Targeting the cgmp Pathway to Treat Colorectal Cancer

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Thomas Jefferson University Jefferson Digital Commons Department of Pharmacology and Experimental Therapeutics Faculty Papers Department of Pharmacology and Experimental Therapeutics 29 Targeting the

1 Thomas Jefferson University Jefferson Digital Commons Department of Pharmacology and Experimental Therapeutics Faculty Papers Department of Pharmacology and Experimental Therapeutics 29 Targeting the cgmp Pathway to Treat Colorectal Cancer Giovanni Mario Pitari Thomas Jefferson University, Let us know how access to this document benefits you Follow this and additional works at: Part of the Medical Pharmacology Commons, and the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Pitari, Giovanni Mario, "Targeting the cgmp Pathway to Treat Colorectal Cancer" (29). Department of Pharmacology and Experimental Therapeutics Faculty Papers. Paper This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion in Department of Pharmacology and Experimental Therapeutics Faculty Papers by an authorized administrator of the Jefferson Digital Commons. For more information, please contact: JeffersonDigitalCommons@jefferson.edu.

2 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 1917

3 Translational Medicine Molecular Biology Cell Culture Laboratory Organ Culture Animal Models Clinical Trials Clinic

4 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

5 Cyclic GMP Signaling General Model for cgmp Signaling Agonist Pituitary Cells Ca 2+ Ca 2+ CNG cgmp GC GTP PDE1 + Ca 2+ - CNG + PKG cgmp - Ca 2+ + L-type Ca 2+ Channels Plasma membrane cnos PKA PKG PDE sgc + NO Modified from Li, T. et al. (23) Curr. Topics Biochem. Res. 5: Tiyyagura, S.R. et al. (24) Vit. Hormones 69:69-94

6 Lucas, et al. (2) Pharmacol. Rev. 52: Guanylyl Cyclases

Guanylyl Cyclase C (GCC) GCC is selectively expressed at brushborder membranes of

O H 2 O NaCl NaCl NaCl NaCl NaCl Crypt and Villus Enterocytes Gartner L.P.

Color Textbook of Histology Brush Border Microvilli Keeton W.T. and Gould, J.L.

7 Guanylyl Cyclase C (GCC) GCC is selectively expressed at brushborder membranes of intestinal epithelial cells and regulates fluid homeostasis H 2 O H 2 O H 2 O H 2 O H 2 O NaCl NaCl NaCl NaCl NaCl Crypt and Villus Enterocytes Gartner L.P. (1997) Williams & Wilkins Pub. Co., Baltimore and Hiatt, J.L. Color Textbook of Histology 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. (2) Pharmacol. Rev. 52:

8 Antiproliferative cgmp Signaling Targets Cyclic Nucleotide-Gated Channel Pitari, G.M. et al. (23) Proc. Natl. Acad. Sci. USA 1:

9 Cell Number, % DNA Synthesis, % Antiproliferative cgmp Signaling Undergoes Negative Feedback Regulation A 125 Preincubation 75 8-br-cGMP Preincubation B C PBS +ZAP+RP8pCPT 5 G /G Sub- G 1 S G 2 /M Proliferation, % Pitari, G.M. et al. (25) Cancer Res. 65:

10 The Antiproliferative cgmp Signaling Pathway in Intestinal Epithelial Cells CNG (+) GCC (-) PKG (+) cgmp (+) PDE5 Ca 2+ (-) (+) (-) M G 1 M G 1 G 2 S G 2 S Cell Cycle Pitari, G.M. et al. (25) Cancer Res. 65:

11 Cyclic GMP Signaling by GCC Controls The Crypt-Villus Homeostasis Differentiation Migration Inactive fibroblast Guanylin Uroguanylin Proliferation Active fibroblast Pitari, G.M. et al. (27) Clin. Pharmacol. Ther. 82:441-7

12 Colon Cancer: the 2nd Most Deadly Cancer in Developed Nations

13 Incidence Reversibility Cancer Risk The Pathological Sequence of Colorectal Cancer Carcinomas Dysplastic Adenomas Adenomatous Polyps Aberrant Crypt Foci Early Genetic Mutations

14 Colon Cancer: Diagnosis and Therapy Stage I Invasion up to the muscularis propria Stage II Invasion of the serosa and adjacent organs Stage III Invasion of regional lymph nodes Stage IV Distant Metastasis Surgery Surgery Surgery Surgery Chemiotherapy? Chemiotherapy Chemiotherapy 5-years survival ~ 95% ~ 8% ~ 65% ~ 7%

15 ETEC Infections Confer Resistance to Colon Cancer Pitari, G.M. et al. (23) Proc. Natl. Acad. Sci. USA 1:

16 GLN-stimulated thymidine incorporation (%) 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 Pitari GM, et al. (21) Proc. Natl. Acad. Sci. USA 98: CTR TJU URO 8-BrcGMP N D D C E L C V N V A C T G C L P G T C E I C A Y A A C T G C uroguanylin guanylin Carrithers S, et al. (1996) Proc. Natl. Acad. Sci. USA 93:

17 GCC is a Novel Intestinal Tumor Suppressor Li, P. et al. (27) Gastroenterology 133:599-67

18 % Inhibition of Proliferation % Inhibition of Proliferation Proliferation, % % Inhibition of Proliferation GCC Signaling through cgmp Potentiates Cytostatic Calcium Effects PBS , 24 h +, 3 h , 24 h, 3 h [Ca 2+ ] o, mm [Ca 2+ ] o, mm 75 T84 Caco-2 SW Pitari, G.M. et al. (28) Carcinogenesis 29:161-7

PBS CaR Merge h 3 h 24 h CaR Villin PBS PBS PBS Membrane GCC -/-

19 GCC Regulates the Function of Calcium-Sensing Receptor (CaR) in the Intestine CaR GAPDH h 3 h 24 h Cytosol GCC +/+ DAPI Villin PBS PBS PBS CaR Merge h 3 h 24 h CaR Villin PBS PBS PBS Membrane GCC -/- DAPI Villin Pitari, G.M. et al. (28) Carcinogenesis 29:161-7 CaR Merge

20 Inhibition of Proliferation, % Proliferation, % GCC-Targeted Therapy in Combination with Dietary Calcium 75 + Ca PBS Vector AS-CaR S-CaR Control Ca 2+ Mg 2+ Gd 3+ Spermine Pitari, G.M. et al. (28) Carcinogenesis 29:161-7

21 A Tumor Suppressor cgmp Signaling Pathway in Colon Cancer Uroguanylin Guanylin Spermine Ca 2+ Mg 2+ GCC CNG CaR Membrane GTP cgmp Ca 2+ Tumor Suppression Pitari, G.M. et al. (28) Carcinogenesis 29:161-7

5-Year Survival Rate, % Colon Cancer Mortality Reflects Metastatic Disease Progression 1 75 5 Local 39% 61% Metastasis 25 Local Disease

22 5-Year Survival Rate, % Colon Cancer Mortality Reflects Metastatic Disease Progression Local 39% 61% Metastasis 25 Local Disease Regional Metastasis Tumor Stage Distant Metastasis Lubbe, W.J. (26) Clin. Cancer Res. 12: Fidler IJ. (23) Nat. Rev. Cancer 3:

23 Relative Levels of MMP-9 mrna MMP-9-Dependent Gelatinolytic Activity, % MMP-9, % Relative Levels of MMP Protein Lubbe, W.J. et al. (29) Cancer Res. On Line First Cyclic GMP Induces Functional Remodeling of Cancer Cell MMP-9 A 2 1 PBS C prommp-9 Active MMP pCPT cgmp PBS 8-brcGMP 8-brcGMP 8-pCPT cgmp B prommp-9 (92-kDa) PBS kda T84 8-pCPT cgmp 8-brcGMP D prommp-9 GAPDH PBS PBS MMP-9 Caco-2 PBS MMP-2 MMP-9 + Primary Neoplasm Growth Vascularization Invasion Detachment Migration Extravasation Proliferation/angiogenesis Metastasis

24 Relative Levels of MMP-9 mrna Relative Levels of MMP-9 mrna Relative Levels of MMP-9 mrna MMP-9 Promotes Metastasis in Colon Cancer Tumor Epithelium Tumor Stroma LCM NAT Tumor 5 NAT Tumor + LCM Pro-MMP T84 Cell CM NTC T84 CaCo2 SW48-92 kda Stroma Epithelium Lubbe, W.J. et al. (26) Clin. Cancer Res. 12:

(ng/ml) 5 immp-9 TIMP-1 4 3 2 1 18 15 12 9 6 3 Control (n=4) MMP-9 (n=4) BB94 (n=4) TIMP-1

25 Inhibition of Cell Spreading, % Tumor Seeding (cells/field) MMP-9 Activity, % Colon Cancer Cell MMP-9 Induces Metastatic Seeding 12 Control (-)MMP Log TIMP-1 (ng/ml) 5 immp-9 TIMP Control (n=4) MMP-9 (n=4) BB94 (n=4) TIMP-1 (n=3) immp-9 (n=4) immp-9 + MMP-9 (n=3) Lubbe, W.J. et al. (26) Clin. Cancer Res. 12:

26 Inhibition of Cell Spreading, % Inhibition of Cell Spreading, % Inhibition of Cell Spreading, % GCC and cgmp Signaling through MMP-9 Regulates Colon Cancer Cell Shape and Spreading DIC DAPI β-actin MMP-9 Merge PBS br-cGMP Lubbe, W.J. et al. (29) Cancer Res. On Line First

27 Tumor Seeding, % GCC and cgmp Signaling through MMP-9 Suppresses Metastatic Seeding by Colon Cancer Cells T84 T84-V T84-MMP-9 12 PBS Lubbe, W.J. et al. (29) Cancer Res. On Line First

28 The Antimetastatic cgmp Signaling Pathway in Colon Cancer Cells (-) cgmp Pathway (+) cgmp Pathway GCC Matrix Degradation cgmp MMP-9 secretion Cell Spreading Metastasis Metastatic Seeding Tumor Containment / Vascular Clearance Lubbe, W.J. et al. (29) Cancer Res. On Line First

29 Summary The cgmp pathway in intestinal epithelial cells regulates the crypt-villus axis and opposes colorectal tumorigenesis 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 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 Cancer cell MMP-9 regulates metastatic functions, including actin polymerization and cell spreading, and in vivo seeding of target organs

30 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 Combinatorial strategies with GCC ligands and dietary calcium may provide a novel paradigm for the treatment of colon cancer Cancer cell MMP-9 is a highly selective and effective molecular target for preventing metastatic progression of colorectal cancer

Enterotoxin preconditioning restores calciumsensing receptor-mediated cytostasis in colon cancer cells

Enterotoxin preconditioning restores calciumsensing receptor-mediated cytostasis in colon cancer cells Thomas Jefferson University Jefferson Digital Commons Department of Pharmacology and Experimental Therapeutics Faculty Papers Department of Pharmacology and Experimental Therapeutics 6-19-2008 Enterotoxin