Tumor Suppressor Genes Robert A. Weinberg The Biology of Cancer First Edition Chapter 7: Tumor Suppressor Genes Copyright © Garland Science 2007

II. Neurofibromatosis Malignant Benign Small, subcutaneous nodules in the back of the patients Nodules in iris of the eye Café au lait spots Malignant Benign

Neurofibromin (NF1) Haploinsufficiency Other examples 1) Smad4 2) p27 Kip1 3) PTEN 4) p19 ARF Figure 7.21 The Biology of Cancer (© Garland Science 2007)

III. Familial adenomatous polyporis Adenomatous polyporis coli Figure 7.22 The Biology of Cancer (© Garland Science 2007)

Figure 7.24a The Biology of Cancer (© Garland Science 2007)

The formation of stem cell compartment near the bottom of the crypts Staining marker : Ki67 Figure 7.24b The Biology of Cancer (© Garland Science 2007)

Apc, b-catenin, and Tcf/Lef primary structure of APC

III. von Hippel-Lindau (VHL) Disease Hereditary cancer syndrome (100 years ago) retinal angioma : Eugene von Hippel blood vessel tumors of the retina von Hippel, E. Graefe. Arch. Ophthalmol. 59, 83-106 (1904) haemangioblastoma : Arvind Lindau blood vessel tumors of the brain and spinal cord Lindau, A. Acta. Ophthalmol. 4, 193-226 (1927) renal cell carcinoma kidney cancer 2. VHL Disease 1/35,000 relatively rare

IV. Von Hippel-Lindau Syndrome Retinal angioma hemangioblastoma Figure 7.28b The Biology of Cancer (© Garland Science 2007)

von Hippel-Lindau (VHL) Disease 3. VHL gene and product - locus : chromosome 3p25 (1993) - gene product : pVHL - 3 exons, 4.5 kb mRNA - proteins : pVHL30, pVHL19 (1996) - internal translational initiation : different location - a and b domain - no homology with other proteins - knockout : embryonically lethal 4. Tumor suppressor

Ubiquitylation and proteasomes in VHL Figure 7.26a The Biology of Cancer (© Garland Science 2007)

Ubiquitin and proteasomes Regulatory subunits Catalytic subunits Regulatory subunits Figure 7.26b The Biology of Cancer (© Garland Science 2007)

Table 7.3 The Biology of Cancer (© Garland Science 2007)

HIF-1 and its regualtion by pVHL Figure 7.28a The Biology of Cancer (© Garland Science 2007)

Molecular details of the HIF-1a –pVHL interaction Figure 7.29a The Biology of Cancer (© Garland Science 2007)

Molecular details of the HIF-1a –pVHL interaction Figure 7.29b The Biology of Cancer (© Garland Science 2007)

How Does VHL Serve As a Tumor Suppressor ? pVHL serves as an ubiquitin ligase complex of HIF-1a Crystal Structure of HIF1a-VCB Complex

Direct HIF-1 Target Genes I. Glucose/Energy Metabolism and Cell Proliferation/Viability Adenylate Kinase 3, Aldolase A, Aldolase C, Enolase 1 (ENO1), Glucose Transporter 1, Glucose Transporter 3, Glyceraldehyde-3-phosphate Dehydrogenase, Hexokinase 1, Hexokinase 2, Insulin-like Growth Factor 2 (IGF-2), IGF Binding Protein 1 (IGFBP-1), IGFBP-3, Lactate Dehydrogenase A, Phosphoglycerate Kinase 1, Pyruvate Kinase M, p21, Transforming Growth Factor b3(TGFb3) II. Erythropoiesis and Iron Metabolism Ceruloplasmin, Erythropoietin, Transferrin, Transferrin Receptor III. Vascular Development/Remodeling a1B-Adrenergic Receptor, Adrenomedullin, Endothelin-1, Heme Oxygenase 1, Nitric Oxide Synthase 2, Plasminogen Activator Inhibitor 1, Vascular Endothelial Growth Factor (VEGF), VEGF Receptor FLT-1

Metabolic Enzymes in Cancer 1. Succinate dehydrogenase (SDH) 2. Fumarase/fumarate dehydratase (FH) - Increasing of succinate - feedback inhibitor of prolyl hydroxylase - HIF stabilization 3. Isocitrate dehydrogenase (IDH) - Decreasing of 2-oxoglutarate - CpG DNA methylation - Histone (H3K9 and H3K27) methylation FIGURE 16-7 Reactions of the citric acid cycle. The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are not the carbons released as CO2 in the first turn. Note that in succinate and fumarate, the two-carbon group derived from acetate can no longer be specifically denoted; because succinate and fumarate are symmetric molecules, C-1 and C-2 are indistinguishable from C-4 and C-3. The number beside each reaction step corresponds to a numbered heading on pages 622–628. The red arrows show where energy is conserved by electron transfer to FAD or NAD+, forming FADH2 or NADH + H+. Steps 1, 3, and 4 are essentially irreversible in the cell; all other steps are reversible. The product of step 5 may be either ATP or GTP, depending on which succinyl-CoA synthetase isozyme is the catalyst.

1. Succinate Dehydrogenase (SDH) and Fumarate Hydratase (FH) mutations in subunits B, C or D of SDH : paraganglioma or phaeochromocytoma mutations in FH : leiomyoma, leiomyosarcoma or renal cell carcinoma

Loss of SDH & FH causes pseudo-hypoxia and HIF induction Mutations in SDH & FH Accumulation of TCA intermediates: succinate & fumarate Competitive inhibitors of a-ketoglutarate for PHD activity

2. Isocitrate Dehydrogenase (IDH) Genomic analysis of human glioblastoma multiforme (GBM) Mutations in the active site of isocitrate dehydrogenase-1 (IDH1): IDH1(R132H) (Parsons et al., Science, 2008) cf: IDH2 mutant (R172H) was found in AML (Ward et al., Cancer Cell, 2010) B. IDH: a-ketoglutarate production IDH1: cytoplasm, NADPH-dependent IDH2: mitochondria, NAD-dependent

C. Oncometabolite: 2-hydroxyglutarate Metabolite Profiling Using LC-MS scanning Produced by IDH mutants (IDH1, IDH2) Dang et al., Nature, 2009

a-ketoglutarate-dependent dioxygenases D. 2-Hydroxyglutamate is a competitive inhibitor of a-ketoglutarate-dependent dioxygenases a-ketoglutarate-dependent dioxygenases Prolyl hydroxylase: Hydroxylation of HIF-1: Reduction of HIF1 Tet1&2 (5-methylcytosine hydroxylase): Hydroxylation of 5-methyl cytosine : Reduction of CpG methylation KDM7A (jhdm1da): demethylation for di-methyl H3-K9 and H3-K27 ( Xu et al., Cancer Cell, 2011)

Expression of the VEGF target of HIF-1 In situ hybridization of VEGF A large area of the necrotic cells are found tin the center of the tumor In situ hybridization of VEGF Figure 7.30 The Biology of Cancer (© Garland Science 2007)