pathobiochemistry of malignant transformation

Doc. MUDr. Zdenek Kleibl , Ph.D. ([email protected])

Inst. of Biochemistry and Experimental Oncology,

First Faculty of Medicine, Charles University Prague

Pathobiochemistry of tumorigenesis

Pathobiochemistry of malignant transformation

Affection of genomic

DNA by various

pathogenetic events

Cancer intro:

Malignant cancer – the genetic disease

Normal tissue

malignant

transformation

MaLigNant tumor formation


DNA by various

pathogenetic events

Cancer intro:



Normal tissue

malignant

transformation

WHICH GENES?

WHICH PROCESSES?

WHICH CELLS?

A few introductory remarks:

- cells in multicellular organism

- known facts about malignant transformation

- cell renewal

Organism Organs Tissues Tissue clusters Cells

Growth control in multicellular organism:

Considered hierarchical building plan

1 102 103 108 1014

[entities]

107 x population



Regulated by hierarchical chemical signalling

Growth factors

Cytokines

Apoptotic regulators

Etc.

Hormones

Local signals



Regulated by hierarchical chemical signalling

Immune system

Surveillance by continuous control

Hormones

Local signals

Self?

Proper?

Controlled?

Hormones

Local signals



Regulated by chemical signalling at the cellular level

Considering the intracellular conditions and

local environment in a tissue cluster

Hormones

Local signals



Regulated by chemical signalling at the cellular level

Considering the intracellular conditions and

local environment in tissue cluster

Cells (not the organism) autonomously regulate the

absolute amount of entities in a tissue clusters

Cellular growth control:

Interactions of local and distant factors

chemical

signalling

intracellular

conditions

local environment

in tissue cluster

Hormones

Local signals

„Immune“ signals

& & Cell integrity

DNA integrity

Energetics

Resources

Space, contacts

Stromal interaction


Represented by biochemical interactions

chemical

signalling

intracellular

conditions

local environment

in tissue cluster

Hormones

Local signals


& & Cell integrity

DNA integrity

Energetics

Resources

Space, contacts

Stromal interaction

signal ligands

receptors

signal transducers

IC signaling

networks

transcription factors

executive proteins

cell membrane

mitochondrion

damage sensors

transducers

DNA repair

telomere

maintenance

ATP production

nutrients

oxygen

integrins

cell-cell interaction

molecules




- cell renewal


Represented by biochemical interactions regulating

chemical

signalling

intracellular

conditions

local environment

in tissue cluster

Hormones

Local signals


& & Cell integrity

DNA integrity

Energetics

Resources

Space, contacts

Stromal interaction

signal ligands

receptors

signal transducers

IC signaling

networks

transcription factors

executive proteins

cell membrane

mitochondrion

damage sensors

transducers

DNA repair

telomere

maintenance

ATP production

nutrients

oxygen

integrins

cell-cell interaction

molecules

Cell

replication

Senescence Differentiation Migration

Cell death


Physiologically - balanced state in tissues

Cell

replication

Senescence Differentiation Migration

Cell death

Tissue homeostasis

Regression Progression

Cellular


Critical events for tissue homeostasis maintenance

Cell

replication Cell death


Cellular



Cell


Mitotic signaling pathways

Cell cycle regulation

Apoptotic pathways

Cellular



Cell



Critical conditions:

- healthy (intact) DNA

- competent immunity system

Cellular



Cell



Critical conditions:

- healthy (intact) DNA

- competent immunity system

DNA repair mechanisms

Cell cycle checkpoints

Differentiation


Pathologically – deterioration of tissue homeostasis

Cell

replication Senescence Differentiation Migration

Cell

death

Tissue homeostasis

Cancer:

Specific deterioration of tissue homeostasis

Cell

replication

Cell death

↑Mitotic signaling pathways

↑Cell cycle regulation

↓Apoptotic pathways

Cancer:


Cell

replication

Cell death

Enabled by:

- Damaged DNA

- Uncompetent immunity system

DNA repair mechanisms

Cell cycle checkpoints

Differentiation

Malignant transformation:


Mitotic hyperstimulation

Cell cycle upregulation

Evasion of apoptosis

Deterioration of DNA repair

Impaired differentiation

Growth of malignant tissue

Insensitivity to local and

systemic growth control

Growth across the tissue

architecture

Immortalization

Toleration of genetic alterations

Clonal variability

Immature phenotype

Metastatic potential

Escape from immune

surveillance




- cell renewal

Cell growth control in tissues:

Majority of tissues have self renewal capacity

Stem

cell

Progenitor

1

Matured

cells 108

[entities in hypothetical tissue]

103

101

Progenitor

2

provided by gradual maturation from tissue stem/progenitor cells



Stem

cell

Progenitor

1

Matured

cells 108


103

101

Progenitor

2


Stimulated by many (?),

mainly local, factors ?

? ?

? ?

?

? ? ? ?



Stem

cell

Progenitor

1

Matured

cells 108


103

101

Progenitor

2


: Supplementation of progenitors -Divisions: unlimited

-Mitotic activity: low

-Phenotype: unmaturated



Stem

cell

Progenitor

1

Matured

cells 108


103

101

Progenitor

2





: Supplement desired cell types -Divisions: limited (100x)

-Mitotic activity: high

-Phenotype: partialy maturated



Stem

cell

Progenitor

1

Matured

cells 108


103

101

Progenitor

2








: Providing specific tissue functions -Divisions: very limited (10x)

-Mitotic activity: variable

-Phenotype: maturated

Origin of cancer cells:

Genetic abberations in stem /progenitor cells

Stem

cell

Progenitor

1

Matured

cells

Progenitor

2

provide propagation of cancer-prone mutations to downstream cells







: Providing specific tissue functions -Divisions: very limited (10x)

-Mitotic activity: variable

-Phenotype: maturated

Cancer stem cells / progenitors

Examples of:

mitotic hyperstimulation

defects of DNA repair mechanisms

evasion of apoptosis

Part 2: Signaling pathways failure in oncogenesis

Signals of cascades pathways for mitotic stimulation

Growth factors cytokines+ lipophilic hormones

Their receptors

Signal transducers

Intracytoplasmic kinases

Nuclear receptors and transcription factors

Cell cycle regulators Gene expression changes

DNA:

Transmission of mitotic signals to the nucleus

Mitotic signal

Signal recognition and its transport

accross cell membrane

Transmission of mitogenic signal from

receptors to intracelular transducers

Amplification and propagation of the signal

toward the cell nucleus

Growth factors

Receptors

Ras Jak

Raf

MEK

ERK

PI3K

PDK1

Akt/PKB

Cytokines

Receptors

Transcription factors

Cell cycle regulators expression

DNA:

STAT Lipophilic hormones receptors

Lipophilic hormones

mTOR

Growth factors cytokines

Their receptors

Signal transducers

Intracytoplasmic kinases

Nuclear receptors and transcription factors

Cell cycle regulators

Signals of cascades pathways for mitotic stimulation

Signaling of receptor tyrosine kinases

GAP

GEF Others P

P P

Grb

Sos Ras

GDP Ras

GTP

GDP GTP

Pi

Farnesyl

transferase

Palm

itoyl

transferase

Ras

Raf

Transcription regulatin

Proliferation

Ras

MAPK cascade

AKT/PKB

Apoptosis

PI3K

GDP GDP

SH2 domain

Defects of receptor tyrosine kinases signaling

P P

P

P Raf

MEK

ERK

Transcription regulatin (eg. Ets-2, Elk-1)

PKC

Proliferation

Apoptosis

P

P P

Grb

Sos Ras

AKT/PKB

PI3K

STEP

MAPKKK:

MAPKK:

MAPK:

GTP

nucleus

Hyperproduction of GF

and/or their receptors

P

Many cancer cells

overexpress production

of growth factors

ErbB-2 (EGFR member)

overexpressed (by gene

amplification) in ~25%

breast cancers

ErbB-2 (her2/neu) could

be targeted by specific Ig

(Herceptin)


P P

P

P Raf

MEK

ERK


PKC

Apoptosis

Ras

AKT/PKB

PI3K

MAPKKK:

MAPKK:

MAPK:

GTP

nucleus

Mutations in transducer

Mutation in k-ras

oncogene: one of the

most frequent event in

solid cancers (~30%).

Mostly pancreatic (~80%)

and lung (~40%) cancers.

Triggering the down-

stream pathways

independent on presence

of upstream stimulatory

events

Proliferation

Ras - directed therapy

GAP

GEF Others P

P P

Grb

Sos Ras

GDP Ras

GTP

GDP GTP

Pi

Farnesyl

transferase

Palm

itoyl

transferase

Ras

Raf

Transcription regulatin

Proliferation

Ras

MAPK cascade

AKT/PKB

Apoptosis

PI3K

GDP GDP

SH2 domain

Inhibitors of transferases

Ras molecule fails to

insert in submembranal

space – defective

signaling


P P

P

P Raf

MEK

ERK


PKC

Proliferation

Apoptosis

Ras

AKT/PKB

PI3K

MAPKKK:

MAPKK:

MAPK:

GDP

nucleus

Mutations in

kinases

Mutation in B-raf

oncogene: melanoma

(~70%), papillary

thyroideal cancer (~50%)

and colorectal (~40%)

cancers.

Specific therapy:

antisense oligos and

specific inhibitors of

kinase activity

PI3K signaling

PI3P

Akt

Bad P

PTEN

Caspase 9 P

FOXO P

CD95L/FasL

IKK mTOR P

P

Regulation of

translation

PDK1

IkB

NFkB Proliferation Apoptosis

P

P P

Grb

Sos Ras

GDP PI3K

[p85/p110]

P P

P

P

O O

OH HO

PI2P Pi

Defects of PI3K signaling

PI3P

Akt

Bad P

PTEN

Caspase 9 P

FOXO P

CD95L/FasL

IKK mTOR P

P

Regulation of

translation

PDK1

IkB

NFkB Proliferation Apoptosis

P

P P

Grb

Sos Ras

GDP PI3K

[p85/p110]

PI2P Pi

Overexpression

in kinase

Mutations /amplification

in kinase

Mutations in

phosphatase

Mutations in PI3K (CA) –

ovarian cancer

Overexpression in AKT

(PKB) – ER negative breast

cancer

Familial PTEN mutations –

Cowden syndrome

Cytokine signaling canonnical pathway

P P

SOCS

P P

Adaptor proteins SH2

domain

P

SOCS

MAPK cascade

P

STA

T

P P

Gene expression

regulation

Jak

Defects of cytokine signaling

P P

SOCS

P P

Adaptor proteins SH2

domain

P

SOCS

MAPK cascade

P

STA

T

P P

Gene expression

regulation

Jak

Mutations /

amplification in kinase

Overexpression

of TFs

Upregulation of

JAK/STAT pathways

– hallmark of many

lymphoproliferative

diseases

Down-regulation / hyper-

methylation of SOCS

Results of aberrant signaling pathways?

Autonomous hyperstimulation of cellular growth potential

Immaturity

Enhanced migration potential

Cell cycle (CC): aims

Cell cycle – the sequence of consecutive biochemical

events leading to cell replication

Highly precise copying and even distribution of genetic material

between the new daughter cells

Creation of two identical daughter cells from mother cell

Cell differentiation in some cases

The aims of CC:

Cell cycle: overview

R

G1

S

M

G2 R

G0 Terminal differentiation

Cell cycle entry.

Preparing to gDNA

replication

Replication of gDNA.

Synthesis of organelles.

Symetrical gDNA and cell

contens. Formation of

doughter cells

S phase completion control.

gDNA reparation.

Preparing for mitosis .

2n 4n

Restriction point.

Divide or die!

Restriction point.

DNA complete or die!

Cell cycle: overview

R

G1

S

M

G2 R

G0 Terminal differentiation

Cell cycle entry.

Preparing to gDNA

replication

Replication of gDNA.

Synthesis of organelles.

Symetrical gDNA and cell

contens. Formation of

doughter cells

S phase completion control.

gDNA reparation.

Preparing for mitosis .

2n 4n

Restriction point.

Divide or die!

Restriction point.

DNA complete or die!

Mitotic

hyperstimulation

Checkpoint

failure

Checkpoint /

DNA repair

failure

Regulation of cell cycle entry

E2F-1 Rb

E2F-1

P P

P P Rb

Cdk4/6

Cyclin D

Cdk2

Cyclin E

Expresion

G1 phase:

R

S phase:

Regulation of cell cycle entry

E2F-1 Rb

E2F-1

P P

P P Rb

Cdk4/6

Cyklin D

Cdk2

Cyklin E

Expresion

p15, p16 p21 p27

TGFβ

SMAD p53

ATM DNA damage

G1 phase:

Contact inhibition

R

S phase:

Perturbances of proper cell cycle entry

E2F-1 Rb

E2F-1

P P

P P Rb

Cdk4/6

Cyklin D

Cdk2

Cyklin E

Expresion

p15, p16 p21 p27

TGFβ

SMAD p53

ATM DNA damage

G1 phase:

Contact inhibition

R

Activation/

amplification of

oncogenes

Inactivation/

mutation of TSG

Inactivation/

mutation of TSG S phase:

Inactivation/

mutation of CKI

Core DNA repair pathways (DSBR)

Ku70

Ku80

MRE11 Rad51

NBS1 Rad17

Rad1

Rad26

Rad9

HUS1 ATM

ATR

ATM BRCA1 BRCA2

Rad51

PARP

PARP

Poly ADP-ribose

Proteins of chromatin


Poly ADP-ribose

XRCC4

LigaseIV

NHEJ (G1/S phase)

Homologous recombination (G2 phase)

Sister chromatide

DNA-PKcs

p53

Cell cycle block

NHEJ (G1/S phase)

Homologous recombination (G2 phase)

Cell cycle block

Deterioration of DNA repair pathways (DSBR)

Ku70

Ku80

MRE11 Rad51

NBS1 Rad17

Rad1

Rad26

Rad9

HUS1 ATM

ATR

ATM BRCA1 BRCA2

Rad51

PARP

PARP Proteins of chromatin


XRCC4

LigázaIV

DNA-PKcs

p53

Hereditary

breast cancer

Li-Fraumeni

syndrome

Ataxia telangi-

ectasia (AT)

AT-like

Nijmegen

breakage

syndrome

Poly ADP-ribose

Poly ADP-ribose

Sister chromatide

Hereditary

breast cancer

Inborn: Hereditary cancer syndromes

Somatic: Genomic instability

Apoptosis pathways: overview

Caspases of proximal

Apoptotic pathway

Executive caspases

Substrates (enzymes, structural proteins, DNA)

Bcl-2 family

Mitochondrion

Death receptors

Init

iati

on

C

on

tro

ll E

ffec

tor

Extrinsic

pathway

Intrinsic

pathway

Caspases of proximal

Apoptotic pathway

APOPTOSIS

APOPTOSIS

Substrates (Lamin, gelsolin, actin, ICAD)

Mitochondrion CD95R

Caspase-8 NFkB

TRAF-2 FADD

Procaspase-9

DISC

CFLAR

Procaspase-3 Caspase-3

APAF1

APAF1

- Cytochrom C

Procaspase-3

CD95L

Caspase-9

Apoptosome Procaspase-8 Bcl-2

Bax

Bid

BIRC5

DIABLO

Apoptosis pathways: players

APOPTOSIS

Substrates (Lamin, gelsolin, actin, ICAD)

Mitochondrion CD95R

Caspase-8 NFkB

TRAF-2 FADD

Procaspase-9

DISC

CFLAR

Procaspase-3 Caspase-3

APAF1

APAF1

- Cytochrom C

Procaspase-3

CD95L

Caspase-9

Apoptosome Procaspase-8 Bcl-2

Bax

Bid

BIRC5

DIABLO

Apoptosis pathways: defects

Inhibition

of caspase

activation

Lost of activation

signaling

Changes in

Bcl-2 members

Signal

switch

Inhibition

of caspase

activation

p53 – the most frequently mutated TSG

p53

mdm2 Ub

p53

Ub

Ub

Ub

Proteasomal degradation

Ub

Ub Ub

TP53 stably expressed under physiological conditions

Low IC concentration of p53 maintained by its targeting for

ubiquitin mediated proteasomal degradation my mdm2 E3 activity

p53 – the most frequently mutated TSG

P P

P

P

P

P

P ATM

CHEK2

p53 mdm2

p21

GADD45

14-3-3

CDC25 Cdk1

Cyclin B

G2 M phase

DNA damage (DSB)

Bax PUMA

Bcl-2

,

p53 – transcriptional regulation of CC & apoptosis following DNA damage


DNA by various

pathogenetic events

Malignant transformation: summary



Normal tissue

malignant

transformation

WHICH GENES?

WHICH PROCESSES?

WHICH CELLS?


DNA by various

pathogenetic events




Normal tissue

WHICH GENES?

WHICH PROCESSES?

WHICH CELLS?

Up-regulation of oncogenes

(genes promoting cell cycle or

inhibiting apoptosis)

Downregulation / silencing of

tumor supressor genes

(genes halting cell cycle or

facilitating apoptosis)

Inactivation of genes coding

for DNA repair proteins main-

taining DNA integrity


DNA by various

pathogenetic events




Normal tissue

WHICH GENES?

WHICH PROCESSES?

WHICH CELLS?

Up-regulation of oncogenes

(genes promoting cell cycle or

inhibiting apoptosis)

Downregulation / silencing of

tumor supressor genes

(genes halting cell cycle or

facilitating apoptosis)

Inactivation of genes coding

for DNA repair proteins main-

taining DNA integrity

WHICH MUTATIONS?


DNA by various

pathogenetic events




Normal tissue

WHICH GENES?

WHICH PROCESSES?

WHICH CELLS?

WHICH MUTATIONS? Drivers

- mutations „driving“ malignant

transformations

Passengers

- mutations that arise from disordered

DNA repair processes that are NOT

involved in tumorigenesis


DNA by various

pathogenetic events



Normal tissue

WHICH GENES?

WHICH PROCESSES?

WHICH CELLS?

Activation of cell cycle

(including the promitotic

pathways)

Evasion of apoptosis

Impairment of DNA repair

mechanisms

Decreased maturation and

senescence



DNA by various

pathogenetic events



Normal tissue

WHICH GENES?

WHICH PROCESSES?

WHICH CELLS? Stem / progenitor cells rather

than matured cells in tissues


Cancer pathways:

Why we should know them?

Classification of tumors

Identification of cancer genes and „driving“

mutations

Improved, optimized treatment

Targeting of tumor cells – not somatic cells

Individualization of therapy based on

molecular cancer profile

pathobiochemistry of malignant transformation

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