Mice lacking epidermal PPARγ exhibit a marked augmentation in photocarcinogenesis associated with increased UVB-induced apoptosis, inflammation and barrier dysfunction

Recent studies suggest that peroxisome proliferator-activated receptor gamma (PPARγ) agonists may have cancer chemopreventive activity. Other studies have shown that loss of epidermal PPARγ results in enhanced chemical carcinogenesis in mice via unknown mechanisms. However, ultraviolet B (UVB) exposure represents the primary etiological agent for skin cancer formation and the role of PPARγ in photobiology and photocarcinogenesis is unknown. In previous studies, we demonstrated that UVB irradiation of cells results in the formation of oxidized glycerophosphocholines that exhibit PPARγ ligand activity. We therefore hypothesized that PPARγ would prove to be a chemopreventive target in photocarcinogenesis. We first showed that UVB irradiation of mouse skin causes generation of PPARγ agonist species in vivo. We then generated SKH-1 hairless, albino mice deficient in epidermal Pparg (Pparg-/-(epi)) using a cytokeratin 14 driven Cre-LoxP strategy. Using a chronic model of UVB photocarcinogenesis, we next showed that Pparg-/-(epi) mice exhibit an earlier onset of tumor formation, increased tumor burden and tumor progression. Increased tumor burden in Pparg-/-(epi) mice was accompanied by a significant increase in epidermal hyperplasia and p53 positive epidermal cells in surrounding skin lacking tumors. After acute UVB irradiation, Pparg-/-(epi) mice exhibited an augmentation of both UVB-induced Caspase 3/7 activity and inflammation. Increased apoptosis and inflammation was also observed after treatment with the PPARγ antagonist GW9662. With chronic UVB irradiation, Pparg-/-(epi) mice exhibited a sustained increase in erythema and transepidermal water loss relative to wildtype littermates. This suggests that PPARγ agonists could have possible chemopreventive activity in non-melanoma skin cancer

Inhibition of epidermal growth factor receptor signalling reduces hypercalcaemia induced by human lung squamous-cell carcinoma in athymic mice

The purpose of this study was to evaluate the role of the epidermal growth factor receptor (EGFR) in parathyroid hormone-related protein (PTHrP) expression and humoral hypercalcaemia of malignancy (HHM), using two different human squamous-cell carcinoma (SCC) xenograft models. A randomised controlled study in which nude mice with RWGT2 and HARA xenografts received either placebo or gefitinib 200 mg kg−1 for 3 days after developing HHM. Effectiveness of therapy was evaluated by measuring plasma calcium and PTHrP, urine cyclic AMP/creatinine ratios, and tumour volumes. The study end point was at 78 h. The lung SCC lines, RWGT2 and HARA, expressed high levels of PTHrP mRNA as well as abundant EGFR protein, but very little erbB2 or erbB3. Both lines expressed high transcript levels for the EGFR ligand, amphiregulin (AREG), as well as, substantially lower levels of transforming growth factor-α (TGF-α), and heparin binding-epidermal growth factor (HB-EGF) mRNA. Parathyroid hormone-related protein gene expression in both lines was reduced 40–80% after treatment with 1 μM of EGFR tyrosine kinase inhibitor PD153035 and precipitating antibodies to AREG. Gefitinib treatment of hypercalcaemic mice with RWGT2 and HARA xenografts resulted in a significant reduction of plasma total calcium concentrations by 78 h. Autocrine AREG stimulated the EGFR and increased PTHrP gene expression in the RWGT2 and HARA lung SCC lines. Inhibition of the EGFR pathway in two human SCC models of HHM by an anilinoquinazoline demonstrated that the EGFR tyrosine kinase is a potential target for antihypercalcaemic therapy

Topical application of a platelet activating factor receptor agonist suppresses phorbol ester-induced acute and chronic inflammation and has cancer chemopreventive activity in mouse skin.

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-KitW-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-KitW-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development

Dose and time related acute ear inflammation changes in response to topical CPAF.

<p><i>1A</i>. <i>Topical CPAF dose-dependently induces rapid inflammatory responses as measured by ear thickness measurements.</i> One ear of WT and <i>Ptafr</i> (-/-) were treated with one of three doses of CPAF (20 µl of a 0.1, 0.3, and 1.0 mM solution for a total treatment dose of 2, 6, or 20 nmole CPAF per ear). The contralateral ear was treated with acetone alone (VEH). Ear thickness was measured prior to treatment and 2 hours after treatment. After the pretreatment ear thickness values were subtracted, the mean and SEM were plotted (n = 4 for 20 nmole and n = 8 for 2 & 6 nmole CPAF & VEH treated mouse ears). <i>1B</i>. <i>Topical CPAF treatment induces a rapid, but transient increase in inflammation as measured by ear thickness changes.</i> One ear of wildtype (WT) and <i>Ptafr</i> (-/-) mice was treated with 20 µl of CPAF (20 nmoles of a 0.1 mM solution in acetone) and 20 µl of acetone (VEH) was applied to the contralateral ear. Ear thickness was measured just prior to reagent application and at 1, 2, 4, and 8 hours after application. Results represent the mean and SEM (n = 4 mice) after subtracting the pretreatment ear thickness. CPAF induced a significant increase in ear thickness in WT mice relative to the WT+VEH treated ears. *, <i>p</i><0.05; **, <i>p</i><0.01; ***; <i>p</i><0.001; 2-tailed <i>t</i>-test.</p

CPAF induces transient ear thickness changes are blocked in c-Kit^W-sh/W-sh mast cell deficient mice.

<p>WT and c-Kit^W-sh/W-sh mice were treated with vehicle (VEH) alone on one ear, and 20 ml of 0.3 mM CPAF (6 nmole) on the contralateral ear. Ear thickness was measured both prior to and 2 hrs after reagent application. After subtraction of the ear thickness at time 0, the mean and SEM were plotted (n = 5 for WT mice, n = 4 for Kit^W-sh/W-sh mice). *, <i>p</i><0.05; 2-tailed <i>t</i>-test.</p

Topical application of CPAF suppresses DMBA/PMA-induced tumorigenesis.

<p><i>3A. Topical treatment with CPAF suppresses DMBA/PMA-induced tumor multiplicity.</i> SKH-1 mice were treated once with DMBA+/- CPAF, then with PMA or PMA+CPAF for 25 weeks. Durable tumors were counted on a weekly basis. Tumor multiplicity (Avg tumor number per mouse was plotted at each week. Results represent the mean and SEM for n = 19–20 mice/group. <i>p</i><0.05 for weeks 9–12,14–25; <i>p</i><0.01 for week 13; Mann-Whitney U test. <i>3B. Topical CPAF delayed tumor incidence in mice treated with DMBA/PMA.</i> The percent of mice remaining tumor free over the 25 week chemical tumorigenesis study were plotted using a survival curve. The treatment with CPAF resulted in a significant change in the tumor incidence, with a median time until first tumor occurrence of 8 weeks for DMBA/PMA treated and 9 weeks for DMBA/PMA+CPAF treated mice. *, <i>p</i><0.05; Log-rank (Mantel-Cox) Test. <i>3C</i>. <i>Topical CPAF treatment results in a smaller number of large tumors (≥3 mm in greatest diameter) after 25 weeks of treatment.</i> Tumor size distribution was plotted as the number of tumors in each size distribution for each treatment group. Histopathologic exam showed no significant difference in the rates of papilloma and SCC formation between the treatment groups (not shown). ***, <i>p</i> = 0.0001 Fisher's exact test. <i>3D</i>. <i>DMBA/PMA-induced MPO activity is suppressed by CPAF.</i> After the mice were euthanized following 25 weeks of DMBA/PMA +/- CPAF treatment, tumor free areas of skin were excised and MPO activity was assessed in tissue lysates. After normalization to total protein, MPO activity was plotted as the mean and SEM (n = 5–9 mice per group). **, <i>p</i><0.05; ***, <i>p</i><0.001; 2-tailed <i>t</i>-test.</p

Effect of CPAF treatment on ear thickness changes over an 18 day course of thrice weekly PMA applications in WT and <i>Ptafr</i>-/- mice.

<p><i>4A. Topical CPAF treatment suppresses PMA-induced changes in ear thickness in WT mice.</i> CPAF (6 nmoles) alone or PMA +/- CPAF were applied to WT mouse ears thrice weekly for 18 days. Skin thickness measurements were taken at time 0 and just prior to each reagent application. After subtraction of the time 0 ear thickness, ear thickness changes were plotted as the mean and SEM (n = 4–5 mice per group). PMA relative to PMA+CPAF (^a), PMA+CPAF relative to CPAF(^b); *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; 2-tailed <i>t</i>-test. <i>4B. Topical CPAF treatment is ineffective in altering PMA-induced ear thickness changes in Ptafr-/- mice. Ptafr</i>-/- mice were treated and assessed as in 4A above. For the sake of comparison, the data for the PMA + CPAF treatment in WT mice is included. (mean and SEM; n = 4–5 mice per group). WT mice treated with PMA + CPAF exhibit a significant decrease in ear thickness measurements relative to <i>Ptafr</i>-/- mice treated with PMA + CPAF (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; 2-tailed <i>t</i>-test). <i>4C. Topical CPAF treatment suppresses PMA-induced skin thickness increases in dorsal epidermis following 18 days of treatment.</i> The dorsal epidermis of SKH-1 mice was treated thrice weekly with vehicle, CPAF, PMA, or PMA + CPAF. Doses of PMA and CPAF were the same as that used for the tumorigenesis studies in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111608#pone-0111608-g003" target="_blank">Fig 3</a>. ***, <i>p</i><0.05 relative to PMA treated skin; 2-tailed <i>t</i>-test (n = 3 per group).</p

Kit^W-sh/W-sh mice exhibit a reduction in PMA-induced acute inflammation but an augmented chronic or sustained inflammatory response to multiple PMA applications.

<p>For all experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111608#pone-0111608-g007" target="_blank">Fig 7A–C</a>, ear thickness measurements were made just prior to reagent application as well as at the indicated time points up to 18 days. Results represent the mean and SEM of ear thickness (n = 4–14 mice per group). *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.01; 2-tailed <i>t</i>-test. <i>7A. Mast cell deficient mice have a blunted initial inflammatory response to a single application of PMA.</i> WT and c-<i>Kit^W-sh/W-sh</i> mice were treated with VEH or PMA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111608#pone-0111608-g004" target="_blank">Fig 4A&B</a>. After subtracting the ear thickness at time 0 prior to PMA application, ear thickness changes were plotted (n = 12–14 mice/group). Ear thickness changes were significantly reduced in c-<i>Kit^W-sh/W-sh</i> mice treated with PMA relative to WT + PMA mice at 2 and 8 hrs following PMA application. ***, <i>p</i><0.001. <i>7B. Following a single application of PMA, topical CPAF treatment has no effect on PMA-induced ear thickness changes in c-Kit^W-sh/W-sh mice.</i> PMA-induced ear thickness changes were assessed in WT or c-<i>Kit^W-sh/W-sh</i> mice treated with PMA or PMA + CPAF. After subtracting the ear thickness at time 0, the ability of CPAF to suppress PMA-induced ear thickness changes was calculated as a percentage inhibition of PMA-induced ear thickness increases. CPAF treatment resulted in a significant inhibition of PMA-induced ear thickness changes at all time points (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111608#pone-0111608-g006" target="_blank">fig 6B</a>). CPAF treatment had no significant effect on PMA-induced ear thickness changes in c-<i>Kit^W-sh/W-sh</i> mice (Wilcoxon Signed Rank Test). The percent inhibition of PMA-induced ear thickness changes by CPAF in WT mice was also significantly different than that seen in c-<i>Kit^W-sh/W-sh</i> mice (**, <i>p</i><0.01, ***, <i>p</i><0.001; <i>t</i>-test). <i>7C. Topical CPAF is inactive in Kit^W-sh/W-sh mice while these mice also exhibit a reduction in acute inflammatory ear thickness changes observed in the first 4 days, but exhibit a significant increase in chronic sustained ear thickness changes.</i> Significant differences were noted at the indicated time points in WT mice treated with PMA relative to Kit^W-sh/W-sh mice treated with PMA (^a) as well as in WT mice treated with PMA/CPAF relative to Kit^W-sh/W-sh mice treated with PMA/CPAF (^b).</p

Following a single application of PMA, <i>Ptafr-</i>/- mice exhibit a reduction in PMA-induced ear thickness while CPAF treatment also induces a paradoxical PAF-R dependent decrease in PMA-induced ear thickness.

<p>For all studies in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111608#pone-0111608-g006" target="_blank">Figs 6</a>, WT and <i>Ptafr</i>-/- mice were treated with VEH, PMA, CPAF (6 nmole) or PMA + CPAF as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111608#pone-0111608-g004" target="_blank">Fig 4A&B</a>. Ear thickness measurements were made just prior to reagent application as well as at the indicated time points up to 48 hrs. <i>6A. Ptafr-/- mice exhibit a reduction in early and delayed ear thickness increases following a single PMA application.</i> Ear thickness plots for WT and <i>Ptafr</i>-/- mice treated with and without PMA are shown. Statistically significant changes are noted to WT mice treated with PMA relative to <i>Ptafr</i>-/- mice treated with PMA. Results represent the mean and SEM of ear thickness after subtracting the ear thickness at time 0 (n = 4–12 mice per group). *, <i>p</i><0.05; **, <i>p</i><0.01; <i>t</i>-test. <i>6B. Coadministration of topical CPAF blocks PMA-induced increases in ear thickness at all time points (2–48 hrs).</i> PMA-induced ear thickness changes were assessed in WT or <i>Ptafr</i>-/- mice treated with PMA or PMA + CPAF. After subtracting the ear thickness at time 0, the ability of CPAF to suppress PMA-induced ear thickness changes was calculated as a percentage inhibition of PMA-induced ear thickness increases. CPAF treatment resulted in a significant inhibition of PMA-induced ear thickness changes at all time points (p<0.01–0.05; % inhibition significantly different from no inhibition, Wilcoxon Signed Rank Test). CPAF treatment had no significant effect on PMA-induced ear thickness changes in <i>Ptafr</i>-/- mice (One sample analysis, Wilcoxon Signed Rank Test). The percent inhibition of PMA-induced ear thickness changes by CPAF in WT mice was also significantly different than that seen in <i>Ptafr</i>-/- mice (*, <i>p</i><0.05; **, <i>p</i><0.01; <i>t</i>-test).</p