Introduction: IGF-1 overexpression has been associated with tumorigenesis. IGF-1Ea, IGF-1Eb and IGF-1Ec isoforms were shown to be regulated differently in cancer. IGF-1Ec and IGF-1Eb overexpression has been positively associated with cell survival and proliferation in various tumors. Elevated IGF1Eb/IGF-1Ea ratio has been suggested as a biomarker of cervical cancer prognosis. Herein, we aimed to examine the expression of IGF-1 isoforms in human adrenocortical carcinomas (ACCs), adrenocortical adenomas (ACAs), pheochromocytomas (Pheo) and compare to normal adrenal gland (NAG). Additionally, we investigated the role of IGF1-Ec and IGF1-Eb peptides in proliferation and migration of adrenal cortex carcinoma cell lines elucidating the underlying mechanism.
Methods: mRNA levels of the isoforms were evaluated by qPCR in fresh frozen tissues (ACA=8, ACC=6, Pheo=11). Immunohistochemical (IHC) analysis (ACA=15, ACC=13, Pheo=8, NAG=5) was performed using IGF-1Ec, IGF-1Eb and IGF-1Ea antibodies. SW−13 and H295R adrenal cortex carcinoma cells were incubated with IGF-1Ec and IGF-1Eb peptides at various concentrations alone or in combination with ERK inhibitor. XTT cell proliferation assay and scratching assay were also performed.
Results: IGF-1Ec, IGF-1Eb, IGF-1Ea mRNA levels were increased in adrenal cortex neoplasms (ACC+ACA) compared to medulla (Pheo). Additionally, the mRNA levels of IGF-1Ec and IGF-1Eb were significantly higher in ACCs as compared to ACAs. These findings were confirmed at protein level, since 100% of ACCs (IRS=6.62) and 73.3% of ACAs (IRS=3.6) expressed IGF-1Ec, 67% of ACCs (IRS=5.57) and 25% of ACAs (IRS=1.25) expressed IGF-1Eb and 58% of ACCs (IRS=4.5) and 66% of ACAs (IRS=3.1) expressed IGF-1Ea. More importantly, protein expression of IGF-1Eb (IRS=6.4) was higher while IGF-Ec expression was attenuated in NAG (IRS=4.42) compared to ACCs. Interestingly, no expression of any isoform was detected in the Pheo group. A borderline negative correlation between IGF-1Eb (P=0.03) levels as well as IGF-1Eb/IGF-1Ea ratio and Ki-67 (P=0.07) was observed in ACC group. Incubation of SW-13 and H295R cells with IGF-1Ec resulted in significantly increased cell proliferation and migration compared to untreated cells, while ERK inhibitor attenuated this effect, indicating the involvement of ERK1/2 pathway.
Conclusion: Higher expression of IGF-1Ec and IGF-1Eb is linked to increased malignant potential of adrenal neoplasms. IGF-1Ec may play a causative role in ACC tumorigenesis acting -at least in part- through activation of ERK1/2 pathway, while higher expression of IGF-Eb may have a protective effect. Further studies are warranted in order to address the possible prognostic role and/or the use of IGf-1Ec and IGF-1Eb as therapeutic targets.
18 - 21 May 2019
European Society of Endocrinology