The IGF pathway is activated in insulinomas but downregulated in metastatic disease

in Endocrine-Related Cancer
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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.

Downloadable materials

  • 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.

 

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    Representative examples of immunohistochemical EGFR, p-AKT, p-ERK, IGF2, IGF1R and INSR protein expression in human tissue: normal pancreas (B, D, G, J and M), a premalignant laryngeal lesion (A) and insulinomas (C, E, F, H, I, K, L, N and O). Strong membranous EGFR expression in a premalignant laryngeal lesion (A), weakly positive expression in normal acinar cells, and no detectable expression in the islets (B) and in insulinomas (C). Moderate-to-strong nuclear p-AKT expression in normal pancreatic islets (D) and moderate-to-strong nuclear and moderate cytoplasmic expression in an insulinoma (E). Strong nuclear p-ERK immunostaining in an insulinoma (F). Strong aggregate like, extracellular IGF2 localization in normal acinar cells, and a moderate, diffuse granular cytoplasmic staining pattern in pancreatic islets (G). Strong, granular cytoplasmic IGF2 expression in a non-metastatic insulinoma and weakly positive, diffuse cytoplasmic expression in a metastatic insulinoma (H and I respectively). Strongly positive, membranous IGF1R expression in normal acinar cells and moderately positive, granular cytoplasmic expression pattern in islet cells (J). In insulinomas a strong, cytoplasmic expression (K) or combined cytoplasmic and membranous IGF1R expression pattern (L) is seen. Moderate-to-strong cytoplasmic, perinuclear INSR expression in normal acinar cells with a granular, cytoplasmic pattern in islet cells (M). Moderate, diffuse granular and strong perinuclear INSR expression (N) or weak to moderate, diffuse expression (O) in insulinomas. Original magnifications 200×.

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    Representative examples of immunohistochemical p-S6K and p-4EBP1 protein expression in normal human pancreatic tissue and insulinomas. Strong, perinuclear expression pattern of p-S6K in normal acinar cells, while the islets cells show a very weak, diffuse cytoplasmic expression (A). Weak cytoplasmic and moderate to strong nuclear expression of p-S6K in insulinoma (B). Moderately to strongly positive diffuse cytoplasmic and nuclear p-4EBP1 immunostaining in normal exocrine pancreas cells at the periphery of a lobule (C). No detectable p-4EBP1 expression in normal pancreatic islet (D). Strong nuclear and weak-to-moderate cytoplasmic p-4EBP1 expression in tumor adjacent exocrine tissue (E). Moderate-to-strong nuclear and weak cytoplasmic p-4EBP1 expression in insulinoma (F).

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    Kaplan–Meier analysis showing 10-year disease-free survival rates of insulinoma patients with regard to mRNA expression (A, B, C and D) and clinicopathological parameters (E, F, G and 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 vs Grade 2 + 3), (F) Disease (non-metastatic vs metastatic), (G) Tumor size (<2 cm vs ≥2 cm) and (H) Disease stage (Stage I + IIa vs IV). Dotted lines in plot (A, B, C and D) refer to low expression.

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    Kaplan–Meier analysis showing 10-year disease-free survival rates of insulinoma patients with regard to protein expression (A, B and C) and clinicopathological parameters (D, E, F and 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 vs Grade 2 + 3), (E) Disease (non-metastatic vs metastatic), (F) Tumor size (<2 cm vs ≥2 cm) and (G) Disease stage.

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