Cancer,

Oncogenes, Tumor Suppressor Genes

Cancer is formed by tumor cells

Cancer - A group of diseases

Cancer is a disorder that occurs at a

cellular level

Cancer occurs when genetic

alterations result in the unregulated

proliferation of cells

Most cancers derive from a single

abnormal cell.

Scanning electron micrographs reveal the organizational and morphological

differences between normal and transformed 3T3 cells

Loss of contact inhibition by cancer cells in cell culture.

Steps in the process of metastasis

Cell transformation

Physical factors

chemical factors

biological factorsaccumulation of

gene mutation

transformationnormal cell cancer cell

immortalization

invasion

metastasis

the development of cancer is resulted from

interaction between hereditary factors and

environmental factors.

Two general classes of genes play

key roles in cancer induction

1. proto-oncogenes: positive control gene,

gain-of-function mutations, act in a

dominant manner

2. Tumor suppressor genes: negative control

gene, loss-of-function mutations, act in a

recessive manner.

正负调控信号平衡的重要性

细胞正常正调信号

负调信号

抑制增殖，促进分化、

成熟、衰老和凋亡

促进细胞生长增

殖,阻止分化

Section Ⅰ oncogene

What is an oncogene?

an oncogene is any gene that encodes a

protein able to transform cells in culture or

to induce cancer in animals, including v-onc

and c-onc. Most oncogenes are mutant

forms of normal genes (proto-onc) involved

in the control of cell growth or division.

Peyton Rous

(1879-1970)

• 1911，Rockefeller institute

• Suggested that spontaneous

chicken tumor caused by a

virus—Rous sarcoma virus

• 1966， Nobel Prize in

Physiology or Medicine

Discovery of oncogene began from the Rouse sarcoma virus research

Rous sarcoma

Experiment by Rouse

Isolate chicken sarcoma

Grind tissue cells

Centrifuge

Separate supernatant

Filter sterilization

Inoculated it on healthy chickens

Experimental results and conclusions by Rouse

Result: The recipients developed tumors

of the same type as seen in the original

animal

Conclusions: Chicken tumor can be

transmitted between individuals; media

may be filterable virus

Confirm that the retroviral with RNA genome has

led to chicken tumors, which was named the

Rouse sarcoma virus (RSV)

1969，oncogene theory： Carcinogenesis is

caused by the oncogene

1970， the first oncogene src was isolated and

identified from RSV

1971， to determine that

the Src in the 3'end of RSV

genome

Progress after Rouse

Genome structures of two forms of RSV

Str

uct

ure

and g

ene p

roduct

s of

an inte

gra

ted r

etr

oviral

genom

e.

RSV: strong tumorigenesis role, lead to

tumors after 1-2 weeks of animals

infection, strong transformation effects

on cultured cells

slow-transforming retrovirus: weakly

tumorigenesis role, lead to tumors after

several months of animals infection, no

transformation effects on cultured cells

•Two types of RSV：

The resulting problem

What is the origin of Virus oncogene ?

Answer for this question

1976，Bishop

1.Prepare the radioisotope labeled v-src gene probe

nuclease S1

V-src cDNA

Full length cDNA of

wild RSV

Genome RNA of

slow-acting

retrovirus

+

2. Hybridization of v-src gene probe and chicken

cell genomic DNA

Result：In the genome of normal chicken cells, found

the homologous sequences of v-src—c-src, and then to

find that the split gene c-src exsit in from primitive

eukaryote genome to normal human genome.

Conclusion: c-src is inherent normal gene within the

cell genome.v-src is originated from the c-src—

Proposed the concept of proto-oncogene, which

specifically refers to the corresponding sequences in

normal cells, oncogenes are mutant forms of proto-

oncogene that is activated in cancer cells

Experimental results and conclusions by Bishop

The relationship between the v- src and the c-src

proto-oncogene from which it has been derived

Next question

How do virus get proto-oncogene

src from normal cells and then

convert it into a oncogene?

Retroviral life cycle

蹲睛视被砻挛醮扯艋溲茔舂嗤兔驰绌滏述秽腭睫鲺该剧苑唾叫伤排驯嘟竿乃踱袒扒葑矽悖恬獍

病毒癌基因v-src来源于宿主细胞的C-SRC基因

Retroviral Oncogenes (partial list)

Oncogene(v-onc) Prototype Retrovirus

src Rous sarcoma virus

myc Avian myelocytomatosis virus

erb A, erb B Avian erythroblastosis virus

myb Avian myeloblastosis virus

H-ras Harvey rat sarcoma virus

K-ras Kirsten murine sarcoma virus

abl Abelson murine leukemia virus

fes Feline sarcoma virus

sis Simian sarcoma virus

Isolation and identification of c-onc

Tumor retrovirus is only responsible

for less than 20% of human cancers.

Transformation is due to the

abnormal genes (i.e. onc) generated

for a variety of reasons (chemical,

biological and physical)

?

?

Oncogenes

arise in

humans

through two

distinct routes:

viral route and

inherited route

distribute widely from Yeast to Human beings;

Because most proto-oncogenes are basic to animal life,

they have been highly conserved over eons of

evolutionary time and have low expression level;

Whose products participate in cellular growth

controlling pathways; have not carcinogenic function in

normal cells and play important roles in cell growth,

development and differentiation;

When inappropriately activated, these genes

convert into oncogenes and lead to fault in growth

control and contribute to the development of cancer.

Characteristics of proto-oncogenes

Proto-oncogenes

Classification of proto-oncogenes (family)

1. src family src,abl,fgr,fes,yes,fps, tyrosine protein kinase

lck,kek,fym,lyn,tkl

2. ras family H-ras,K-ras,N-ras GTP binding proteins(p21)

3. myc family c-myc,N-myc,L-myc ,fos DNA binding proteins

4. sis family sis PDGF derived protein(p28)

5. myb family myb, myb-ets Transcription factor

Family Oncogen Function

Several families of oncogene

• How do the proto-oncogenes turn into ‘enemies within’?

• What are the functions of oncogene products?

Two questions about oncogene

At least four mechanisms can produce

onc from the corresponding proto-onc:

Point mutation

Chromosomal translocation

Promoter/enhancer insertion

Gene amplification

Point mutation

in a proto-onc that results in a constitutively

active protein product

single base change leads to one code

change, which further lead to one AA

change, which further lead to increase of

oncoprotein activity

Normal H-ras

GGC GTC

Cancer H-ras

Gly Val

e.g. Point mutation in c-H-Ras in bladder cancer: 12th coden (13,61)

Mutant Ras can continuously transduce

growth signal and cause cell transformation。

Transduce

growth signal

Mutant Ras

protein

maintain

active status

This mutation affect the conformation of ras protein and

diminish its activity as a GTPase. The lower activity of

GTPase could result in chronic stimulation of the activity

of ras protein, which can result in a number of effects on

cellular metabolism for ras mediate a wide variety of

signal transduction pathways.

Promoter/enhancer insertion

The strong promoter / enhancer in the

retrovirus LTR can activate some

proto-oncogene and its overexpressing

when retrovirus integrate into cell

chromosome.

Chromosome translocation is that

one part of one chromosome

exchange with another chromosome .

Chromosome translocation can

cause changes in gene structure

and gene expression.

Proto-oncogene can be activated

and overexpression if it is close to

a strong promoter / enhancer.

Chromosomal translocation

Burkitt淋巴瘤是1964年Epstein首先从非洲儿童

Burkitt淋巴组织中分离出EB病毒而命名。病变特

点为肿瘤常发生于颌骨、颅面骨、腹腔器官和中

枢神经系统等。目

前认为本病的发

病与EB病毒感染

密切有关，90%的

病人瘤细胞有异

常核型

Chromosomal translocation in Burkitt’s lymphoma

As a result of a translocation between chromosomes 8

and 14, the c-myc gene is placed adjacent to the gene

for part of the antibody heavy chain (CH), leading to

overproduction of the Myc transcription factor in

lymphocytes and hence their growth into a lymphoma.

慢性粒细胞白血病是一种影响血液及骨髓的恶性肿

瘤，它的特点是产生大量不成熟的白细胞，这些白

细胞在骨髓内聚集，抑制骨髓的正常造血；并且能

够通过血液在全身扩散，导致病人出现贫血、容易

出血、感染及器官浸润等。

慢性粒细胞白血病的病因仍未

明确，但认为费城染色体与该

病密切相关，大约有90至95%

的病人出现费城染色体。

Chromosomal translocation in chronic myeloid leukemia

Philadelphia chromosome

The conversion of the Abl proto-oncogene into an oncogene in patients with CML.

How Bcr-Abl protein causes chronic myelogenous leukemia.

Gene amplification

of a DNA segment including a proto-onc,

so that numerous copies exist, leading to

overproduction of the encoded protein

This anomaly may take either of two forms: the

duplicated DNA may be tandemly organized at a

single site on a chromosome, or it may exist as

small, independent mini-chromosome-like

structures.

DNA amplifications in stained chromosomes take two forms

Four ways in which a proto-onc can be

made overactive to convert it into an onc

The major character of cancer cells is

uncontrolled growth, function of oncoproteins

is to promote cell growth and transformation

Growth factors act on the cell cycle and

mitosis via transmembrane signal

transduction

How do the proto-oncogenesturn into ‘enemies within’?

4. Transcription

factors

Growth gene

3. Intracellular

signaling protein

1. Growth factors

2.Growth factor receptor

FUNCTION OF ONCOGENE PROTEINS

Growth factor

◆ GF: polypeptides, which can bind to its

receptor and promote cell growth, such

as EGF, FGF, PDGF

◆ oncogenes: e.g. sis (P28) ～ B chain of

PDGF, overexpression of sis can induce

cell transformation .

Growth factor receptor

◆ RTK, GPCR

◆ e.g. c-erb B ～ EGFR, can not bind to

GF due to gene mutation, can

continuously promote cell growth and

cell transformation.

Oncogene-encoded defective EGF receptor

吉非替尼—易瑞莎

EGFR=ErbB-1=HER1

Intracellular signal molecules

non-receptor TPK: such as src, abl, fes,

they bind with cytokine-R/GF-R,

phosphorylate downstream protein and

transfer growth signal.

Regulation of Src activity and its activation by

an oncogenic mutation. (a) Domain structure of

c-Src and v-Src. (b) Effect of phosphorylation

on c-Src conformation.

Csk: C-terminal Src kinase

Mec

han

ism

of

au

toin

hib

itio

no

f S

rc

Imatin

ibfo

r th

era

py o

f C

ML

G-protein: Ras family (K-ras, N-ras, H-ras ),

play a central role in signal transduction

Ser/Thr protein kinase: such as raf, mos,

they are downstream the ras and

phosphrylate its downstream protein.

Val→Glu, V600E

Zelboraf (vemurafenib) for advanced melanoma

Transcriptional factor

◆Including: c-myc, c-myb, c-fos, c-jun

◆ they are final member of growth

signal transduction pathway.

◆ immediate-early genes

◆ their protein form transcriptional

factor (homodimer/heterodimer)

The path from growth factors

to cell division leads through

the enzyme cascade that

activates MAPK;

phosphorylation of the

nuclear transcription

factors Jun and Fos; and the

activity of the transcription

factor E2F, which promotes

synthesis of several enzymes

essential for DNA synthesis

Regulation of cell division

by growth factors

Protein products of oncogenes

Oncoproteins

Resemble the normal products of proto-

oncogenes

Devoid of important regulatory elements

Independent on growth factors or other signals

Some anticancer drugsand drug targets in the Ras–MAPK signaling pathway.

Section II

Tumor Suppressor Genes

A kind of negative regulatory gene ,the

normal function of these genes is to suppress

the proliferation-stimulating activities of

cellular oncogenes.

That have a negative, suppressing effect on

tumor creation and thus help to prevent

formation of tumors.

1. Definition of tumor suppressor gene

2. Evidence for tumor suppressor gene

Cell fusion experiment

Single chromosome transfection experiment

two hit hypothesis

Detection of LOH (loss of heterozygosity)

Normal cell

fusion

hybrid cell (normal,but instability)

passage

tumor cell

chromosome 11 loss

Import chromosome 11

Cell normal

tumor cell

Knudson: two hit hypothesis

The genetic mechanisms underlying retinoblastoma

Hereditary versus Sporadic Retinoblastoma

Retinoblastoma(视网膜母细胞瘤）is a juvenile eye

cancer that is caused by a mutation in the Rb gene

located on chromosome 13 of humans.

3. Cells proliferate in the of cell cycle

cyclin dependent PK (CDK): 1~6, activated by cyclin, inhibited by CKI,

cyclins: activate CDK, A, B, C , D, E, etc.

different cyclin-CDK complex regulate different phase of cell cycle

CDK inhibitor protein (CKI): inhibit CDK, p15, p16. p21, p25, p27, etc.

Cell proliferation in cell cycle way

3 types of proteins regulate cell cycle directly

The control of the cell cycle

Whether cell

cycle can start and

run depends on two

critical check

points:

The former is more

important. If cell can

pass G1/S, the cell

can divide

G1/S and G2/M

Cyclin-Cdk complexes of the cell-cyclec ontrol system

Variations in the activities of specific CDKs

during the cell cycle in animals

The inhibition of a cyclin-Cdk complex by a CKl

4. Tumor suppressor gene p53 and Rb function

to inhibit cell cycle

① Retinoblastoma susceptibility gene (Rb):

the first identified tumor suppressor gene

forms of RB :

Rb gene locate in 13q14, has 27 extrons, 4.7kb mRA

encode nuclear protein P105

Active unphosphorylated/ hypophosphorylated form

Inactive hyperphosphorylated form

function: control cell cycle via interaction with

transcription factor E2F

RB is a negative regulator of progression in the cell

cycle at the G1 to S transition

PRb functions as a switch for conversion of

mitogenic and antimitogenic signals at the

transcription level in the cell cycle, which is mediated

by phosphorylation status.

The pathway by which Rb controls cell cycle

Figure. Regulation of Rb

and E2F activities in late G1.

Interaction of E2Fs with

nonphosphorylated Rb

protein initially inhibits E2F

activity. When signaling from

mitogens is sustained, the

resulting initiate the

phosphorylation of Rb,

converting some E2F to the

active form, which activate

target genes whose products

are essential for S phase.

In G0/G1, un/hypo-phosphorylated RB binds to a E2F and inactivates E2F.

At the end of G1, RB is phosphorylated, releasing (activating) E2F.

Active E2F stimulates the expression of genes required for the entry into S-phase (DNA replication).

At the end of M, RB is dephosphorylated. Active RB binds E2F and inhibits the expression of genes required for DNA synthesis.

② p53 gene

• p53 gene located in 17p13, is

another gene with tumor

suppressor activities. This

protein contains 393 amino

acid residues and a single

amino acid substitution can

lead to loss of function of the

gene. P53四聚体形式

Three regions of p53 protein:

N-terminus (1-80) transcriptional activation domain. Sites for phosphorylation.

Core section (102-290) which binds to DNA in a sequence specific manner.

C-terminus(319-393) by sequences involved in the oligomerization of the protein. Sites for phosphorylation.

1. p53 activates p21 when damaged DNA emerge, cell cycle stops at G1 phase

2. Inhibiting the activity of helicase

3. DNA replication or repaired with replication factor A.

4. If above processing donesn’t work, p53 will induce cell apoptosis (programmed cell death)

This genetic function of this gene is to prevent cell division of cells with damaged DNA.

How DNA damage arrests the

cell cycle in G1 by P53 protein

Figure. Damage to DNA

activates p53. The outcome

depends on the stage of the

cell cycle. Early in the

cycle, p53 activates a

checkpoint that prevents

further progress until the

damage has been repaired.

If it is too late to exercise

the checkpoint, p53

triggers apoptosis.

When p53 is inactive (loss-of-function), this

regulatory pathway does not function

• The RB gene product is always phosphorylated

(inactive), cells continue to replicate DNA and

divide

• About 50% of human cancers can be

associated with a p53 mutation including

cancers of the bladder, breast, cervix, colon,

lung, liver, prostate, and skin. p53 related

cancers are also more aggressive and have a

higher degree of fatalities.

If p53 gene mutation

Regulation of passage from G1 to S

by phosphorylation of pRb

Properties Oncogene Tumor suppressor gene

Mutational events involved One Two

in cancer

Function of mutation Gain of function Loss of function

(“dominant”) (“recessive”)

Germ-line inheritance No Yes

Somatic mutations Yes Yes

Effect on growth control Activate cell proliferation Negatively regulate

growth promoting genes

Effects of gene transfection Transform partly abnormal Suppress malignant

fibroblasts (e.g., NIH3T3) phenotype in malignant cells

Genetic alterations Point mutations, Deletions,

gene rearrangements point mutations

amplification

Properties of Oncogenes and Tumor Suppressor Genes

Test 3

It is known that norepinephrine, as a ligand for the β3-adrenergic

receptor (one member of the GPCR family) in adipocyte plasma

membranes，up-regulate the expression of the uncoupling protein 1（UCP1）by activating PKA, please explain the molecular mechanism.

Based on this, one experiment is designed to study the effect of

norepinephrine treatment on mouse adipocytes which cultured in vitro

and to observe: whether UCP1 gene expression is up-regulated at

transcription and translation levels, respectively, before and after

treatment. What molecular biology techniques can be used to detect

UCP1 gene expression? Please list the techniques and describe the

experimental principle and technical process, respectively.

NOTE: 1. time mark: 20171221

2. the question must be copied on the answer paper before

you answer.