IGF2R: A NEW PLAYER IN THE INSULIN LIKE GROWTHFACTOR 2 (IGF2) PATHWAY SUSTAINING ADRENOCORTICAL TUMOURS CELL GROWTH

Adrenocortical tumours (ACT) are a highly heterogeneous class of endocrine cancers, ranging from the common adrenocortical adenoma (ACA) to the rare and aggressive adrenocortical carcinoma (ACC). ACT management is currently relying on the surgical approach; however, the identification of novel therapeutic strategies constitutes an urgent need especially in the management of advanced and metastatic ACC. During the years, several approaches directed at different overexpressed target have constituted the core of clinical and preclinical studies. Of particular interest, the insulin-like growth factor 2 (IGF2), overexpressed in the majority of ACC, is a growth factor responsible for the autocrine proliferative loop that sustains ACC growth. In the IGF2 system, two well addressed tyrosine-kinase receptors, IGF1R and IR, were found responsible for the IGF2 pro-mitotic signal transduction. Unfortunately, even if promising in in vitro settings, the inhibition of these receptors was not successful in prolonging the overall survival of ACC patients in clinical trial. Furthermore, in the IGF2 system is involved another specific IGF2 receptor, IGF2R, which was historically addressed as anti-oncogenic, due to its role in promoting IGF2 lysosomal degradation. However, recent studies in other cancers determined a possible tumorigenic action of IGF2R. Thus, the main aim of this PhD project was to uncover the presence, role, and mechanism of action of the IGF2R in the ACT context. The project was conducted in vitro, using 4 different ACC cell lines, derived from either primary or metastatic tumours, to better reflect ACC heterogeneity. Moreover, data were corroborated using several ACA, uncertain malignant potential tumours (UMP), and ACC primary cultures. Firstly, IGF2R was found overexpressed in ACT compared to normal adrenal tissue (NA), and we confirmed IGF2 overexpression in ACC versus ACA. Then we proceeded to assess whether the role of IGF2R was pro or anti-oncogenic, both on stabilized models and primary cultured cells. Knockdown and inhibition of IGF2R at its IGF2-binding site confirmed a reduction of proliferative activity and coherently, IGF2R transient transfection promoted both proliferation and viability increase. This evidence of a pro-tumorigenic role for IGF2R was a complete novelty in ACT context, thus it needed a supporting mechanism of action. Indeed, we investigated the IGF2-IGF2R binding as well as the possible downstream effectors of IGF2R. The evaluation of the IGF2 impact on IGF2R mitogenic action was conducted in the only cell line, H295R, where IGF2 extracellular levels were affected by IGF2R presence. Indeed, we administer retinoic acid (RA), another IGF2R ligand, known to facilitate IGF2 internalization and stimulate apoptosis. Surprisingly, RA treatment increased IGF2 extracellular levels, and even though this accumulation was potentially able to promote cellular proliferation via IGF1R and IR, we observed a decrease in cellular proliferation. This anti-mitotic effect was confirmed to be mediated by IGF2R and due to the increase of IGF binding proteins (IGFBP2 and 6) induced by RA, that determined the extracellular sequestering of IGF2. We also confirmed the pro-apoptotic effect of RA, even though it was not attributable only to IGF2R. The downstream mechanism of IGF2R was object of study in other cellular models, mainly by the group of El Shewy, who identified two enzymes, the sphingosine kinases (SphKs), and their product S1P, as responsible for the activation of PI-3K/Akt and MAPK pathways. Indeed, we confirmed the overexpression of SphK, mainly the isoform SphK1, in ACT compared to NA. Moreover, this was the first project to correlate IGF2R with SphK in ACT, proving that in case of IGF2R knockdown or inhibition the activity of SphK is hampered. The identification of these novel actors in the ACT pathogenesis opened the possibility to investigate SphKs as therapeutic targets. Indeed, we tested two different inhibitors, safingol and fingolimod. The two drugs were able to impair cell proliferation, viability, and cortisol secretion, both on ACC cell lines and primary cultures. Moreover, we compared their antitumoral activity with the current therapeutic strategy for advanced ACC, the EDP-M scheme, and the combination of safingol or fingolimod with EDP-M resulted additive in controlling cell viability. Overall, in this PhD project a novel pro-oncogenic role of IGF2R in the ACT context was demonstrated. In particular, we proved its connection with IGF2 and most importantly with SphK, able to activate a downstream proliferative cascade sustaining tumorigenesis. Remarkably, we successfully tested two SphK inhibitors in vitro, laying the foundations of new preclinical pharmacological studies.