LGD1069, a selective retinoid X receptor agonist, is used in patients for cutaneous T-cell lymphoma , and is also demonstrated that it is an efficacious chemopreventive and chemotherapeutic agent in a number of preclinical rodent models breast cancer [7, 16–18]. Furthermore, LGD1069 was shown recently to prevent tumor-induced angiogenesis and metastasis. Yen and his colleagues determined the influence of this retinoid X receptor agonist on angiogenesis and metastasis in solid tumors . Moreover, in previous study, we also demonstrated that LGD1069 decreased tumor-induced angiogenesis. In that study we found LGD1069 impair the capacity of vessel formation induced by tumor cells via suppression of VEGF secretion in human non-small cell cancers. Thus, we sought to determine whether LGD1069 exerts its anti-angiogenic ability by directly impairing activation of human endothelial cells and to identify the underlying story here. In the current study, we found that LGD1069 inhibited proliferation and survival of endothelial cells, and destroyed vessel-network formation on Matrigel. In addition, in vitro analysis indicated that LGD1069 attenuated endothelial cell adhesion, migration and inhibited cell invasion through reconstituted extracellular matrix (ECM).
Angiogenesis denotes the formation of new blood vessels from pre-existing vessels. Angiogenetic factors, such as fibroblast growth factors (FGFs) and vascular endothelial growth factors (VEGFs), regulate endothelial cells to secrete several proteases (such as MMPs and TIMPs) and plasminogen activators (such as uPA), resulting in the degradation of the vessel basement membrane, and the formation of stable new vessels[20–22]. Besides activating endothelial cells, VEGFs binding to their specific receptors expressed on cell surface, also regulate proliferation and survival of endothelial cells, and microvascular permeability [19; 23]. Led by these findings and our published study, we tested whether there are effects on biological behaviors of ECM related enzymes. Our data showed that at the presence of VEGF (2 ng/ml), LGD1069 inhibited expression of MMP-2, MMP-9 in endothelial cells. Collectively, these results indicated that the anti-angiogenetic and anti-metastatic properties of LGD1069 may be due to the inhibition of expression of matrix-related proteases in endothelial cells, resulting in inhibiting endothelial cell adhesion, migration, invasion and proliferation. These data suggest that LGD1069 may impede the activation of endothelial cells directly, and its effects on angiogenesis and metastasis may occur through activation of protease expression and events that regulate angiogenesis.
Runx2 is a member of the runt family of DNA-binding transcription factor which possesses a highly conserved DNA binding and protein-protein interaction domain. It plays essential roles for the formation of skeleton. Qiao et al. reported that Runx2 DNA-binding activity is elevated in proliferating endothelial cells . Recent Studies showed that Runx2 mediates endothelial cell migration and invasion during tumor angiogenesis . Furthermore, it was found that Runx2 is ectopically expressed in metastatic cancer cells but not in non-metastatic cancer cells, and that several genes required for bone development and turnover, such as opn, bsp, mmp12 etc., are activated by Runx2 . It' well known that this cohort regulated by Runx2 is essential mediators of tumor invasion and metastasis. Pratap et al. also demonstrated that Runx2 activates the "vicious cycle" of TGF- β-mediated tumor growth and metastatic bone disease . Therefore, Harada and Rodan et al. presumed that Runx2 is a "master" transcription factor in the development of metastatic foci in several solid tumor . Given the important role of Runx2 in cancer-induced angiogenesis and metastasis, we wondered whether it plays a role in the inhibition of MMP-2 and MMP-9 expression induced by LGD1069. The following specific studies will test this hypothesis. Data from current study suggested that LGD1069 inhibited expression of Runx2 and phosphorylation of Smad2/3, but no effects were found on expression of total Smad2/3.
In conclusion, we demonstrated here that LGD1069 may impairs metastatic potential in solid tumor, and the inhibitory effects of LGD1069 were due to its ability to inhibit endothelial cell growth and interfere with adhesion, migration and invasion of endothelial cells directly. These effects of LGD1069 on endothelial cells may be associated with inhibition of activation of Runx2 protein by interacting with the TGF-β/Smad family. These findings have important implications for patients with malignant diseases. For example, LGD1069 can be used alone or incorporated with other conventional modalities such as radiation and chemotherapy to improve the efficacy of these treatments as well as to prevent development of distant metastasis.
However, there are several questions arising from our data. Such as, whether is there a direct interaction between Runx2 and RXRs, which both are DNA-binding transcription factors? Whether are the effects on cell behaviors and expression of molecules of LGD1069 coincidental or directly causes and effects? Recently, the work from Hoover et al. may provide a little bit clues, but not all. They reported that retinoids, such as LGD1069 and 9-cis RA, directly regulated Smad activation and sub-cellular accumulation via RXR-α. From these data, it seems that there exist indirect association between RXRs and Runx2. Therefore, it is worthwhile to further explore the role and regulation in of Runx2 in endothelial cells, and to figure out the cross-talk between Retinoic receptors and Runx2 activity. On-going research is directed towards understanding the underlying story of effects of LGD1069 on Runx2 in endothelial cells and tumor cells. Information obtained from these studies will have significant impact on the therapeutic use of LGD1069 in treatment of those malignant diseases.