Cancer Biology Course

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1 Cancer Biology Course Tumor suppressor and Apoptosis: Master Guardian and Executioner 徐欣伶 (Hsin-Ling Hsu) 國家衛生研究院 03/31/2016 本簡報內容部份取材自 Garland Science, Taylor & Francis Group 出版社所出版之 The Biology of Cancer, 僅供本課程教學使用, 除上課目的外, 請勿以任何形式使用全部或部分內容

2 Cancer Pathogenesis Bozcuk et al., Breast Cancer Res Treat (3): Hyper-activation of oncogenes Dysfunction of suppressor genes Loss of cell differentiation Augmentation of proliferate activity Alteration of hormone receptor status Increment of metastatic potential

3 Oncogene (proliferation) Cell cycle progression Differentiation Apoptosis Senescence Genomic instability Tumorigenesis Tumor suppressor (prevention)

4 Cell growth Differentiation Apoptosis Senescence Tumor suppressor (prevention) Oncogene (proliferation) Genomic instability Tumorigenesis

5 Discovery of p53 p53 was identified in 1979 by Lionel Crawford, David P. Lane, Arnold Levine, and Lloyd Old, It had been hypothesized to exist before as the target of the SV40 virus, a strain that induced development of tumors. The TP53 gene from the mouse was first cloned by Peter Chumakov of the Russian Academy of Sciences in 1982, and independently in 1983 by Moshe Oren (Weizmann Institute). The human TP53 gene was cloned in p53 was initially presumed to be an oncogene due to the use of mutated cdna following purification of tumor cell mrna. Its character as a tumor suppressor gene was finally revealed in 1989 by Bert Vogelstein working at Johns Hopkins School of Medicine. p53 is The Guardian Angel of the Genome -is a gene that, when a cell's DNA is damaged, acts as an "emergency brake" to halt the resulting cycle of cell division that can lead to tumor growth and cancer.

6 SV40-transformed cells express large T antigen in nuclei SV40 transformed human mammary epithelial cells (MECs) normal stroma cells Tumor xenografts T-antigen Ab staining Linking Ab with peroxidase enzyme The transformed MECs form ducts Figure 9.1 The Biology of Cancer ( Garland Science 2007)

7 SV40 transformed 3T3 hamster cells and F9 mouse embryonal carcinoma express p53 protein 35 S-methionine labeled lysates of 3T3 cells anti-tumor serum immunoprecipitation (IP) 94 kda (SV40 large T) and 53 kda (p53) 35 S-methionine labeled lysates of F9 carcinoma cells anti-tumor serum IP 53 kda (p53) No physical interaction between p94 and p53 proteins. N, normal hamster serum T, hamster serum reactive with SV40-transformed hamster cells Elevation of p53 levels in SV40 transformed cells and the F9 carcinoma cells. Figure 9.2 The Biology of Cancer ( Garland Science 2007)

8 Table 9.1 The Biology of Cancer ( Garland Science 2007)

9 Specialized domains of p53 and the consensus p53-binding DNA sequence p53 phosphorylation blocks MDM2 binding and saves p53 from ubiquitylation and degradation Immunoprecipitation of p53-dna complex p53-ip assays identify 452 sites in the human genome which binds p53 Figure 9.12 The Biology of Cancer ( Garland Science 2007)

10 Multiple types of post-translational modifications of p53 Figure 9.35 The Biology of Cancer ( Garland Science 2007)

11 Anti-cancer function of p53 The normal p53 works through several mechanisms: --- Activates DNA repair proteins in the response to DNA damage --- Induces growth arrest by holding the cell cycle at the G 1 /S checkpoint --- Allow DNA repair lesions to be fixed before returning to the cell cycle --- Initiates apoptosis, if the DNA damage is irreparable --- Mitotic aberrations and chromosomal missegregation The mutation of p53 can lose its suppressive powers or have the devastating effect of actually promoting abnormal cell growth Discovering a compound that could restore function to a mutant p53 may benefit to the development of anti-cancer drugs

12 標靶 p53 定位於線粒體可能作為癌症治療策略 (Targeting p53 to mitochondria for cancer therapy) (Mitochondrial outer membrane permeabilization) release of cytochrome C and Ca 2+ from the mitochondria to trigger apoptosis Cell Cycle Volume 7, Issue 13, 2008

13 DNA damage Response 細胞內基因損壞時促使 p53 表達及活性提升 p53 休止細胞生長週期 p53 幫助基因修復或啟動基因極度損壞的細胞進入死亡和老化途徑 Ref:Molecular Mechanisms of Cellular Senescence 2013;25-50

14 p53-activating signals and p53 downstream effects Under the cell-physiologic stress p53 accumulation p53 post-translational modification p53 oligomerization induces a number of cellular responses Figure 9.8 The Biology of Cancer ( Garland Science 2007)

15 The cell cycle is controlled at three checkpoints p53 p53 p53

16 The major steps of the G1 & G2 DNA damage checkpoint Front Genet. 2011; 4: 117.

17 p53 在線粒體 ( 製造能量 ) 扮演重要功能細胞試驗 : p53 轉換癌症細胞趨向正常細胞之特性 高表現 p53 降低細胞內過氧化物的生成 提升細胞內過氧化物促使癌細胞的爬行能力和侵襲能力 調節 p53 活性能抑制癌細胞轉移和侵犯能力 動物試驗與臨床證據 : 動物試驗發現,p53 能抑制癌細胞的轉移並降低癌細胞侵襲血管的能力 從癌症患者檢體中發現 ROS 的生成增強癌細胞的侵襲能力 了解 p53 在抑制癌症形成的新角色, 將利於針對 p53 有突變的癌症患者選擇一個最有效的治療策略 Oncotarget 2014; 5(18):

18 Carcinogenesis effect vs. ROS level at various stages of carcinogenic process Chem Biol Interact. 2006, 160: 1-40.

19 Tumor microenvironment ( 腫瘤微環境 ) influences angiogenesis ( 血管生成 ) and tumor progression ( 腫瘤增生 ) Ageing Research Reviews, 12: , 2013 Tumor-associated Macrophage (M2)

20 CCL2/MCP-1 and IL-6 affect tumor progression ( 腫瘤進展 ) and microenvironment ( 腫瘤微環境 ) J Biol Chem. 2009, 284(49):

21 Table 9.2 The Biology of Cancer ( Garland Science 2007)

22 Natures of gene mutations in different tumor suppressors Figure 9.6a The Biology of Cancer ( Garland Science 2007) Missense mutation is a point mutation in which a single nucleotide is changed, resulting in a codon that codes for a different amino acid. This can render the resulting protein nonfunctional. Mutations that change an amino acid to a stop codon are considered nonsense mutations. The locations and frequencies of p53 mutations Figure 9.6b The Biology of Cancer ( Garland Science 2007) More than 15,000 mutant alleles have been identified in human tumors, most of which are point mutations..

23 Frequency of mutant p53 alleles in human tumor cell genomes IARC release data, ,689 somatic mutations and 225 germ-line mutations Figure 9.4 The Biology of Cancer ( Garland Science 2007)

24 Accumulation of p53 in p53-mutant epithelial cells of ovarian carcinoma (abnormality of development cells, early neoplastic process) Figure 9.17 The Biology of Cancer ( Garland Science 2007) (ovarian surface epithelial)

25 Familiar cancer susceptibility due to mutant p53 germ-line alleles Li-Fraumeni Syndrome: 李 - 佛美尼症候群 autosomal dominant hereditary disorder ( 體染色體顯性遺傳性疾病 ) several kinds of cancer are involved cancer often appears at a young age cancer reoccurs throughout the life Breast cancer Lung cancer Pancreatic carcinoma Leukemia Sarcoma * cancer of the connective tissue (bone, cartilage, fat) The pedigree of family suffering Li-Fraumeni syndrome Figure 9.20 The Biology of Cancer ( Garland Science 2007) Wilms tumor (kidney) Glioblastoma (brain)

26 In vitro cell-based study Functional p53 suppresses Ras-induced rat embryo fibroblast transformation Ras is an oncogene p53 is a tumor suppressor Figure 9.3 The Biology of Cancer ( Garland Science 2007)

27 Induction of p53 and p21 following DNA damage X-irradiation Thymocytes of p53 +/+ mice are more sensitive to X-irradiation than p53 +/- heterozygous and p53 -/- homozygous Loss of viability is attributable to apoptosis CDK inhibition block cell proliferation p53 induces p21 expression and p21 acts as a potent CDK inhibitor of cyclin-cdk complex that are active in late G1, S, G2, M and can thereby halt further cell proliferation at any of these phases of the cell cycle Figure 9.9 The Biology of Cancer ( Garland Science 2007) Figure 9.10 The Biology of Cancer ( Garland Science 2007)

28 No embryonic lethality In vivo animal study Loss-of-function p53 in mice Increase mortality in early life, causing by sarcomas and leukemias in the p53 -/- homozygous mutant Wild-type Heterozygous Homozygous sarcomas & leukemias Figure 9.5 The Biology of Cancer ( Garland Science 2007)

29 Tetramerization domain of p53 Mechanism of p53 dominant-negative mutations 1/16 is wild-type 15/16 are mutants Figure 9.7b The Biology of Cancer ( Garland Science 2007) Figure 9.7a The Biology of Cancer ( Garland Science 2007) Negative dominant effect

30 Negative-feedback regulation between MDM2 and p53 MDM2, antagonist of p53 一物剋一物 p53 accumulation induces MDM2 mrna and protein expressions MDM2 binds to p53 molecules and triggers their ubiquitylation and export to the cytoplasm for degradation by proteosomes Figure 9.11 The Biology of Cancer ( Garland Science 2007)

31 Control of apoptosis by ARF Protect p53 from degradation The gene encoding p14/p19 protein uses an alternative transcription promoter Figure 9.14 and 9.15 The Biology of Cancer ( Garland Science 2007)

32 Control of apoptosis by ARF Crossing ARF mutant mice with Em-myc mice Oncogenic signals mediated apoptosis Elevate ARF levels impair ARF lose Myc-induced apoptosis drive fetal tumors decrease animal survival rate Increase p53 amounts Cell death Figure 9.15b and 15c The Biology of Cancer ( Garland Science 2007)

33 Control of p53 levels by various kinases Chk2 ATM ATR Phosphorylation of p53 blocks MDM2 binding Positively regulates p53 AKT/PKB Phosphorylation of MDM2 enhances binding to p53 p53 ubiquitylation and proteosome-mediated destruction Figure 9.13 The Biology of Cancer ( Garland Science 2007) Negatively regulates p53

34 Apoptosis is a cellular response to a cellular "insult" such as UV light, chemical or physical damage or a viral infection. This insult starts a cascade of events which lead to the destruction of the cell. This mechanism is often called "programmed cell death" as it is an innate response of the cell which protects the rest of the organism from a potentially harmful agent.

35 Apoptosis Process Allows a cell to self-degrade in order for the body to eliminate unwanted or dysfunctional cells. The genome of the cell will fracture, the cell will shrink and part of the cell will disintegrate into smaller apoptotic bodies. Unlike necrosis, where the cell dies by swelling and bursting its content in the area, which causes an inflammatory response. The apoptotic cell will be phagocytosed by macrophages before the cell s contents have a chance to leak into the neighborhood to prevent unnecessary inflammatory response. Phagocytosis of apoptotic bodies

36 Alcohol consumption may contribute to the death of liver cells (hepatocytes) by apoptosis ( 細胞凋亡 ) and necrosis ( 壞死 ) through numerous mechanisms. Of these two processes, apoptosis occurs at all stages of alcoholic liver disease whereas necrosis generally is found in advanced stages (i.e., alcoholic hepatitis and cirrhosis). 酒精性肝炎和肝硬化

37 Table 9.3 The Biology of Cancer ( Garland Science 2007)

38 Morphological changes of apoptotic cells Apoptotic bodies

39 Florescence-activated cell sorting (FACS) analyzes E2F1-mediated induction of apoptosis Measure the size of individual cell ((Tamoxifen) Estrogen receptor (ER) and E2F1 fusion protein Apoptotic effects Smaller in cell size than those normal healthy cells Loss of DNA contents Sub G1 populations Apoptosis Figure 9.16 The Biology of Cancer ( Garland Science 2007)

40 Scanning electron microscopy Diverse manifestations of the apoptotic program Chromatin condensation and nuclear collapse DNA laddering Golgi (green) body fragmentation and nuclear PARP cleavage (orange) Phospho-histone 2B (serine 14) stains in apoptotic nuclei Figure 9.18a-f The Biology of Cancer ( Garland Science 2007)

41 Anoikis triggered by loss of anchorage to extracellular matrix Anoikis ( 凋亡 ): programmed cell death which is induced by anchoragedependent cells detaching from the surrounding extracellular matrix (ECM). Usually cells stay close to the tissue to which they belong since the communication between proximal cells as well as between cells and ECM provide essential signals for growth or survival. When cells are detached from the ECM, i.e. there is a loss of normal cell-matrix interactions, they may undergo anoikis. However, metastatic tumor cells may escape from anoikis and invade other organs. Immortalized but non-tumorigenic human mammary epithelial cells grow in an anchorage-dependent fashion DNA Integrin Caspase 3 Figure 9.21 The Biology of Cancer ( Garland Science 2007)

42 Aberrant acinar formation induced by overexpressing MCT-1 in MCF-10A cells control MCT-1 A DNA B EGFR E DNA F EGFR C D G H Round Organized nuclei Robust cell - cell adhesion Acinar formation at day 10 In vitro mammary tumorigenesis assay Grape - like Disorganized nuclei Poor cell - cell adhesion

43 Apoptosis and development Apoptosis is essential to embryonic development and the maintenance of homeostasis in multicellular organisms. In humans, for example, the rate of cell growth and cell death is balanced to maintain the weight of the body. During fetal development, cell death helps sculpt body shape, separating digits and making the right neuronal connections. In the immune system, cell death eliminates B cells and T cells that elicit autoimmune response and selects the most efficient lymphocytes to encounter an antigen in the process of affinity maturation. The webs tissue between the future fingers of a embryonic mouse paw Apoptosis is preferentially labeled with TUNEL assay Apoptosis and morphogenesis Incomplete differentiation in two toes (syndactyly) due to lack of apoptosis Figure 9.19 The Biology of Cancer ( Garland Science 2007)

44 Apoptosis pathways Death-receptor pathway The extrinsic pathway is initiated through the stimulation of the transmembrane death receptors, such as the Fas receptors, located on the cell membrane. Mitochondria pathway The intrinsic pathway is initiated through the release of signal factors by mitochondria within the cell.

45 FasL (Fas receptor ligand) is used by cytotoxic T lymphocyte to kill cancer cells Death-receptor pathway Colorized scanning electron micrograph Cytotoxic T lymphocyte Apoptotic cancer cells (as evidenced by numerous blebs on its surface) Figure 9.31c The Biology of Cancer ( Garland Science 2007)

46 Mitochondria in human liver cell Function of mitochondria Oxidative phosphorylation Biosynthesis of metabolites Power producers Figure 9.23a-b The Biology of Cancer ( Garland Science 2007)

47 Cytochrome C detected by a specific fluorescence-labeled Ab (green) counter-stained with nuclei (red) During apoptosis, cytochrome C releases from the space of outermost mitochondria membrane and the inner membrane (A) Cytochrome C coincides with mitochondria in cytoplam (B-D) Apoptosis: Cytochrome C release to cytoplam Nuclear fragmentation Figure 9.24 The Biology of Cancer ( Garland Science 2007)

48 Important role of mitochondria in apoptosis Directly activated by apoptotic signals such as cell stress, free radical damage or growth factor deprivation Mitochondria contain many pro-apoptotic proteins such as Apoptosis Inducing Factor (AIF), Smac/DIABLO and cytochrome C Release these factors from the mitochondria following the formation of the Permeability Transition pore, or PT pore Pores are formed through the action of the pro-apoptotic members of the bcl- 2 family of proteins

49 Apoptosome assembly Bcl-2 family proteins act as sensors of cellular damage or stress in the cytoplasm Relocate to the surface of the mitochondria Interact between pro- and anti-apoptotic proteins Disrupt the normal function of the anti-apoptotic bcl-2 proteins Form the pores in the mitochondria Release cytochrome C and other pro-apoptotic molecules from the intermembrane space Release Cytochrome C into the cytosol from mitochondria Interact with Apaf-1 protein and then recruits pro-caspase 9 to form a multiprotein complex with cytochrome C and Apaf-1 called the apoptosome Formation of the apoptosome leads to activation of caspase 9 and the induction of apoptosis. Activate the caspase cascade

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51 Figure 9.28 The Biology of Cancer ( Garland Science 2007) The wheel of death (apoptosome) Cytochrome C release from mitochondria into cytoplasm and associates with Apaf-1 Attracts procaspase 9 to the hub of the wheel Cleaves and activates other caspase molecules Triggers the apoptotic cascade

52 The apoptotic caspase cascade Apoptosome Figure 9.29 The Biology of Cancer ( Garland Science 2007)