis of microvascular endothelial cells in vitro as well as in vivo. However, the potential therapeutic implications of LGALS3BP function blockade have not been explored yet. Here, we tested the ability of an anti-LGALS3BP mouse monoclonal antibody, SP-2, to antagonize LGALS3BP-induced angiogenesis and tumor growth. The antibody was found to inhibit endothelial cell tubulogenesis induced by either conditioned medium of breast cancer and melanoma cells or human recombinant LGALS3BP. In addition, SP-2 inhibited phosphorylation of FAK and its recruitment to membrane sites as well as AKT and ERK phosphorylation promoted by LGALS3BP. When used in vivo, the antibody restrained LGALS3BP-stimulated angiogenesis and growth of tumor xenografts. Furthermore, the combination of SP-2 and low-dose bevacizumab was more effective than either agent alone. Taken together, these results lead to consideration of SP-2 as a promising candidate for LGALS3BP-targeted therapy. Mol Cancer Ther; 13(4); 916–25. ©2014 AACR.

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The 90K protein, also known as Mac-2 BP or LGALS3BP, can activate the immune response in part by increasing major histocompatibility (MHC) class I levels. In studies on a non-immune cell model, the rat FRTL-5 cell line, we observed that transforming growth factor (TGF)-β1, like γ-interferon (IFN), increased 90K levels, despite its immunosuppressive functions and the ability to decrease MHC class I. To explain this paradoxical result, we investigated the mechanisms involved in the TGF-β1 regulation of 90K expression with the aim to demonstrate that TGF-β1 utilizes different molecular pathways to regulate the two genes. We found that TGF-β1 was able to increase the binding of Upstream Stimulatory Factors, USF1 and USF2, to an E-box element, CANNTG, at −1926 to −1921 bp, upstream of the interferon response element (IRE) in the 90K promoter. Thyrotropin (TSH) suppressed constitutive and γ-IFN-induced 90K expression by decreasing USF binding to the E-box. TGF-β1 was able to overcome TSH suppression at the transcriptional level by increasing USF binding to the E-box. We suggest that the ability of TGF-β1 to increase 90K did not result in an increase in MHC class I because of a separate suppressive action of TGF-β1 directly on the MHC class I gene. We propose that the increased levels of 90K may play a role, rather than in immune response, in the context of the TGF-β1-induced changing of the cellular microenvironment that predisposes to cell motility and cancer progression. Consistently, analyzing the publicly available cancer patient data sets cBioPortal, we found that 90K expression directly correlated with TGF-β1 and USFs and that high levels of 90K were significantly associated with increased mortality in patients affected by different types of cancer.

Accumulating evidence indicates that serum and tissue levels of lectin, galactoside-binding soluble 3 binding protein (LGALS3BP), a secreted glycoprotein, are elevated in human cancers. Recently, we have identified LGALS3BP as a factor capable of stimulating angiogenesis of microvascular endothelial cells in vitro as well as in vivo. However, the potential therapeutic implications of LGALS3BP function blockade have not been explored yet. Here, we tested the ability of an anti-LGALS3BP mouse monoclonal antibody, SP-2, to antagonize LGALS3BP-induced angiogenesis and tumor growth. The antibody was found to inhibit endothelial cell tubulogenesis induced by either conditioned medium of breast cancer and melanoma cells or human recombinant LGALS3BP. In addition, SP-2 inhibited phosphorylation of FAK and its recruitment to membrane sites as well as AKT and ERK phosphorylation promoted by LGALS3BP. When used in vivo, the antibody restrained LGALS3BP-stimulated angiogenesis and growth of tumor xenografts. Furthermore, the combination of SP-2 and low-dose bevacizumab was more effective than either agent alone. Taken together, these results lead to consideration of SP-2 as a promising candidate for LGALS3BP-targeted therapy. Mol Cancer Ther; 13(4); 916–25. ©2014 AACR.

Elevated serum or tissue levels of lectin galactoside-binding soluble 3 binding protein (LGALS3BP) have been associated with short survival and development of metastasis in a variety of human cancers. However, the role of LGALS3BP, particularly in the context of tumor-host relationships, is still missing. Here, we show that LGALS3BP knockdown in MDA-MB-231 human breast cancer cells leads to a decreased adhesion to fibronectin, a reduced transendothelial migration and, more importantly, a reduced expression of vascular endothelial growth factor (VEGF). Production of VEGF, that was restored by exposure of silenced cells to recombinant LGALS3BP, required an intact PI3k/Akt signaling. Furthermore, we show that LGALS3BP was able to directly stimulate HUVEC tubulogenesis in a VEGF-independent, galectin-3-dependent manner. Immunohistochemical analysis of human breast cancer tissues revealed a correlation among LGALS3BP expression, VEGF expression, and blood vessel density. We propose that in addition to its prometastatic role, LGALS3BP secreted by breast cancer cells functions critically as a pro-angiogenic factor through a dual mechanism, i.e by induction of tumor VEGF and stimulation of endothelial cell tubulogenesis.

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and characterization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of mALCAM showed that the CD6 binding site is located in the N-terminal Ig-like domain and that mALCAM is capable of binding both human and mouse CD6. Mutagenesis studies on hALCAM suggested that residues critical for CD6 binding map to the predicted A'GFCC'C" beta-sheet of ALCAM's N-terminal binding domain. Residue differences in the N-terminal domains of mALCAM and hALCAM were analyzed with the aid of a molecular model of ALCAM. All residues critical for CD6 binding are conserved in both mALCAM and hALCAM, whereas residue differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale for the observed cross-species CD6/ALCAM interaction and the apparent inability to generate monoclonal antibodies (mAb) against the CD6 binding site. RNA blot analysis showed that mRNA transcripts encoding mALCAM are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two-color immunofluorescence studies was shown to bind to both activated CD4+ and CD8+ T cells.

We investigated the possibility that a secreted glycoprotein of approximately 90,000 daltons, termed 90K and identified as a member of the protein superfamily characterized by the scavenger receptor cysteine-rich (SRCR) domain, might have value as a predictor of progression to acquired immunodeficiency syndrome (AIDS) in subjects infected with the human immunodeficiency virus (HIV). Among 488 HIV-seropositive intravenous drug users with a median follow-up of 32.5 months, high levels of serum 90K at baseline proved to be a significant predictor of faster progression to AIDS, either as a continuous variable (log 90K; p0.0001) or as a dichotomous variable with an optimized cutoff point of 30 U/ml (p0.00001). Analysis of 90K in relation to known prognostic factors found an association with CD4 count, beta 2-microglobulin, and p24 antigen but none with neopterin. In multivariate analysis, the baseline 90K level was an independent predictor of AIDS. As compared with subjects with low levels of 90K, the relative risk of developing AIDS was 3.5 (95% CI 1.9-6.5) among those with high levels of 90K. The predictive value of 90K was maintained after stratification by baseline CD4 count: among subjects withor = 500 x 10(6)/L CD4 cells, the proportion in whom AIDS developed was 10.5% for those with 90K levelsor = 30 U/ml as compared with 20% for those with 90K above the cutoff point (p = 0.006). Serum 90K is an independent predictor of the risk for progression to AIDS in HIV-infected subjects, including those whose CD4 counts have not fallen.

Monoclonal antibody SP-2 to the tumour-associated antigen 90K was generated by immunisation with conditioned medium of human breast cancer cells. We investigated whether circulating levels of 90K can influence the prognosis of patients with breast cancer. Serum samples were obtained from 425 patients with histologically proven breast cancer with no clinical evidence of disease after surgery (NED) and in 310 patients with metastatic disease. Serum 90K was determined by a new immunoradiometric assay (IRMA). Antigen levels in NED patients were elevated in 18.5% of cases, mean levels being higher than in healthy controls (P = 0.001). Among 375 evaluable patients, the 75-month overall survival for 90K-negative (< or = 11 U ml-1) and 90K-positive (> 11 U ml-1) patients was 78% and 53% respectively (P = 0.004). The prognostic value of 90K appeared to be limited to patients with node-positive disease. Number of metastatic axillary lymph nodes and level of 90K antigen were the only independent variables for predicting overall survival. Patients with metastatic breast cancer had elevated 90K in 51.3% of cases. High 90K levels were significantly associated with the presence of metastases to liver, shorter disease-free interval and younger age. We conclude that an elevated 90K antigen level in serum is a predictor of poor prognosis in breast cancer.

The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance, which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand that abrogates receptor-mediated signaling. Toward this end, we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. Assessment of human tumor cell lines, as well as early passage tumor cells revealed that MP-RM-1 effectively inhibited both NRG-1β-dependent and -independent ErbB-3 activation. The antagonizing effect of MP-RM-1 was of non-competitive type, as binding of [(125)I]-labeled NRG-1β to ErbB-3 was not influenced by the antibody. MP-RM-1 treatment led, in most instances, to decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in two human tumor xenograft nude mouse models. Treatment with the antibody was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Collectively, these results indicate that MP-RM-1 has the potential to interfere with signaling by ErbB-3 and reinforce the notion that ErbB-3 could be a key target in cancer-drug design.

Galectins, a family of structurally related carbohydrate-binding proteins, contribute to different events associated with cancer biology, including apoptosis, homotypic cell aggregation, angiogenesis and tumor-immune escape. To interfere with galectin-carbohydrate interactions during tumor progression, a current challenge is the design of specific galectin inhibitors for therapeutic purposes. Here, we report the synthesis of three novel low molecular weight synthetic lactulose amines (SLA): (1) N-lactulose-octamethylenediamine (LDO), (2) N,N´-dilactulose-octamethylenediamine (D-LDO), and (3) N,N´-dilactulose-dodecamethylenediamine (D-LDD). These compounds showed a differential ability to inhibit binding of galectin-1 and/or galectin-3 to the highly glycosylated protein 90K in solid-phase assays. In addition, each compound demonstrated selective regulatory effects in different events linked to tumor progression including tumor-cell apoptosis, homotypic cell aggregation, and endothelial cell morphogenesis. Our results suggest that galectin inhibitors with subtle differences in their carbohydrate structures may be potentially used to specifically block different steps of tumor growth and metastasis Fil: Rabinovich, Gabriel A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Cumashi, Albana. Università degli Studi G. D´Annunzio; Italia Fil: Bianco, German Ariel. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ciavardelli, Domenico. Università degli Studi G. D´Annunzio; Italia Fil: Iurisci, Ida. Università degli Studi G. D´Annunzio; Italia Fil: D’Egidio, Maurizia. Università degli Studi G. D´Annunzio; Italia Fil: Piccolo, Enza. Università degli Studi G. D´Annunzio; Italia Fil: Tinari, Nicola. Università degli Studi G. D´Annunzio; Italia Fil: Nifantiev, Nikolay. Academia de Ciencias Rusa; Rusia Fil: Iacobelli, Stefano. Università degli Studi G. D´Annunzio; Italia

Background: Accumulating evidence indicates that serum and tissue levels of the lectin galactoside-binding soluble 3 binding protein (LGALS3BP), a secreted glycoprotein, are elevated in human cancer. Recently, we have identified LGALS3BP as a factor capable of stimulating angiogenesis of microvascular endothelial cells in culture as well as in vivo. However, the potential therapeutic implications of LGALS3BP function blockade, has not been explored yet. Methods: Here we tested the ability of an anti-LGALS3BP monoclonal antibody, named SP-2, to antagonize LGALS3BP-induced angiogenesis and tumor growth. Results: SP-2 dose-dependently inhibited HUVEC tubulogenesis induced by either conditioned medium of breast cancer and melanoma cells or purified recombinant LGALS3BP. This was accompanied by inhibition of FAK, Akt and ERKs phosphorylation as well as FAK recruitment to membrane sites and actin remodeling. In vivo, SP-2 inhibited LGALS3BP-stimulated angiogenesis and restrained growth of different xenograft models of human cancer. Finally, the combination of SP-2 with low-dose Bevacizumab was more effective than either agent alone in suppressing angiogenesis and tumor growth. Conclusions: Targeting LGALS3BP with SP-2 could represent a promising therapeutic approach for LGALS3BP expressing tumors. Citation Format: Enza Piccolo, Sara Trani, Nicola Tinari, Cosmo Rossi, Rossana La Sorda, Rossano Lattanzio, Maurizia D'Egidio, Annalisa Di Risio, Antonino Grassadonia, Francesca Spinella, Anna Bagnato, Mauro Piantelli, Clara Natoli, Stefano Iacobelli. Inhibition of tumor growth and angiogenesis by SP-2, an anti-LGALS3BP antibody. [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 2684. doi:10.1158/1538-7445.AM2014-2684

The tumor antigen 90K (Mac-2BP, L3 antigen), which has been shown to have T cell costimulatory activity, is a approximately 90 kDa secreted protein found in high levels in plasma, saliva, breast milk and other human fluids. The 90K antigen can be divided into three domains: an amino terminal scavenger receptor cysteine-rich (SRCR)-like domain (D1), followed by a heavily glycosylated mucin-like domain (D2) and a approximately 27 kDa carboxy-terminal domain (D3). In this study we report on the construction of six different 90K immunoglobulin (Ig) fusion proteins containing different 90K domain combinations. Initially these fusion proteins were used to identify which 90K domain contains the epitopes recognized by the anti-90K monoclonal antibodies (mAb) SP2 and L3. Both of these mAbs were found to recognize 90K-D2. A new panel of anti-90K mAb was then generated by immunizing mice with ascites derived 90K protein. The 90K domain specific fusion proteins were then used to identify novel anti-90K mAbs which recognize the amino terminal SRCR domain and the carboxy terminal approximately 27 kDa domain of 90K. Two novel anti-90K SRCR (D1) and one anti-90 27 kDa domain (D3) mAbs were obtained. These 90K-Ig fusion proteins, as well as the novel and existing anti-90K mAbs, provide a set of tools which will allow further dissection of the structure and function of this immune modulatory protein.

Background: The clinical outcome of hepatitis virus infections is thought to depend on the complex interplay between the host immune response profile and virus factors. 90K/MAC-2 BP is a novel member of the Scavenger Receptor Cysteine Rich protein superfamily that functions as a molecular alarm signal for the cellular immune system against both cancer cells and virus infections. Methods: To assess the significance and the potential clinical usefulness of testing for serum levels of 90K/MAC-2 BP in chronic viral hepatitis patients we studied 115 consecutive patients with chronic HCV hepatitis, 28 HBsAg chronic hepatitis patients, 12 asymptomatic HCV carriers and 11 asymptomatic HBV carriers. 103 out of the 115 HCV patients have been treated with recombinant α2a-interferon at the dose of 3 Mega Units (MU) t.i.w. for 6 months followed by 1.5 MU t.i.w. for 6 months, and have been followed up for a further 12 months. Serum levels of 90K/MAC-2 BP were measured by an immunoradiometric assay based on the specific SP-2 monoclonal antibody. Results and Conclusions: Serum 90K/MAC-2 BP levels are increased in chronic vital hepatitis patients, being significantly higher in HCV than in HBV patients. In chronic HCV hepatitis, serum 90K/MAC-2 BP levels are related to both the degree of disease severity and duration of infection. Moreover, elevated 90K/MAC-2 BP serum levels are an independent predictor of failure to respond to α-interferon treatment in a cohort of community-acquired chronic hepatitis C patients.

e13538 Background: The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targe...

Levels of a 90-kDa protein (90K), recently reported as a possible marker of HIV-1 infection, were serially examined in a group of HIV-1-infected (HIV-1+) and uninfected (HIV-1-) subjects drawn from the same cohort of homosexual men. The first phase of the study included 61 HIV-1+ AIDS-free subjects 4 years (+/- 6 months) postseroconversion and 75 contemporaneous unifected subjects. Two years later, a subset of 35 HIV-1+ AIDS-free subjects and 72 HIV-1- controls was examined. Mean 90K levels for HIV-1+ subjects were significantly higher than for contemporaneous HIV-1- subjects both 4 and 6 years postseroconversion (p0.0001). A significantly more rapid progression to AIDS was seen in HIV-1+ subjects with high 90K levels both 4 years (p = 0.01) and 6 years (p = 0.003) postseroconversion. Four years postseroconversion, 90K was significantly correlated with CD8 cell percent, interferon, neopterin, and beta 2-microglobulin (p0.05). Two years later, significant correlations were seen between 90K levels and CD4 cell percent, CD4 cell number, and beta 2-microglobulin (p0.05). Stepwise-stepdown regression modeling using 90K, CD4 cell percent, interferon, and beta 2-microglobulin levels 4 years postseroconversion showed that the predictive value of a trivariate model of 90K-interferon-CD4 percent was better than any univariate or bivariate model. We conclude that the 90K protein may be a useful predictor of progression to AIDS in HIV-1+ patients, particularly in combination with the established markers of CD4 cell percent and interferon.

We have previously shown that a short course of recombinant interferon-alpha-2b (rIFN-alpha-2b) (3 million units day for 5 days) for patients with primary gynaecologic malignancies was able to increase the circulating levels of a newly discovered tumour associated antigen, termed 90K. In this study, we have investigated the effects of the same modality of administration of rIFN-alpha-2b in 62 patients with breast and colorectal cancer whose primary tumour was surgically removed 1 month before and who were without evidence of disease (NED) at the time of the study. A significant increase of 90K serum concentration was already observed 24 h after the first r-IFN-alpha-2b injection and persisted throughout the investigational period. The increase was more pronounced in patients with a basal 90K-negative than a 90K-positive assay. Of 54 patients who started the test with a 90K negative assay, 17 (31%) shifted to a positive assay after rIFN-alpha-2b. Twenty-eight of 62 (45%) patients exhibited a 90K value above the mean increment of the whole population. The serum levels of CEA, CA-15-3, CA 19-9, and alpha-fetoprotein measured in the same serum samples were not modified. After 2 years of follow-up, ten patients relapsed. Six of them showed a 90K increase above the mean increment of the whole population. As with ovarian cancer, the increase of 90K following r-IFN-alpha-2b administration might be of importance for the early detection of disease recurrence in clinically NED breast and colon cancer patients.

A novel tumor-associated protein, termed 90K, and recognized by mAb SP-2 was purified from serum of breast cancer patients, ovarian cancer ascitic fluid and conditioned medium of human breast cancer cells. In these three sources, native 90K is present as a high molecular weight complex that was dissociated by SDS-PAGE into a major band of approximately 90,000 Da. On the basis of electrophoretic mobility, buoyant density value, amino acid composition, and immunoreactivity, the 90K from the different sources appeared to be identical. NH 2 -tenninal amino acid sequence revealed no homology to known protein.

Background A previous report has shown that LGALS3BP (also known as 90K or Mac-2 BP) has antitumor activity in colorectal cancer (CRC) via suppression of Wnt signalling with a novel mechanism of ISGylation-dependent ubiquitination of β-catenin. The role of LGALS3BP in CRC prognosis was investigated. Methods The role of LGALS3BP on CRC progression and clinical prognosis was analyzed by combining cell cultures, in vitro assays, and immunohistochemistry. Results Silencing of LGALS3BP in HCT-116 human colon cancer cells resulted in enhanced β-catenin expression that was reversed by addition of human recombinant LGALS3BP. Moreover, intra-tumor delivery of LGALS3BP reduced tumor growth of xenografts originating from LGALS3BP-silenced HCT-116 cells. Finally, in a series of 196 CRC patients, LGALS3BP expression in tumor tissue associated with clinical outcome. Patients with high LGALS3BP expression had lower risk of relapse and a longer overall survival time than those with low LGALS3BP expression. Multivariate analyses confirmed LGALS3BP expression status as the only independent prognostic factor of survival. Conclusions These results provide evidence that low expression of LGALS3BP participates in malignant progression of CRC and implicates poor prognosis, highlighting its augmentation as a potential therapeutic approach.

ErbB-3 (HER-3) receptor is involved in tumor progression and resistance to therapy. Development of specific inhibitors impairing the activity of ErbB-3 is an attractive tool for cancer therapeutics. MP-RM-1, a murine monoclonal antibody targeting human ErbB-3, has shown anticancer activity in preclinical models. With the aim to provide novel candidates for clinical use, we have successfully generated a humanized version of MP-RM-1. The humanized antibody, named EV20, abrogates both ligand-dependent and ligand-independent receptor signaling of several tumor cell types, strongly promotes ErbB-3 down-regulation, and efficiently and rapidly internalizes into tumor cells. Furthermore, treatment with EV20 significantly inhibits growth of xenografts originating from prostatic, ovarian, and pancreatic cancers as well as melanoma in nude mice. In conclusion, we provide a novel candidate for ErbB-3-targeted cancer therapy.