Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia

Abstract

Hypoxia-inducible factors are transcription factors controlling energy, iron metabolism, erythropoiesis, and development. When these proteins are dysregulated, they contribute to tumorigenesis and cancer progression. However, mutations in genes encoding α subunits of hypoxia-inducible factors (HIF-α) have not previously been identified in any cancer. Here we report two novel somatic gain-of-function mutations in the gene encoding hypoxia-inducible factor 2α (HIF2A) in two patients, one presenting with paraganglioma and the other with paraganglioma and somatostatinoma, both of whom had polycythemia. The two mutations were associated with increased HIF-2α activity and increased protein half-life.

Figure 1

Panel A: Histopathological findings of pheochromocytoma (middle) and paraganglioma (left). Multiple pheochromocytoma masses were well encapsulated, solid, and pink-tan in color. Morphologically, they were composed of large spindle and round cells with prominent nuclei and granular cytoplasm. Some cells had a large irregular and hyperchromatic nucleous with large nucleoli. The tumor cells were arranged, forming nests or growing in a diffuse pattern, and appeared to be embedded in a rich vascularized stroma. Occasionally a brown pigment was identified in the tumor. Immunohistochemistry showed positive staining for chromogranin A and synaptophysin. MiB1 staining was weak, consistent with a low proliferative index. Neuroendocrine somatostatinoma involved the duodenal wall and was composed of round uniform cells with small compact nucleoli and scant cytoplasm. The cells were arranged in nests and cords that infiltrated the lamina propria and superficial muscularis. These cells stained positive for chromogranin A and synaptophysin and somatostatin receptor (right). Panels B, C, and D present evidence of pseudohypoxic signaling and HIF2α mutations in tumor specimens from the patients. Panel B: Quantitative mRNA expression measurement of four hypoxia related genes - EDN1, EPO, GLUT1, and VEGFA - performed by qPCR assay. Panel C: Genomic DNA sequencing of nucleotides 1580 to 1597 in exon 12 of HIF2α. Heterozygous G1588A (patient 1) and C1589T (patient 2) mutations were identified. A similar mutation was not found in germline DNA obtained from the peripheral blood. Panel D: Alignment of amino acid sequences of HIF2α residues 524 to 542 (human HIF2α nomenclature) in human, mouse, rat, and Xenopus laevis, as well as the same conserved domain in human HIF1α and HIF3α.

Figure 2

Functional characterization of the Ala530 mutation in the HIF2α protein. Panel A: Reduced hydroxylation of peptides encoding HIF2α mutant sequences demonstrated through matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Biotinylated HIF2α peptides were incubated with recombinant EGLN1 and purified through streptavidin magnetic beads. Native peptides (downward pointing arrows) incubated with EGLN1 (labeled +) were hydroxylated (downward pointing arrowhead), and this was recorded as an increase in the molecular mass of the peptide. Panel B: Binding assay demonstrating the reduced association of the VHL protein with mutant HIF2α peptides when compared with a peptide encoding the wild type HIF2α sequence. Input represents 10% of the total amount of [³⁵S]-labeled recombinant VHL protein. VHL binding to the HIF2α peptides was analyzed by autoradiography. Panel C: Ubiquitination of the HIF2α mutant protein. The binding of VHL and HIF2α ubiquitination were analyzed by immunoprecipitation assay. After incubating the various His-tagged HIF2α proteins with VHL, comparable immunoprecipitations were performed with an anti HA antibody, as shown in the middle blot. The upper blot shows greater ubiquitination of the wild type HIF2α protein, coinciding with the greater association of the VHL protein shown in the lower blot. Panel D: Enhanced stability of mutant HIF2α proteins evidenced by the higher levels seen on Western blot analysis over time following the addition of cycloheximide (CHX). Panel E: Quantification and half-life of the HIF2α mutants based on data in panel D.