Biology of Cancer

Sofia Mubarika
Kuliah S2 FK UGM

Biomolecular and cancer

Basic

of molecules of life
How cancer developed
Molecular application in cancer diagnosis
Molecular application in cancer treatment
Cancer biomarkers

Basic molecules of life

DOGMA CENTRAL

DNA

RNA

Protein

Mathematical Biosciences Institute (Ohio State Univ), 2 October 2003

Gen : DNA T-A C-G

Thymine (T)
Cytosine (C)

Adenine (A)
Guanine (G)

Over 3 billion base pairs, 30,000 genes

Humans are over 99.9% identical

Central Dogma of Molecular Biology

DNA
Translation
Transcription

RNA

A gene is expressed in 3 steps:

Protein

1) Transcription: RNA synthesis

2) Splicing: removal of intron sequence from RNA
3) Translation: Protein synthesis

Gene Expression
DNA RNA Protein
RNA Transcription

Protein Translation

How much DNA do we have?

humans have 2 x 23
chromosomes
EACH cells contains 6
billion bases DNA
that is 1 meter of DNA
a human being has
>100.000.000.000.000
cells
that is 100 billion km
of DNA

6 billion bases = 6
Gigabyte
30.000 - 50.000 genes
a lot of junk-DNA
contains no code bus
has a different function

Cause of cancer?

Multiple genetic abnormalities

Internal factors : defect genetic/inherited
External factors :

X RAYS
Environment : Viral infections : HPV, EBV, HBV,
HIV
Carcinogenic compounds :

food, working area,

lifestyle: smoking , alcohol, HIV, HBV, HPV, EBV

Retroviruses

Retroviruses family of RNA viruses that cause cancer

in variety of animals and humans.
The Retrovirus : 3 main genes gag, pol & env required
for virus replication, not play role in cell transformation.
a retrovirus can transform cells from normal to cancer if
they include a specific gene that is capable of inducing
cell transformation this gene is known as Oncogene.

Retrovirus
Cancerous
Retrovirus

Oncogene

Retrovirus oncogene

Two main types of oncogenes:

Viral oncogene: gene from the retrovirus itself
Non-Viral oncogene (Cellular oncogene): genes
derived from the genes of the host cell that are in
an inactive form usually. Occasionally if the gene
incorporates with the viral genome will form a
highly oncogenic virus.
Proto-oncogenes: are the form of cellular genes that
inactive normally but can incorporate with the viral
genome to produce a highly oncogenic virus.

EBV-NPC in Indonesia

Central Java map

Late stage

Most are Javanese, the 1st among men

Hot Spot areas way of life? Salted fish?
Special tobacco ingredients?

EBV-associated tumors
in man
Epithelial tissues

Lymphoid tissues
Burkitts lymphoma, endemic

98%

Oral hairy leukoplakia

Burkitts lymphoma, sporadic

25%

Gastric adenocarcinoma

100%
5-10%

AIDS-immunoblastic lymphoma 60%

-in CNS
100%

Nasopharyngeal carcinoma,
undifferentiated
100%

Post-transplant lymphoma

Salivary gland carcinomas <100%

Hodgkins lymphoma
T-cell lymphomas
--lethal midline granuloma

100%
50%

Muscle tissue

10-30%
>90%

Leiomyosarcoma in
immunosuppressed

100%?

Epstein-Barr virus
Latent Membrane Protein 1 (LMP 1 )
TRAF-1

TRAF-3
TRADD/TANK

p65/p50

- Cell aggregation - Cycline D2

- CD 23
- EGFR
- CD 18, 21,39,40,44 - tumorigenicity
- LFA 1
- CAM (Ca2+ pr
- LFA 3
kinase G1
- ICAM 1
- vimentin?
- MHC Class II

-epithelial cell
differentiation

EBNA 2

Bcl-2

PU1

A20

LMP1

+/cellular genes

N C

LRS

blocks apoptosis

W Y F HQ

M E

K B G D

Jk

EBV latent membrane protein (LMP1)

as an oncoprotein
-Transform rodent cells, loss of contact inhibition
Anchorage-independent growth
-Morphological transformation of human keratinocytes
tumour in nude mice
-Inhibit human epithelial cells differentiation
-Induction of bcl-2 to protect infected B cells from apoptosis
-A20 in epithelial cells
-Essential for B-lymphocyte growth transformation

LMP 1
NF-kB
EGFR

NF-kB
Cox-2
Prostaglandin E2

VEGF
(angiogenesis)

MMP9
(invasiveness
IV collagenase)
IL-8
(angiogenic factor)

So what .. ( from results??)

Clinical application ??
Potential to be developed and further explored
Better diagnostic approach
Non invasive for follow up
Field epidemiology for early screening/ high risk
Molecular epidemiology The profil EBV- NPC
to develop vaccine, immunotherapy (LMP1,
LMP2, EBNAs)
Serology markers.

EBV cycle in vivo

Virus replication in
oropharyngeal epithelium

IgA?
Corresponds
To neuronal
Axon of
neurotropic
Herpesvirus

Infected resting B
cells
Latent reservoir of EBV

Infection of
circulating B cells
Ag?
T cell mediated control of
proliferation
CTL
Proliferation of EBV
activated blasts

Aim:
Develop simple and cheap non-invasive
diagnostic and prognostic methods for NPC
by monitoring EBV related parameters
Serology:
anti-EBV IgA
- Serum/plasma
- Finger prick
- Dried blood spot

- Simple and cheap procedure

(ELISA, Dipstick, filter)
- Screening + Monitoring assay

NA-based diagnostics:
EBV DNA load and/or
EBV RNA profile
- Blood/plasma
- Nasal swab

- Simple sampling/extraction (GuSCN)

- Real-time PCR/NASBA methods
- Confirmation & monitoring

Molecular Diversity of
Epstein-Barr virus IgG
and IgA Antibody
Responses in
Nasopharyngeal Carcinoma:
Jajah Fachiroh1, et al.
J.Infect.Dis 189 (15-06-04)

NPC-STAGE II

NPC-STAGE I
MW (kD)

IgG IgA
56

IgG

8 9 16 21

IgA

8 9 16 21

EA-p138 (138 kD)

106.0
81.0

EBNA1 (72 kD)

TK (65 kD)
DNAse (55+57 kD)

47.5

EA(d) (47-54 kD)

VCA-p40 (40 kD)
ZEBRA (36+38 kD)

35.5
28.2

VCA-p18 (18 kD)

20.8

NPC- STAGE III

IgG

MW (kD) 14 17 34 43 47

106.0
81.0
47.5
35.5

IgA

14 17 34 43 47
EA-p138 (138 kD)
EBNA1 (72 kD)
TK (65 kD)
DNAse (55+57 kD)
EA(d) (47-54 kD)
VCA-p40 (40 kD)
ZEBRA (36+38 kD)

28.2
20.8

Fig.2

VCA-p18 (18 kD)

IgG

NPC-STAGE IV

5 11 19 27 31

IgA

5 11 19 27 31

EBNA1
combination

VCA-p18

control

NPC

0
normal

control

NPC

normal

EBNA1

VCA-p18

combination

Cut-off
value

0.1205

0.2233

0.3536

Sensitivity
(%)

88.49

79.76

85.32

Specificity
(%)

80.13

70.86

90.07

control

NPC

Case: Female caucasian (age 16): T4N1M0

Treatment Chemoradiation + IFN-

> 2 years follow-up

R.Mertens et al. Cancer. (1997);80(5):951-9.

Immunoblot
1

9*
BALF2 (138 kD)
BKRF1 (72 kD)
BXLF1 (68 kD)
BGLF5 (58 kD)
BMRF1 (47/54 kD)
BdRF1 (40 kD)
BZLF1 (36+38 kD)

E L IS A O D 4 5 0 v a lu e

BFRF3 (18 kD)

Date

Days

1: 12-09-2001
2: 03-12-2001
3: 15-03-2002
4: 17-10-2002
5: 16-12-2002
6: 01-05-2003
7: 02-07-2003
8: 05-11-2003
9: 19-11-2003*

1: 0
2: 82
3: 184 CR
4: 400
5: 460
6: 596
7: 658
8: 784
9: 798* Mx

* Lung metastasis (CT/MRI)

EBV IgA ELISA

3,5
3,0

VCA-p18 peptide

2,5

EAd-extract

2,0
1,5
1,0
0,5
0,0

Sample number

EBV-serology predictive
at >6 months pre-metastasis
Note: No increased EBV-DNA in circulation

EBV Polymorphism polytope vaccine

(future)

LMP1, 2 and EBNAs) polytope vaccine

Immunotherapy
Polytope vaccine Decrease the mass tumor of
Non Hodgkin lymphoma - clinical trial phase I
(Denis Moss et al 2002)

The genome is an organisms complete set of DNA.

a bacteria contains about 600,000 DNA base
pairs
human and mouse genomes have some 3 billion.
human genome has 24 distinct chromosomes.
Each chromosome contains many genes.
Gene
basic physical and functional units of heredity.
specific sequences of DNA bases that encode
instructions on how to make proteins.
Proteins
Make up the cellular structure
large, complex molecules made up of smaller
subunits called amino acids.

Proto-Oncogene

Oncogene

The proto-oncogene become oncogene by:

Mutation:

1.

Example: mutation in Ras gene

Continuous
activation of Ras by (constitutively in the GTP-bound
conformation )
Unregulated cell proliferation
Cell transformation.

Proto-Oncogene

Oncogene

2. Abnormal Activity:
Example: Removal of the Regulatory domain in the Raf gene
and replaced by gag gene
Raf kinase domain
consciously active
Cell transformation

Raf Proto-oncogene

Regulatory Domain Protein Kinase Domain

Raf oncogene
gag

Protein Kinase Domain

Proto-Oncogene
3. Gene translocation:
Example: c-myc gene is
translocated from
chromosome 8 to the IgH on
the chromosome 14 resulting
in abnormal c-myc
expression
Cell
transformation

Oncogene

Proto-Oncogene

Oncogene

4. Amplification:
Example: Amplification of n-myc
neuroblastoma. Amplification of erbB-2
Breast & ovarian carcinomas

How does a Proto-oncogene

become an Oncogene?
Proto-Oncogene

1.Mutation

2. Abnormal
Activity

Oncogene

3.Gene Translocation 4. Amplification

Abnormal Activity

Functions of oncogene
1.

2.
3.

4.

Growth Factor (example, Epithelium growth

factor EGF , and platelet derived growth factor
PDGF)
Growth Factor Receptor (Example; PDGFR)
Signal transudation (example; Ras, Raf, &
MEK)
Transcription Factor (example; Jun, Fos, Elk-1
& myc)

Oncogenes

Oncogene causes cancer by affecting:

1.
Cell Proliferation: (example; Ras, Raf, EGF)
2.
Cell differentiation (example, PML/RAR that
inhibits the differentiation of promyelocyte to
granulocyte which will maintain the cell in its
active proliferate state)
3.
Cell Survival (example; Pl-3/AKT which will
activate BCL-2
inhibit Apoptosis &
maintain cell survival.

Tumour Suppressor Genes

Tumour Suppressor genes: are genes that act to

inhibit cell proliferation and tumour
development.
If Tumor Suppresor Gene was
Mutated

OR

Inactivated

It will lead to cell transformation

Hereditary
Mutation

Nonhereditary
Mutation

Function of Tumour Suppressor gene

1.

Antagonize the action of oncogene. (ex.PTEN which

converts PIPIII to PIPII because PIPIII will activate Pl3/AKT which will activate BCL-2 that will inhibit apoptosis
and induce cell transformation)

PIPII

PTEN
PI-3

PIPIII
AKT

BCL-2

Inhibit apoptosis & induce

cell transformation

Function of Tumour Suppressor

gene
2. Transcription factors
Repressor transcription factors: example; WT1 is
a repressor that appears to suppress transcription
factor ( Insulin like growth factor) which will
contribute in the development of tumour.
Activator transcription factors: example; SMAD
family that are activated by TGF-, leading to
inhibition of cell proliferation.

Function of Tumour Suppressor

gene
3. Regulate cell cycle :

Rb gene: that inhibits the cell cycle in the G1

phase
decrease cell proliferation.

INK-4 gene: that produces P16

that
inhibits cdk4/cyclin D action ( to
phosphorylate Rb gene to inactivate its
action)

P53: that produces P21 that has the same

action of P16 in inhibiting the action of
cdk4/cyclin D

Function of Tumour Suppressor

gene
4. Induce apoptosis:

P53 release will

increase Bax
holes in the mitochondria
release
cytochrom c
activate apoptosis

form

Cancer Detection

Cancer detection :
Clinical detection by mammogram,
coloscopy etc
Molecular detection by

Serotype
Restriction

fragment length
polymorphism (RFLP)
PCR
Western Blot

Cancer Treatment

Chemotherapy:

Inhibiting Angiogenesis

Inhibit blood flow/supply to the tumour cells

Decrease franesylation of Ras

Deals with DNA damage, & has affinity to all

proliferating cells not specifying if it was a cancer cell
or not.

Decrease activation of Ras, because Ras mutation

causes most cancers.

Monoclonal Antibody

Biomarkers and Prevention?

Folate

and vit B12 decreased breast Ca

risk (Canc Epid Biomarkers Prev 2006;15(3):443-448)

Genetic variation of Nucleotide excission
repair (NER) and cancer risk ( Canc Epid
Biomarkers Prev 2006;15:536-542)
Lipid

profile :Monosaturates lipid elevated

and low ratio w6/w3 fatty acid decreased
breast ca risk ( Canc Epid Biomarkers Prev 2006;15:416421)

Molecular target in cancer therapy

Anti

tyrosine kinase: Gleevec, Iressa

Anti VEGF
EGFR inhibitor etc

Need to enhance translational research into early

IRT-MTA (Interdiscilinary Research Teams) for
Molecular Target assesment

The same genes The changed diet

Paleolithic era

Modern Times

1.200.000 Generations between

feast en famine

% Energy

100

50

2-3 Generations in energy abundance

% Energy
Low-fat meat
Chicken
Eggs
Fish

Fruit
Vegetables (carrots)
Nuts
Honey

100

50

Grain
Milk/-products
Isolated Carbohydrates
Isolated Fat/Oil
Alcohol
Meat
Chicken
Fish
Fruit
Vegetables
Beans

Nutrigenomics and nutrigenetics:

two sides of a coin

For personalized
nutrition:

effects of diet on
body-metabolism

influence of
genotype on
nutritionally related
diseases

must be considered
Mutch, FASEB 2005

Nutrigenomics
Target Genes
Mechanisms
Pathways

Foods
Nutrition

Molecular Nutrition
& Genomics

Signatures
Profiles
Biomarkers
Nutritional
Systems Biology

Identification of dietary signals

Identification of dietary sensors
Identification of target genes
Reconstruction of signaling pathways

Measurement of stress signatures

Identification of early biomarkers
Large research consortia
Big money

Small research groups

Small budgets
Complexity
04/11/15

45

Problem 1: Nutrition tasty + complex

Indonesia ?? - no change life style

2030 obesity predicted about 50%
04/11/15

50% of those DM

46

Bioactive Components
Cellular
Division
Compound
Activation
DNA
Repair
Apoptosis

Digestion

Bioactive
Components

Cellular
Energetics
Hormonal
Homeostasis
Inflammation

Differentiation

Immunity

Problem 2:
Our gene passports and nutrition
Individual genotype
Functional phenotype
AA
AB
BB

Optimal Nutrition

Lifestyle

Improvement
of Health
Maintenance
Eat right for your genotype??
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04/11/15

Nutritional Systems Biology

Gene

Sample Types:

protein index

metabolite index

Protein

10 ApoE3 mice
10 wildtype mice
liver tissue
plasma
urine

Biostatistics
Biostatistics
Bioinfomatics
Bioinfomatics

Metabolite
9

0 ppm

Targets
Targets
and
and
Biomarkers
Biomarkers

Figure 1. A typical Systems Biology strategy for study of atherosclerosis [1] using
a transgenic ApoE3 Leiden mouse model.

Onset of
disease

Predisposition
Genotype

Surrogate
Biomarkers
Late biomarkers
of disease
Early biomarkers
of disease
Diagnostic
markers
Prognostic
markers

Changes in pathway dynamics

to maintain homeostasis

04/11/15

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