Cancer Biology Course - nhri.org.t · p53 was identified in 1979 by Lionel Crawford, ... Figure 9.10 The Biology of Cancer ... Allows a cell to self-degrade in order for the body

Cancer Biology Course

Tumor suppressor and Apoptosis:

Master Guardian and Executioner

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

徐欣伶 (Hsin-Ling Hsu)

[email protected]

國家衛生研究院

03/31/2016

Cancer Pathogenesis Bozcuk et al., Breast Cancer Res Treat. 2001. 68(3):239-48.

• 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

Cell cycle progression

Differentiation

Apoptosis

Senescence

Genomic instability

Tumorigenesis

Oncogene

(proliferation) Tumor suppressor

(prevention)

Oncogene

(proliferation)

Cell growth

Differentiation

Apoptosis

Senescence

Genomic instability

Tumorigenesis

Tumor suppressor

(prevention)

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 1984.

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.

Discovery of p53

Figure 9.1 The Biology of Cancer (© Garland Science 2007)

SV40-transformed cells express large T antigen in nuclei

SV40 transformed

human mammary

epithelial cells

(MECs)

T-antigen Ab staining

Linking Ab with

peroxidase enzyme

Tumor xenografts

The transformed

MECs form ducts

normal stroma cells


SV40 transformed 3T3 hamster

cells and F9 mouse embryonal

carcinoma express p53 protein

35S-methionine labeled lysates of 3T3 cells

anti-tumor serum immunoprecipitation (IP)

94 kDa (SV40 large T) and 53 kDa (p53)

35S-methionine labeled lysates of F9 carcinoma cells

anti-tumor serum IP

53 kDa (p53)

• No physical interaction between p94 and p53 proteins.

• Elevation of p53 levels in SV40 transformed cells and

the F9 carcinoma cells.

N, normal hamster serum

T, hamster serum reactive

with SV40-transformed

hamster cells

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


Specialized domains of p53

and the consensus p53-binding DNA sequence

p53-IP assays identify 452 sites in the human genome which binds p53

p53 phosphorylation blocks MDM2

binding and saves p53 from

ubiquitylation and degradation

Immunoprecipitation

of p53-DNA complex


Multiple types of post-translational modifications of p53

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 G1/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

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)

Cell Cycle Volume 7, Issue 13, 2008

release of cytochrome C and

Ca2+ from the mitochondria

to trigger apoptosis

(Mitochondrial

outer membrane

permeabilization)

Ref：Molecular Mechanisms of Cellular Senescence 2013；25-50

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

DNA damage Response


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

The cell cycle is controlled at three checkpoints

p53

p53

p53

The major steps of the G1 & G2 DNA damage checkpoint

Front Genet. 2011; 4: 117.

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

Oncotarget 2014; 5(18):8452-65.

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。

p53 在線粒體(製造能量)扮演重要功能

Chem Biol Interact. 2006, 160: 1-40.

Carcinogenesis effect vs. ROS level

at various stages of carcinogenic process

Ageing Research Reviews, 12: 376 – 390, 2013 Tumor-associated

Macrophage (M2)

Tumor microenvironment (腫瘤微環境) influences

angiogenesis (血管生成) and tumor progression (腫瘤增生)

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):34342-54

http://www.ncbi.nlm.nih.gov/pubmed/19833726





+

-

+

+

Figure 9.6a The Biology of Cancer (© Garland Science 2007)

Natures of gene mutations in different tumor suppressors

Figure 9.6b The Biology of Cancer (© Garland Science 2007)

The locations and

frequencies of p53

mutations

More than 15,000 mutant

alleles have been identified in

human tumors, most of which

are point mutations..

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.

http://en.wikipedia.org/wiki/Codon


Frequency of mutant p53 alleles in human tumor cell genomes

17,689 somatic mutations

and 225 germ-line mutations

IARC release data, 2002


Accumulation of p53 in p53-mutant epithelial cells

of ovarian carcinoma

(ovarian surface epithelial)

(abnormality of development cells, early neoplastic process)


Familiar cancer susceptibility due to mutant p53 germ-line alleles

The pedigree of family suffering Li-Fraumeni syndrome

Breast cancer

Sarcoma

Lung cancer

Leukemia

Pancreatic

carcinoma

Wilms tumor (kidney) Glioblastoma (brain)

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

* cancer of the connective

tissue (bone, cartilage, fat)


Functional p53 suppresses Ras-induced rat embryo

fibroblast transformation

In vitro cell-based study

Ras is an oncogene p53 is a tumor suppressor


Induction of p53 and p21

following DNA damage


Thymocytes of p53+/+ mice are more

sensitive to X-irradiation than p53+/-

heterozygous and p53 -/- homozygous

X-irradiation

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

Loss of viability is attributable to apoptosis

CDK inhibition

block cell proliferation


Loss-of-function p53 in mice

No embryonic lethality

Increase mortality in early life, causing by sarcomas and leukemias

in the p53-/- homozygous mutant

In vivo animal study

Wild-type

Heterozygous

Homozygous

sarcomas & leukemias

Figure 9.7a The Biology of Cancer (© Garland Science 2007)

Tetramerization domain of p53

Figure 9.7b The Biology of Cancer (© Garland Science 2007)

Mechanism of p53 dominant-negative mutations

15/16 are mutants

1/16 is wild-type

Negative dominant effect


• 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

Negative-feedback regulation between MDM2 and p53

MDM2, antagonist of p53

一物剋一物

Figure 9.14 and 9.15 The Biology of Cancer (© Garland Science 2007)

Control of apoptosis by ARF

The gene encoding p14/p19 protein uses

an alternative transcription promoter

Protect p53

from degradation

Figure 9.15b and 15c The Biology of Cancer (© Garland Science 2007)

Control of apoptosis by ARF

Oncogenic signals

mediated

apoptosis

Elevate ARF levels

Increase p53 amounts

Crossing ARF mutant mice with Em-myc mice

• impair ARF

• lose Myc-induced apoptosis

• drive fetal tumors

• decrease animal survival rate

Cell death


Control of p53 levels by

various kinases

Chk2

ATM

ATR

Phosphorylation

of p53 blocks

MDM2 binding

AKT/PKB

Phosphorylation

of MDM2

enhances

binding to p53

p53 ubiquitylation and

proteosome-mediated

destruction

Positively regulates p53

Negatively regulates p53

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.

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

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


Morphological changes of apoptotic cells

Apoptotic bodies


Florescence-activated cell sorting (FACS)

analyzes E2F1-mediated induction of apoptosis

((Tamoxifen)

Measure the size

of individual cell

Apoptosis

Apoptotic effects • Smaller in cell size than

those normal healthy cells

• Loss of DNA contents

• Sub G1 populations

Estrogen

receptor (ER)

and E2F1

fusion protein

Figure 9.18a-f The Biology of Cancer (© Garland Science 2007)

Diverse manifestations of the apoptotic program Scanning electron microscopy

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


Anoikis triggered by loss of anchorage to extracellular matrix

Immortalized but non-tumorigenic

human mammary epithelial cells grow

in an anchorage-dependent fashion

Integrin

DNA

Caspase 3

Anoikis ( 凋亡 ): programmed cell

death which is induced by anchorage-

dependent 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.

Aberrant acinar formation induced by overexpressing MCT-1

in MCF-10A cells

E F

G H

A B

C D

control MCT-1

DNA EGFR DNA EGFR

Acinar formation at day 10

Round

Organized nuclei

Robust cell - cell adhesion

- Grape like

Disorganized nuclei

Poor cell - cell adhesion In vitro mammary tumorigenesis assay

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.

Apoptosis and development

Apoptosis and morphogenesis

The webs tissue between the future fingers

of a embryonic mouse paw

Incomplete differentiation in two toes

(syndactyly) due to lack of apoptosis


Apoptosis is

preferentially

labeled with

TUNEL assay

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.

Figure 9.31c The Biology of Cancer (© Garland Science 2007)

FasL (Fas receptor ligand) is used by cytotoxic T lymphocyte to kill cancer cells

Cytotoxic T lymphocyte

Apoptotic cancer cells

(as evidenced by numerous

blebs on its surface)

Colorized

scanning

electron

micrograph

Death-receptor pathway

Figure 9.23a-b The Biology of Cancer (© Garland Science 2007)

Mitochondria in human liver cell

Function of mitochondria

• Oxidative phosphorylation

• Biosynthesis of metabolites

• Power producers


Cytochrome C detected by a specific fluorescence-labeled Ab (green)

counter-stained with nuclei (red)

Cytochrome C coincides with

mitochondria in cytoplam

(A)

(B-D)

Apoptosis:

• Cytochrome C release to

cytoplam

• Nuclear fragmentation

During apoptosis, cytochrome C releases from the space of

outermost mitochondria membrane and the inner membrane

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

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 multi-

protein 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


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


The apoptotic caspase cascade

Apoptosome

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Cancer Biology Course - nhri.org.t · p53 was identified in 1979 by Lionel Crawford, ... Figure 9.10 The Biology of Cancer ... Allows a cell to self-degrade in order for the body