Cancer is the leading cause of death worldwide. Despite advances in tumor biology and novel cancer therapies, cancer remains a global health problem. It is therefore imperative to search for new ways to improve cancer therapy and to further investigate tumor biology to identify molecular mechanisms that can be exploited to develop new therapies.
Drug delivery to tumors is necessary for chemotherapy to be effective. Tumor vasculature consists of abnormal vessels which limit drug perfusion into tumor tissues. We hypothesized that targeting integrin receptors on endothelial cells with synthetic disintegrins would increase tumor vascular permeability and improve anti-cancer effects of cyclophosphamide. We synthesized disintegrins by conjugating integrin-binding peptides containing arginine-glycine-aspartate to ends of 3.4, 6 or 10 kDa polyethylene glycol polymers. The largest synthetic disintegrin, G10, inhibited endothelial cell attachment in vitro. G10 also increased tumor vascular permeability of EMT6 tumors to Evans Blue dye, which binds albumin, compared to its control. A single intratumoral dose of G10 alone or in combination with a single systemic dose of cyclophosphamide reduced tumor growth compared to respective controls. These data indicate that a divalent integrin-targeting agent of sufficient size can increase tumor vascular permeability and improve effectiveness of a chemotherapeutic agent.
We also examined effects of the peptidyl-proline isomerase (PPIase), Protein Never in Mitosis Gene A Interacting-1 (PIN1), on tumor growth. PIN1 is the only PPIase that specifically recognizes and isomerizes the prolyl bond between cis and trans of phosphorylated Ser/Thr-Pro motifs. PIN1 is overexpressed in many human cancers and is associated with poor prognosis. As expected, stable PIN1 depletion (KD) using lentivirally-delivered shRNA reduced EMT6 mammary carcinoma and Lewis Lung Carcinoma (LLC1) growth in culture. In contrast, EMT6 and LLC1 tumors lacking PIN1 grew faster in mice. Additionally, LLC1 KD tumors grew even faster in pin1 knockout mice, whereas control tumors were unaffected by host pin1 status. The faster growing EMT6 KD tumors contained lower cleaved caspase-3 levels and higher vascular endothelial growth factor levels compared to control tumors, suggesting that PIN1 depletion reduced apoptosis and increased angiogenesis.
To mimic the tumor environment, cultured EMT6 cells were treated with hypoxia (1% oxygen). Hypoxia treatment increased cleaved caspase-3 levels in both control and KD cells, however, levels were reduced by PIN1 depletion similar to that in tumors. As expected, hypoxia increased HIF-1α and VEGF levels in EMT6 cells, but levels were further increased in KD cells. Treatment with exogenous VEGF increased growth of KD but not control EMT6 cells. These results suggest that EMT6 tumors lacking PIN1 have an exaggerated response to hypoxia and a reduced level of apoptosis that may account for their elevated growth in mice.
Overall, these results suggest that synthetic disintegrins can increase tumor vascular permeability and anti-cancer effects of a chemotherapeutic agent. Future investigations could yield novel adjuvant chemotherapeutic agents. The studies on PIN1 in cancer suggest that PIN1 is directly involved in tumor growth and responses to hypoxia. Understanding how PIN1 functions to limit HIF-1α and VEGF expression could lead to novel anti-angiogenic therapies.