Session SS05 - Cell Death Mechanism

Soo-Youl Kim, Ph.D. Division of Cancer Biology

National Cancer Center, Korea

kimsooyoul@gmail.com

Apoptotic Cell Death : Opportunity of Oncological Application

What is Apoptosis? A Greek word meaning

“falling off” as leaves from a tree

The term apoptosis was first used in a paper by Kerr, Wyllie, and Currie to

describe a morphologically distinct form of cell death.

Br J Cancer 26, 239–57, 1972

Dr. Horvitz found that programmed cell death occurs during the development

of the nematode Caenorhabditis elegans (Horvitz, Cancer Res 59, 1701s–

1706s,1999)

In this organism 1090 somatic cells are generated in the formation of the

adult worm, of which 131 of these cells undergo apoptosis or “programmed

cell death.”

The Nobel Prize in Physiology or Medicine

2002

Prof. H. Robert Horvitz

Apoptosis

(A) Large nuclei and scant cytoplasm / (B) Early during the chromatin condensation phase, arrow is a

fragmented section of nucleus and the arrowhead indicates an apoptotic body/ (C) Extensive plasma

membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic

bodies during a process called “budding”/ (D) These bodies are subsequently phagocytosed by

macrophages, parenchymal cells, or neoplastic cells and degraded within phagolysosomes.

Lymphocytes TEM

B

A C

D

Morphology of Apoptosis

Apoptosis vs Necrosis It is mixed in the tumor tissues.

Intracellular ATP Concentration:

A Switch in the Decision Between

Apoptosis and Necrosis

J Exp Med. 185(8):1481-6, 1997

Apoptosis: Point of No Return to Life

(1) apoptotic cells do not release their cellular

constituents into the surrounding interstitial

tissue; (2) they are quickly phagocytosed by

surrounding cells thus likely preventing

secondary necrosis; and, (3) the engulfing cells

do not produce anti-inflammatory cytokines.

Most Confused Idea

Mitochondria outer membrane permeabilization

Toxicologic Pathology, 35:495–516, 2007

Major Pathways of Apoptosis

DISK: death inducing signaling complex MPT: mitochondrial perfusion transformation SET: nucleosome assembly protein

Intrinsic Apoptosis

Intrinsic Apoptosis

The intrinsic signaling pathways

that initiate apoptosis involve a

diverse array of non receptor

mediated stimuli that initiates

mitochondrial events.

Negative signals: the absence of certain

growth factors, hormones and cytokines that

can lead to failure of suppression of death

programs, thereby triggering apoptosis.

Positive signals: radiation, toxins, hypoxia,

hyperthermia, viral infections, and free

radicals.

Intrinsic Apoptosis

mitochondrial permeability transition

All of these stimuli cause

changes in the inner

mitochondrial membrane that

results in an opening of the

mitochondrial permeability

transition pore, loss of the

mitochondrial

transmembrane potential and

release of two main pro-

apoptotic proteins including

cytochrome c and Smac

from the intermembrane

space into the cytosol.

Mitochondria outer

membrane

permeabilization (MOMP)

is considered the “point of

no return” for apoptosis.

The steps leading up to

MOMP can be stopped in

their tracks by inhibitor

molecules, but once

MOMP has been achieved,

the cell will complete the

death process.

Intrinsic Apoptosis

BAX (Green) forms ring like structure On the fragmented mitochondria

Cytochrome-C in the cell

cytoplasm prompts the

formation of the

“apoptosome” with APAF1

that performs the final step

to beginning cellular

breakdown.

The apoptosome turns pro-

caspase-9 into caspase-9,

which triggers a cascade of

caspases activation.

Intrinsic Apoptosis

Cytochrome c

Inactive APAF1 Active apoptosome

Intrinsic Pathway Proteins

Cancer cells employ this molecules

Extrinsic Apoptosis

Extrinsic Apoptosis

Extrinsic Pathways initiate apoptosis involved with transmembrane receptor-mediated

Interactions such as FasL/FasR, TNF-α/TNFR1, Apo3L/DR3, Apo2L/DR4 and

Apo2L/DR5. After signaling, a death-inducing signaling complex (DISC) is formed,

resulting in the autocatalytic activation of procaspase-8. Once caspase-8 is activated, the

execution phase of apoptosis is triggered.

cFLIP is an Inhibitor of Apoptosis

Death receptor-mediated apoptosis can be inhibited

by a protein called c-FLIP which will bind to FADD

and caspase-8, rendering them ineffective.

Extrinsic Pathway Proteins

TNF-Induced Apoptosis

TNFα, cachexin, or cachectin: discovered in 1975

Discovery of tumor necrosis

factor (TNF), a key immune

signaling molecule (cytokine)

that, in addition to its promise

for the treatment of cancer

and other diseases, has

provided a powerful research

tool in biomedicine.

Dr. Lloyd J. Old, M.D., c. 1995

Lloyd John Old

TNF inhibitor

• autoimmune and immune-mediated disorders such as rheumatoid

arthritis, ankylosing spondylitis, inflammatory bowel disease,

psoriasis, hidradenitis suppurativa and refractory asthma.

• The drugs inhibiting TNF include Remicade (infliximab), Enbrel

(etanercept), Humira (adalimumab), Cimzia (certolizumab pegol) and

Simponi (golimumab).

TRAIL and FAS Ligand Induced Apoptosis

TNF-related apoptosis-inducing ligand

(TRAIL)

The FAS receptor (FasR), also known as

apoptosis antigen 1 (APO-1 or APT), cluster

of differentiation 95 (CD95) or tumor necrosis

factor receptor superfamily member 6

(TNFRSF6)

Perforin/Granzyme Pathway

Cytotoxic T lymphocytes

(CTLs) are able to kill

target cells via the

extrinsic pathway and the

FasL/FasR interaction is

the predominant method.

CTL Induced Apoptosis

Perforin/Granzyme Pathway

A novel pathway involves secretion

of the transmembrane pore-forming

molecule perforin with a

subsequent exophytic release of

cytoplasmic granules including

granzymes A and B through the

pore and into the target cell.

Granzyme B can directly activate

Caspase 3, BID, and ICAD which

triggers apoptosis.

Granzyme A can activates

DNAse NM23-H1, which blocks

the maintenance of chromatin

structure integrity.

ICAD: Inhibitor of Caspase Activated DNAse

Cancer PD-L1 Expression Avoids TCR Activated FasL/TRAIL by SHP2

T cell death

Activation of cytotoxic T cells (Tc) is an antigen-specific process requiring the interaction of the TCR–CD3

complex with a processed tumor antigen–derived peptide bound to a MHC class I molecule. Tc activation

Induces FASL and TRAIL to kill cancer cells. However, PD-1 activation by cancer PD-L1 stops Tc activation through SHP2 and triggers T cell death.

How to Cure Cancer with Apoptosis Inducers ?

Treatment with Coley’s toxins

A patient with round cell sarcoma of the jaw and

abdominal metastases seen by coley in 1899.

Photograph after 63 injections with coley’s toxins;

tumour had diminished to about half its original

size.

Mouse bearing subcutaneous

human tumour xenograft

Wild-type mouse treated with the

carcinogen DMBA and the

tumour promoter TPA

Tnf–/– mouse

TNF treat

TNF-a Between Promotion and Suppression in Cancer

Coley’s toxins : a mixture consisting

of killed bacteria of species

Streptococcus pyogenes and Serratia

marcescens

Apoptosis Inducers

Oncogenic Apoptosis

Oncogenic Apoptosis

Apoptosis can also cause unwanted effects that may even promote cancer.

Understanding this may help maximizing anti-cancer apoptosis while

minimizing pro-tumorigenic effects.

Enhancing Apoptosis

pink boxes: Possible enhancers of apoptosis

blue boxes: inhibitors of unwanted effects

Nat Rev Cancer. 2016 Aug;16(8):539-48.

MOMP: mitochondria outer membrane permeabilization

SMAC: second mitochondria-derived activator of

Caspases.

XIAP: X-linked inhibitor of apoptosis protein

(caspase inhibitor)

CAD: caspase-activated Dnase

TAM: tumour associated macrophage

celecoxib

Novel Approach to Apoptosis Induction Via p53 Stabilization

p53 is a Key Regulator in Cancer

Nature Reviews Cancer 9, 749-758 (2009)

Regulator of p53

- + -

IB: p53

170 130

95 70

55 43 34 26

17

+ + + + P53

TGase2 p53

TGase2

BSA

Coomassie

p53

monomer

+ +

p53

polymer

IP: p53, IB: TGase2

INPUT: TGase2

p53

1-3

93

1-3

56

1-3

23

Δ3

23

-356

Δ1

02

-292

55

43

34 28

IP: IgG, IB: TGase2

INPUT: p53

Caki-1/p53-/-

1-50 63-97 102-292 323-356 363-393

Trans-

activation SH3 DNA binding domain Tetramer-

ization

Regulatory

domain

K Q K Q

p53 is the Major Target of Transglutaminase 2

132 144 164 192

FASEB J. 2013 Sep;27(9):3487-95.

By Microarray of TGase 2 using NCC 72 Cell Lines

Transglutaminase 2 is Universally Increased in RCC

RCC

TGase 2

b-actin

Oncogene. 30, 4780-4790, 2011

FASEB J. 2013 Sep;27(9):3487-95

A New Regulator of p53

p53 Mutation is Only 4% in ccRCC TGase 2 is highly increased in ccRCC

p62/SQSTM1 (p-value=0.00618) TGM2 (p-value=0.00579)

TGM2 (p-value=3.5708×10-15)

mR

NA

Expre

ssio

n

(RN

A S

eq v

2 R

SE

M)

Normal Tumor

Frequent mutations in clear cell renal cell carcinoma In COSMIC database

Cell Death and Disease (2016) 7, e2163

Instability of p53 in RCC is not associated with mutations because the COSMIC database showed only 4% mutation of p53 in clear cell RCC.

+ high + low

+ high + low

National Cancer Center, KOREA

p53 MDM 2

Proteasome degradation

p53 TGase 2

Autophagy degradation

1) Normal cells

2) RCC MDM 2

TGase 2

Mechanism of TGase 2-p53 Regulation in RCC

Nutlin-3 KN383

Cell Death and Disease (2016) 7, e2163

FASEB J. 2013 Sep;27(9):3487-95

Autophagosome

p53-polymer

Ca++

Autolysosome

Lysosome

Phagophore

LC3II

LC3II

N’

C’

p62 p53

C’ C’

N’

N’

LC3 binding

N’

C’ p62 p53

TG2

C’

C’

N’

N’

PB1 UBA

Ca++

LC3

N’

C’

p53 TG2

C’

N’

CD DBD

Ca++

C’

CD DBD

C’

CD DBD C’

CD DBD

C’

CD DBD

TG2

National Cancer Center, KOREA

p53 H&E BrdU

CT

L

GK

92

1

TGase 2 Inhibitor Reverses RCC via p53 Stabilization

J Cancer Res Clin Oncol. 140(5):757-67, 2014

By collaboration with Prof. Gong Y.D

β-actin

p53

p-p53(S15)

TG2

Doxo (hr)

siTG2

0 8 0 8

- - + +

CAKI-1

p21

Bad

Bax

Puma

Control GK921

Doxorubicin GK921+Doxorubicin

3.57 23.52

9.52 53.64

DNA intercalation of Doxo

Inhibits topo II

ATM/ATR

P-p53 apoptosis

p53

HDM2

TG2 GK921

DNA damage + TGase 2 Inhibition = Synergy

Cell Death and Disease (2016) 7, e2163

Control Doxorubicin 1mg/kg

GK921 2mg/kg Combination

32

12

0

500

1000

1500

2000

0 1 2 3 4 5 week

Tu

mo

r v

olu

me(m

m3)

(1 mM)

(1 mM)

Novel Approach to Apoptosis Induction Via ATP Depletion

Cancer

receptors Signaling

mTOR

mTOR produces

survival signal molecules

& nutrient uptake/catabolic

Enzymes for proliferation

receptor tyrosine

kinase EGFR, VEGFR, IGFR, PDGFR

RAS

RAF

ME

K

ERK

PI3

K

AKT

mTO

R

Gefitinib, Erlotinib, Imatinib, Sutent

Rapamycin, Temsirolimus

Sorafenib

transcription

Translation

Survival Proliferation

Conventional Anti-Cancer Drug Development

Transcription

regulation

Translation

regulation

Kinase

inhibition

Rapamycin

Ribavirin

Pateamine A

Targeting Anabolism

41

ALDH Is Required for ATP Production in Cancer

Oncotarget. 7:49397-49410, 2016

Experimental & Molecular Medicine (2016) 48, e272

ALDH1L1

low

ALDH1L1

high

(P-value = 0.01135)

Cancer Normal

ALDH1L1

KN817

ALDH Inhibitor + Mito Complex I Inhibitor

A549

(P-value = 0.01135)

ALDH1L1

ALDH1L1 low

ALDH1L1 high

Cancer Normal

ALDH1L1

Xenograft mice model

Oncotarget. 7:49397-49410, 2016

-300

200

700

1200

1700

0 5 9 12 15 19 22 25 29 32 35

★ ★

★

★ ★

★

★ ★

KN817

Cancer

cNADH ATP

mTOR

Cytosolic NADH

production increase

via eg. ALDH

ATP

Production

via ETC

ATP suppresses

AMPK & activates

mTOR

mTOR produces

survival signal molecules

& nutrient uptake/catabolic

Enzymes for proliferation

Opportunity for anti-cancer drug development via regulation of oxidative cancer energy metabolism

receptor tyrosine kinase

EGFR, VEGFR, IGFR, PDGFR

RAS

RAF

MEK

ERK

PI3K

AKT

mTOR

Gefitinib, Erlotinib, Imatinib, Sutent

Rapamycin, Temsirolimus

Sorafenib

transcription

Translation

Survival Proliferation

NADH

transport

Conventional anti-cancer drug development

ATP

Summary : New Approach for Drug Dev

Cell death

Cancer energy metabolism