Endogenous peptides with antiangiogenic activity have emerged as one of the leading tools to treat cancer in addition to polypeptides, small molecules, and antibodies [29
]. We have previously introduced a systematic methodology for the identification of antiangiogenic peptide sequences and demonstrated that a high percentage of the predicted peptides indeed inhibit the proliferation and migration of HUVECs in vitro
. On the basis of this in vitro
screening, we selected one molecule from three protein classes—pentastatin-1, a collagen IV-derived peptide; chemokinostatin-1, a CXC chemokine-derived peptide; and properdistatin, a TSP-1 domain-containing peptide—and tested them in vitro
on additional cell lines and in vivo
in an orthotopic MDA-MB-231 breast xenograft model.
We demonstrated in vitro
that pentastatin-1 has anticellular effects on MDA-MB-231 cells and decreases 3T3 fibroblast viability; however, chemokinostatin-1 and properdistatin do not exhibit these effects. The latter peptides have been previously shown to exhibit antiproliferative and antimigratory properties on HUVECs [26,27
]. Pentastatin-1 has also been previously shown to inhibit proliferation and migration of endothelial cells in vitro
], and we demonstrated inhibition of tumor growth in vivo
. Supplementary flow cytometry data in Figure W2
show receptor identification of β1
-integrins for MDA-MB-231 cells. Pentastatin-1 has been previously shown to bind to β1
], which we show are present in high percentages for both HUVECs at 85% of cells and MDA-MB-231 at 79% of cells, indicating that the peptide is capable of directly inhibiting tumor cell proliferation in addition to being antiangiogenic. Chemokinostatin-1 was similarly shown to bind to CXCR3 through antibody neutralization experiments, and properdistatin was shown to bind to the CD36 receptor [24
]. Both of these peptides inhibit the proliferation of HUVECs; however, they did not inhibit tumor cell proliferation or proliferation of 3T3 fibroblasts. Flow cytometry data indicate a low percentage of these receptors in MDA-MB-231 cells, suggesting that the activity of these peptides is likely monomodal and localized only to proliferating endothelial cells.
The three endogenous peptides show significant tumor suppression at the concentrations of 10 mg/kg for pentastatin-1 and 20 mg/kg for both chemokinostatin-1 and properdistatin. The scrambled peptide controls strongly suggest the efficacy of the peptides is sequence-dependent. Although randomly mutating peptide amino acid sequences does not guarantee the resulting sequence will not retain some binding interactions among receptor families, in this case the scrambled sequences only retain nominal activity, which was not statistically significant from the experimental controls.
Immunohistochemistry was performed using the CD31 antibody, which is specific for endothelial cells. In pentastatin-1- and chemokinostatin-1-treated tumors, there is a statistically significant inhibition of endothelial cells and a reduction in microvessel volume, indicating that the peptides are antiangiogenic. Chemokinostatin-1-treated tumors grew more rapidly toward the end of the experiment, indicating possible endothelial cell resistance to the peptide. In our H&E staining of tumor cross sections on day 13, we found that chemokinostatin-1 is most similar to the experimental control, containing minimal core necrotic regions, suggesting the peptide's less deleterious effect on the tumor microenvironment. We also show that in chemokinostatin-1-treated mice, their tumors have a higher percentage of proliferating cells than the pentastatin-1- or properdistatin-treated tumors. The significant quantity of mouse stromal cells suggests a high infiltration of these cells into the tumor microenvironment owing to the presence of the chemokine-derived peptide. Chemokinostatin-1 is notably derived from the proangiogenic ELR-positive CXC chemokines. The cancer biology of CXC chemokines is complex because these peptides may stimulate the infiltration of various stromal cells into the tumor microenvironment [30
]. Although the SCID mice used in this experiment lack T and B lymphocytes, there may be an influx of other mouse stromal cells into the tumor owing to the presence of the chemokine-derived peptide.
The experiments were terminated after day 13 of treatment because some peptide-treated tumors had begun to escape and grew as rapidly as the controls, indicating tumor resistance to the applied peptides. Recently, it has been proposed that there can be at least two underlying mechanisms for resistance to antiangiogenic therapies: adaptive or evasive resistance and intrinsic nonresponsiveness [31
]. On the basis of the initial response phase of peptide treatment we observed in the case of each peptide, we hypothesize a form of adaptive resistance developed within the tumor microenvironment in response to the peptide treatment. Recent systematic investigation of resistance to endogenous antiangiogenic agents (TSP-1, endostatin, and tumstatin) demonstrates that tumors escape the treatments after several days, and a number of proangiogenic factors (e.g., VEGF, FGF-2, transforming growth factor-β) are upregulated [4
]. Other general mechanisms of resistance include the recruitment of bone marrow-derived proangiogenic cells, increased pericyte coverage in the tumor vasculature, and increased activation of invasion and metastasis for access to normal tissue vasculature [31
]. Future studies should be performed to determine the modes of resistance for each peptide and whether treatment efficacy can be further sustained. Despite these initial limitations, the peptides have a potential to regulate and suppress vessel growth in vivo
and in translational studies possibly when combined with cytotoxic agents or in combinations with other antiangiogenic agents.
Breast cancer has been shown to be angiogenesis-dependent; thus, advances in the development of antiangiogenic agents should contribute to the arsenal of therapeutic tools available for treatment of the disease. The presented results indicate the selected peptides may have the potential for translational clinical therapeutics in breast cancer. In summary, we have demonstrated that systematically identified peptides derived from type IV collagen, CXC chemokines, and TSP-1 inhibit neovascularization in MDA-MB-231 breast xenografts and are candidates for translational therapeutics in breast cancer.