Growth factors in cancer cell signaling
Karel Souček
ksoucek@ibp. cz
Eva Lincová Andrea Staršíchová Zuzana Pernicová
Department of Cytokinetics
Institute of Biophysics AS CR, v.v.i., Brno, Czech Republic
Growth factors in cancer cell signaling
■  cancer is not single cell disease;
■  tissue microenvironment plays an important role in tumor initiation and progression;
■  growth factors - cytokines - play crucial role in cancer development and some of them belong to the significant autocrine/paracrine factors produced by various cell types in tumor microenvironment;
■  modulation of their signal transduction represent potential target for therapy.
Growth factors in cancer cell signaling
■ What is a role of TGF-ß family cytokines in chemopreventive action of inhibitors of arachidonic acid metabolim?
■ How we can effectively modify neuroendocrine differentiation of the cancer cells?
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What is a role of TGF-ß family cytokines in chemopreventive action of inhibitors of arachidonic acid metabolim?
Eva Lincová
Growth Differentiation factor - 15 (GDF-15)
NAG-1 (Non Steroidal Anti-Inflammatory Drugs (NSAIDs)-Activated Gene)
placental transforming growth factor beta (PTGF-ß)
macrophage inhibitory cytokine-1 (MIC-1)                                    __ _ _          _           ..                         ,
placental bone morphogenetic protein                                 |bl"-p T3íTllly moíTIDOľ
Prostate-derived factor (PDF)
Cancer progression inhibitor
-  Inductor of apoptosis
-  Inhibitor of proliferation?
■
■
Figure 14.1  The Biology of Cancer (© Garland Science 2007)
MU ■■■    ■ ■
31
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5.               probability of eventually developing metastasis (%)
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primary tumor	localized	intravasation	transport	arrest in	extravasation
formation	invasion		through circulation	microvessels of various organs	
interaction with
platelets, lymphocytes,
and other blood
components
formation of a micrometastasis
colonization -formation of a macrometastasis
Figure 14.4 The Biology of Cancer (© Garland Science 2007)
Genetic determinants of cancer metastasis
Oon X. Nguyen orrd Jocsn Mrtsscujtic
Tumorigenic genes
I
Metastasis initiation genes
Metastasis progression genes       Metastasis virulence genes
1
Proliferative autonomy Genomic instabiity Self-renewal Evasion of death Evasion of cytostasis Evasion of immunity Resistance to hypoxia
Detachment Motility > Invasion
Marrow p rage n i tor-ceil recruitment Angiogenesis Intravasalen
• Survival in circulation
• Embolism
• Capillary adhesion
• Extravasation
• Adaption to new environment
• Emergence from dormancy
• Organ-specific colonization
NATURE REVIEWS  GENETICS
VOLUME 8 | MAY 2007 | 343
prostate
liinnc             J
b   -                     lungs             -W                bone marrow
liver
.
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pancreas                    breast                      colon
Figure 14.42 The Biology of Cancer (© Garland Science 2007)
Cyclooxygenases (COX-1, -1 b, -2)
■ key enzymes of arachidonic acid metabolism
Bradykinin
PLA2 (inhibited by steroids)
G-pratan''                                           ^arachidonic acid +
cyclooxygenase^------(2)02        lysophospholipids
PGGj
peroxidase
t£zL
PGH
prostacyclin    j«»   /   . i 2 synthase
(2) GSH (2) GSSG
thromboxane synthase
PGI
PGD„      PGF2a
TXB,
copyright 1996 M.W.King
... and its inhibitors
■  Nonsteroidal anti-inflammatory drugs (NSAIDs)
■  COX-2 specific inhibitors
Inflammations Increase in cyclooxygenases
Increase in and binding of prostaglandin E2
Accumulation of    — QaZ ß-catenin and entry into the nucleus
/          Q
ß -eaten in
'V
GSK-3ß
Transcription ofß-caten n target genes
Cell proliferation and colorectal cancer
NSAIDs -#- Decrease in cyclooxygenases Decrease in prostaglandin E2
A PC         Ax i i
GSK-3/3
■"I
ß-catenin  ^-^>
Degradation of
phosphorylated
ß-catenin
"
&.
f\&

\.
No transcription ofß-catenin target genes
\
X
No cell proliferation H. Clevers, "Colon cancer-understanding howNSAIDs work," NEnglJMed, vol. 354, no. 7, pp. 761-763,2006.
Nonsteroidal anti-inflammatory drugs (NSAIDs)
■ variety of mechanisms           ____________
I BAYER.               5ind for )
http://www.home.duq.edu/~harr old/Chem3D/NSAID_binding_pa gel .html                                                               http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=27194                         http://en.wikipedia.org/wiki/Aspirin
Some effects of NSAIDs are independent on inhibition of prostaglandin synthesis
Table I. A list of the 10 gene* most highly induced by 5 mM aspirin treatment, together with their GeneBank accession numbers and the mean fold induction
Table II. A list of the 10 genes most highly repressed by 5 mM aspirin treatment, together with their GeneBank accession numbers and the mean fold repression
Gene name
Mean fold induction
Gen Ban k ace. no.
Gene name
DNA array analysis of the effects of aspirin on colon cancer cells: involvement of Racl
James C.II.IIardwick . Marije van Sauten, (iijs K.van den Brink, Sander J.II.van Deventer and Maikel P.Peppelenbosch
Mean fold re press!tin
JÍHH9770	Prostate differentiation factor	2.6	X535S7 X92106 M92287	HLA-G histocompatibility antigen, class 1, G Cyclin D3 Serine proteinase inhibitor	1.69 1 56
M29870	Small G'ľľ binding protein Raci				1.49
MS0563	S100 calcium-binding protein A4 (metastasin)	2.5	SS5655	Glutathione transferase omega	1.45
S40706	DNA-d am age-inducible transcript 3	2.5	M35543	Cell division cycle 42 (GTP-binding protein.	1.39
X54941	CDC28 protein kinase 1	2.1		25 kDa)	
M60S54	Ribosomal protein S16	2.1	U65410	Heat shock protein 70 kDa	1.33
J04111	v-jun avian sarcoma virus 17 oncogene homol ogue	2.1	X51521	Mitotic feedback control protein MADP2 homolog ue	1.30
XI6277	Ornithine decarboxylase 1	2.0	U5SMS	Neurotrophic tyrosine kinase receptor type 1	1.30
K02770	Interleukin 1, beta	1.8	X56681	NF-kB (pi 05)	1.28
M27364	Eukaryotic translation elongation factor 1 alpha 1	1.7	M93056	Mitogen-activated protein kinase kinase 1	1.28
Carcinogenesis vol.25 no.7 pp.1293-1298, 2004					
DO!: 10.109:Vciircin/bghlia					
Growth Differentiation Factor -15 (GDF-15)
NSAID-Activated Gene (NAG-1) macrophage inhibitory cytokine-1 (MIC-1) placental transforming growth factor beta (PTGF-ß) placental bone morphogenetic protein Prostate-derived factor (PDF)
NSAIDs Antioxidants
activation
Retinoids
Vitamin D3 P PAR; ligands
latent
GDF-15
GDF-15
Transcription Factor: p53, SP-1,etc.
fflÖ
Transcription Factor:
AP-1,SP-1,etc.
Target Genes -
Regulating
Cytokineties
Inhibition of Proliferation ?
Induction of Proliferation
Cytoplasm
GSK30      CHFRqI      Mytí Q
Plk I
G2/M
TrBS
TRENDS in Bioch emicai Sciences   Vo ľ 29 N o > 5 M ay 2 0 04
Phosphoinositide-dependent protein kinase-1
Inactive PKB
Substrates
TŕBS
TRENDS in Bioch emical Sciences   Vo ľ 29 N o > 5 M ay 2 0 04
Negative regulators of PI3K
■   PTEN - phosphatase and tensin homolog
-  Tumor suppressor;
-  dual-specificity protein phosphatase;
-   lipid phosphatase, removing the phosphate in the D3 position of the inositol ring from PI3P
■   SHIP1/2- inositol polyphosphate 5-phosphatases
-  Regulators of proliferation
-  key role in insulin metabolism and obesity
PI3K
- Ptdnap.4Pz
Fetty actis
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Nature Ravŕews I Cancer
Presumptions
■ NSAIDs induce expression of GDF-15.
■ GDF-15 is an autocrine mediator of proapoptotic effects of NSAIDs.
Hypotheses
■ GDF-15 is a mediator of antiproliferative effects of NSAIDs;
■ GDF-15 induction is one of the key determinant of different sensitivity of cancer cells to the antiproliferative effects of NSAIDs;
■ PKB/Akt activation is part of the mechanism of NSAIDs effects.
Experimental models
■ Prostate cancer cells
- LNCaP - androgen sensitive, p53 wt, PTEN -/-, SHIP2 -/-
- PC3 - AR -/-, p53 -/-, PTEN -/-
■ Colon cancer cells
-HCT-116Neo
-HCT-116PTEN-/-
LNCaP cells are more sensitive to the anti-proliferative effects of NSAIDs
1.4 2  1.2
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■ LNCaP n PC3
T

LNCaP
Go/G,
■  COfilroI
- INDO 13,0 pM
■  INDO 25 jiM
■  INDO 50 jiM
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- Cúťilrol
■  INDO 12.5 jjM
■  INDCi25;iM
■  INDQSOilM
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12 hours
'2 hours
Expression of p21 and GDF-15 proteins is induced by INDO in LNCaP cells
A)
LNCaP                                                          PC3
u <?£>   kDa
0   12,5 25   50 nM      0     6     12    24 h           0   12,5 25   50 ^iM      0     6     12   24 h      <?
4-35 4-53 «-21
4-20
MDM2                                                                                                                                     ^- 90
GDF-15
p53 p21
Bax
INDO effects on LNCaP cells are
independent on COX inhibition
A)
GDF-15

1       0,6      0,5       0,5      2,0     2,3     2,7      2,3
p21
Cipl/Wafl
1        1,4      1,0      0,7      1,3     1,3     1,7      1,4

ß-actin

PGE2 (nM) INDO
B)
20
15
10
	X		I		I		T		T				T			
1	10	50	-	1	10	50	PGE2 [.uM)
_	_	_	+	+	+	+	INDO
1       10    50      -        1      10     50
+       +       +
GDF-15 siRNA does not affect INDO-
induced cell cycle arrest in LNCaP cells
A)
B)
scramble siRNA               GDF-15 siRNA
Control          INDO 50 \.M        Control          INDO 50 |jM
nM siRNA     10   20    30     10   20    30       10   20    30     10   20    30
GDF-15
ß-actin
1.2
A   1.0 o
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Q. CO,
ö   0.6
•5   °A
0.8
0.2 00
Scramble siRNA GßF-'5 siRNA
T
I
T
0                25               50
INDO (nM)
INDO delays a decrease of p21WAP1/CIP1 level induced by ActD and/or CHX


1.0   0.9     0.3   0.1    1.4    1.5     0.7 0.07
P  Cl\_ LI I I      ■^"™*                    ~
time (h) 12   1     6    12    12   1    6   12       time (h) 12    1    6   12   12   1    6   12
ActD     -     +     +    +     -      4-4-4-                     CHX    -       +      +      4-       -4-4-4-
INDO    -     -      -       -     4-     +      4-     4-             INDO   -       -       -       -       4-     4-      4-     4-
PKB/Akt phosphorylation is induced by INDO in LNCaP cells
time(h)    0   0.5   1    3    6    12   24        kDa
cyclin Dl
1.0    2.1    2.0   1.9   3.4     4.4    4.S
1.0    Q.S     0.7   0.4   0.6     0.7    0.8
®-S473-Akt------------------— — — +60

1.0    0.9     1.0   1.4   2.4     2.6    2.2
Akt
4" 60
1.0    0.9     0.S   0.8   0.7     0.7    0.S
Is SHIP2 expression responsible for resistance to NSAIDs in PTEN -/-cells?
■ Prostate cancer cells
- LNCaP - PTEN -/-, SHIP2 -/-
- PC3 - PTEN -/-
■ Colon cancer cells
-HCT-116Neo
-HCT-116PTEN-/-
SHIP2 siRNA sensitizes PC3 and HCTl 16 PTEN -/■ cells to the antiproliferative effects of INDO
1.2
1.0
~   0.8
o
o
?   0.6
O) co CO
CO
0.4
0.2
0.0
PC3
HCT-116 Neo124
HCT-116 K022
i scramble siRNA/ EtOH i scramble siRNA/INDO i SHIP2 siRNA/EtOH : SHIP2 siRNA/INDO
Summary I
■  GDF-15 is not mediator of early induction of G0/G1 arrest induced by NSAIDs;
■  Posttranslational stabilization of p21WAF1/CIP1 through PKB/AKT activation is a novel mechanism of NSAIDs effects;
■  Multiple defects in negative regulation of PI3K/Akt activity sensitize cancer cells to the effects of NSAIDs.
How we can effectively modify neuroendocrine differentiation of the cancer cells?
Andrea Staršíchová Zuzana Pernicová
Neuroendocrine cells	Produced factors
■„hybrid" epithelial/nerve/endocrine cells Secretion of various factors	■Chromogranins ■Serotonin ■Gastrin releasing peptide (bombesin) ■Calcitonin gene family
	■Somatostatin
	■Parathyroid hormone-related protein
	■Neuropeptide Y
	■Vascular endothelial growth factor (VEGF)
	■Cholecystokinin
	■Proadrenomedullin N-terminal peptide
	■TSH-like peptide
	■Histamine
Neuroendocrine prostate cancer
■  develops as an aggressive disease that does not respond to androgen ablation therapy;
■  paracrine action of NE cells promote the progression of androgen dependent adenocarcinoma to an androgen independent state;
■  significant role for NE cells during failure of androgen ablation therapy.
//
normaľ prostate epithelium
■  stem cells
■  basal epithelial cells
■  transitamplifying cells
■  neuroendocrine cells
■  secretory luminalepithelial cells
Lam, J.S. et ah, 2006
LUMINAL
BASAL
K5/K14 Stem «II
Differentiated Intermediate    secretory differentiating       KS/18
KH/IS
Neu roeadoc r i ne
KS/K 19, K5/K17. K8
T r» n vi l amplifying
Hudson, D.L. et al, 2004
Neuroendocrine cells
■
Androgen depletion-induced neuroendocrine differentiation of prostate cancer cells
Differentiation markers
10%FBS                 5% CS
days     2    4     8    16      2    4    8    16
cytokeratin 18 ■■f •
cytokeratin 19 1_ m
ß-actin   «m m
«t
Inflammation & Cancer
■   Chronic    inflammation    plays    important    role    in development of several type of cancer.
■   Pro   inflammatory  cytokines  such  as   IL-6   induce neuroendocrine differentiation.
Polyunsaturated fatty acids (PUFAs)
■   Modulators of carcinogenesis
■   n-6 - pro-inflammatory
■   n-3 - anti-inflammatory
■   Docosahexaenoic acid (DHA (n-3)) - modulates signal transduction of various cytokines through lipid rafts.
Modulation of IL-6 signaling pathway
Ara ch i d on i c
acid
?   DHA
Transcription Factor: NF-kB.AP-1. etc.
Transcription Factor:
NF-kB, SRE. etc.
Inhibition of Apoptosis
Induction of Proliferation
Modulation of IL-pathway by DHA
A)
Control     DHA 5 MM  DHA 25 ^M
IL-6 (5 ng/ml)   ftEi^aflS __^^tf _^^M| ©-Y705-STAT3        - •#      * ~W        -*■ •
STAT3   -——-__------—-----------
p-actin *ww w^^jva» v w •
6 signaling
Summary II
■ We have characterized model of NED of LNCaP cells;
■ DHA has potential to modulate signal pathway of I L-6.
Acknowledgement
■   Pavel Krčmár, Miroslav Machala, Veterinary Research Institute, Brno
■   Todd Waldman, Georgetown University, Washington DC