Tyrosine kinase receptors as molecular targets in pheochromocytomas and paragangliomas

Abstract

Pheochromocytomas and paragangliomas are neuroendocrine tumors shown to be responsive to multitargeted tyrosine kinase inhibitor (TKI) treatment. Despite growing knowledge regarding their genetic basis, the ability to predict behavior in these tumors remains challenging. There is also limited knowledge of their tyrosine kinase receptor expression and whether the clinical response observed to the TKI sunitinib relates only to its anti-angiogenic properties or also due to a direct effect on tumor cells. To answer these questions, an in vitro model of sunitinib treatment of a pheochromocytoma cell line was created. Sunitinib targets (VEGFRs, PDGFRs, and C-KIT), FGFRs, and cell cycle regulatory proteins were investigated in human tissue microarrays. SDHB immunohistochemistry was used as a surrogate marker for the presence of succinate dehydrogenase mutations. The FGFR4 G388R single nucleotide polymorphism was also investigated. Sunitinib treatment in vitro decreases cell proliferation mainly by targeting cell cycle, DNA metabolism, and cell organization genes. FGFR1, -2, and -4, VEGFR2, PDGFRα, and p16 were overexpressed in primary human pheochromocytomas and paragangliomas. Discordant results were observed for VEGFR1, p27, and p21 overexpressed in paragangliomas but underexpressed in pheochromocytomas; PDGFRβ, Rb, and Cyclin D1 overexpressed in paragangliomas only; and FGFR3 overexpressed in pheochromocytomas and underexpressed in paragangliomas. Low expression of C-KIT, p53, and Aurora kinase A and B was observed. Nuclear FGFR2 expression was associated with increased risk of metastasis (odds ratio (OR)=7.61, P=0.008), as was membranous PDGFRα (OR=13.71, P=0.015), membranous VEGFR1 (OR=8.01, P=0.037), nuclear MIB1 (OR=1.26, P=0.008), and cytoplasmic p27 (OR=1.037, P=0.030). FGFR3, VEGFR2, and C-KIT levels were associated with decreased risk of metastasis. We provide new insights into the mechanistic actions of sunitinib in pheochromocytomas and paragangliomas, and support current evidence that multitargeted TKIs might be a suitable treatment alternative for these tumors.

Figure 1. Sunitinib treatment of the mouse pheochromocytoma MPC 4/30 cell line in vitro

A. Cell cycle analyses of MPC 4/30 cells treated with 2.5 and 5 μM of sunitinib show a sigificant inhibition of replication compared with controls receiving 0 μM (* p< 0.05). Values shown represent means derived from 3 different experiments. B. More than 50% of cell cycle proteins >2X down regulated by sunitinib affect the M phase of mitosis. C. The major biological processes affected by sunitinib in MPC 4.30 cells are demonstrated.

Figure 2. Differential expression of RTKs and cell cycle markers between SDHB-intact and SDHB-deficient tumors

SDHB-deficient tumors (n=46) are considered to be SDH-related. Only significant results (p <0.05) are shown, along with representative tissue microarray spots. Score refers to the overall staining score (values range: 0–300).

Figure 3. Expression of VEGFRs PDGFRs and FGFRs in pheochromocytomas, paragangliomas and normal adrenal medulla

Representative tissue microarray spots are shown and staining score values ranging from 0–300 are graphed. Pheos – pheochromocytomas; paras – paragangliomas.

Figure 4. Heat Map Summary of Immunohistochemical expression of selected markers in Paragangliomas and Pheochromocytomas

Rows represent the type of tumor and its genetic background. Columns represent the immunohistochemical stains. Green indicates the lowest expression, black indicates intermediate expression, and red indicates the highest expression of the overall immunohistochemistry score given for each tumor sample. Pheo –pheochromocytoma; para – paraganglioma.

Figure 5. Differential expression of cell cycle markers in pheochromocytomas, paragangliomas and normal adrenal medulla

39 pheochromocytomas, 76 paragangliomas, and 35 normal human adult adrenomedullary tissue samples were compared. Unless otherwise specified, the heading “score” refers to the overal staining score (values range 0–300). MIB1 index refers to the percentage of nuclei staining for MIB1. Pheos – pheochromocytomas; paras – paragangliomas.