Cancer development and progression is a complex process involving tumor cell mechanisms as well as tumor-host cell interactions. Using autocrine and paracrine mechanisms the cytokine TGF-b1 can influence cancer progression, in part through promoting the synthesis and secretion of extracellular matrix molecules. TGF-β1 strongly upregulates the gene TGFBI, which encodes a secretory protein named BIGH3. BIGH3 is an extracellular matrix, adhesion-class proapoptotic protein characterized by four fasciclin-1 (FAS1)-like domains, a cysteine-rich region and several different integrin-binding sequences. The C-terminus of BIGH3 encodes the integrin-binding peptides EPDIM and RGD. These sequences, and the integrin α3β1, have been implicated as important components of BIGH3-induced apoptosis. Breast cancer cells express the integrin α3β1, which is reported to play various roles including signaling, invasion and metastasis. Breast cancer cells and tumor-host cells express BIGH3 protein, which is expected to accumulate in the presence of TGF-β1. The above findings highlight the molecules TGF-β1 and α3β1 in tumor progression and are the basis for our hypothesis that BIGH3 protein itself is sufficient to induce an increase in breast cancer cell apoptosis. In order to test this hypothesis we utilized human breast cancer MCF-7 cells, human recombinant BIGH3 and TUNEL assays. Here, we show for the first time that exogenous human recombinant BIGH3 in cancer cell growth medium evokes a statistically significant increase in breast cancer cell apoptosis. A concentration curve shows that there is a direct relationship between the quantity of BIGH3 in the cells’ milieu and the number of cells undergoing apoptosis. This finding is consistent with receptor-ligand binding and indicates that BIGH3 is a potential target for breast cancer treatment. Importantly, in-house developed anti-BIGH3 antibody completely blocked apoptosis when compared to apoptotic cells in negative control conditions. Stimulation of BIGH3 gene expression in breast cancer cells, and providing exogenous BIGH3 to breast cancer cells, are expected to diminish breast cancer progression. Along this line, BIGH3 may be valuable as an agent for neoadjuvant or adjuvant therapy for human breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-02-03.

Numerous studies have demonstrated a relationship between the extracellular matrix protein BIGH3 and variations in the malignant properties of different cancer cell types, including osteosarcoma cells. BIGH3 protein can suppress and promote tumor growth, even on the same cancer cell type, indicating that contextual cues regulate BIGH3-mediated divergent outcomes. We employed a multicellular tumor spheroid model to study the effects of BIGH3 with respect to physical and molecular features of three-dimensional tumor growth. The results demonstrated that exogenous recombinant BIGH3 blocked the development of multicellular large tumor spheroids so that only small spheroids formed. The effect was dependent on the BIGH3 concentration in the growth medium and the time of incubation of BIGH3 with the osteosarcoma cells in the spheroid model. TGF-β1 signaling induced multicellular tumor spheroids to synthesize a greater quantity of BIGH3 relative to non-treated spheroids. The TGF-β1-mediated increase in BIGH3 protein antagonized the development of multicellular large spheroids. Anti-BIGH3 antibody, and an inhibitor of TGF-β1 signaling, blocked the antagonistic effect induced through TGF-β1 stimulation and BIGH3 protein expression, resulting in the formation of multicellular large spheroids. Immunohistochemistry detected BIGH3 at cell bodies within the spheroid stroma, suggesting osteosarcoma cell-surface proteins bind BIGH3. Flow cytometry demonstrates that osteosarcoma cells interact with soluble BIGH3, and solid-phase cell adhesion assays show that osteosarcoma adhesion to BIGH3 substratum is mediated by integrin α4β1. However, anti-α4 antibody did not attenuate the BIGH3-mediated antagonism toward formation of multicellular large spheroids. We conclude that TGFβ1 and BIGH3 suppress the development of large osteosarcoma tumors.