S100A4, a member of the S100 calcium-binding protein family secreted by tumor and stromal cells, supports tumorigenesis by stimulating angiogenesis. We demonstrated that S100A4 synergizes with vascular endothelial growth factor (VEGF), via the RAGE receptor, in promoting endothelial cell migration by increasing KDR expression and MMP-9 activity. In vivo overexpression of S100A4 led to a significant increase in tumor growth and vascularization in a human melanoma xenograft M21 model. Conversely, when silencing S100A4 by shRNA technology, a dramatic decrease in tumor development of the pancreatic MiaPACA-2 cell line was observed. Based on these results we developed 5C3, a neutralizing monoclonal antibody against S100A4. This antibody abolished endothelial cell migration, tumor growth and angiogenesis in immunodeficient mouse xenograft models of MiaPACA-2 and M21-S100A4 cells. It is concluded that extracellular S100A4 inhibition is an attractive approach for the treatment of human cancer.

Despite progresses in diagnosis and treatment, pancreatic cancer continues to have the worst prognosis of all solid malignant tumors. Recent evidences suggest that the metastasis-promoting protein S100P stimulates pancreatic tumor proliferation, survival, invasion and metastasis progression through extracellular functions. Moreover, its expression is strongly correlated with poor prognosis in patients with several types of cancer although the entire molecular mechanism responsible for the diverse biological functions is not fully understood. We showed that extracellular S100P stimulates pancreatic carcinoma BxPC3 cell line by promoting cell proliferation. We also demonstrated that S100P induces, in this cell line, the phosphorylation of IκBα and the secretion of matrix metalloproteinase 9 (MMP-9). In addition, treatment with S100P protected cells from injuries induced by the cytotoxic agent Gemcitabine. On the basis of these results, we developed function-blocking anti-S100P monoclonal antibodies (mAbs) that abolished all of its in vitro activities. Furthermore, in vivo treatment with the candidate 2H8 antibody decreased tumor growth and liver metastasis formation in a subcutaneous and orthotopic BxPC3 tumor model. We conclude here that a therapeutic strategy blocking the extracellular activity of S100P by means of specific mAbs could be an attractive therapeutic approach as a single agent or in combination with target-directed or chemotherapeutic drugs to treat pancreatic cancer.

The antiapoptotic protein survivin is an attractive target in cancer therapy because it is expressed differently in tumors and normal tissues and it is potentially required for cancer cells to remain viable. Given that survivin is also overexpressed in endothelial cells (ECs) of newly formed blood vessels found in tumors, its RNA targeting might compromise EC viability and interfere with tumor angiogenesis. We used two antisense strategies against survivin expression, antisense oligonucleotides (aODN) and small interfering RNA (siRNA), to study in ECs the contribution of survivin in various steps leading to tumor angiogenesis. A 21-mer phosphorothioate aODN and two siRNA oligonucleotides against survivin mRNA were designed to downregulate survivin expression. Survivin targeting caused (1) a strong growth-inhibitory effect, (2) a 4-fold increase in apoptosis, (3) an accumulation of cells in the S phase and a decrease in G2/M phase, (4) a dose-dependent inhibition of EC migration on Vitronectin, and (5) a decrease in capillary formation. Control oligonucleotides, an unrelated oligonucleotide, and one with four mismatches, had no significant effect. All these results show that survivin is a suitable target in cancer therapy because its inhibition in EC causes both a proapoptotic effect and an interruption of tumor angiogenesis. The two strategies used, classic aODN and siRNA technology, were very effective. Moreover, the latter can be used in the low nanomolar range, thus increasing the sensitivity of the treatment.

Within the tumor, malignant and stromal cells support each other by secreting a wide variety of growth factors and cytokines, allowing tumor growth and disease progression. The identification and regulation of those key factors in this crosstalk has opened the opportunity to develop new therapeutic strategies that not only act on the tumor cells but also on the stroma. Among these factors, S100A7 protein has gained interest in the last years. With key roles in cell motility its expression correlates with increased tumor growth, angiogenesis and metastatic potential. This work aims to deepen in the role played by extracellular S100A7 in the tumor microenvironment, offering a new integrative insight of its mechanism of action on each cellular compartment (tumor, endothelial, immune and fibroblast). As a result, we demonstrate its implication in cell migration and invasion, and its important contribution to the formation of a proinflammatory and proangiogenic environment that favors tumor progression and metastasis. Furthermore, we define its possible role in the pre-metastatic niche formation. Considering the relevance of S100A7 in cancer progression, we have developed neutralizing monoclonal antibodies, reporting for the first time the proof of principle of this promising therapeutic strategy for cancer treatment.

A series of murine monoclonal antibodies were raised against purified human alpha v beta 3 integrin and against M21 human melanoma cells. Five notable hybridomas were identified by ELISA on purified integrins, and the isolated antibodies bound the alpha v-chain. These antibodies, 17E6, 20A9, 23G5, 14D9.F8 and 10G2, recognised the extracellular domains of the integrin, and were shown to be reactive in FACS, immunoprecipitation, ELISA, and ELISA on fixed cells with M21, M21-L4, and UCLA-P3, but not with the alpha v-deficient M21-L or M21-L-IIb (M21-L transfected with GpIIb integrin). One antibody, 17E6, strongly perturbed cell attachment mediated by alpha v integrins, reacting at least with alpha v beta 3, alpha v beta 5, and alpha v beta 1, and strongly inhibiting cell attachment to alpha v-ligands vitronectin and fibronectin with an IC50 of approximately 0.1 microgram ml-1. Furthermore, 17E6 at this concentration could induce cell retraction from the substrate, while LM609 (anti-alpha v beta 3) and control antibody 14E2 (anti-200 kDa melanoma surface protein) at 1,000-fold higher concentrations had minimal effects on cell morphology. The action of 17E6 was reversible and was not due to toxic effects: in vitro 17E6 at 0.1 mg ml-1 did not affect either cell proliferation or DNA synthesis. In two nude-mouse tumour models, subcutaneous tumour development and a lung colonisation (‘experimental metastasis’) assay, injection of 17E6 strongly inhibited tumour development, while isotype-matched controls had no effect. There was no obvious mechanism of cell or of complement-mediated tumour cytotoxicity; the antibody did not mediate ADCC or AECDC, or complement fixation. The data strongly support previous studies which have indicated the importance of alpha v-integrins, and especially alpha v beta 3, in the tumour progression of human melanoma.