The present study shows that the 2ME2-analog ENMD-1198 interferes with growth factor-induced signaling in hepatocellular carcinoma and effectively inhibits HIF-1α and STAT3 activation. This disruption in cell signaling leads to a significant reduction of migratory and invasive properties of cancer cells and diminishes VEGF mRNA expression.
Tissue hypoxia constitutes as one of the main characteristics of the tumor microenvironment and has been shown to be implicated in the progression and metastasis of various solid malignancies [37
]. One key regulatory protein in the cell response to changing oxygen levels is the hypoxia inducible factor-1α (HIF-1α). Interestingly, HIF-1α activation appears to be a very early event in carcinogenesis and this protein is expressed before histological evidence of angiogenesis or invasion even occur [40
]. In regards to HCC, overexpression of HIF-1α has been reported, which has been associated with a poor prognosis [41
]. Recently, both HIF-1α and VEGF were identified to be involved in the malignant transformation of dysplastic liver nodules and additionally a hypoxia-independent overexpression of HIF-1α has been shown to be involved in a model of mouse hepatocarcinogenesis [4
]. Overall, these studies suggest that anti-HIF-1α therapy could prove valuable for treatment of HCC, or its pre-malignant lesions. However, specific inhibitors to HIF-1α are still under development, thus demanding the evaluation of alternative strategies for inhibiting HIF-1α in cancers. Interestingly, recent studies have demonstrated that 2ME2, an endogenous metabolite of estrogen, elicits antineoplastic efficacy which in part is mediated by an anti-HIF-1α activity [13
]. The substance used in our study, ENMD-1198, is based on a modified chemical structure of 2ME2. However, ENMD-1198 has previously been evaluated in models of HCC. Indeed, we now demonstrate that ENMD-1198 effectively inhibits activation of HIF-1α in HCC, ultimately leading to a reduction of tumor growth and vascularization in vivo
. However, the exact mechanism by which 2ME2 and ENMD-1198 inhibit HIF-1α is not fully understood. Recent studies suggested that 2ME2 treatment induces superoxide radicals in tumors [44
], which in turn could down-regulate HIF-1α. Another potential mechanism for reducing HIF-1α activity could be mediated through interference with STAT3 phosphorylation, as STAT3 is required for forming an active HIF-1 complex [46
]. In HCC, STAT3 appears to play a central role, as this transcription factor is implicated in oncogenesis and metastasis [7
]. Furthermore, STAT3 appears to be necessary for a PI-3K/Akt-mediated hypoxia-independent HIF-1α protein synthesis [46
]. Importantly, our results indicate that ENMD-1198 effectively diminishes phosphorylation of STAT3 in HCC cells, a finding which has not been reported for 2ME2 compounds to date. Hence, we now provide evidence for another mechanism for impairing HIF-1α function in cancer cells by 2ME2-like inhibitors in terms of diminishing STAT3 activation. Moreover, ENMD-1198 also reduced phosphorylation of Akt, a signaling intermediate that is up-stream of HIF-1α, thus resulting in further impairment of HIF-1α activity. This was also reflected in Western blot results for HIF-1α expression analysis in tumor tissues, which were markedly reduced in the ENMD-1198 treated tumors. In fact, one has to realize that tumor sizes differ significantly among these treatment groups, which per se could affect HIF-1α expression. Nevertheless, we also detected a diminished STAT3 activation by Western blotting of tumor tissues in the ENMD-1198 treatment group (data not shown), suggesting that the drug also effectively inhibits both targets in vivo
Another important aspect is the observed anti-metastatic property of ENMD-1198 in vitro, as treatment of HCC cells led to reduced phosphorylation of the pro-migratory signaling component FAK. In in vitro assays, ENMD-1198 substantially abrogated EGF- and HGF-mediated cancer cell migration and invasiveness, suggesting that this substance could be valuable for reducing HCC metastasis. Development of intra-hepatic metastases is a common problem in HCC patients who underwent liver resection for HCC, or are on the waiting list for transplantation. Since HIF-1α and STAT3 both promote metastasis formation, treatment with ENMD-1198 appears to be reasonable and warrants further investigation. ENMD-1198 is already being investigated in a phase I trial in advanced cancers and results from these trials will be important for further defining the suitability of this agent in cancer therapy.