Tumor Angiogenesis and Metabolism Cancer Biology Fall 2024 PDF

Tumor Angiogenesis and Metabolism Cancer Biology Fall, 2024 1 Essential changes for the development of cancer Cancer hallmarks 1) Self-sufficiency in growth signals 2) Insensitivity to growth inhi...

Tumor Angiogenesis and Metabolism Cancer Biology Fall, 2024 1 Essential changes for the development of cancer Cancer hallmarks 1) Self-sufficiency in growth signals 2) Insensitivity to growth inhibition 3) Immortalization 4) Evasion of death (apoptosis) 5) Angiogenesis 6) Tissue invasion and metastasis (7) Metabolic reprogramming (8) Evasion of immune system 2 1  Viability of cancer cells and distance to the blood vessel 3 Visualization of hypoxic areas by EF5 staining EF5 O2 [H] EF5-S-Cys-protein adduct Immunostaining Green – capillaries, Red – EF5 localization (hypoxic marker) 4 2  Nutrient supply to tumors: old and current views Old view Current view Initially: the blood supply reached tumors simply because pre-existing blood vessels dilated. Now: angiogenesis is necessary for tumors to grow and spread. 5 Tumor growth requires angiogenesis To find out whether cancer growth can continue without angiogenesis, the behavior of cancer cells in two regions of the eye was compared. Both iris and anterior chamber had nutrients available, but only iris could support angiogenesis. 6 3  Cancer in angiogenesis-deficient mice Id1+/- Id3-/- mice Normal mice Angiogenesis-deficient (adult mice) Inject breast cancer cells Inject breast cancer cells Tumors No tumors 7 Tumor blood vessel growth via upregulation of VEGF W/out new vessels, tumors are small (1-2 mm in diameter) Tumors cause ­ angiogenesis: new blood vessels from pre-existing capillaries New blood supply causes ­ tumor growth 8 4  Angiogenesis: recruitment of endothelial cell precursors from bone marrow Human endothelial markers: Transplant nude mice with Implant mouse PECAM, CD34, VEGFR2 human hematopoietic tumor cells stem cells 9 Mosaic structure of tumor vessels and metastasis Colon cancer Tumor cells within the vessel wall Arrow - tumor cell inside the blood vessel wall (human colon carcinoma) 10 5  Mechanisms of blood vessel formation c d EPS – endothelial progenitor cells, EC – endothelial cells 11 Differences between normal and tumor vasculature Normal tissue Tumor tissue Linear vessels Irregularly shaped vessels Smooth layers of endothelium Many dead-ends Supported by pericytes Too few pericytes - less stable Leaky More thick and rigid matrix 12 6  Hypoxia-induced transcription responses via HIF-1 and HIF-2 pH O2 HIF-1/2a Glycolysis CA IX/XII VEGF GLUT1,3 HIF - hypoxia-inducible factor, CA – carbonic anhydrase, GLUT – glucose transporter, VEGF – vascular endothelial growth factor, HRE - hypoxia-responsive element 13 PHDs and FIH: O2-sensitive regulators of HIF-a via hydroxylation O2 O2 PHD - prolyl hydroxylase domain-containing proteins FIH - factor inhibiting HIF Asn – asparagine, Pro - proline 14 7  Loss of VHL: in ~90% renal clear cell (RCC) carcinoma (the most common form of kidney cancer) VHL gene (chr. 3p) - mutated in von Hippel-Lindau syndrome Incidence: 1:30,000 RCC carcinoma (usually both kidneys) Hemangioblastomas - blood vessel tumors in the brain, spinal cord, and eye Pheochromocytoma (tumor of the adrenal gland) Pancreatic neuroendocrine tumors Most common loss of VHL in spontaneous tumors: chromothripsis combining chr. 3 and 5 (initiating event for this cancer) (occurring during the first 2 decades of life) 15 HIF2a is a key oncogenic target of VHL PAX8 ↑Cyclin D1 Proliferation Mutated VHL ↑↑↑HIF2a Transformation PAX8 ↑MYC Belzutifan (oral drug, FDA-2021) PAX8 - kidney lineage specific transcription factor, heterodimer formation with HIF2a establishes permissible chromatin structure for transcription 16 8  2019 Nobel Prize in Physiology or Medicine William Kaelin Peter Ratcliffe Gregg Semenza Gregg Semenza (Johns Hopkins): identification of HIF-1a William Kaelin (Harvard/Dana-Farber Cancer Institute): hypoxia-like state of VHL mutations, role of HIF hydroxylation Peter Ratcliffe (Oxford University): VHL-dependent HIF degradation, role of HIF hydroxylation 17 Oncogene-driven expression of pro-angiogenic proteins (HER2) VEGF - vascular endothelial growth factor Pro-angiogenic bFGF - basic fibroblast growth factor IL-8 - interleukin 8 Anti-angiogenic TSP - thrombospondin 18 9  Major mechanisms regulating HIF-1a /2a levels 1. O2 availability controls HIF-1/2a degradation: ¯O2 ¯OH-HIF-a ­HIF-a 2. Control via protein synthesis: Growth signaling-dependent: ­signaling, ­HIF-1a translation (oncogene-dependent) 3. ROS-mediated inhibition of PHDs: ¯O2 ­mt-ROS PHDs ¯OH-HIF-a (Cys-SH oxidation) 4. Inhibition of PHDs by oncometabolites (by mutated Krebs cycle enzymes) Oncometabolites PHDs ¯OH-HIF-a 19 Hypoxic gene expression signature in tumors = usually bad prognosis RTX – radiation therapy, LN – lymph node, SCC – squamous cell carcinoma 20 10  Other solutions to tumor hypoxia 1. Spreading along existing blood vessels (glioma/glioblastoma) 2. Invasion into surrounding tissues with normal O2 and nutrients (HIF1-dependent response) 21 Rationale for anti-angiogenic therapy Majority of tumors require angiogenesis for growth acute ® chronic disease Adult tissues: no need for new blood vessels Avoidance of drug resistance 22 11  Inhibitors of angiogenesis: clinical tests Endostatin Avastin Endostatin - natural angiogenesis inhibitor, failed in trials Avastin - anti-VEGF antibody (proven efficacy for colon and renal cancer), side effects: cardiovascular 23 Endothelial cells as a target of the radiation therapy? ASMase-/- Only the bone marrow is different: 1) ASMase-/- mice 2) ASMase+/+ mice IR ASMase+/+ ASMase sphingomyelin ® ceramide ® apoptosis (mouse fibrosarcoma) ASM - acidic sphingomyelinase 24 12  Antitumor effects of radiation: direct + indirect effects Radiation Direct effect Indirect effect Endothelial Tumor cells cells Tumor shrinkage 25 Clinical results with anti-VEGF therapy Approval withdrawn later No tumor dormancy (initial predictions) (no survival benefit) Ineffective alone X and colon cancers, renal) Works in combination with chemotherapy (metastatic lung, breast Initial delay in tumor growth, but not survival benefit (ovarian cancer, glioblastoma) Selection for compensatory metastatic/invasion programs Therapy: MRI anti-VEGF antibody plus irinotecan Before therapy After Spreading and progression therapy in other brain regions 26 13  Can tumor angiogenesis be clinically beneficial? ? Initial shrinkage Improved drug delivery Regrowth of aggressive cancer Better drug response Immune access Less aggressive tumors 27 Anti-VEGF therapy: reducing immunosuppression Tumor-infiltrating CD8+ T cells exhaustion and loss of activity VEGF-R Tumor cells VEGF Therapy (Avastin mAb) 28 14  Cancer hallmark: Metabolic reprogramming 29 Glucose metabolism in normal human cells glycolysis 30 15  Warburg effect: a key metabolic hallmark of cancer Warburg effect: aerobic glycolysis in cancers Glucose 2 ATP Pyruvate acetyl-CoA Krebs cycle O2 36 ATP Lactate mitochondrion Pasteur effect: inhibition of glycolysis by O2 Warburg effect: metabolism of glucose through lactic acid in the presence of O2 (aerobic glycolysis) 31 Toxic lactic acid: price for NAD+ recycling and running aerobic glycolysis Glucose Glucose Pyruvate Lactate metabolism by ↑↑ HIF1 NAD+ NADH NADH NAD+ 32 16  Metabolic addiction of cancer #1: high glucose usage ↓ OxPhos ↑glucose uptake Aerobic glycolysis ↑ PI3K-AKT (Warburg effect) 33 Positron-emission tomography (PET) imaging of lymphoma with radioactive glucose (FDG) (PET-FDG) 18 F-fluorodeoxyglucose (FDG) detection by positron emission PET-FDG performance: 90% specificity and sensitivity for primary/metastatic cancers Limits: Size: 5-6 mm High background in the brain (bladder-excretion) 34 17  Decreased glucose metabolism as a measure of clinical responses PET + CT merged images Sunitinib - 1 month tyrosine kinase inhibitor, broad spectrum, approved drug Loss of PET signals Still CT-detectable abnormalities CT scan: gray PET signal: yellow T - tumor in the liver K - kidney Label excretion B - bladder 35 Warburg effect and cancer aggressiveness Glucose consumption W– Warburg effect P – Pasteur effect MCF-7 MDA-MB-231 Non-invasive Metastatic 36 18  Cancer cells have constitutively active glycolysis - Why? Not hypoxia – cells in culture/leukemic cells in vivo/lung tumors Not favorable energetics – 36 ATP for Krebs cycle vs. 2 ATP for glycolysis Not a better environment – acidification of extracellular environment Extracellular pH 7.0 PH changes overtime pH pO2 6.4 37 Aerobic glycolysis as a growth advantage for cancer cells Competition for resources: GLUT1 – high Vmax, GLUT3 – low Km Adaptation: rapid cycles of hypoxia/normoxia (­ carbonic anhydrases CA9/12) Lactate as another energy source for cancers in low-glucose conditions (uptake of lactate via MCT1, and LDHA converts it into pyruvate) -Bidirectionality of MCT1 transport and LDHA activity(NAD+/NADH-controlled) Immune escape: high acidity ® impaired cytolytic activity of CD8+ T-cells Invasiveness: low pH kills neighbors, more ECM degradation 38 19  Cancers cells are biosynthetically hungry: “fetal” metabolism Biosynthesis: Fatty acids Nucleotides 39 Control of glycolytic flux in cancer: PKM2 isoform PKM2 – pyruvate kinase M2 (slow activity), overexpressed in tumors, fetal tissues and stem cells phosphate transfer from PEP to ADP to generate ATP PEP - phosphoenolpyruvate PPP – pentose phosphate pathways 40 20  Molecular basis of the Warburg effect: HIF-1 and more p53 TIGAR LDHA Mitochondrion AKT: ↑glucose uptake, HK activity to trap glucose PDK1 – kinase, PDH – pyruvate dehydrogenase MYC: ↑glycolytic gene expression HIF1 targets: PDK1, GLUT1/3 LDHA and other glycolytic enzymes 41 Mutated isocitrate dehydrogenase (IDH) in cancers (glioma, AML) Majority of gliomas: mutated IDH1/2 Vorasidenib (FDA approval- 8/2024): IDH inhibitor Classification of gliomas based on IDH status: 1. IDHmut and chromosome 1p/19q codeletion 2. IDHmut (mutated p53) 3. IDHwt/wt IDH a-Ketoglutarate Succinate IDHmut 2-Ketoglutarate (oncometabolite) 42 21  Oncometabolites from mutations in Krebs cycle IDH SDH FH a-Ketoglutarate Succinate Fumarate Malate mut IDHmut SDH FHmut 2-Ketoglutarate ↑↑ Succinate ↑↑ Fumarate Inhibitors of aKG-dependent dioxygenases 1. HIF-hydroxylating PHDs (↑HIFs in mutants) 2. Histone-Lys demethylases (differentiation block in mutants) 3. 5-methylC demethylation by TET enzymes (differentiation block in mutants) IDH - isocitrate dehydrogenase, SDH – citrate dehydrogenase, FH – fumarate hydratase 43 Metabolic addiction of cancer #2: glutamine 1. Glucose (Warburg effect): carbon source 2. Glutamine: nitrogen source (ammonia) + alternative energy source High uptake: cellular Gln for uptake of essential amino acids (leucine) Glutamine anaplerosis: 2 deamination reactions (glutaminase, glutamate dehydrogenase): - nitrogen source for biosynthetic reactions - Glu to α-ketoglutarate: feeding Krebs cycle MYC éUptake Gln Glu α-ketoglutarate Krebs cycle ATP NH4+ NH4+ Leu uptake Gln – the most abundant amino acid in the body (plasma) Anaplerosis – replenishment of Krebs cycle intermediates depleted through their use in biosynthetic reactions 44 22  PET-Gln imaging of brain tumors (glioma) PET-Glucose PET-Gln High background Incomplete detection Dashed white line – previous surgical region Venneti S. Sci. Transl. Med. (2015) 45 Regulation of intracellular pH in hypoxic cells +H2O H+ + HCO3- H2CO3 (bicarbonate) 1- Na+/H+ exchanger 2- MCT (MCT4- hypoxia-inducible) -monocarboxylate transporters (H+/lactate cotransporters) 3- CA IX - Carbonic anhydrase 9 4- Cl-/HCO3- exchangers 46 23  Impaired xenograft tumor growth w/out carbonic anhydrases Control Tumor size (mm3) sh-CA9 sh-CA9 + sh-CA12 Chiche J. et al. Cancer Res. (2009) 47 Metabolism of cancer cells: summary of causes, advantages and disadvantages Hypoxia: HIF-1 D. Hypoxia resistance Amino acids 48 24