Clinical and molecular studies have implicated epidermal growth factor receptor (EGFR), insulin-like growth factor (IGF) and target of rapamycin (mTOR) signaling pathways in the regulation of pancreatic neuroendocrine tumor (PanNET) growth. Interpretation and comparison of these studies is complex due to clinical and molecular tumor heterogeneity. We therefore focused in this study on insulinomas, which we examined for mRNA and protein expression of EGFR, IGF and mTOR signaling pathway components by quantitative real-time PCR (n = 48) and immunohistochemistry (n = 86). Findings were compared with normal pancreatic islets and correlated with histopathological data and clinical outcome. Insulinomas showed low EGFR and high IGF2 expression. IGFBP2, IGFBP3 and IGFBP6 mRNA levels were 2- to 4-folds higher than those in islets. High protein expression of IGF2, IGF1R and INSR (in 51–92% of the tumors) and low-to-moderate expression of mTORC1 pathway proteins p-S6k and p-4EBP1 (7–28% of the tumors) were observed. Correlations were found between (1) ERK1 mRNA expression and that of numerous IGF pathway genes, (2) p-ERK and IGF1R protein expression and (3) decrease of IGF pathway components and both metastatic disease and shorter 10-year disease-free survival. In conclusion, our observations suggest that high expression of IGF signaling pathway components is a hallmark of insulinomas, but does not necessarily lead to increased mTOR signaling. Reduced expression of IGF pathway components may be an adverse prognostic factor in insulinomas.
Supplementary Material 1
Supplementary Material 2
Supplementary Table 1 Clinicopathological data of insulinoma patients
Supplementary Table 2 Primer sequences used in quantitative RT-PCR
Supplementary table 3 Antibody characteristics and immunohistochemistry methods
Supplementary table 4 Pearson correlation between mRNA expression levels in insulinomas
Supplementary table 5 Pearson correlation between protein expression levels in insulinomas
Supplementary Table 6 Clinicopathological data of patient with P-NETS other than insulinomas
Supplementary Table 7 Immunohistochemical expression of proteins in the EGFR / MAPK / AKT / IGF and mTOR pathways in different PanNET subtypes, presented as percentage of the samples with a moderately or strongly positive staining intensity in ≥ 10% of the cells.
Supplementary Table 8 Correlation of protein expression levels with grade, tumor size and metastatic disease in PanNETs other than insulinomas
Suppl. Figure 1. Control immunoperoxidase stainings for p-AKT, p-ERK, p-S6K and p4-EBP1.Moderate to strong nuclear and weak cytoplasmic expression of p-AKT (A) and moderate nuclear expression of p-ERK (B) in a lung tumor harboring a K-ras exon 2 mutation. Moderate nuclear p-S6K expression (C) and moderate cytoplasmic p-4EBP1 expression (D) in glandular normal colon cells . A low percentage of cells also shows a moderate to strong nuclear p-4EBP1 staining pattern (D). Original magnifications 200X.
Suppl. Figure 2. Kaplan-Meier analysis showing 10 year overall survival rates of insulinoma patients with regard to mRNA expression (A-D) and clinicopathological parameters (E-H). Correlation between survival and mRNA expression, A: INS (cut off 25th percentile), B: IGF1R (cut off 25th percentile), C: INSR-A (cut off 25th percentile), D: IGFBP3 (cut off 25th percentile), E: Grade (Grade1 versus Grade 2+3), F: Disease (non-metastatic versus metastatic) G: Tumor size (<2 cm versus ≥2 cm) and H: Disease stage (Stage I+IIa versus IV). Dotted lines in plots A-D refer to low expression.
Suppl. Figure 3. Kaplan-Meier analysis showing 10 year overall survival rates of insulinoma patients with regard to protein expression (A-C) and clinicopathological parameters (D-G). Correlation between survival and protein expression, A: IGF2 (moderate (2) vs high (3) expression), B: cytoplasmic IGF1R (low vs high expression), C: cytoplasmic INSR (low vs high expression), D: Grade (Grade1 versus Grade 2+3), E: Disease (non-metastatic versus metastatic), F: Tumor size (<2 cm versus ≥2 cm) and G: Disease stage. Dotted lines in plots A-C refer to low expression.