Abstract
Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvβ3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvβ3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvβ3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a KrasG12D-LSLp53fl/fl subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.
Highlights
OPN is a multifunctional protein involved in several pathological processes such as inflammation and cancer [1]
Species specificity of anti-OPN monoclonal antibody (AOM1) was determined by surface plasmon resonance (SPR) using OPN immobilized on a Biacore chip
The ability of AOM1 to inhibit OPN binding to integrin avb3 which is considered to be the major receptor by which OPN regulates cancer cell migration and proliferation, and to prevent thrombin-mediated cleavage of OPN was characterized in an ELISA-based and western blot assays, respectively
Summary
OPN is a multifunctional protein involved in several pathological processes such as inflammation and cancer [1]. Binding of OPN to the above receptors on tumor cells triggers downstream signaling pathways including Ras, Akt, MAPK, Src, FAK and NF-KB [1] that collectively lead to the following in tumor cells: i) invasion to ECM (extracellular matrix) mainly via upregulation of MMPs [19] (matrix metalloproteinases) and uPAs [20] (urokinase plasminogen activator) by OPN; ii) increased migration and adhesion of tumor cells [21]; iii) inhibition of cell death likely through upregulation of antiapoptosis mediators such as GAS6 [22]; and iv) development of pre-metastatic niche [23] Tumor stroma such as endothelial cells [18] and immune infiltrating cells [24,25] ( monocytes) express OPN receptors. VEGF may accelerate thrombin activity to generate cleaved-OPN that in turn results in increased migration of endothelial cells [34]
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