Analysis of the interaction between the IGF system and HIF: rationale for cotargeting the two systems in breast cancer.

The observation that several RTKs can act as promoters of tumor development and growth has brought a revolution in the study of new targets, in the hope of developing more specific anticancer drugs: the Insulin-like Growth Factors system is among the most studied. The role of this system in the development and progression of a large number of malignancies is well documented. Hence the development of different classes of compounds that can affect this system, in particular directly targeting the type 1 receptor (IGF-1R). Some of these compounds, both monoclonal antibodies (mAbs) against the receptor or small molecules that inhibit the TK activity of IGF-1R, are already undergoing clinical trials, because of their effectiveness in inhibiting the signal. Unfortunately, cancer cells show a high degree of plasticity and redundancy in the proliferative and anti-apoptotic signals, which may contribute to the creation of resistance to specific inhibitors: the identification of these mechanisms, and their overcome, are two key goals in the development of these drugs. Within this study, we encountered a similar problem: our experiments show that the compound in use, NVP-AEW541 (a specific, low molecular weight inhibitor of IGF-1R catalytic activity), is perfectly capable of inhibiting the signal transduced by the receptor following stimulation with IGF-1, but it appears to lose effectiveness following stimulation with IGF-2. Starting from the observation that IGF- 2 seems to be the key factor in this resistance, literature search showed us a growing number of experimental evidences in support of the role of variant A of the insulin receptor (IR-A) in the maintenance of a proliferative signal similar to that classically attributed to IGF-1R: our experiments confirmed the activation of this receptor following exposure to IGF-2, justifying the loss of effectiveness of NVP-AEW541 in regard of the migratory potential of MDA-231 cell line. While the dual inhibition of IGF-1R and IR could overcome this redundancy in the signal, it remains to consider the problems that may occur due to the inhibition of a receptor as essential for cellular metabolism as IR. A different and probably more practicable strategy would be the inhibition of the key player in the dual activation, IGF-2. The one proposed is a strategy that reflects the physiological mechanism of controlling the levels of IGF-2: in fact normal cells are able to control the levels of circulating IGF-2 by binding the ligand to IGF-2R, followed by its subsequent transport to lysosomes for degradation. During this study we then concentrated also on IGF-2R, observing no significative differences between the cell lines in study. Following the ligand-based targeting of the IGF system, which effectiveness seems to be proven by an ongoing phase I clinical trial using MEDI-573, actually the only mAb in clinical testing that exerts its effects by neutralizing not only IGF-2 but also IGF-1, we used a neutralizing antibody against IGF- 2 (MAB292), making it impossible for it to bind both IGF-1R and IR-A. The use of MAB292 proved to be effective both in combination with NVP-AEW541 and alone: MDA-231 cells seem in fact unable to respond to stimulation with IGF-2 and almost completely lose the ability to migrate. This strategy could be particularly effective especially considering that, in hypoxic conditions (pO2 <2.5%), stabilization of HIF-1α leads to an increase in the levels of IGF-2. This increase in fact results in the resistance of MDA-231 to NVP-AEW541, while MAB292 still retain its effectiveness. We also observed the lack of a direct and defined relationship between HIF-1α and IGF-2R levels: cells seemed not able to compensate to IGF-2 increase by increasing also the receptor levels. From this observation it’s therefore evident that another strategy to overcome the resistance to IGF-1R inhibitors may be the combination with HIF-1α inhibitors. At the present days, the fundamental problem is the lack of specific inhibitors: many chemotherapic drugs, in fact, have a complete or partial inhibitory activity of HIF-1α, that is however accompanied by a number of off-target effects. One of these compounds is Topotecan, a Top-1 poison in use in the treatment of ovarian and small cell lung cancer that inhibits HIF-1α translation by a Top1-dependent but DNA damage-independent mechanism, suggesting that its effect as HIF-1α inhibitor could be mechanistically distinguished from those characterizing its cytotoxic activity. In our case the dual inhibition was obtained with success through the use of Topotecan in combination with NVP-AEW541. We also introduced the combination between NVP-AEW541 and Luteolin, a compound of natural origin belonging to the flavonoid class that possess anti-proliferative and anti-migratory activity. Despite the more complex comprehension of its effect on HIF-1, which resulted only slightly decreased, the most interesting feature of this compound resulted to be its anti-migratory potential: the use of Luteolin in fact completely inhibited migration in MDA-231. As a last point in our study, we focused our attention on the metabolic alteration determined by the modulation of insulin/IGF system. Since deregulation of cellular energy metabolism is considered an increasingly important hallmark of cancer, IR and its related metabolic syndromes have become another major focus in the breast cancer research and treatment field. Preliminary analysis of the oxidative and glycolitic profiles of three cell lines showed in first stance an interesting difference at the basal level as well as a modulation of the two processes after treatment with NVP-AEW541 together with MAB292 or with Luteolin. Further experiments would be needed to assess the effective impact of these compounds on cellular metabolism, as well as the metabolic processes and responses in hypoxia. In conclusion we can say that the dual targeting of IGF-1R and HIF-1 appears to be an interesting therapeutic strategy in breast cancer, especially given the close relationship between these two systems.