What is Osteopontin?

1. What is Osteopontin? Effect of Anti-Osteopontin Monoclonal Antibodies on the Growth of Cancer Cells in Soft AgarMegha Rajpal*, Tanya Gordonov*, Dana Cifelli*, Yacov Ron**, Esben Sørensen***, Larry Steinman**** and David Denhardt* *Rutgers, The State University of New Jersey; **Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey; ***Aarhus University, Denmark; ****Stanford Univ, California Following are the pictures of the colonies and their areas as calculated by the Image J Program. All pictures are of the same magnification. It is intriguing that many of the mAbs tested bind to the overlap region of peptide 15/16 (depicted with * in Figure 1 in the introduction), but that only 3 of those tested have an effect on colony formation. SEE SUPPLEMENTARY FILE! This peptide may be part of the sequence that engages CD44 or its variants. The interaction of CD44 with OPN has been implicated in the migration of macrophages and tumor cells. Some research has shown that OPN increases expression of CD44 (2). The other “sticky” peptide, #11, is interesting in that it contains the SVVYGLR sequence at its N-terminal end, which is the same sequence that is found at the C-terminal end of thrombin-cleaved OPN. It was a surprise to find that 1H3F7 and 3D9 stimulated the formation of colonies by the mouse melanoma cells. We are presently trying to confirm these results. Since all three mAbs, 3D9, 1H3F7 and 21B1, are binding to roughly the same region of OPN, the opposite effect of stimulation and inhibition might be explained by the different epitopes these mAbs could be binding to in that region. Also, differences in amount of OPN produced by these cells might be a factor. In another study we investigated the non-specific binding of mouse IgG to the “sticky” peptides (see poster by Kadia et al.). In standard ELISA assays we have found 1) that human (native and recombinant) OPN binds very strongly to mouse IgG and 2) that native mouse OPN binds strongly to goat IgG. For more detailed data, see the appended figure. An issue we are trying to address is whether in some cases one (both) of the “sticky” peptides is (are) actual epitopes for binding the Fab portion of the mAb. Osteopontin, OPN, is a phosphoglycoprotein that is found in various tissues and body fluids. Its various functions include bone remodeling, cell signaling, inflammatory and stress responses. It also plays a role in autoimmune diseases, bone resorption, and cancer cell metastasis. Many of these actions are likely the result of OPN providing cell survival signals because it engages integrins and CD44(v) similar to components of the extracellular matrix (1). OPN is known to stimulate colony formation of many cancer cell lines in soft agar. In the past several years we have isolated a set of hybridomas producing anti-OPN monoclonal antibodies to mouse and human OPN (4).The purpose of this study was to test the action of the monoclonals on the growth of cancer cells in soft agar. Figure 1: Proposed structure of human osteopontin (2). * is the peptide overlap region 15/16 (see supplementary data). GOAL We are trying to identify which mAbs, if any, can inhibit OPN function (formation of colonies in soft agar in this case) using mouse melanoma cells (B16F10), human breast cancer cells (MDA MB 435),and human melanoma cells (YUSITI and YURIF) (a generous gift from Dr. Ruth Halaban, Yale University). MATERIALS AND METHODS • Monoclonal antibodies were purified from ascites tumors generated by the hybridomas using ammonium sulfate precipitation and chromatography on a Protein A column. The soft agar assay entailed the following: • 1. Prepare bottom agar layer • a. Mix equal volume of growth medium with 1% agarose at 50oC. • b. Add 1ml of this mixture into each well of a 12-well plate; allow agarose to solidify for about 1 hour at RT. • 2. Trypsinize cells and count them. Cells should be sub-confluent and growing; add 2500 cells/ well, without or with the desired monoclonal antibody – 100 µg/well and 10 µg/well. (Lower concentration usually without effect.) • 3. Prepare top layer • a. Each well consists of 250 µl of the growth medium and 250 µl of 0.7% agarose and 100 µl of the cells and the desired amount of the antibody. After the agarose hardens transfer the plate to the incubator. • b. After 30-60 min add 1 ml of growth medium with the appropriate antibody, if present. REFERENCES • Denhardt D.T., Noda M., O’Regan A.W., Pavlin D., and Berman, J.S. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. The Journal of Clinical Investigation. 2001; 107: 1055-61. • Kazanecki C.C., Uzwiak D.J., Denhardt D.T. Control of Osteopontin Signaling and Function by Post- Translational Phosphorylation and Protein Folding. Journal of Cellular Biochemistry. 2007; 102: 912-924. • Price J.E., Polyzos A., Zhang R.D., et al. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res. 1990;51:717–721. • Wang K.X., Shi Y.F., Ron Y., Kazanecki C.C., and Denhardt D.T. Plasma Osteopontin Modulates Chronic Restraint Stress-Induced Thymus Atrophy by Regulating Stress Hormones: Inhibition by an Anti-Osteopontin Monoclonal Antibody. The Journal of Immunology. 2009; 182: 2485-2491. • Zhou Y., Dai D.L., Martinka M., et al. Osteopontin Expression Correlates with Melanoma Invasion. The Journal of Investigative Dermatology. 2005; 124: 1044-1052. Figure 2: Coomassie Blue-Stained SDS PAGE Gel of the mAbs purified from ascites. The heavy and light chains are indicated by the arrows at about 55 kDa and 25 KDa respectively. The 3D9 and 1H3F7 are fairly pure IgGs. 1H3 consists of proteins (we believe) produced by the uncloned hybridoma; 1H3F7 is a clone of 1H3. The proteins produced by 21B1, which were purified from an ascites using a standard ammonium sulfate precipitation followed by chromatography on Protein A, are atypical. Clones of 21B1 generate much the same pattern, which we are currently trying to characterize. RESULTS Effect of mAbs on colony formation in soft agar: We hypothesized that some of the mAbs would inhibit colony formation in soft agar. We found, amongst the subset of mAbs tested so far, that mAb 21B1 inhibits colony formation of MDA MB 435 (human breast cancer cells) and a human melanoma cell line (Yusiti). We also found that the mAbs 1H3F7 and 3D9 enhance colony formation of B16F10 mouse melanoma cells. After analyzing the digital pictures of the colonies using the Image J program, we found that the inhibition or stimulation was statistically significant in repeated experiments. The table below shows the results that we have so far. Peptide binding studies (see Poster by Kadia et al. and the Supplementary Data for more detail) We have analyzed binding of the mAbs to the peptides in SBF (Simulated Body Fluid- which has the salt composition as plasma). We are still in the process of testing more mAbs. As the table below shows, all the mAbs, 21B1, 3D9 and 1H3F7, seem to be binding to the same overlap region- peptide 15/16.See Figure 1 (the OPN molecule above) in the introduction for the location of this region on OPN. DISCUSSION Substantial data have linked OPN with tumor progression and metastatic spread. Studies have shown that MDA-MB-435 (human breast cancer cells) metastasize from the mammary fat pad but rarely from the subcutis. These cells form progressively growing tumors that metastasize to lungs and regional lymph nodes (3). Melanoma is one of the most aggressive cancers affecting humans. Research has shown that OPN expression may increase early in melanoma development and may enhance tumor cell growth in invasive melanoma (5). Our major goal is to identify one or more anti- OPN mAbs that might be therapeutically useful in the treatment of metastatic diseases. OPN is known to promote tumor progression and exists in various isoforms with differing post- translational modifications. So far we have confirmed one monoclonal, 21B1, that inhibits growth of human breast cancer cells and one line of human melanoma cells. We have also confirmed that 3D9,1H3, and 1H3F7 stimulate B16F10 colony formation in soft agar. ACKNOWLEDGEMENTS This research was supported by funds from the National Multiple Sclerosis Society, the National Institutes of Health, the Aresty Research Center and a gift from Mrs. J. G. Harrar. MR and TG thank Dean Justine Levine for her support.