A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas

Abstract

Pheochromocytomas are neural crest-derived tumors that arise from inherited or sporadic mutations in at least six independent genes. The proteins encoded by these multiple genes regulate distinct functions. We show here a functional link between tumors with VHL mutations and those with disruption of the genes encoding for succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD). A transcription profile of reduced oxidoreductase is detected in all three of these tumor types, together with an angiogenesis/hypoxia profile typical of VHL dysfunction. The oxidoreductase defect, not previously detected in VHL-null tumors, is explained by suppression of the SDHB protein, a component of mitochondrial complex II. The decrease in SDHB is also noted in tumors with SDHD mutations. Gain-of-function and loss-of-function analyses show that the link between hypoxia signals (via VHL) and mitochondrial signals (via SDH) is mediated by HIF1alpha. These findings explain the shared features of pheochromocytomas with VHL and SDH mutations and suggest an additional mechanism for increased HIF1alpha activity in tumors.

Figure 1. Unsupervised Analysis of Pheochromocytomas Links Tumors with VHL and SDHB or SDHD Mutations

Unsupervised hierarchical clustering identifies two major clusters in pheochromocytomas: Cluster 1 contains VHL (V), SDHD (D), and SDHB (B) tumors; Cluster 2 contains MEN2 (M) and NF1 (N) pheochromocytomas. Multiple tumors from seven independent unclassified families with recurrent pheochromocytoma (numbered 1–7) and also sporadic tumors (S) are distributed between the two clusters. Letters or numbers on the first row indicate the various tumor classes, as described above. The second row identifies tumor location as adrenal (A) or extra-adrenal (E). Mutations were later detected in samples marked with an asterisk, guided by cluster distribution (see text and Table 1 for details).

Figure 2. Similarity between VHL and SDH Tumors from Cluster 1 by Supervised Learning Methods

Supervised analysis reveals an extensive overlap between genes that discriminate MEN2 from VHL (left) and MEN2 from SDH tumors (right). Samples are shown in columns and genes are represented in rows. Expression levels are normalized for each gene, where the mean is zero. Red indicates high-level expression and blue, low-level expression. The color scale at the bottom indicates relative expression and standard deviations from the mean. Some representative genes are displayed in a color-coded manner according to their functional classes (green, kinase receptor signaling and adrenergic metabolism; pink, oxidative response; blue, hypoxia-responsive/angiogenesis genes). Within each of these functional classes the order of gene appearance in the heat map has been maintained for each class comparison. Complete gene lists are available as Dataset S4.

Figure 3. Low Expression of SDHB Is a General Feature of Cluster 1 Tumors

(A) Expression of SDHB protein in pheochromocytomas with SDHB or SDHD mutations. Western blot analysis of SDHB of whole cell lysates from primary tumors was performed as described in Methods. Lane 1 is normal adrenal medulla used as control and lanes 2–6 are tumors 140, 158, 136, 58, and 220, respectively, from Figure 1 and Table 1. β-actin was used as a loading control. (B) SDHB expression segregates with cluster membership. Cluster 2 tumors, comprising MEN2, NF1, and other sporadic tumors, are shown in lanes 2–4 (tumors 105, 91, and 196, respectively, from Figure 1). Cluster 1 contains tumors with VHL and SDHB mutations and a subset of sporadic samples (lanes 5–7 are tumors 16, 85, 101, and 152, respectively, from Figure 1). Lane 1 is normal adrenal medulla. β-actin was used as a loading control. (C) Immunostaining of SDHB protein in pheochromocytomas or paragangliomas with various genetic backgrounds. A MEN2-related pheochromocytoma is shown on the top row, followed by tumors with mutations in NF1, SDHB, SDHD, and VHL genes. Corresponding hematoxylin/eosin staining is shown on the left.

Figure 4. HIF1α Attenuates SDHB Levels

(A) HIF1α expression was induced by treatment of mouse pheochromocytoma MPC 9/3L cells with 150 μM cobalt chloride for the indicated times. SDHB expression decreased in treated cells. Glut1 indicates increased activity of HIF1α, and β-actin was used as a loading control. (B) Transient expression in HEK293 cells of a HIF1α double mutant PA (P402A/P564A) that is resistant to VHL-mediated degradation reduced expression of SDHB. (C) A2058 cell lines stably expressing HIF1α shRNA do not show change in SDHB after cobalt chloride exposure, while SDHB is downregulated in control GFP shRNA cells treated with cobalt chloride. (D) Proposed model of HIF1α and SDHB interregulation. HIF1α downregulates SDHB, which leads to complex II dysfunction. High succinate levels resulting from loss of complex II, in turn, inhibit prolyl hydroxylase (PHD) activity [19]. Non-hydroxylated HIF1α is resistant to VHL-mediated targeting for degradation and can therefore activate downstream genes, such as angiogenic factors. “E3 complex” indicates the E3 ubiquitin ligase complex for which VHL is the substrate recognition factor.