RAGE signaling sustains inflammation and promotes tumor development

Abstract

A broad range of experimental and clinical evidence has highlighted the central role of chronic inflammation in promoting tumor development. However, the molecular mechanisms converting a transient inflammatory tissue reaction into a tumor-promoting microenvironment remain largely elusive. We show that mice deficient for the receptor for advanced glycation end-products (RAGE) are resistant to DMBA/TPA-induced skin carcinogenesis and exhibit a severe defect in sustaining inflammation during the promotion phase. Accordingly, RAGE is required for TPA-induced up-regulation of proinflammatory mediators, maintenance of immune cell infiltration, and epidermal hyperplasia. RAGE-dependent up-regulation of its potential ligands S100a8 and S100a9 supports the existence of an S100/RAGE-driven feed-forward loop in chronic inflammation and tumor promotion. Finally, bone marrow chimera experiments revealed that RAGE expression on immune cells, but not keratinocytes or endothelial cells, is essential for TPA-induced dermal infiltration and epidermal hyperplasia. We show that RAGE signaling drives the strength and maintenance of an inflammatory reaction during tumor promotion and provide direct genetic evidence for a novel role for RAGE in linking chronic inflammation and cancer.

Figure 1.

Rage^−/− mice are protected against DMBA/TPA-induced tumor development. (A) wt and Rage^−/− mice were subjected to the DMBA/TPA skin carcinogenesis protocol and were assessed and statistically analyzed as described in Materials and methods. The graphs display the number of tumor-bearing mice (left, incidence) and the mean number of tumors per mouse (right, multiplicity) at the indicated time points after DMBA treatment. (B) Representative macroscopic pictures (top) and bright-field microscopic views of hematoxylin-eosin–stained sections (bottom) of wt and Rage^−/− tumors. Bars: (top) 1 mm; (bottom) 200 μm. (C) The percent total of Ki67-labeled tumor cells of three tumors from each genotype was calculated as described in Materials and methods. (D) Representative pictures of wt and Rage^−/− tumor sections that were analyzed by TUNEL assay (top, red signal) and by indirect IF staining of phospho-p65 protein (bottom, red signal), as described in Materials and methods. Nuclear staining was done with H33342 (blue signal). Inset shows a higher magnification of phospho-p65 stained nuclei. Bar, 100 μm. (E) The percent total of phospho-p65–labeled tumor cells within three tumors from each genotype was calculated as described in Materials and methods. Error bars represent the SEM.

Figure 2.

Impaired inflammatory response in Rage^−/− mice. (A) Innate immune cells within the stroma of at least six wt and Rage^−/− tumors were counted based on indirect IF stainings for neutrophils (antineutrophils, black bars) and macrophages (anti-CD68, gray bars), or on toluidine blue staining for mast cells (white bars), and are given as cells per square millimeter. Values represent means ± SEM from three mice from each genotype. (B) Representative pictures of hematoxylin-eosin–stained wt and Rage^−/− skin specimens that were collected at the indicated time points after a single topical treatment of acetone (co) or 10 nmol TPA in acetone. Dashed lines indicate the border between the epidermis and dermis. Bar, 20 μm. (C) Innate immune cells within the dermis of at least three wt and Rage^−/− mice treated as described in B were counted based on specific stainings and analyzed as described in A.

Figure 3.

Expression of proinflammatory genes in TPA-treated mice. (A) RQ-PCR was performed with cDNA from total wt and Rage^−/− skin samples treated once with acetone (0 h) or with 10 nmol TPA in acetone and collected at the indicated time points and by using primers for Mip-1α, Mip-1β, Mip-2, Ptgs2, S100a8, S100a9, Tnf-α, and Tgf-β3. Each cDNA from three mice of three separate animal experiments was analyzed in triplicates, and Hprt transcripts served as an internal reference. Error bars represent the SEM. (B) Representative pictures of sections derived from tissue specimens as described in A and analyzed for S100a9 and Mip-1α protein expression by IHC staining (brown signal) and followed by counterstaining with hematoxylin. Dashed lines indicate the border between the epidermis and dermis. co, acetone-treated controls. Bar, 20 μm.

Figure 4.

RAGE expression in immune cells is required for TPA-induced dermal infiltration and epidermal hyperplasia. (A) wt and Rage^−/− mice were treated with acetone (co) or 10 nmol of TPA three times every 48 h. Representative images are shown of hematoxylin-eosin–stained skin sections derived from specimens collected 3 d after the final treatment. Dashed lines indicate the border between the epidermis and dermis. Bar, 20 μm. (B) Representative images are shown of hematoxylin-eosin–stained skin sections derived from sublethally irradiated wt mice reconstituted with bone marrow cells from wt or Rage^−/− mice (wt→wt or Rage^−/−→wt), or sublethally irradiated Rage^−/− mice reconstituted with bone marrow cells from wt or Rage^−/− mice (wt→Rage^−/− or Rage^−/−→Rage^−/−), and were treated as described in A (top two rows). Representative images of sections that were analyzed for S100a8 protein expression by IHC (brown staining; bottom two rows) and that were counterstained with hematoxylin. Dashed lines indicate the border between the epidermis and dermis. Bar, 20 μm. (C) Innate immune cells within the dermis of at least six chimeric mice of each group that were treated as described in B were counted based on specific stainings as described in Fig. 2 C. (D) Percent totals of Ki67-positive keratinocytes of skin specimens from at least three chimera of each group that were treated as described in B, measured, and analyzed as described in Fig. 1 C. Error bars represent the SEM.

Figure 5.

Epidermal hyperplasia and tumor promotion after accelerated TPA treatment. (A) wt and Rage^−/− mice were treated with acetone (co) or 10 nmol TPA three times every 48 or 24 h, respectively, on the shaved back skin. Shown are the percent totals of Ki67-positive keratinocytes of three skin specimens from each genotype that were measured and analyzed as described in Fig. 1 C. (B) Representative images of skin sections from mice treated as described in A that were analyzed for S100a8, S100a9, and Tnf-α protein expression by IHC (brown staining) and counterstained with hematoxylin. Bar, 20 μm. (C) wt and Rage^−/− mice were subjected to a modified DMBA/TPA skin carcinogenesis protocol with TPA treatment every 24 h and were analyzed as described in Materials and methods.