Pempti 4 Patrignani

6th Summer School of the Hellenic Atherosclerosis Society

Pharmacotherapy of the prevention and management of cardiovascular disease in special populations

June 27 – 29, 2013, Crowne Plaza Hotel, Athens, Greece

Low-dose aspirin, atherothrombosis and cancer Paola Patrignani Professor of Pharmacology Department of Neuroscience and Imaging – Section of Cardiovascular and Pharmacological Sciences and Ce.S.I., “G. d’Annunzio” University, School of Medicine, Chieti, Italy E-mail:ppatrignani@unich.it

Outline Platelet angiogenesis and growth factor proteome Role of COXs in intestinal tumorigenesis Efficacy of aspirin in cardiovascular disease and colorectal cancer: a shared mechanism of action?

Enhanced TXA2 generation in colon neoplasia Platelets and metastasis Conclusions

Outline Platelet angiogenesis and growth factor proteome Role of COXs in intestinal tumorigenesis Efficacy of aspirin in cardiovascular disease and colorectal cancer: a shared mechanism of action?

Enhanced TXA2 generation in colon neoplasia Platelets and metastasis Conclusions

Platelet angiogenesis proteome When microscopic tumors began to grow new blood vessels and grow, angiogenesis regulatory proteins began to appear in the plasma as well as in the platelets.

Platelets can up-take proteins from plasma by mechanisms quite unknown.

Judah Folkman, 2007

Contribution of platelets to angiogenesis. Different stimuli can release selectively different subsets of a-granules from platelets Italiano et al Blood. 2008 1111:1227-33

VEGF = vascular endothelial growth factor; PDGF = platelet-derived growth factor; bFGF = basic fibroblast growth factor; HGF = hepatocyte growth factor; EGF = epidermal growth factor; IGF = insulin-like growth factor; TSP-1 = thrombospondin-1; PAI-1 = plasminogen activator inhibitor-1; PF-4 = platelet factor 4; Hka = high molecular weight kininogen domain 5, TGF- 1 = transforming growth factor- 1; TIMPs = tissue inhibitor of metalloproteinases.

Aspirin decreases agonist-induced release of platelet angiogenesis and growth factors stored in a-granules Aspirin Control

(growth regulating growth factor)

(oncostatin)

Coppinger et al. Blood 2007;109: 4786-92

Outline Platelet angiogenesis and growth factor proteome Role of COXs in intestinal tumorigenesis Efficacy of aspirin in cardiovascular disease and colorectal cancer: a shared mechanism of action?

Enhanced TXA2 generation in colon neoplasia Platelets and metastasis Conclusions

Phospholipids – Arachidonic Acid Phospholipases (cPLA2/sPLA2) activated by physical, hormonal, inflammatory, mitogenic stimuli

Arachidonic Acid COX-1

tNSAIDs

Arachidonic Acid tNSAIDs

COX-2

Coxibs

PGH2 cPGE

PGH

TX synthase PGI synthase synthase

2 mPGE-1 TX synthase PGI synthase synthase

TXA2 PGI2 PGE2

TXA2 PGI2 PGE2

Specific Prostanoid Receptors (TP, EPs, IP)

The molecular mechanism of irreversible inactivation of the COX activity of COX-1 and COX-2 by aspirin Acetylated COX-2 by aspirin may generate the epi-LXs (AspirinTriggered Lipoxins, ALT) that have antiproliferative and antiinflammatory actions (Serhan 2005) However, convincing evidences that these lipid mediators triggered by aspirin are generated in vivo in humans are lacking Systemic concentrations of aspirin, reached after dosing with low-doses, are inadequate to significantly acetylate COX-2

Inhibitory effects of COX-1 and COX-2 by aspirin and salicylic acid in human whole blood assays in vitro

% inhibition

80

100

Platelet COX-1 IC50 M: 18

Monocyte COX-2 at 5000 M: 54%

60 40

60

Platelet COX-1 IC50 M: 1400

40 20

20 0 0.1

Monocyte COX-2 IC50 M: 1100

80

% inhibition

100

1

10

100

1000

10000

Aspirin (M)

0 0.1

1

10

100

1000 10000 100000

Salicylic Acid (M)

Aspirin

Aspirin Cmax

Salicylate Cmax

Antiplatelet dose 75 mg/day Solution Controlled release

7.31 µM 0.29-0.54 µM

15 µM 4 µM

Analgesic doses 325-600 mg/4-6 h

28-80 µM

500 µM (1 g single dose)

Anti-inflammatory doses 1.2 g/4-6 h

142 µM

1500-2500 µM

Assessing systemic biosynthesis of prostanoids in humans Index of vascular PGI2 COX-2-dependent (by 60%)

Urine

PGI-M: 2,3-dinor-6-keto-PGF1a Index of PGI-M platelet TXA2 COX-1-dependent TX-M PGE-M (by 75%) TX-M: 11-dehydro-TXB2 and 2,3-dinor-TXB2

PGD-M

Index of inflammatory and tumoral PGE2 COX-2-dependent (by 60%) PGE-M: 7-hydroxy-5,11-diketotetranorprostane-1,16-dioic acid

In Vivo

Differential dose-response relationships to inhibit platelet COX-1 ex vivo and systemic PGI2 in vivo (mainly from vascular COX-2) by aspirin The inhibition of platelet COX-1 activity is maximal at low-doses (100 mg/day) Similarly reduced incidence of vascular events is maximal at low-doses (75-150 mg/day) Differently, the gastrointestinal (GI) side-effects increase at higher doses

5

PGI-M COX-2 (in vivo)

80

4

60

3

40

2

20

1

Reduced incidence vascular events

% inhibition

100

0 1

10

100

1000

GI bleeding

ODDS RATIO

Platelet COX-1 (ex vivo)

0 10000

Aspirin dose (mg daily) Patrono et al. JCI 1982;69:1366-72; FitzGerald et al JCI 1983;71:676-88; ATTC BMJ 2002; 324:71-86; Weil et al. BMJ. 1995;310:827-30

RCTs of Aspirin or COX-2 Inhibitors in Patients with Previous Polyps or Colorectal Cancer • 4 RCTs of aspirin

– Baron JA et al. N Engl J Med. 2003;348:891-899 – Sandler RS et al. N Engl J Med. 2003;348:883-890 – Benamouzig R et al. Gastroenterology 2003;125:328-336

– Logan RFA et al. Gastroenterology 2008; 134: 29-38.

• 3 RCTs of COX-2 inhibitors – Bertagnolli MM et al. N Engl J Med. 2006; 355: 873-84 – Arber N et al. N Engl J Med 2006; 355: 885-95 – Baron JA et al. Gastroenterology 2006; 131:1674-82

Relative Risk of Any Colorectal Adenoma at Follow-up Endoscopic Examination RR (95% CI)

Drug/dose

Trial

Celecoxib 400 mg bid

APC, 2006

Celecoxib 200 mg bid

APC, 2006

Rofecoxib 25 mg

APPROVe, 2006

Aspirin 325 mg

Sandler et al, 2003

Aspirin 325 mg

Baron et al, 2003

Aspirin 81 mg

Baron et al, 2003

0.0

0.2

0.4

0.6

0.8

COX-Inhibitor better

1.0

1.2

1.4

1.6

Placebo better

1.8

Modified from Markowitz.

NEJM 2007;356:2195-8

Arachidonic Acid Aspirin

COX-1 COX-2

NSAIDs

PGH2 Prostaglandin synthases

Prostanoids

PGD2,PGF2a, PGI2, TXA2

EP1, EP2, EP3, EP4

Prostaglandin E2 receptors

PPARd

PGE2

b-Catenin

EGF-R

PI3K/AKT

Bcl-2

VEGF

transcriptional activity

Target genes Biologic activities

Cyclin D1

Growth

Migration & invasion Anti-apoptosis

Angiogenesis

Min mice

A mutant mouse, Min, was found with multiple intestinal neoplasia in 1990 by Moser, Pitot & Dove .

It was shown to have a mutated Apc gene (a nonsense mutation at codon 850 of Apc gene).

Truncated Apc in position 716 (Apc716 mutant mice) causes even more adenomas in the gut.

This promising animal model mimics the rapid development of adenomatous polyps that affect humans with germline inactivation of one Apc gene.

The results with knockout mice of either COX-1 or COX-2 suggest that both COXisoforms play a role in colon tumorigenesis “Both COX1- and COX2- null Min mice had an 8090% reduction of intestinal polyps at 4,6, and 8 months of age and were long-lived when compared to Min/+ mice.�

Chulada et al. Cancer Res 2000; 60:4705-08

Sequential role of COX-1 and COX-2 in colon tumorigenesis

Growth factors, prostanoids

Dovizio M, Bruno A, Tacconelli S, Patrignani P. Mode of action of aspirin as a chemopreventive agent. Recent Results Cancer Res. 2012;191:39-65

Outline Platelet angiogenesis and growth factor proteome Role of COXs in intestinal tumorigenesis Efficacy of aspirin in cardiovascular disease and colorectal cancer: a shared mechanism of action?

Enhanced TXA2 generation in colon neoplasia Platelets and metastasis Conclusions

Acetylation of Platelet COX-1, Inhibition of TXA2 Production and Reduction of Vascular Events by Aspirin are Saturable at Low Doses Clinical Pharmacology of ATT Collaboration Platelet COX-1 Meta-Analysis of Aspirin Trials in High100 Risk Patients (14) % inhibition of TXB2 production

Mechanism of Action

80

Comparison

Aspirin Control

Reduction

Asp 75-150

11.0%

15.2%

32%±6

Asp 160-325

11.5%

14.8%

26%±3

Asp 500-1500

14.5%

17.2%

19%±3

Any aspirin

12.9%

16.1%

(5) 60

Mean ± SD (n) (4)

40 (5) 20

J Clin Invest 1982;69:1366-72

23%±2 (2P<0.00001)

(5) 0.0

0

1

10

100

Oral Aspirin dose

1000 mg

0.5

1.0

BMJ 2002;324:71-86

1.5

2.0

Aspirin and Colorectal Cancer â&#x20AC;˘

If aspirin does indeed prevent the early development of an adenomatous lesion, one would require a long-term follow-up of aspirintreated patients in order to detect a beneficial effect on the risk of colorectal cancer (CRC) and CRC-related death.

Long-Term Effect of Aspirin on Colorectal Cancer Incidence and Mortality: 20-Year Follow-Up of Five Randomised Trials.

Rothwell PM et al. Lancet 2010;376:1741-50

Pooled Analysis of the Effect of Low-Dose (75300mg) Aspirin (thick line) versus Control (thin line) on Subsequent Incidence and Mortality Due to Colorectal Cancer in TPT, SALT and UK-TIA Incidence

Mortality 4

3

p=0.04

% Risk

% Risk

4

2

1

0

0 5

10

15

20

0

Time to notification (years)

A: 785 C: 814

2

1

0

p=0.006

3

5

10

15

20

Time to death (years)

4030

3618

3095

2552

779

4030

3629

3114

2575

4043

3645

3149

2545

806

4043

3653

3164

2566

Rothwell PM et al, Lancet 2010; 376:1741-50

Aspirin and Colorectal Cancer â&#x20AC;˘

If the chemopreventive effect of aspirin is related - directly or indirectly - to its antiplatelet action, then one would expert saturability of cancer prevention at low doses (ie 75-100 mg) given once daily.

Death Due to Colorectal Cancer on Long-Term FollowUp After Randomization in Trials of Aspirin vs Control Deaths due to cancerOdds Aspirin Control Ratio 95% CI 500-1200mg daily British Doctors Study (500mg) UK-TIA (1200mg)

59/3429 40/1710

0.73

0.49-1.10

11/821

0.68

0.31-1.47

SUBTOTAL

70/4250 56/2527

0.72

0.50-1.03

UK-TIA (300mg)

8/811

0.50

0.21-1.17

TPT (75mg)

34/2545 55/2540

0.61

0.40-0.94

SALT (75mg)

7/676

0.71

0.27-1.86

SUBTOTAL

49/4032 81/4041

0.60

0.42-0.86

TOTAL

119/8282 137/6568

0.66

0.51-0.84

16/817

75 - 300mg daily

Heterogeneity: p=0.84

Rothwell PM et al, Lancet 2010; 376:1741-50

16/817

10/684

p=0.001 0

1

2

Odds Ratio (95% CI)

Data窶電riven hypothesis 窶｢ The benefit of low-dose aspirin in colorectal cancer and atherothrombosis convincingly supports that they share a common mechanism of disease, i.e. platelet activation in response to epithelial (in tumorigeneis) and endothelial (in tumorigenesis and atherothrombosis) injury

Atherothrombosis and intestinal tumorigenesis may share a common mechanism: platelet activation in response to altered endothelial (EC) and epithelial functions Platelet-dependent induction of atherosclerosis

Platelet-dependent induction of tumorigenesis COX-2

Altered TXA EC functions ADP2

WNT signalling

PDGF

KRAS BRAF

TP53 TGFb

18qLOH SMAD4

COX-2

PL T

TXA2 ADP PDGF

ADP PDGF IL-1b

Stromal cell

PL T

COX-2

° °

PL T

° ° ° ° °

PL T

° °

Platelet-dependent induction of thrombosis

Normal Adenomatous Intermediate Late Carcinoma adenoma adenoma epithelium polyp PGE2 Growth Factors TXA2

° °

° ° ° ° °

° °

PL T

Altered EC functions

Adapted from Ahnen et al. Am J Gastroenterol 2011;106:190-8; Kulendran et al. Cancers 2011;3:1622-38; Gawaz et al. JCI 2005;115:3378–84; Prescott JCI 2000;105:1511–3; Patrono et al 2001;108:7–13

Outline Platelet angiogenesis and growth factor proteome Role of COXs in intestinal tumorigenesis Efficacy of aspirin in cardiovascular disease and colorectal cancer: a shared mechanism of action?

Enhanced TXA2 generation in colon neoplasia Platelets and metastasis Conclusions

Familial Adenomatous Polyposis (FAP): natural history • • • •

Adenomas begin to develop in early adolescence 100-5000 colorectal adenomas Cancer risk increases with number of adenomas If untreated

• •

100% colorectal cancer risk Median life expectancy – 42 years

*

100

75

50 B

25

0

WT

Min/+

Apc

PGE-M ng/mg creatinine

dinor-TX-M ng/mg creatinine

Biosynthesis of prostanoids in vivo in ApcMin/+mice versus wild type A 7.5

** 5.0

2.5

0.0

WT

ApcMin/+

PGI-M ng/mg creatinine

7.5

5.0

2.5

0.0

WT

ApcMin/+

Dovizio et al J Pharmacol Exp Ther.

2012; 341:242-50

Enhanced generation of TXA2 is detected in FAP patients through a COX-1-depedendent pathway

TXM ng/mg creatinine

2.0

*

1.5

*

1.0 0.5 0.0 Controls

FAP

*p<0.05 vs controls

FAP+cele 400 mg BID for 1 week

Dovizio et al J Pharmacol Exp Ther. 2012; 341:242-50

PGIM ng/mg creatinine

0.20 0.15 0.10

*

0.05 0.00 Controls

FAP

FAP+cele

PGEM ng/mg creatinine

In FAP, systemic generation of PGI2 and PGE2 are due to COX-2: celecoxib (400 mg BID for 1 week) inhibits it 40

*

30

*

20 10 0 Controls

FAP

Dovizio et al J Pharmacol Exp Ther. 2012; 341:242-50

FAP+cele

6000

B

Effects of low-dose aspirin (50 mg/day) on TXA2 biosynthesis ex vivo and in vivo in patients with colorectal cancer

5000 4000 3000

Aspirin

ex vivo whole blood

500

2000 1000 0 Patients with Matched Colon Cancer Controls

Aspirin 12000

11-dehydro-TXB 2 pg/mg creatinine

750

Serum TXB 2 (ng/ml)

A

Urinary 11-dehydro-TXB2 (pg/mg creatinine)

Enhanced biosynthesis of TXA2 in patients with colorectal cancer (A) and its cumulative inhibition by low-dose aspirin (50 mg/day)(B)

250

0

9000

in vivo systemic biosynthesis

6000

3000

0

Baseline

1

3

5

Baseline

1

3

5 days

Sciulli et al. Prostaglandins Leukot Essent Fatty Acids 2005;72:79-83

Platelets adhere rapidly to HT29 human colon carcinoma cells and form platelet aggregates 0h

2h

4h

20h

COX-1

HT29 human colon carcinoma cells

trasmission

Adhesion > aggregation

Aggregation

Dovizio et al Mol Pharmacol April 2013

Cancer cells induced time-dependent generation of TXB2 and PDGF-BB by platelets during co-culture

150

TXB2 ng/ml

125

HT-29+PLT PLT HT

**

100

** ** **

75 50 25

**

** ** ** 0 0 4

**

**

8 12 Time (hours)

**

**

16

20

PDGF-BB pg/ml

2000

HT-Plt Plt HT

1500 1000

**

**

**

**

500 * *

0 0

4

Dovizio et al Mol Pharmacol April 2013

ยง

*

**ยง

8 12 Time (hours)

**ยง

**ยง

16

20

Summary I Low-dose aspirin (at doses normally used for cardioprotection) was associated with a reduction in the incidence and mortality of colorectal cancer;1 this indicates that the antiplatelet effect of aspirin is at the center of its antitumor efficacy.

The chemopreventive benefit was also detected with a controlled-release formulation of aspirin 75 mg with negligible systemic bioavailability.2 At low-doses given once daily, aspirin acts mainly by a preferential and irreversible inactivation of platelet COX-1 thus causing a profound and persistent inhibition of platelet function.3,4 1. Rothwell et al. Lancet 2010;376:1741â&#x20AC;&#x201C;50 ; 2. Clarke et al. NEJM 1991;325:1137-41; 3. Patrignani et al. JCI 1982;69:1366-72; 4. DavĂŹ et al N Engl J Med 2007;357:2482-94

Outline Platelet angiogenesis and growth factor proteome Role of COXs in intestinal tumorigenesis Efficacy of aspirin in cardiovascular disease and colorectal cancer: a shared mechanism of action?

Enhanced TXA2 generation in colon neoplasia Platelets and metastasis Conclusions

Role of platelets in tumor metastasis, tumor growth and angiogenesis

BAMBACE & HOLMES. Journal of Thrombosis and Haemostasis 2011; 9: 237â&#x20AC;&#x201C;249

Platelets contribute in tumor metastasis through several mechanisms: Formation of platelet aggregates surrounding tumor cells which may support tumor cell survival and protection from immune elimination.

Enhancement of the adhesion of tumor cells to the endothelium thus leading to tumor cell arrest and extravasation.

Synthesis of lipid products[such as thromboxane(TX)A2] and the release of proteins from ι-granules[such as transforming growth factor(TGF)-β and platelet-derived growth factor(PDGF)] during platelet activation that may affect tumor vascularization and facilitate tumor cell dissemination into the bloodstream.

Gay LJ et al., Nat Rev Cancer. 2011;11:123-34

Steps in metastasis that are critically supported by platelets

Labelle et al., (2011) Cancer Cell 20, 576â&#x20AC;&#x201C;590.

An EMT involves a functional transition of polarized epithelial cells into mobile and ECM componentâ&#x20AC;&#x201C;secreting mesenchymal cells

Kalluri & Weinberg JCI 2009; 119: 1420-28

Adhesion protein expressed on platelet surface possibly involved in platelet-tumor cell cross-talk GPVI

Revacept

Galectin-3

aIIb1

GPIb

Platelet

P-selectin

unique among the galectin family of lectins because it contains a â&#x20AC;&#x153;collagen-likeâ&#x20AC;? domain

Gal-3 blocker, ie Gal-3C

Mucines syacylated (LS180cells)

Heparin

Mannori et al., Cancer Res 1995;55:4425-4431..

GPIIb/IIIa-like protein GPIIbIIIa

(melanoma cells)

GPIIb/IIIa antagonist

Boukerche et al., Blood. 74(2):658-63.

Cyclooxygenase(COX)â&#x20AC;&#x201C;2â&#x20AC;&#x201C;derived prostanoids can influence several processes that are linked to carcinogenesis.

We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelial-mesenchymal transition(EMT).

HT29 human colon carcinoma cells were co-cultured with human platelets

Platelets

HT-29 cells

The kinetics of platelet-HT-29 cell interactions in cocultures up to 20h COX-2

COX-1

trasmission

merge

0h

Adhesion > aggregation

2h

4h

Aggregation

20h

Dovizio et al Mol Pharmacol, April 2013

Cancer cells induced time-dependent expression of COX-2 0

4

8

12

16

20 h

16

20

COX-2 b-actin HT+Plt

COX-2/b-actin(OD) ratio

HT 2.5 2.0 1.5 1.0 0.5 0.0 0

4

8

12

Time (hours)

Possible molecular determinants of the interaction of platelets with cancer cells and their pharmacological inhibition Dense granules

PSGL-1

agranules

Revacept P-selectin

GPVI

aIIb1

Revacept as a dimeric Fc fusion protein with the IgG part and the extracellular domain of the human glycoprotein VI (GPVI) platelet receptor

P-selectin antagonist Inhibitors of galectin-3 function (β-lactose, a dominant negative form of galectin-3,Gal-3C, and anti-galectin-3 antibody M3/38)

GPIb

Galectin-3 [collagen like domain Pro-Gly-Ala-Tyr-Pro-Gly-X-XX] Gal-3 b-actin

Tumor cells

HT

Effects of galectin-3 functional blockers on COX-2 protein expression in HT-29 cells co-cultured with platelets for 20h

Dovizio et al Mol Pharmacol, April 2013

Effects of soluble dimeric GPVI receptor-Fc fusion protein, revacept, on COX-2 protein expression in HT-29 cells cocultured for 20h with platelets COX-2

b-actin HT

HT+Plt

HT+Plt HT+Plt

4

vehicle

40

HT+Plt

400 g/ml

revacept COX-2/b -actin ratio %C (HT)

600

§

400

200

0

*

°

@

HT HT+Plt HT+Plt HT+Plt HT+Plt Vehicle

4

40 400 g/ml Revacept

Dovizio et al Mol Pharmacol, April 2013

Effects of a P-selectin antagonist on COX-2 protein expression in HT-29 cells co-cultured with platelets for 20h

COX-2 b-actin HT

HT+Plt HT+Plt

vehicle

10

HT+Plt 100 M

P-selectin ant

COX-2/b -actin ratio %C (HT)

300

§

**

°

200

100

0

HT HT+Plt HT+Plt HT+Plt Vehicle

Dovizio et al Mol Pharmacol, April 2013

10 100 M P-selectin antagonist

The overepression of COX-2 in HT29 cells interferred with the expression of proteins involved in cell-cycle progression p21/b -actin ratio

2.0 1.5

** 1.0

p21WAF1/CIP1

*

b-actin

0.5

HT 0.0 HT

HT+Plt HT+Plt

rofecoxib 0.3 M

HT+Plt HT+Plt rofecoxib 0.3M

cyclinB1/b -actin ratio

2.5

G0 Cyclin B1

M

**

2.0

cyclinB1

1.5

b-actin

**

1.0 0.5

HT

0.0

CDK1

HT

HT+Plt

HT+Plt

HT+Plt rofecoxib 0.3 M

HT+Plt

rofecoxib 0.3M

p21CIP1/WAF1

G1

STOP

G2 S

p21CIP1/WAF1

Dovizio et al Mol Pharmacol, April 2013

Platelet adhesion to HT29 cells and upregulation of COX-2 protein expression are involved in the induction of genes involved in the epithelial-mesenchymal transition

Dovizio et al Mol Pharmacol, April 2013

Summary II We have unraveled the role of platelets in inducing COX-2 upregulation in HT29 cells, considered as a key event in carcinogenesis.

This program of malignancy is primed by transient plateletcancer cell contact, involving tumor galectin-3 and platelet collagen receptors.

These findings also reveal that inhibitors of this program such as blockers of collagen binding sites, ie revacept, and galectin-3, ie Gal-3C, may represent innovative strategies in colon cancer chemotherapy which should be tested in experimental animals followed by randomized clinical trials in colon cancer patients.

Aspirin and cancer metastasis â&#x20AC;˘

One would not expect short-term effects of aspirin on cancer incidence and mortality, unless the drug also interferes with cancer metastasis.

Cancer Incidence During Six Randomised Trials of Daily LowDose Aspirin in Primary Prevention of Vascular Events Trial Follow-up

Events/Subjects Aspirin Control

Odds Ratio

95%CI

0-2.9 years AAA 50/1675 49/1675 TPT 72/2545 78/2540 POPADAD 23/638 23/638 JPAD 12/1262 12/1277 HOT 219/9399 255/9391 PPP 69/2226 55/2269 TOTAL 445/17745 442/17790

1.02 0.92 1.00 1.01 0.97 1.29 1.01

0.68-1.52 0.66-1.27 0.56-1.80 0.45-2.26 0.81-1.17 0.90-1.84 0.88-1.15

ď&#x201A;ł3 years AAA TPT POPADAD JPAD HOT PPP

0.79 0.74 0.58 0.44 0.87 0.71

0.61-1.02 0.56-0.99 0.34-1.00 0.11-1.69 0.64-1.18 0.42-1.21

TOTAL

116/1593 145/1599 84/2431 112/2433 22/532 37/593 3/1095 7/1117 75/9063 86/9029 24/1689 34/1713

324/16463 421/16484

Rothwell et al, Lancet 21 March 2012

p=0.81 (het) p=0.92 (sig)

p=0.79 (het) p=0.0003 (sig)

0.76 0.66-0.88 0

1

Odds Ratio (95% CI)

2

In platelet-HT29 cell co-cultures, the release of PGE2 derives from platelet COX-1 and tumor COX-2 PGE2 levels were reduced by selective inhibition of platelet COX-1 by aspirin

PGE2 (pg/ml)

400

**

0

4

8

12

300

COX-2

200

*

b-actin

100 0 HT

vehicle

HT+PLT

HT+PLT

HT

aspirin pretreated platelets

Dovizio et al Mol Pharmacol, April 2013

HT+Plt

16

20 h

Twist-1 plays a key role in epithelial-mesenchymal transition(EMT) by down-regulating E-cadherin expression and promoting an invasive and metastatic phenotype

Exogenous PGE2 induces Twist1 mRNA associated with down-regulation of E-cadherin mRNA A

B

E-cadherin gene

** 2

1

0

Vesuna et al. BBRC 2008; 367: 235â&#x20AC;&#x201C;241

%fold change vs HT

1.2

E-cadherin

%fold change vs HT

Twist

Transcriptional repression

TWIST1

3

1.0 0.8

*

0.6 0.4 0.2 0.0

HT

HT

HT

PGE2 5nM

CONFIDENTIAL - NOT FOR CITATION OR PUBLICATION

HT PGE2 5nM

The interaction of platelets with HT29 cells is associated with enhanced Twist1 mRNA and reduced E-cadherin mRNA levels These changes were reversed by aspirin-pretreatment of platelets and rescued by exogenous PGE2 A

PGE2 5nM

B

%fold change vs HT

TWIST1

*

4

2

°

%fold change vs HT

*

6

E-cadherin

1.2 1.0 0.8

**,§

**,§

0.6 0.4 0.2 0.0

0

HT

HT+Plt

HT+Plt (asa)

HT+Plt (asa)

HT

HT+Plt

HT+Plt (asa)

HT+Plt (asa) PGE2 5nM

*P<0.05 vs HT; °P<0.05 vs HT+PlT and HT+Plt(asa)+PGE2

**P<0.01 vs HT; §P<0.01 vs HT+Plt(asa)

CONFIDENTIAL - NOT FOR CITATION OR PUBLICATION

SummaryII In platelet-cancer cell co-cultures, platelet release of PGE2 contributed to enhance expression of Twist1 and down-regulation of E-cadherin.

Selective inhibition of platelet COX-1-dependent PGE2 by aspirin abrogated these changes of EMT marker genes.

Further studies are required to confirm in animal models whether this mechanism is involved in the anti-metastatic effect of aspirin detected in clinical trials with low-dose aspirin.

Outline Platelet angiogenesis and growth factor proteome Role of COXs in intestinal tumorigenesis Efficacy of aspirin in cardiovascular disease and colorectal cancer: a shared mechanism of action?

Enhanced TXA2 generation in colon neoplasia Platelets and metastasis Conclusions

Conclusions The benefit of low-dose aspirin in atherothrombosis and colorectal cancer (CRC) suggests that arterial occlusion and adenoma formation represent different phenotypes of the same abnormal repair process mediated by platelet activation at distinct sites of injury.

The study of the successful paradigm of CRC chemoprevention by aspirin may allow the characterization of novel mechanisms of disease and the development of biomarkers for early diagnosis and individualized prevention.

The Human Activated Platelet Neurodegeneration?

Amyloid b-peptide amyloid precursor peptide Inflammation

Growth Factors

COX-2 Induction

Colo-rectal carcinogenesis

Prostanoids

Inflammatory cytokines & oxygen radicals Patrono, Patrignani & Garcia Rodriguez JCI 2001;108:7-13 Dovizio & Patrignani, Recent Results Cancer Res. 2012;191:39-65 Patrono, in Michelson Ed. Platelets 2012

Vascular Occlusion

Myocardial Infarction Ischemic Stroke

Acknowledgements

Paola Patrignaniâ&#x20AC;&#x2122;s Lab Melania Dovizio, Post-Doc Stefania Tacconelli , Post-Doc Luigia Di Francesco, Post-Doc Emanuela Marcantoni, PostDoc Paloma Guillen, Post-Doc Sara Alberti, PhD stud Annalisa Bruno, PhD stud Undergraduate Students Angela Sacco Simone Schiavone Victoire Ndong

University of Frankfurt, Germany o Dieter Steinhilber oThorsten Maier (Post-Doc) Technical University Darmstadt oBeatrix Suess Catholic University, Rome, Italy Alessandro Sgambato Carlo Patrono

Acknowledgements Corimmun (Munich, Germany) o Gotz Munch

MandalMed, Inc., San Francisco, C.A., U.S.A. Constance M. John

• Dan A Dixon • University of Kansas Medical Center Kansas City, KS , USA

• Lucio Bertario • Register of Hereditary Colorectal Tumours, National Cancer Institute, Milan, Italy

• Luis A Garcia Rodriguez • Centro Español de Investigacion Farmacoepidemiologica (CEIFE), Madrid, Spain

• • • •

Emanuela Ricciotti John Lawson Garret A FitzGerald University of Pennsylvania, Philadelphia, USA

Finantial Support: Grant Associazione Italiana per la Ricerca sul Cancro (AIRC, IG-12111) and PRIN2011 to P.P.; M.D. fellowship was supported by grants from Frankfurt International Research School for Translational Biomedicine (FIRST) to D.S. and Reti per lâ&#x20AC;&#x2122;alta formazione project, Abruzzo Region, Italy.

Disclosure of Conflict of interests: Paola Patrignani declares no competing financial interests.

Multiple choice questions on the topics of Patrignaniâ&#x20AC;&#x2122;s lecture

What is the mechanism of action of Aspirin at lowdoses?

1. It inhibits the activity of COX-2 in platelets 2. It inhibits the activity of COX-2 in inflammatory cells 3. It inhibits the synthesis of TXB2 in platelets in the systemic circulation 4. It inhibits the synthesis of TXB2 in platelets in the pre-systemic circulation

Response_question 1 â&#x20AC;˘ What is the mechanism of action of Aspirin at low-doses? â&#x20AC;˘ 4. It inhibits the synthesis of TXB2 in platelets in the pre-systemic circulation

The results of randomized clinical trials with aspirin show that: 1. The atheroprotective effect of aspirin is dosedependent 2. The anti-cancer effect is dose-dependent 3. Both the atheroprotective and anti-cancer effects are maximal at low-doses 4. The anti-tumorigenic effect is detected only at anti-inflammatory (high) doses

Response_question 2 The results of randomized clinical trials with aspirin show that: 3. Both the atheroprotective and anti-cancer effects are maximal at low-doses

The preventive effect of aspirin against colorectal cancer mortality in randomized clinical trials is: 1.

2.

3.

4.

detected after one year of treatment detected after at least five years of treatment detected after one month of treatment not detected, in a statistically significant manner

Response_question 3 â&#x20AC;˘ The preventive effect of aspirin against colorectal cancer mortality in randomized clinical trials is 2. detected after at least five years of treatment

COX-independent mechanisms of aspirin

Several evidences have shown that some nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, are able to inhibit the proliferation and to induce apoptosis of colon cancer cells in vitro independently from their inhibitory effect on COX-dependent prostanoid biosynthesis (Hanif et al. Biochem Pharmacol 1996, 52:237-245).

These effects were usually detected in vitro at high concentrations of aspirin, often in the millimolar range.

COX-independent mechanisms of aspirin a IKKa

P

IkBa

NF-kB

IKKb

•

Inhibition of nuclear factor kappa B (NF-kB) signaling1

•

Interruption of extracellular signal-regulated kinases (ERK)2

•

Inhibition of Wnt/b-catenin signaling3

•

Capacity of aspirin to acetylate extra-COX proteins, eg p534

Proteasome degradation

b Ras c-Raf

A S P I R I N

ERK ERK

Substrate in cytosol and cytoskeleton

Gene transcription regulation

c

TCF Wnt

pathway

b-catenin

b-catenin

d DNA

p53

RNA

histone

1Yin

et al. Nature 1998;396:77-80; 2Pan et al. Cell Signal 2008;20:1134-1141; 3Bos et al. Oncogene 2006;25:6447–56; 4Alfonso et al. Int J Oncol 2009;34:597-608

Expression of TP and EPs receptors in HT29 cells

EP2 b-actin

1

n=2

nucleus

HT

HT

HT

Cytosol

Nucleus

0.8

1.0

0.5

0.0

n=2

HT nucleus

HT

EP4/b-actin ratio

EP2/b-actin ratio

2

0

HT

1.5

3

EP1/b-actin ratio

HT PLT

HT

citosol

b-actin

HT

nucleus

TP

citosol

b-actin

EP4 b-actin citosol

EP1

HT Cytosol

HT Nucleus

0.6 0.4 0.2 0.0

n=2

HT

HT

Cytosol

Nucleus

TXA2 TPa/b

TPa/b

Gaq11 Cooperative mitogenic signaling

TXA2

TXA2

TPa/b

Gaq

Ga12/13 Rho

PI3K (via bg)

PLCb

Dense granule Release (ADP) ADP P2Y1 P2Y1

PLCb

PTKs, PLCg

Shape change

ADP P2Y12

Gaq

TPa/b

P2Y12

Gai Adenylate Cyclase: cAMP PI3K (via bg) PLC (via bg)

a-granule release

Revacept structure and mode of action Platelet collagen receptors can interact with collagen binding sites of a vascular lesions or â&#x20AC;&#x153;collagen-likeâ&#x20AC;? domain of galectin-3, a member of carbohydratebinding proteins, highly expressed in tumor cells

Revacept as a dimeric Fc fusion protein with the IgG part and the extracellular domain of the human glycoprotein VI (GPVI) platelet receptor

Revacept can prevent the adhesion of platelets to injured vasculature or cancer cells

Ungerer et al. Circulation. 2011; 123: 1891-1899

Galectin-3 is a member of a family of carbohydrate-binding proteins but uniquely consists of a C-terminal carbohydrate recognition domain(CRD), a collagen-like internal R-domain, and the N-terminal domain, is highly elevated in malignancies including colon cancer(Yang, 2008).

It is localized inside the cells but also on cell surface where it mediates cell– cell and cell–matrix interactions by binding to glycoconjugates that contain βgalactosides via the CRD.

Galectin-3 is unique among galectins because it has the collagen-like domain.

N-terminal collagen-like domain

CRD

Gal-3C is the truncated form of galectin3 lacking 107 amino acids from the Nterminus, but it retains carbohydrate binding to glycoproteins containing glycans but is unable to cross-link them, thus acting as a dominant-negative inhibitor of full-length galectin-3

Inhibitors of galectin-3 function (β-lactose, a dominantnegative form of galectin-3,Gal-3C, and anti-galectin-3 antibody M3/38)

Dovizio et al Mol Pharmacol, April 2013