Malignant astrocytic gliomas, especially GBMs, are characterized by poor prognosis and low patient survival rates . Although these tumors rarely metastasize, they almost always recur locally because of their inherent tendency for diffuse infiltration [52, 53]. In particular, a strong induction of angiogenesis marks the transition from lower-grade tumors to more aggressive and lethal GBMs . Therefore, despite advanced clinical approaches with surgery, radiotherapy and chemotherapy [[54–56]], inhibition of angiogenesis might represent a key strategy in the treatments of gliomas.
Recent preclinical data demonstrated that anti-VEGF agents (e.g. ceradinib, bevacizumab) can transiently normalize the elevated permeability and interstitial pressure of brain tumor vessels, enhancing in this way the penetration of concurrently administered drugs [[38–40, 52, 57, 58]]. Besides direct VEGF or VEGFR2 inhibition for glioblastoma, clinical studies are being conducted or planned with agents targeting further downstream or alternative pathways frequently altered in brain tumors, including the mTOR/Akt and EGFR pathways .
Nevertheless, the success with the existing compounds in the management of brain tumors is very limited. It is likely that combination of therapeutic agents targeting different pathways, especially angiogenic pathways, will produce more significant clinical effects. In this context, we focused on leptin, a multifunctional hormone that is able to exert angiogenic activity in different in vitro and in vivo model systems [[6–8, 10, 11, 18, 29–31]].
Leptin has been implicated in neoplastic processes, especially in obesity-related cancers, where the hormone has been shown to stimulate cancer cells growth, survival [[12, 14, 15, 28, 59]], resistance to different chemotherapeutic agents [60, 61] as well as migration, invasion and angiogenesis [[18–21, 29, 62]].
In the central nervous system (CNS) leptin regulates several physiological brain functions, including hippocampal and cortex-dependent learning, memory and cognitive function, neuronal stem cells maintenance, and neuronal and glial development [63, 64]. In addition, recent research suggests the potential role of this hormone in the progression of brain tumors . We previously demonstrated that the expression of leptin and ObR in human brain tumor tissues correlates with the degree of malignancy, and the highest levels of both markers are detected in GBM. Specifically, and in relevance to the present study, leptin and ObR were expressed in over 80% and 70% of 15 GBM tissues analyzed . Other studies demonstrated leptin mRNA expression in rat glioma tissues and cell lines [33, 36]. Because leptin and ObR in human brain tumors are commonly coexpressed, leptin effects are likely to be mediated by autocrine pathways. Using in vitro models, we found that LN18 and LN229 ObR-positive GBM cells respond to leptin with cell growth and induction of the oncogenic pathways of Akt and STAT3, as well as inactivation of the cell cycle suppressor Rb . However, the potential role of intratumoral leptin in glioma progression, especially in the regulation of angiogenesis, has never been addressed. Here we investigated if the hormone can be expressed by human GBM cell cultures, if it can affect angiogenic and mitogenic potential of endothelial cells, and if its action can be inhibited with specific ObR antagonists. The results were compared with that induced by the best-characterized angiogenic regulator, VEGF.
Our data demonstrated that conditioned media produced by both LN18 and LN229 GBM cell lines enhanced HUVEC tube formation and proliferation. These data are in agreement with previous reports showing that GBM cultures express VEGF and other factors that can induce HUVEC angiogenesis [[65–67]].
We found variable levels of leptin and VEGF mRNA in LN18 and LN229 cell lines cultured under SFM conditions. In general, the abundance of VEGF transcripts in both cell lines was significantly greater that that of leptin mRNA. Secreted leptin and VEGF proteins were found in LN18 CM, while in LN229 CM, leptin was undetectable and VEGF was present at low levels. The reason for lack or minimal presence of these proteins in LN229 CM, despite quite prominent expression of the cognate mRNAs, is unclear. It is possible that it is due to limited sensitivity of ELISA assays unable to detect proteins below the minimal threshold level. We speculate that LN229 cells might produce proteins binding VEGF and leptin, thereby converting them into ELISA-unrecognizable complexes. Alternatively, LN229 CM might contain proteases degrading the angiogenic proteins.
In order to clarify if LN18 CM angiogenic and mitogenic effects are, at least in part, related to leptin secreted by these cells, we used specific ObR inhibitor, Aca1. We have previously demonstrated that this antagonist binds ObR in vitro, inhibits leptin-induced signaling at pM-low nM concentrations in different types of cancer cells, including LN18 and LN229 cells, while its derivative Allo-aca is able to reduce the growth of hormone-receptor positive breast cancer xenografts and enhance survival of animals bearing triple-negative breast cancer xenogranfts [37, 68]. Furthermore, All-aca also inhibits leptin activity in some animal models of rheumatoid arthritis . Interestingly, we also detected CNS activity of Aca1, suggesting that the peptide has the ability to pass the blood-brain barrier [[37, 68, 70]].
In the present work, we found that Aca 1 can abrogate leptin-induced tube formation and mitogenesis of HUVEC at 10 and 25 nM concentrations, respectively. Notably, the peptide alone did not affect cell growth and did not modulate the ability of HUVEC to organize into tube-like structures, suggesting that it acts as a competitive antagonist of ObR. Next, we demonstrated that Aca1 at 10-50 nM concentrations was able to antagonize tube formation and growth effects of LN18 CM. The anti-angiogenic effects of 25 and 50 nM Aca1 were comparable to that obtained with 1 μM SU1498, while anti-mitotic activity of 25 and 50 nM Aca1 was comparable to the action of 5 μM SU1498. Furthermore, the combination of low doses of Aca1 (10 nM) and SU1498 (1 or 5 μM) produced greater inhibition of CM effects than that obtained with single antagonists.
Interestingly, Aca1 or SU1498 appeared to differentially affect the morphology of HUVEC cultures. While Aca1 reverted the organized ES phenotype to the initial appearance of dispersed cell culture, SU1498 disrupted ES structures, reduced cell-matrix attachment and induced cell aggregation. This might suggest that the inhibitors affect different cellular mechanism and that leptin and VEGF control HUVEC biology through different pathways.
Taken together, our data indicated that GBM cells are able to induce endothelial cells proliferation and organization in capillary-like structures through, at least in part, leptin- and VEGF-dependent mechanisms. Thus, leptin might contribute to the progression of GBM through the stimulation of new vessel formation. Leptin action can be direct or indirect, through upregulation of VEGF expression. Indeed, we observed that leptin can transiently increase VEGF mRNA levels in GBM cells at 6-8 h of treatment (data not shown). In this context, effective reduction of tube formation and mitogenic activity of endothelial cells by ObR antagonist, especially in the combination with VEGFR2 inhibitor, suggest that targeting both leptin and VEGF pathways might represent a new therapeutic strategy to treat GBM.