Targeted deletion and lipidomic analysis identify epithelial cell COX-2 as a major driver of chemically induced skin cancer

Abstract

Pharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX-2) plays a critical role in DMBA/TPA-induced skin tumor induction. Although many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell type-specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared with littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2-expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell type-specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biologic responses.

Implications: Cox-2 gene deletion demonstrates that intrinsic COX-2 expression in initiated keratinocytes is a principal driver of skin carcinogenesis; lipidomic analysis identifies likely prostanoid effectors.

Figure 1. Epidermal keratinocyte-specific Cox-2 gene deletion reduces DMBA/TPA-induced mouse skin papilloma formation; in contrast myeloid cell-specific Cox-2 deletion has no effect on DMBA/TPA-induced skin tumor induction

(A) DMBA/TPA-induced skin papilloma sections rom a Cox-2^fl/fl mouse stained for H&E, COX-2 or F4/80. Scale bars: 50µm. (B) Cox-2^ΔE mice (n = 20) and Cox-2^fl/fl littermates (n = 17) were subjected to DMBA/TPA skin cancer induction. Left panel, the tumor incidence difference between Cox-2^ΔE and their Cox-2^fl/fl littermate mice was significant by χ² test (p<0.05). Right panel, tumor multiplicities; error bars, SEM. Significant differences when comparing these groups were determined by Mann-Whitney U test (p < 0.05–0.001, week 11–20). (C) DMBA/TPA tumor induction in Cox-2^ΔM (n = 15) and Cox-2^fl/fl littermate mice (n = 15). Error bars, SEM. No significant differences for tumor incidence (χ² test) or tumor multiplicities (Mann-Whitney U test) were detectable between the groups.

Figure 2. DMBA/TPA-induced tumors of Cox-2^ΔE mice are smaller than tumors of Cox-2^fl/fl mice, and do not express COX-2 in epithelial tumor cells

(A) Left panel; DMBA/TPA-induced tumors on Cox-2^ΔE and littermate Cox-2^fl/fl mice (20 weeks of TPA treatment). Right panel; DMBA/TPA-induced tumors on Cox-2^ΔM and littermate Cox-2^fl/fl mice (20 weeks of TPA treatment). (B) Upper panels; Papillomas from Cox-2^ΔE and littermate Cox-2^fl/fl mice were stained for COX-2. Lower panels; Papillomas from Cox-2^ΔM and littermate Cox-2^fl/fl mice were stained for COX-2. Scale bars: 100µm.

Figure 3. Epidermal hyperplasia is reduced in response to DMBA/TPA treatment in Cox-2^ΔE mice, but not in Cox-2^ΔM mice

(A) Micrographs (Top): H&E stained skin sections from Cox-2^ΔE and littermate Cox-2^fl/fl mice, either untreated or after 20 weeks of DMBA/TPA treatment. Graph (bottom): Epidermal thickness quantification. Error bars, SD. **; p<0.01. (B) Micrographs (Top): H&E stained skin sections from Cox-2^ΔM and littermate Cox-2^fl/fl mice, either untreated or after 20 weeks of DMBA/TPA treatment. Graph (bottom): Epidermal thickness quantification. Twenty fields/mouse, four mice/genotype were counted for all fields. Error bars, SD. n.s., p>0.05. Scale bars: 50µm.

Figure 4. DMBA/TPA-induced skin proliferative responses and epidermal cell differentiation in Cox-2^ΔE mice and Cox-2^fl/fl littermate controls

(A) Untreated skin samples and skin samples from Cox-2^fl/fl and Cox-2^ΔE littermates taken 18 h after the last TPA treatment of the DMBA/TPA-tumor induction protocol, immunostained for Ki67. Arrows indicate Ki67-positive cells. Scale bars: 50µm. (B) Percentage of Ki67-positive basal cells in skin samples from untreated mice and DMBA/TPA-treated Cox-2^ΔE and Cox-2^fl/fl littermates (C) Untreated skin samples and skin samples from Cox-2^fl/fl and Cox-2^ΔE littermates taken 18 hours after the last TPA treatment, immunostained for K1. Arrows indicate K1-positive basal cells. Scale bars: 50µm. (D) Percentage of K1-positive basal cells in skin samples from untreated mice and DMBA/TPA-treated Cox-2^ΔE and littermate Cox-2^fl/fl mice. Ki67-positive and K1-positive basal cells were determined on at least 4 random areas for each section, 3 sections/mouse and 3 mice/genotype. Error bars, SD. *, p<0.05.

Figure 5. Proliferation and epithelial cell differentiation in DMBA/TPA-induced papillomas from Cox-2^ΔE mice, Cox-2^ΔM mice, and their Cox-2^fl/fl littermates

DMBA/TPA-induced papillomas from at least four mice of each of the four groups were fixed, paraffin embedded, and immunostained for Ki67 and K1. (A) Micrographs (Left panels): Ki67-stained papilloma sections from Cox-2^ΔE and littermate Cox-2^fl/fl mice. Graphs (right panels): Percentage of Ki67-positive cells in papillomas from Cox-2^ΔE mice and their Cox-2^fl/fl littermates, and from Cox-2^ΔM mice and their Cox-2^fl/fl littermates. Each data point is the Ki67 value for one papilloma. (B) Micrographs (Left panels): K1-stained papilloma sections from Cox-2^ΔE and littermate Cox-2^fl/fl mice. Graphs (right panels): Percentage of K1-positive cells in papillomas from Cox-2^ΔE mice and their Cox-2^fl/fl littermates, and from Cox-2^ΔM mice and their Cox-2^fl/fl littermates. Each data point is the K1 value for one papilloma, ***, p<0.001. Scale bars: 100µm.

Figure 6. Eicosanoids of skin and tumors from mice with DMBA/TPA-induced skin tumors

(A) Total eicosanoids in tumors (n = 12) of DMBA/TPA-treated Cox-2^ΔE mice, their Cox-2^fl/fl littermates (n = 12), Cox-2^ΔM mice (n = 14), and their Cox-2^fl/fl littermates (n = 13). (B) Eicosanoids, separated by pathway of origin (COX-dependent, non-enzymatic, lipoxygenase-dependent and cytochrome P450 dependent), present in the tumor groups. Error bars, SD. *, p<0.05, determined by a two-tailed student’s t-test. (C) Heat maps depicting differences in COX-dependent eicosanoid amounts in Cox-2^ΔE mice, Cox-2^ΔM mice, and their respective Cox-2^fl/fl littermates. For each eicosanoid, the amount in tumors from the Cox-2^ΔE or Cox-2^ΔM mice was divided by the amount in tumors from their control Cox-2^fl/fl mice. Green indicates fold-decrease versus Cox-2^fl/fl control tumors. Red indicates fold increases. Grey, no significant change compared to control Cox-2^fl/fl tumors. Black (ND), the eicosanoid was either not detected or below the detectable limit. Only eicosanoids significantly different (p<0.05) from control littermates are shown. (D) PGF_2α and PGE₂ amounts detected in untreated skin, in skin from mice treated with DMBA/TPA, and in papillomas from Cox-2^ΔE mice, Cox-2^ΔM mice, and their cohort littermate Cox-2^fl/fl control mice (untreated skin from all mice, DMBA/TPA-treated skin of Cox-2^ΔM mice and DMBA/TPA-treated skin of control Cox-2^fl/fl mice, n=6; DMBA/TPA-treated skin of Cox-2^ΔE mice, n=8; DMBA/TPA-treated-skin of control Cox-2^fl/fl mice, n=7). For papillomas, numbers of samples are the same as panels 6A–6C. Error bars, SD. **, p<0.005, determined by a two-tailed Student’s t-test.

Figure 7. Macrophage infiltration and blood vessel density in DMBA/TPA-induced papillomas from Cox-2^ΔE mice, Cox-2^ΔM mice, and their Cox-2^fl/fl littermates

DMBA/TPA-induced papillomas from mice of the four groups were fixed, paraffin embedded, and immunostained for F4/80 and for CD31. (A) Micrographs (Left panels): F4/80-stained papilloma sections from Cox-2^ΔE mice, Cox-2^ΔM mice, and their respective Cox-2^fl/fl littermates. Graphs (right panels): Number of F4/80-positive cells/papilloma tumor area for Cox-2^ΔE mice and their Cox-2^fl/fl littermates, and for Cox-2^ΔM mice and their Cox-2^fl/fl littermates. Each data point is the F4/80 value for one papilloma. (B) Micrographs (Left panels): CD31-stained papilloma sections from Cox-2^ΔE mice, Cox-2^ΔM mice, and their respective Cox-2^fl/fl littermates. Graphs (right panels): Vessel density in papillomas from Cox-2^ΔE mice and their Cox-2^fl/fl littermates, and from Cox-2^ΔM mice and their Cox-2^fl/fl littermates. Each data point is the vessel density value for one papilloma. **, p<0.01. n.s, p>0.05. Scale bars: 100µm.