1. Volume 137, Issue 4, Pages 1358-1366.e3 (October 2009)
Cyclooxygenase-2 Inhibitors Down-regulate Osteopontin and Nr4a2—New Therapeutic Targets for Colorectal Cancers 
Rachid Zagani, Nadim Hamzaoui, Wulfran Cacheux, Aurélien de Reyniès, Benoît Terris, Stanislas Chaussade, Béatrice Romagnolo, Christine Perret, Dominique Lamarque 
Gastroenterology 
Volume 137, Issue 4, Pages e3 (October 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
Inhibition of stromal COX-2 prolongs survival and reduces polyp load of ApcΔ14/+ mice. Adenomas of small intestine (A, original magnification 10×) and colon (B, original magnification 10×) from ApcΔ14/+ mice stained with anti–COX-2 antibody. ApcΔ14/+ mice were treated with parecoxib or saline for 9 weeks, the percentage of surviving animals was analyzed alongside the experience (C), and polyp load was obtained by adding all adenoma diameters (D) (n = 8–11 per group in each experience). (E) PGE metabolite concentrations in intestinal adenomas treated with parecoxib. *P < .05 as compared to H0.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
Down-regulation of OPN expression in intestinal polyps. (A) OPN transcripts from normal mucosa and small intestinal polyps of ApcΔ14/+ mice, treated with parecoxib (2 hours, 6 hours, 24 hours, 5 days, or 9 weeks) or saline (0 hours, control), measured by qRT-PCR. Expression is shown as fold induction relative to saline-treated mice (*P < .05). (B) In situ hybridization of OPN in small intestinal polyps of ApcΔ14/+ mice treated with saline (top, original magnification 4×) or parecoxib (bottom, original magnification 4×) for 9 weeks. (C) In situ hybridization of OPN in small intestinal crypts of wild-type mice (original magnification 4×).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
Repression of the Wnt/β-catenin pathway in adenomas of ApcΔ14/+ mice treated with parecoxib. (A) Axin2, (B) c-Myc, and (C) cyclin D1 transcripts from normal mucosa and small intestinal polyps of ApcΔ14/+ mice, treated with parecoxib (2 hours, 6 hours, 24 hours, 5 days, or 9 weeks) or saline (0 hours, control), measured by qRT-PCR. Expression is shown as fold induction relative to saline-treated mice (*P < .05). (D) Nuclear β-catenin staining of small intestinal adenoma sections (original magnification 10×). ApcΔ14/+ mice were treated with parecoxib (2 hours, 5 days, and 9 weeks [9w]) or saline (nontreated).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
Parecoxib induces Nr4a2 down-regulation in mouse intestinal polyps. (A) Nr4a2 expression in normal mucosa and small intestine polyps from ApcΔ14/+ mice measured by qRT-PCR after 2 hours, 6 hours, 24 hours, 5 days, and 9 weeks of parecoxib treatment. Expression is shown as fold induction relative to saline-treated mice (*P < .05). (B) Staining with anti-NR4A2 antibody of small intestinal adenoma sections (original magnification 10×) from ApcΔ14/+ mice treated with parecoxib (2 hours [2h] and 9 weeks [9w]) or saline (nontreated). (C) Staining of small intestinal crypts from wild-type mice with anti-NR4A2 antibody (original magnification 10×). (For control immunohistochemistry stainings of secondary antibodies, see Supplementary Figure 3).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
Nr4a2 activates OPN promoter. (A) CT26 cells were cultured for 48 hours. Equal amounts of protein were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and visualized with anti-Nr4a2 antibody. (B) CT26 cells were transiently transfected with constructs OPN-LUC, OPN S1mut-LUC, pCMX-Nurr1, Nurr1DN (see text for plasmid description), and pGL3 empty vector. Measures of Nr4a2 messenger RNA by (C) qRT-PCR and (D) OPN-LUC activity after cotransfection of anti-Nr4a2 siRNAs and the reporter plasmid. Data are representative of 3 independent experiments (*P < .05).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
Rofecoxib inhibits NR4A2 expression in human colon adenomas. Staining with anti-NR4A2 antibody was performed on polyps resected in the same patient, before the start of therapy (baseline polyp), and after 3 years (3-year polyp) of treatment with rofecoxib (25 mg/day).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

8. Figure 7
Unlike OPN, NR4A2 increased expression is not correlated to advancing tumor stage. (A and B) OPN and (C and D) NR4A2 expression in (A and C) human and (B and D) mouse intestinal tumors measured by qRT-PCR. Human adenocarcinomas were grouped according to American Joint Committee on Cancer stages (see text for detailed description of the mouse models). OPN gene expression in human and mouse tumors increases concordantly with advancing tumor stage, whereas NR4A2 gene expression is already maximally induced at the adenoma stage. Expression is shown as fold induction relative to control intestinal tissue (see text for description of controls) (*P < .05).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

9. Supplementary Figure 1
Expression of control gene cyclophilin A is not modified by parecoxib treatment. Cyclophilin A transcripts from normal mucosa and small intestinal polyps of ApcΔ14/+ mice, treated with parecoxib (2 hours, 6 hours, 24 hours, 5 days, or 9 weeks) or saline (0 hours, control), measured by qRT-PCR. Expression is shown as fold induction relative to saline-treated mice.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

10. Supplementary Figure 2
Western blot analysis of Wnt targets in treated polyps and controls. Wnt targets cyclin D1 and c-myc are reduced in polyps treated several weeks with parecoxib.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

11. Supplementary Figure 3
Control immunohistochemistry stainings with the secondary antibody alone. (A and C) Mouse and (B) human adenomas were probed with (A and B) anti-rabbit and (C) anti-mouse secondary antibodies (II Ab) conjugated to horseradish peroxidase.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

12. Supplementary Figure 4
OPN overexpression in a human colon adenoma. In situ hybridization revealed a strong expression of OPN in adenomatous glands as compared with normal ones (bottom left) (original magnification 20×). We could not evidence with this technique a reduction of OPN expression in treated polyps (not shown).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions