We demonstrate that exogenous administration of EETs in vivo promote tumor growth, as well as dramatically enhancing metastatic spread in several tumor models known to metastasize poorly. Specifically, in Tie2-CYP transgenic mice, which are engineered to raise endothelial EET levels, tumors that rarely metastasize exhibit extensive metastatic spread into a range of organs, including lung, axillary lymph nodes, liver, and kidney. Exogenous administration of EETs induce multiorgan metastasis and tumor dormancy escape in a variety of transplantable and genetically engineered cancer models to a high degree, providing what we believe to be the first in vivo demonstration that pharmacological modulation of these lipid autacoids affects tumor growth.
EETs promote metastasis by triggering secretion of VEGF by the endothelium, which we show to be critical for EETs’ cancer-stimulating activity. We also demonstrate that stimulation of metastasis by EETs is due to their action at the secondary (metastasis) site and not due to their effect on the cells of the primary tumor. We arrived at this insight by dissecting the two steps of metastasis using a series of parabiosis experiments in which mice transgenically modified to produce either low or high levels of EET in the endothelium were surgically cojoined. Using parabiosis, we have delineated and identified the distinct roles of stromal EET levels in promoting or inhibiting growth of the primary tumor and at the point of metastasis.
Although we show that the endothelium, acting as a source of local EETs, is a critical regulator of primary tumor growth and metastasis, our studies with exogenously administrated 14,15-EET indicate that high levels of systemic EETs can also stimulate tumor growth and metastasis. The levels of EETs after adenoviral CYP expression increase approximately 3- to 5-fold in vitro (11
). Treatment with 14,15-EET via osmotic minipump in our studies yielded a plasma steady-state EET concentration of approximately 3.0 ng/ml, twice that observed in sEH null mice (~1.5 ng/ml), which have high levels of systemic EETs, and 15- to 30-fold higher than that in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr mice (100–200 pg/ml). This may explain why systemic exogenous 14,15-EET was able to stimulate tumor growth, whereas in the parabiosis studies, the high-EET partner (e.g., sEH-null mouse) failed to rescue tumor growth in the low-EET partner. Plasma EET levels were likely diluted in the parabiosed low-EET partner to levels that approximate those in WT mice.
Our work demonstrates a number of important findings on lipid autacoids and in particular EETs in tumor biology. We establish a connection between the disparate fields of lipid autacoids and tumor biology. By manipulating EET levels in vivo, as opposed to CYP levels or activity (11
), we demonstrate the dramatic metastatic potential of EETs on low-metastasizing tumors, thereby opening an avenue for metastasis research, which has suffered from the scarcity of spontaneous metastasis models. EETs in the endothelium may also be a key paracrine mediator of the tumor-promoting role of the stroma, revealing a trophic function for the endothelium in promoting tumor growth in addition to its role in providing circulation. Inflammatory cells such as macrophages may also contribute to EET-mediated tumor growth. We also demonstrate that sEH inhibitors, which elevate endogenous EET levels, promote primary tumor growth and metastasis. Our findings are of potential clinical relevance because drugs that raise EET levels are now in clinical trials for the treatment of hypertension and are being considered for long-term use in other medical conditions (4
). Our finding that sEH inhibitors promote tumor growth, notably explosive metastatic growth from presumably dormant disseminated tumor cells, raises significant concerns about the use of sEH inhibitors, especially their chronic use, which may adversely affect cancer patients. Dormant tumors have been identified at autopsy in normal adults who died of trauma and without prior history of clinical evidence of cancer, including 39% for in situ breast carcinoma, 46% for in situ prostate cancer, and 36% for thyroid carcinoma in people aged 50–70 (55
). The factors that regulate tumor dormancy and escape from it are poorly understood (57
). The escape is critically dependent on the induction of angiogenesis (22
). Our data suggest that the increase in EET levels, due to downregulation of sEH, may support the angiogenic switch in part by an increase in VEGFR2 and loss of thrombospondin.
We also demonstrate the ability of EET antagonists to inhibit tumor growth and metastasis. These results could pave the way for a new strategy for the prevention and treatment of metastatic disease — i.e., inhibition of EET bioactivity. Specific EET antagonists, inhibitors of endothelial CYP epoxygenases, or the overexpression of EET-metabolizing enzymes may represent new strategies for the treatment of angiogenic diseases, including cancer. Manipulation of the EET system is thus a double-edged sword, and further studies are needed to carefully evaluate the benefits as well as the risks in the clinical modulation of these lipid mediators.