PPAR-gamma is expressed and NF-kB pathway is activated and correlates positively with COX-2 expression in stromal myofibroblasts surrounding colon adenocarcinomas

Abstract

Purpose: Accumulated evidence indicates that carcinogenesis is closely associated with the transformation of normal stroma into a 'reactive' stromal phenotype. The present study investigated the role of PPARgamma, COX-2 and p-IkB-alpha--important molecular targets of colon cancer chemoprevention--in this stromal remodeling by evaluating and comparing the expression of these factors in stromal myofibroblasts, macrophages and endothelial cells that surround normal colonic mucosa and colon cancer.

Methods: Immunohistochemical methodology was employed on archived paraffin-embedded sections prepared from tumors and adjacent normal colon from 45 patients with colon adenocarcinomas. Double immunostaining with the universal marker for myofibroblasts (alpha-smooth muscle actin/alpha-SMA) as second primary antibody was also performed.

Results: Stromal macrophages and endothelial cells expressed these factors both in normal colonic mucosa and colon cancer. By contrast, stromal myofibroblasts expressed PPARgamma, COX-2 and p-IkB-alpha only in colon adenocarcinomas (77.7%, 100% and 100% of cases, respectively) and not in normal colon. COX-2 and p-IkB-alpha expressions were strongly correlated in these cells (P < 0.001). PPARgamma, COX-2 and p-IkB-alpha expression did not correlate with the stage or differentiation of the adenocarcinomas.

Conclusions: NF-kB pathway is activated and COX-2 expression is upregulated in stromal myofibroblasts surrounding colon adenocarcinomas compared to normal colon. Induction of COX-2 expression is primarily induced by NF-kB. NSAIDs, selective COX-2 inhibitors and PPARgamma ligands may exert their chemoprophylactic properties through direct actions on these cells.

Fig. 1

Expression of COX-2, p-IkB-α and PPARγ in normal colonic stroma. α-SMA (universal marker of myofibroblasts) staining is also shown for identification of myofibroblasts in normal colonic stroma. a COX-2 is expressed in macrophages (black arrow) and endothelial cells (white arrow) in normal colonic stroma, while there is no staining for COX-2 in myofibroblasts (X40). b α-SMA staining in normal colonic stroma showing myofibroblasts (X40). c p-IkB-α expression in macrophages (black arrows) and endothelial cells (white arrows) in the stroma of normal colon (X40). No staining for myofibroblasts is identified. d PPARγ expression in macrophages (black arrows) and endothelial cells (white arrows) in normal colonic stroma (X40). No obvious staining for myofibroblasts is identified

Fig. 2

a Myofibroblasts (white arrow) and macrophage (black arrow) exhibiting COX-2 expression in a well differentiated adenocarcinoma (X40). b Stroma of a moderately differentiated adenocarcinoma showing myofibroblasts (white arrow) and endothelial cells (black arrow) expressing COX-2 (X40). c COX-2 expression in myofibroblasts (white arrow) and endothelial cells (black arrow) in the stroma of a well-differentiated adenocarcinoma (X40). d Myofibroblasts in the stroma of a moderately differentiated adenocarcinoma exhibiting p-IkB-α expression (X40)

Fig. 3

a Macrophages (white arrow) and endothelial cells (black arrow) exhibiting p-IkB-α expression in a well-differentiated adenocarcinoma (X40). b Stroma of a poorly differentiated adenocarcinoma showing myofibroblasts (white arrows) and macrophage (black arrow) expressing p-IkB-α (X40). c PPARγ expression in stromal myofibroblasts (black arrows), endothelial cells (arrow head) and macrophages (white arrows) (X40). d PPARγ expression in stromal myofibroblasts (black arrows), endothelial cells (arrow head) and macrophages (white arrows) (X40)

Fig. 4

a, b Double immunolabeling of COX-2 (brown) and α-SMA actin (red) reveals stromal myofibroblasts expressing COX-2 (black arrows) (X40). c, d Double immunolabeling of p-IkB-α (brown) and α-SMA actin (red) reveals stromal myofibroblasts expressing p-IkB-α (black arrows) (X40)