IGF2 role in adrenocortical carcinoma biology

Abstract

Purpose: Clinical outcomes of adrenocortical carcinomas (ACC) could be improved by using novel treatment targets based on the recent advances of tumor biology knowledge. Insulin-like growth factor 2 (IGF2) protein expression is usually 8-80 fold higher in ACC when compared to normal adrenal glands (N-AG) or adrenocortical adenomas (ACA), despite the fact that the biological features of high vs. low IGF2 expressing ACC have not yet been well characterized. Our goal was to understand the IGF2 role in ACC biology by focusing in several cancer hallmarks, including cell proliferation, viability, invasion, and metabolism.

Methods: IGF2 immunohistochemistry expression was evaluated in ACC (n = 13), non-functioning adrenocortical adenoma (ACAn) (n = 14), and N-AG (n = 9). The effects of IGF2 (50, 100 ng/mL) in cell proliferation, viability, invasion, and metabolism, as well as in MAPK/ERK and mTOR pathways activation and N-cadherin expression, were evaluated in the ACC human cell line H295R.

Results: IGF2 expression was increased in ACC compared to ACAn and N-AG. Exposure to 100 ng/mL of IGF2 increased H295R cell proliferation and viability. mTOR inhibition reverted IGF2 triggered cell proliferation and viability while MEK/MAPK/ERK inhibition only reverted IGF2 effects on cell proliferation. IGF2 at a 50 ng/mL concentration increased the glycolytic flux and decreased glutamine consumption.

Conclusions: IGF2 is an excellent marker to differentiate ACC from ACAn. In addition, IGF2 was demonstrated to influence adrenocortical cancer cell proliferation, metabolism, and viability, but not the cell invasion. These data support that different IGF2 concentrations in ACC can be responsible for different biological behaviors of ACC.

Keywords: Adrenocortical carcinomas; Adrenocortical tumors; Hallmarks of cancer; IGF2.

Fig. 1

Immunohistochemistry staining for IGF2 (Scale = 20 µm) in adrenocortical carcinoma (ACC) a, non-functioning adrenocortical adenoma (ACAn) b, and normal adrenal gland (N-AG) c. Graphic representation of the percentage of area staining for IGF2 in the studied groups d and ROC curves with the respective area under the curve (AUC) to compare carcinomas with adenomas e (ANOVA: ***p < 0.001)

Fig. 2

H295R cells viability a and proliferation b after incubation without or with IGF2 at the concentrations of 50 and 100 ng/mL for 24 h (ANOVA: *p < 0.05)

Fig. 3

Relative phospho-ERK expression a and phospho-mTOR expression b, after IGF2 incubation at the concentrations of 50 and 100 ng/mL for 5, 10, and 20 min

Fig. 4

Cell viability a and proliferation b after treatment with IGF2 (100 ng/mL) with and without the MAPK and mTOR inhibitors (100 nM of Rapamycin for mTOR pathway inhibition and 10 nM of PD184352 for MAPK pathway inhibition) (ANOVA: *p < 0.05)

Fig. 5

Matrigel membrane invaded with H295R cells a. N-cadherin expression after 24 h incubation with IGF2 at the 50 and 100 ng/mL concentrations evaluated by western blot b and immunofluorescence c

Fig. 6

Glucose a and glutamine b consumption; pyruvate c, lactate d and alanine e production; lactate/pyruvate ratio f after IGF2 incubation at the concentrations of 50 and 100 ng/mL (ANOVA: *p < 0.05; **p < 0.01; p = 0.06: 0 ng/mL vs 100 ng/mL)