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Department of Pharmacology and ExperimentalTherapeutics Faculty Papers

Department of Pharmacology and ExperimentalTherapeutics

2009

Targeting the cGMP Pathway to Treat ColorectalCancerGiovanni Mario PitariThomas Jefferson University, gmpitari@gmail.com

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Recommended CitationPitari, Giovanni Mario, "Targeting the cGMP Pathway to Treat Colorectal Cancer" (2009).Department of Pharmacology and Experimental Therapeutics Faculty Papers. Paper 21.http://jdc.jefferson.edu/petfp/21

Targeting the cGMP Pathway to

Treat Colorectal Cancer

GianMario Pitari, M.D., Ph.D.

Department of Pharmacology and

Experimental Therapeutics

Thomas Jefferson University

Philadelphia, PA 19107

Translational Medicine

Molecular Biology

Cell Culture

Organ Culture

Animal Models

Clinical Trials

Laboratory Clinic

Translational Research Project:

from the cGMP Pathway to

Colorectal Cancer

Targeting Strategies:

1. Cyclic GMP-Dependent Pathway as a Tumor Suppressor System to Prevent Colorectal Tumorigenesis

2. Cyclic GMP-Dependent Pathway as an Antimetastatic Strategy to Disrupt Colorectal Cancer Metastatic Progression

GTP cGMP

PDE PKG PKA

CNG GC

Agonist

Ca2+

cGMP

sGC

Plasma

membrane

Ca2+

CNG

Ca2+

L-type Ca2+

Channels

cNOS

NO

PKG

PDE1

+ Ca2+

Pituitary Cells

+

+

+ -

-

General Model for cGMP Signaling

Cyclic GMP Signaling

Tiyyagura, S.R. et al. (2004) Vit. Hormones 69:69-94 Modified from Li, T. et al. (2003)

Curr. Topics Biochem. Res. 5:181-92

Guanylyl Cyclases

Lucas, et al. (2000)

Pharmacol. Rev. 52: 375-413

H2O H2O

H2O H2O

H2O

NaCl NaCl

NaCl

NaCl NaCl

Brush Border

Microvilli Keeton W.T. and Gould, J.L. Biological Science

(1986) W.W. Norton & Co., Inc. New York: p.139

Lucas, et al. (2000) Pharmacol. Rev. 52: 375-413

GCC is selectively expressed at brush-border membranes of intestinal epithelial cells and regulates fluid homeostasis

Crypt and Villus

Enterocytes Gartner L.P. (1997) Williams & Wilkins Pub. Co., Baltimore

and Hiatt, J.L. Color Textbook of Histology

Guanylyl Cyclase C (GCC)

Pitari, G.M. et al. (2003) Proc. Natl. Acad. Sci. USA 100:2695-99

Antiproliferative cGMP Signaling Targets

Cyclic Nucleotide-Gated Channel

A

B PBS

ST+ZAP+RP8pCPT

0

10

20

30

40

50

Cell N

um

ber,

%

Sub-

G1

G0/G1

S

G2/M

0 20 40 60 80 100

Proliferation, %

** *

**

C

DN

A S

yn

the

sis

, %

0

25

50

75

100

125

ST

Preincubation

* *

**

0

15

30

45

60

75 8-br-cGMP

Preincubation

** **

Antiproliferative cGMP Signaling

Undergoes Negative Feedback

Regulation

Pitari, G.M. et al. (2005) Cancer Res. 65:11129-35

Cell Cycle

G1

S

M

G2

G1

S

M

G2

GCC

cGMP PDE5

CNG

PKG

Ca2+

(+)

(+)

(-)

(-)

(-)

(+)

(+)

The Antiproliferative cGMP Signaling

Pathway in Intestinal Epithelial Cells

Pitari, G.M. et al. (2005) Cancer Res. 65:11129-35

Differentiation

Migration

Inactive fibroblast

Active fibroblast

Proliferation

Guanylin Uroguanylin

Cyclic GMP Signaling by GCC Controls

The Crypt-Villus Homeostasis

Pitari, G.M. et al. (2007)

Clin. Pharmacol. Ther. 82:441-7

Colon Cancer: the 2nd Most Deadly

Cancer in Developed Nations

The Pathological Sequence

of Colorectal Cancer

Adenomatous Polyps

Aberrant Crypt Foci

Early Genetic Mutations

Dysplastic Adenomas

Carcinomas

Reve

rsib

ilit

y

Can

ce

r R

isk

Incid

en

ce

Colon Cancer:

Diagnosis and Therapy

Stage IV

Distant Metastasis

Stage III

Invasion of regional

lymph nodes

Stage II

Invasion of the serosa

and adjacent organs

5-years

survival

Stage I

Invasion up to the

muscularis propria

Surgery Surgery Surgery Surgery

Chemiotherapy

? Chemiotherapy Chemiotherapy

~ 95% ~ 80% ~ 65% ~ 7%

Pitari, G.M. et al. (2003)

Proc. Natl. Acad. Sci. USA 100:2695-99

ETEC Infections Confer

Resistance to Colon Cancer

GCC is a Therapeutic Target

in Colon Cancer

N T F Y C C E L C C N P A C A G C Y

ST

N D D C E L C V N V A C T G C L

uroguanylin

P G T C E I C A Y A A C T G C

guanylin

Carrithers S, et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14827-32

Pitari GM, et al. (2001) Proc. Natl. Acad. Sci. USA 98: 7846-51

GL

N-s

tim

ula

ted

th

ym

idin

e

0

25

50

75

100

CTR TJU ST URO 8-Br- cGMP

inco

rpo

rati

on

(%

)

**

*

*

* *

*

Li, P. et al. (2007) Gastroenterology 133:599-607

GCC is a Novel Intestinal

Tumor Suppressor

1 10 100 0

25

50

75

100

[Ca2+]o, mM

Pro

life

rati

on

, %

**

** *

PBS +ST, 3 h +ST, 24 h

0

25

50

75

100

1 3 10 30 50

% I

nh

ibit

ion

of

Pro

life

rati

on

ST, 24 h

ST, 3 h

[Ca2+]o, mM

0

25

50

75 T84 Caco-2 SW480

% I

nh

ibit

ion

of

Pro

life

rati

on

** *

*

*

*

*

*

*

0

20

40

60

% I

nh

ibit

ion

of

Pro

life

rati

on

ST

*

* * *

Pitari, G.M. et al. (2008)

Carcinogenesis 29:1601-7

GCC Signaling through cGMP

Potentiates Cytostatic

Calcium Effects

Villin

CaR

PBS ST PBS ST PBS ST

0 h 3 h 24 h

Mem

bra

ne

GAPDH

CaR

PBS ST PBS ST PBS ST

0 h 3 h 24 h

Cyto

so

l

GCC-/- DAPI Villin

Merge CaR

GCC+/+ DAPI Villin

Merge CaR

GCC Regulates the Function

of Calcium-Sensing Receptor

(CaR) in the Intestine

Pitari, G.M. et al. (2008)

Carcinogenesis 29:1601-7

0

25

50

75

Vector AS-CaR S-CaR Inh

ibit

ion

of

Pro

life

rati

on

, %

ST + Ca2+

*

*

0

25

50

75

100

125

Ca2+ Mg2+ Gd3+ Spermine

Pro

life

rati

on

, %

PBS ST

**

* * *

Control

GCC-Targeted Therapy

in Combination with

Dietary Calcium

Pitari, G.M. et al. (2008) Carcinogenesis 29:1601-7

GCC

ST

Guanylin

Uroguanylin

CNG

Membrane

cGMP

Ca2+

CaR

Ca2+ Mg2+

Spermine

Tumor

Suppression

GTP

A Tumor Suppressor cGMP Signaling

Pathway in Colon Cancer

Pitari, G.M. et al. (2008)

Carcinogenesis 29:1601-7

Colon Cancer Mortality Reflects

Metastatic Disease Progression

Tumor Stage

0

25

50

75

100

Local

Disease

Regional

Metastasis

Distant

Metastasis

5-Y

ea

r S

urv

iva

l R

ate

, %

Metastasis

Local

61%

39%

Fidler IJ. (2003) Nat. Rev. Cancer 3: 453 -458

Lubbe, W.J. (2006) Clin.

Cancer Res. 12:1876 -82

Primary

Neoplasm

+

Metastasis

Vascularization

Growth

Invasion

Detachment

Migration

Extravasation

Proliferation/angiogenesis

MMP-9

Cyclic GMP Induces Functional

Remodeling of Cancer Cell MMP-9

Lubbe, W.J. et al. (2009) Cancer Res. On Line First

MM

P-9

-Dep

en

de

nt

Gela

tin

oly

tic A

cti

vit

y, %

0

20

40

60

80

100

8-pCPT

cGMP

8-br-

cGMP

ST

* ** *

PBS

proMMP-9

Active MMP-9

kDa

92

84

ST PBS

***

Caco-2

0

20

40

60

80

100

0

20

40

60

80

100

MM

P-9

, %

*** **

8-pCPT

cGMP

8-br-

cGMP

ST

**

PBS

T84

proMMP-9

(92-kDa)

Rela

tive L

evels

of

MM

P-9

mR

NA

2

1

0 8-pCPT

cGMP

8-br-

cGMP

ST PBS

proMMP-9

GAPDH

PBS ST

0

1

2

PBS MMP-9 Re

lative

Le

vels

of M

MP

Pro

tein

MMP-2

* ST

A B

C

D

MMP-9 Promotes Metastasis in

Colon Cancer

NAT Tumor 5

10

15

20

25

NAT Tumor 5

10

15

20

25

Rela

tiv

e L

eve

ls

of

MM

P-9

mR

NA

*** Tumor Stroma Tumor Epithelium

- LCM

+ LCM

0

5

10

15

Rela

tiv

e L

eve

ls

of

MM

P-9

mR

NA

Stroma

Epithelium

0

5

10

15

20

NTC T84 CaCo2 SW480

Rela

tiv

e L

evels

of

MM

P-9

mR

NA

Pro-MMP-9 - 92 kDa

Cell CM

T84

Lubbe, W.J. et al. (2006) Clin. Cancer Res. 12:1876-82

Colon Cancer Cell MMP-9 Induces

Metastatic Seeding

0

20

40

60

80

100

120

0 1 2 3 4

Log TIMP-1 (ng/ml)

MM

P-9

Acti

vit

y,

%

**

***

***

Inh

ibit

ion

of

Cell

Sp

read

ing

, %

*** ***

0

10

20

30

40

50 iMMP-9 TIMP-1

Control (-)MMP-9

Control

(n=4) iMMP-9

(n=4)

TIMP-1

(n=3)

0

3

6

9

12

15

18

Tu

mo

r S

eed

ing

(cell

s/f

ield

)

BB94

(n=4)

iMMP-9

+

MMP-9

(n=3)

*** ***

***

MMP-9

(n=4)

Lubbe, W.J. et al. (2006) Clin. Cancer Res. 12:1876-82

DIC DAPI MMP-9 β-Actin Merge

PBS

ST

0

10

20

30

40 *

*

Inh

ibit

ion

of

Cell

Sp

read

ing

, %

*

0

10

20

30

40

Inh

ibit

ion

of

Cell

Sp

read

ing

, %

*

8-br-cGMP ST

**

Lubbe, W.J. et al. (2009)

Cancer Res. On Line First

GCC and cGMP Signaling through

MMP-9 Regulates Colon Cancer Cell

Shape and Spreading

0

10

20

30

40

Inh

ibit

ion

of

Cell

Sp

read

ing

, %

*

ST

PBS

ST

T84-V T84-MMP-9 T84

0

20

40

60

80

100

120

Tu

mo

r S

ee

din

g,

%

*** ***

***

ST

*** *

Lubbe, W.J. et al. (2009) Cancer Res. On Line First

GCC and cGMP Signaling through

MMP-9 Suppresses Metastatic

Seeding by Colon Cancer Cells

Metastasis

GCC

cGMP MMP-9

secretion Cell

Spreading

Metastatic

Seeding

Matrix

Degradation

Tumor

Containment

/ Vascular

Clearance

The Antimetastatic cGMP Signaling

Pathway in Colon Cancer Cells

Lubbe, W.J. et al. (2009) Cancer Res. On Line First

(-) cGMP Pathway (+) cGMP Pathway

Summary

•The cGMP pathway reduces the metastatic potential of colorectal cancer

cells, in vitro and in vivo, in part by regulating the function of MMP-9

•The cGMP pathway in intestinal epithelial cells regulates the

crypt-villus axis and opposes colorectal tumorigenesis

•Cancer cell MMP-9 regulates metastatic functions, including actin

polymerization and cell spreading, and in vivo seeding of target organs

•GCC, a guanylyl cyclase receptor selectively expressed by

normal and malignant intestinal epithelial cells, coordinates a

paracrine tumor suppressor system in the intestine

•The cGMP pathway potentiates the cytostatic effects of extracellular

calcium by regulating the activity of CaR

Translational Significance

• GCC ligands represents novel agents for the

prevention of primary and metastatic colon cancer

• GCC ligands represents novel agents for the

treatment of primary and metastatic colon cancer

• Cancer cell MMP-9 is a highly selective and

effective molecular target for preventing

metastatic progression of colorectal cancer

• Combinatorial strategies with GCC ligands and

dietary calcium may provide a novel paradigm for

the treatment of colon cancer