The glutathione S-transferase GSTP is overexpressed in many human cancers and chemotherapy-resistant cancer cells, where there is evidence that GSTP may have additional functions beyond its known catalytic role. On the basis of evidence that Gstp-deficient mice have a comparatively higher susceptibility to skin carcinogenesis, we investigated whether this phenotype reflected an alteration in carcinogen detoxification or not. For this study, Gstp−/− mice were interbred with Tg.AC mice that harbor initiating H-ras mutations in the skin. Gstp−/−/Tg.AC mice exposed to the proinflammatory phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) exhibited higher tumor incidence and multiplicity with a significant thickening of skin after treatment, illustrating hyperproliferative growth. Unexpectedly, we observed no difference in cellular proliferation or apoptosis or in markers of oxidative stress, although higher levels of the inflammatory marker nitrotyrosine were found in Gstp−/−/Tg.AC mice. Instead, gene set enrichment analysis of microarray expression data obtained from skin revealed a more proapoptotic and proinflammatory environment shortly after TPA treatment. Within 4 weeks of TPA treatment, Gstp−/−/Tg.AC mice displayed altered lipid/sterol metabolism and Wnt signaling along with aberrant processes of cytoskeletal control and epidermal morphogenesis at both early and late times. In extending the evidence that GSTP has a vital role in normal homeostatic control and cancer prevention, they also strongly encourage the emerging concept that GSTP is a major determinant of the proinflammatory character of the tumor microenvironment. This study shows that the GSTP plays a major role in carcinogenesis distinct from its role in detoxification and provides evidence that the enzyme is a key determinant of the proinflammatory tumor environment. Cancer Res; 71(22); 7048–60. ©2011 AACR.
Glutathione S-transferases (GST) are a multigene family of dimeric enzymes playing an important role in chemical detoxification because of their capacity to catalyze the addition of reduced glutathione to reactive electrophiles (1). GSTP has received particular attention because of its association with carcinogenesis, drug resistance, and chemical toxicity (2–4). To define the in vivo functions of this protein, we have generated Gstp-null mice; these mice develop normally, are fertile, and show no obvious abnormalities (5). When Gstp-null mice are submitted to a 2-stage skin tumorigenesis protocol, involving topical application of the polycyclic aromatic hydrocarbon (PAH) and tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA), followed by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), there was a significant increase in the number of papillomas in null animals, showing that GSTP is an important determinant in PAH-induced skin cancer susceptibility (5). Similarly, increased adenoma formation in the lungs of Gstp-null mice was observed following the administration of benzo[a]pyrene, 3-methylcholanthene (3-MC), and urethane. In these studies, no increase in pulmonary DNA adducts was found in 3-MC–treated tissue from Gstp-null mice relative to wild-type controls, suggesting that GSTP may be acting in a manner distinct from its role as a detoxification enzyme (6). In this regard, we have also recently reported a marked increase in colon adenoma formation, when APCMin mice are on a Gstp-null background (7). GSTP has now been linked to a wide range of cellular functions, including modulation of c-jun-NH2-kinase and TRAF2 signaling (8, 9), glutathionylation (10) and inflammation (7). Also, mice nulled at the Gstp locus exhibit significant changes in mRNA expression profiles in liver, lung, and colon (6, 7), and the fact that the GSTP gene becomes hypermethylated, and as a consequence inactivated, in certain human cancers all point to novel functions of this protein (11, 12).
To further explore the role(s) of Gstp in carcinogenesis, we have crossed the Gstp-null mouse with the Tg.AC line which is predisposed to the development of skin cancer. The Tg.AC mouse contains the oncogenic v-Ha-ras transgene (mutated at codons 12 and 59; ref. 13) which when treated topically with variety of tumor promoters develops multiple papillomas (14). Because the tumorigenic response observed in Tg.AC mice occurs independently of the initiation step, the Tg.AC mouse has been characterized as a “genetically initiated” model for mouse skin tumorigenesis, allowing us to determine whether GSTP is involved in the initiation or promotion steps, or both.
Materials and Methods
All chemicals were of the highest grade available and were purchased from Sigma or Fisher Scientific Ltd.
All experiments were undertaken in accordance with the Animals (Scientific Procedures) Act 1986 and approved by the Animal Ethics Committees of the University of Dundee and Cancer Research UK. Gstp1/p2-null and wild-type mouse lines, on a 129 × MF1 background, were generated and maintained by random intercrossing as previously reported (5). Tg.AC mice, on an FVB background, were purchased from Taconic and were crossed with Gstp-null or Gstp-wt mice to generate Gstp+/+/Tg.AC or Gstp−/−/Tg.AC mice.
Gstp genotyping was carried out as previously described (7). Tg.AC genotype was determined by Southern blotting (Taconic); only those mice with the Tg.AC responder genotype were used.
Chemical carcinogenesis protocol
TPA (6 μg) was dissolved in acetone (200 μL) and applied twice weekly to the shaved backs of 6- to 9-week-old mice. Matched cohorts of mice were treated with acetone alone or left untreated. All mice were monitored for papilloma growth twice weekly. The date of first papilloma incidence was recorded; papillomas that grew to 1 mm or more in size were counted. All animals entered into the study were included in the final analysis.
Tumors were fixed in PBS-formalin (10%), transferred to 80% ethanol, and processed to wax for sectioning. Tissue sections were stained with hematoxylin and eosin and examined by a pathologist blinded to sample identity.
Mice were sacrificed by a rising concentration of CO2 and skin immediately removed for preparation of RNA using TRIzol (Invitrogen) and an RNeasy Mini kit (Qiagen). RNA was pooled from 2 animals of each genotype, and subsequent hybridizations were carried out in triplicate. A260/280 ratio of total RNA was typically 1.9 or more. RNA quality was assessed on an Agilent 2100 Bioanalyzer.
Total RNA (1 μg) was labeled with Cyanine 3 (Cy3)-CTP (Agilent One-Colour Microarray-Based Gene Expression Analysis protocol, v5.0.1) using the Low Input RNA Fluorescent Linear Amplification Kit (Agilent). Agilent 4 × 44K Whole Mouse Genome Oligo Microarray slides were hybridized, washed, and scanned at 5 μm resolution on an Agilent Microarray Scanner. Scanner images were processed using Agilent Feature Extraction Software v9.1. The microarray scanned image and intensity files were imported into Rosetta Resolver gene expression analysis software v184.108.40.206.1. Individual expression profiles from the various genotypes (Gstp+/+/Tg.AC and Gstp−/−/Tg.AC) and treatments (no treatment, acetone, and TPA) were pooled in silico by calculating an error-weighted mean and compared with build ratios. Data were analyzed using the bioinformatics platform Bioconductor 2.7, running on R 2.12.1. Chip expression data were quantile normalized and a linear model fitted to determine the effects of genotype, time, and stimulation using the LIMMA package (15). Differential genes were selected by applying a 0.05 false discovery rate threshold to P values corrected using the Benjamini and Hochberg method. Differential genes were used for enrichment analysis using Genego's Metacore pathway tool to identify enriched pathways and biological processes.
Gene set enrichment of Biocarta pathways was determined by using the function “geneSetTest” within the LIMMA package to see if pathways are either up- or downregulated using the t-statistics for each factor. Pathways with a false discovery rate of less than 0.05 were called enriched. Gene Ontology annotation enrichments were also determined to see which biological processes and molecular functions were enriched using the same method.
Sections were dewaxed and rehydrated and then underwent microwave antigen retrieval using 10 mmol/L citrate buffer for 10 minutes. Immunohistochemistry was carried out using the Dako Envision Staining System and an anti-BrdUrd antibody (Becton Dickinson) or antinitrotyrosine antibody (Millipore). Signals were developed by standard techniques.
The terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay to determine levels of apoptosis was carried out using the In Situ Cell Death–Detection Kit, POD (Roche). For analysis, the total number of cells and the number of labeled cells for 3 separate fields per slide were counted.
Statistical analysis was conducted using GraphPad Quickcalcs Online statistical calculator. Significant differences when comparing 2 groups were determined by unpaired t test. P values were considered significant if they were less than 0.05.
Papilloma pathology and growth
Wild-type mice on a Tg.AC background (Gstp+/+/Tg.AC) and Gstp-null mice on a Tg.AC background (Gstp−/−/Tg.AC) were born with normal Mendelian frequency and did not differ in size, weight, or growth characteristics. To assess the role of Gstp in the promotion stage of tumorigenesis, we treated Gstp+/+/Tg.AC and Gstp−/−/Tg.AC mice with TPA. Both genotypes developed papillomas, which were indistinguishable either in gross anatomic appearance or after histologic examination (Fig. 1). The first detectable tumors (>1 mm) on both Gstp+/+/Tg.AC and Gstp−/−/Tg.AC mice were seen at 5 weeks (Fig. 2A). However, by week 6, whereas only 18% of Gstp+/+/Tg.AC mice had papillomas, in 55% of Gstp−/−/Tg.AC mice, tumors were observed. This significantly accelerated appearance of papillomas in Gstp−/−/Tg.AC mice continued through weeks 6 to 9. By week 15, all mice of both genotypes had papillomas. Tumor multiplicity was also significantly higher in Gstp−/−/Tg.AC animals, this difference reaching statistical significance after 9 weeks of treatment (Fig. 2B). These results showed that loss of Gstp markedly increased both the incidence and multiplicity of skin papillomas in Gstp−/−/Tg.AC mice.
Effect of Gstp-null genotype on skin thickness, cell proliferation, apoptosis, and oxidative stress
To evaluate the effects of the Gstp-null genotype, we looked for changes in skin morphology and epidermal proliferation rates following TPA exposure. Following 6-week treatment with TPA, a marked increase in the thickness of the epidermis of both genotypes was observed, consistent with the known ability of TPA to induce cellular proliferation (Fig. 3A). This increase was much more marked (28% higher) in Gstp−/−/Tg.AC mice relative to Gstp+/+/Tg.AC animals (Fig. 3A and B). These effects were not observed in acetone-treated controls. Investigation of increased cellular proliferation rates using in vivo bromodeoxyuridine (BrdUrd) labeling experiments showed that there was no difference in epidermal cellular proliferation rate between mouse lines (Fig. 3C). Although the percentage of cells in apoptosis seemed to be slightly higher in Gstp+/+/Tg.AC mice than in null animals (69% ± 5% vs. 58% ± 3%, respectively), this difference was not statistically significant. Western blotting of skin tissue from Gstp+/+/Tg.AC and Gstp−/−/Tg.AC mice was carried out for markers of oxidative stress (Supplementary Fig. S1); heme oxygenase-1 (HO-1) expression was marginally induced and NADH quinone oxidoreductase 1 (NQO1) suppressed, 6 hours after TPA treatment in Gstp+/+/Tg.AC mice, but both returned to untreated levels at 4 weeks. GSTP behaved in a similar manner to NQO1. Similar changes were observed in Gstp−/−/Tg.AC mice for NQO1 and HO-1, and the alterations in protein expression were confirmed by reverse transcriptase PCR, along with Nrf2, a transcription factor considered as a master regulator of the antioxidant response (Supplementary Fig. S2).
Gene expression changes in the skin of Gstp-null and Gstp-WT mice
Despite no histologic differences being evident between untreated wild-type and Gstp-null mouse skin (Fig. 1), the expression of 169 genes was increased and 175 genes reduced, by a factor of 2-fold or greater (Supplementary Table S1). Ten of the 30 genes increased to the greatest extent were unannotated; of the remainder, the most induced was armadillo repeat-containing 10 (Armc10; ∼12-fold), followed by murinoglobulin 1 (Mug1) and mitogen-activated protein kinase kinase kinase kinase 2 (Map4k2; both ∼11-fold). Among the genes, whose expression was most reduced, approximately one third were unannotated; the most suppressed gene (∼10-fold) was Fin15 (fibroblast growth factor inducible 15). Further analysis using Metacore pathway software failed to find enrichment of pathways in either set of genes, although within the set of induced genes, a number of biological processes, rather than pathways, were significantly enriched in Gstp-null mouse skin, including cell inflammation and cell adhesion (data not shown).
Gene expression changes in the skin of Gstp−/−/Tg.AC mice following TPA treatment
To evaluate the effects of TPA on gene expression in the skin in the presence or absence of Gstp, we carried out gene expression profiling following both short- (6 hours) and long-term (4 weeks) exposure of Gstp−/−/Tg.AC and Gstp+/+/Tg.AC mice to TPA.
Genes whose expression was significantly changed in Gstp−/−/Tg.AC and Gstp+/+/Tg.AC mice after 6 hours of TPA treatment were compared with vehicle (acetone)-treated animals. The expression of more than 3,900 genes was changed in both groups. At 6 hours, within the top 50 induced or suppressed genes, there was a high degree of similarity, 45 genes being found in both genotypes (Supplementary Table S2). However, some variation in the extent of induction was observed; expression of 27 genes induced in the skin of both Gstp−/−/Tg.AC and Gstp+/+/Tg.AC mice differed in fold change by a factor of 3 or more, 16 to a greater extent in Gstp−/−/Tg.AC mice—Chi3l3, Trpm2, AK048833, Nrg1, Prg4, Clec4d, AK031717, Ccl22, Klk9, Tnc, Slc26a4, Ccl3, Ccl17, Serpinb1c, IL19, and Il1rl1—several of which are associated with inflammatory response. Eleven genes were induced to a greater extent in the skin of Gstp+/+/Tg.AC mice—Zdhhc21, CCl24, Ier2, Tnip3, AK078295, Lce3f, RSG2, Mmp10, A330043J11Rik, Gfpt1, and Mcpt8.
The expression of many common genes was also reduced, with the majority of the most suppressed genes present in both genotypes (Supplementary Table S2). Similar to the induced genes, the fold change varied between the genotypes. The expression of 13 genes was decreased in both genotypes with fold change ratios differing by a factor of 3 or more, 2 to a greater extent in Gstp−/−/Tg.AC (Pla2g2d and Pamr1), and 11 in Gstp+/+/Tg.AC mice (Adh7, Eml16, Edar, 3830408C21Rik, Cttnbp2, Cldn8, Dlg2, 2130005G13Rik, Pramef12, AK089297, and BC034902). A single gene, Bclp2, was induced in Gstp+/+/Tg.AC mice (3.2-fold) and suppressed in Gstp−/−/Tg.AC mice (2.6-fold).
In addition to the similarities, there were many significant differences between the effects of TPA on gene expression between Gstp−/−/Tg.AC and Gstp+/+/Tg.AC mice. After 6 hours of TPA treatment, a total of 1,750 genes were altered uniquely in Gstp+/+/Tg.AC mice relative to acetone-treated controls and TPA-treated Gstp−/−/Tg.AC animals, that is, changes in the Gstp+/+/Tg.AC were observed which did not occur in the Gstp−/−/Tg.AC mice. The expression of 840 genes was reduced and 910 genes increased by more than 2-fold. The top 50 genes in each category are shown in Table 1. Similarly, the expression of 2,170 genes was uniquely altered in Gstp−/−/Tg.AC mice 6 hours after TPA treatment; of these, 1,058 were expressed at higher levels and 1,112 at lower levels by more than 2-fold; the top 50 genes in each category are shown in Table 2.
After 4 weeks of TPA treatment, 950 genes were altered in both Gstp−/−/Tg.AC and Gstp+/+/Tg.AC mice, compared with control animals; the top 50 induced or repressed genes are shown in Supplementary Table S4. Again, there was a very strong similarity between the Gstp+/+/Tg.AC and Gstp−/−/Tg.AC groups. As with the 6-hour time point, there were, however, differences in the fold changes in the expression of these genes between the groups. Fifteen genes were increased in the skin of both mouse strains and differed in fold change ratio by a factor of 3 or more, 14 to a greater extent in Gstp−/−/Tg.AC mice (Krtap 31-1, Stfa2, Krt84, 2310033E01Rik, Spink12, Lrrc15, Lce3f, Oca2, Sprrl1, Sprr2d, Krt16, Scrg1, Uox, and Dlx4), and 1 in Gstp+/+/Tg.AC mice (Prss51). Two genes were suppressed in both genotypes with a fold change ratio greater than 3, 1 to a greater extent in Gstp−/−/Tg.AC mice (Kel), and 1 to a greater extent in Gstp+/+/Tg.AC mice (1500015O10Rik). Five genes were induced in Gstp+/+/Tg.AC mice but suppressed in Gstp−/−/Tg.AC mice (Cacnb2, Dmrtc2, Olf346, Mpp1, and A630077J23Rik).
In addition to the similarities to the gene expression profiles at 4 weeks, there were also significant differences. In Gstp+/+/Tg.AC mice, the expression of 1,868 genes was uniquely altered in their expression as compared with controls and Gstp-null animals; 937 were higher and 931 lower, by more than 2-fold. The top 50 genes in each category are shown in Table 3. In Gstp−/−/Tg.AC mice, a total of 1,772 genes were uniquely altered 4 weeks after TPA treatment, 910 genes being increased and 862 decreased by more than 2-fold. The top 50 genes in each category are shown in Table 4. It is interesting to note that in Gstp−/−/Tg.AC mice, 40 of the top 50 genes that were found to be increased were keratins or keratin-associated proteins (induced 200- to 2,000-fold).
To define the pathways that were uniquely up- or downregulated in Gstp−/−/Tg.AC mice and identify those biological processes or molecular functions that were statistically enriched at both time points, the unfiltered microarray data from Gstp−/−/Tg.AC and Gstp+/+/Tg.AC mice treated with TPA for 6 hours or 4 weeks were subject to ANOVA and subsequent gene set enrichment analysis (GSEA). There were 101 pathways upregulated in Gstp−/−/Tg.AC compared with Gstp+/+/Tg.AC mice at 6 hours after TPA treatment, and the same number upregulated at the 4-week time point. Seventeen pathways were uniquely upregulated at each time point: at 6 hours, pathways were associated with apoptosis, cell-mediated immunity, T-cell activation and differentiation, inflammation, and cytokine production, whereas at 4 weeks, apoptotic pathways were essentially absent, although pathways related to cell proliferation and the immune system were still upregulated in Gstp−/−/Tg.AC mice compared with Gstp+/+/Tg.AC mice (Supplementary Table S3). With regard to pathways uniquely downregulated in Gstp−/−/Tg.AC mice, there were a smaller number at both 6 hours (n = 8) and 4 weeks (n = 1), and none in common; keratinocyte differentiation was represented at both time points, whereas pathways downregulated at 6 hours also included those involved in cell cycle, inflammatory response, and T-cell activation (Supplementary Table S5). Gene Ontology gene sets were analyzed using GSEA to identify biological processes or molecular functions that were statistically enriched (up or down) in Gstp−/−/Tg.AC compared with Gstp+/+/Tg.AC mice at both time points (Supplementary Table S8). Interestingly, with regard to upregulation, there was a good deal of conformity between the 2 time points; in both cases, there were biological processes and molecular functions associated with the actin cytoskeleton, chromatin remodeling, and transcriptional regulation, as well as kinase signaling, blood vessel development, and epithelial/endothelial cell proliferation and differentiation. However, while at 6 hours, there was a predominance of antiapoptotic biological processes; at the later time point, pro- and antiapoptotic processes were more balanced. Furthermore, cytokine production and cytokine-mediated signaling observed at 6 hours were absent at 4 weeks, whereas altered lipid and sterol metabolic processes, and Wnt receptor signaling, present at 4 weeks were not found at the earlier time point (Supplementary Table S8). For downregulated Gene Ontology annotations, there were relatively few, and little or no overlap between time points: at 6 hours, downregulated molecular functions included aromatase and oxidoreductase activities, whereas at 4 weeks, signal transduction, G-protein–coupled receptor activity, and a number of processes related to pheromones and olfaction were downregulated (data not shown).
Gene expression changes in papillomas from Gstp−/−/Tg.AC mice
To investigate the molecular mechanism(s) underlying the increased tumor incidence and multiplicity in Gstp−/−/Tg.AC mice, mRNA profiles for papillomas from each genotype were determined. In Gstp−/−/Tg.AC mice, only 87 genes were expressed at higher and 127 genes at lower levels, by a factor of 2-fold or greater relative to Gstp+/+/Tg.AC mice (see Supplementary Table S6). The genes that were expressed at higher levels included the β-galactoside–binding proteins galectin-4 (lgals4, 3.4-fold) and galectin-6 (lgals6, 8.5-fold) and Mug1 (3.3-fold). Genes repressed in Gstp−/−/Tg.AC mice versus Gstp+/+/Tg.AC mice were chitinase-like proteins Chi3l4 (17-fold down) and Chi3l3 (6-fold down); Clec2g (C-type lectin domain family 2, member g, 3.8-fold down); and the tumor suppressor Dbc1 (deleted in bladder cancer protein 1, 3.7-fold; ref. 16). Unbiased analysis of the gene expression profiles in Gstp−/−/Tg.AC mice compared with Gstp+/+/Tg.AC mice failed to identify specific biochemical pathways significantly altered in mice lacking Gstp. However, a number of biological processes were found to be enriched in Gstp−/−/Tg.AC mice, including keratinization, keratinocyte differentiation, epidermal morphogenesis, development, and differentiation, as well as several processes related to steroid and lipid metabolism (Supplementary Table S7), all of which could potentially be mechanistically associated with the increased rate of papilloma development observed in the Gstp−/−/Tg.AC mice following TPA treatment (Fig. 2).
Our previous work with the Gstp-null mouse and skin carcinogenesis showed that Gstp can play an important role in the etiology of skin and lung tumors (5, 6). We have also recently reported that by crossing Gstp−/− mice with APCMin mice enhanced the incidence of adenomas arising in the colon (7). However, the mechanism(s) by which GSTP protected against tumor formation was unclear and did not seem to be solely because of its role as a detoxification enzyme. In this study, we have showed that GSTP can have a profound effect on skin tumorigenesis and initiated by the presence of the mutant Ras oncogene, that is, in the absence of a chemical carcinogen. There is now also a growing body of evidence suggesting that Gstp is also an important determinant in inflammatory disease (17–19).
In the experiments described here, there are a number of other ways in which Gstp could influence carcinogenesis. It could alter the signaling pathways induced by the presence of mutant Ras protein or the promotional effects of TPA. Alternatively, because TPA and Ras act, at least in part, through AP1 signaling, Gstp could act through a combination of the 2 mechanisms. However, it is also important to consider the role of Gstp in the detoxification of endogenous toxins as a possible explanation for the increased tumorigenesis noted in the null animals. The generation of endogenous reactive oxygen species by NADPH oxidase-1 (Nox1), which catalyzes the reduction of molecular oxygen to superoxide, has been recently found to be required for Ras-mediated oncogenic transformation (20, 21). In addition, Nox1 is thought to be responsible for the generation of reactive oxygen species in human keratinocytes following UVA irradiation (22). Given the well-known activity of Gstp in the detoxification of genotoxic lipid peroxidation products such as acrolein (23), it is possible that the increased tumorigenesis noted in Gstp-null animals is a direct consequence of their reduced ability to deal with endogenously produced toxic metabolites of oxidative stress. However, expression of oxidative stress markers, such as HO-1 and NQO1, and the transcription factor Nrf2, although altered at 6 hours after TPA, was essentially unchanged from control levels at the 4-week time point (Supplementary Figs. S1 and S2), indicating that while oxidative stress may play a role in the early stages of tumorigenesis, it seems to have less influence in the longer term. The Gstp-null background seems to potentiate the pathways activated by TPA and is manifested in the increased skin thickness observed in the Gstp-null animals. However, these effects do not seem to be mediated by an increased cell proliferation rate, as measured by BrdUrd, or by changes in rates of apoptosis; rationalization of these effects requires further study.
To gain further insights into the mechanism of action of Gstp in skin tumorigenesis, we carried out a detailed gene expression profile analysis on mice where Gstp is either present or absent. In normal skin, the annotated mRNA most induced in Gstp−/−/Tg.AC mice was Armc10 (∼12-fold); this protein has been implicated in cell survival and growth, has been reported to suppress p53 activity and thus apoptosis (24), and is also upregulated in hepatocellular carcinoma (25). We also found that Armc10 expression is elevated (∼4-fold) in the lungs of Gstp-null mice that had a significantly higher incidence of chemically induced pulmonary adenomas (6). Also elevated significantly (∼11-fold) was Map4k2, a member of the serine/threonine protein kinase family and which can be activated by TNFα, copolymer of polyinosinic and polycytidylic acids [poly(I)-poly(C)], lipopolysaccharide, and interleukin (IL)-1, thus mediating an array of inflammatory responses, and couples TNF signaling to the p38 MAPK and stress-activated protein kinase (SAPK) pathways (26, 27). Deletion of this gene in mice renders them resistant to endotoxin-mediated lethality (28). Interestingly, serum amyloid A protein 2 (Saa2) was also expressed at significantly higher levels (6.8-fold) in Gstp−/−/Tg.AC mice; Saa2 is a member of a family of highly conserved acute-phase proteins, secreted during inflammation in response to the cytokines IL-1, IL-6, and TNFα, and which aid in the recruitment of immune cells to inflammatory sites. Saa2 is expressed in a variety of tissues, including skin, primarily in epithelial cells (29). Similarly to Armc10, another Saa family member, Saa3, was significantly induced (∼6-fold) in the lungs of Gstp-null mice, relative to wild type (6). Interestingly, skin sections from mice treated with TPA and stained for the inflammatory marker nitrotyrosine show higher levels of expression in the absence of Gstp (Supplementary Fig. S3). The presence of genes associated with inflammation was also observed in the colonic epithelium of Gstp−/−: APCMin. mice (7). Together, these data add weight to the argument that the absence of Gstp creates an elevated level of basal inflammation in a number of different tissues—skin (this study), lung (6), and colon (7). However, such an inflammatory environment, although protumorigenic, is clearly not sufficient for tumor development, as Gstp-null mice do not develop cancer spontaneously (30).
Many genes induced in the skin by TPA on a Tg.AC background were also induced in the Gstp−/−/Tg.AC background. These data are reassuring and provide confidence about the significance of the differences between the Gstp+/+/Tg.AC and Gstp−/−/Tg.AC genotypes. It is interesting to note that in many cases the genes were induced to a higher level in the Gstp−/− mice than in mice carrying the Tg.AC alone. Among the most prominent genes induced by TPA administration in both genotypes (but to a significantly greater extent in Gstp−/-/Tg.AC mice) at 6 hours or 4 weeks were s100A8, Sprr2a, Sprr2d, and Stfa1, Stfa2, and Stfa3 (Supplementary Tables S2 and S4), previously described as being present in clusters of differentially expressed genes from the skin of Tg.AC mice in which papillomagenesis had been induced by abrasion (31).
Strikingly, more than 90% of the top 50 genes encoded for keratins or keratin-associated proteins and were expressed at several orders of magnitude higher in Gstp−/−/Tg.AC mice than in wild type at the 4-week time point (Table 4). Interestingly, a recent study by Quigley and colleagues (32) in which the authors mapped genetic loci contributing to skin tumor susceptibility in mice identified a network of 62 genes involved in control of the hair follicle containing 37 keratins or keratin-associated proteins, all of which were found on our list of genes uniquely upregulated in Gstp−/−/Tg.AC mice 4 weeks after TPA treatment (Table 4). Furthermore, of the remaining genes in this network, all but 3 (Lyg2, Pdzm3, and Vsig8), were also upregulated (3- to 227-fold) only in Gstp−/−/Tg.AC mice 4 weeks after TPA treatment, strongly suggesting that dysregulation of hair follicle function occurs to a considerably greater extent in the absence of Gstp and may play a significant role in the accelerated development of papillomas in this genotype. Quigley and colleagues (32) also identified the intestinal stem cell marker Lgr5 (leucine-rich repeat containing G-protein–coupled receptor 5) as the best candidate for a master regulator of the hair follicle network; interestingly, although Lgr5 expression is downregulated in both Gstp+/+/Tg.AC and Gstp−/−/Tg.AC mice 6 hours after TPA treatment (12- and 9-fold, respectively), it is upregulated (3.2-fold) uniquely in Gstp−/−/Tg.AC mice 4 weeks after treatment.
Also of note, one α-defensin (Defa-rs12, down 3.2-fold) and two β-defensin proteins (Defb14, up 4-fold; Defb15, down 6-fold) were altered in their expression in Gstp−/−/Tg.AC mice relative to Gstp+/+/Tg.AC; these antimicrobial peptides are involved in the defense response to bacterial infection (33), and interestingly, other proteins from this family (α-defensins) were found be significantly downregulated in both normal colonic tissue and adenomas from Gstp-null mice which also carried the APCMin+ mutation (7). Interestingly, at this stage, rather than key induced pathways, many genes associated with inflammatory responses seemed to be repressed in the Gstp−/−/Tg.AC mice relative to the Gstp+/+/Tg.AC animals.
At both time points after TPA treatment, a large number of genes were either induced or repressed in the Gstp−/−/Tg.AC mouse skin relative to Gstp+/+/Tg.AC mice alone (Supplementary Tables S2 and S4). Although, there were a handful of genes found in the top 50 upregulated genes at both time points (Sprr2d, Uox, Lcn2, S100A8, and S100A9); the transcript profiles of skin at 4 weeks after treatment were markedly different from that at 6 hours; this probably reflects the emerging complexity and cross talk between many regulatory networks prior to overt tumor formation. However, the experimental design used in the current study allowed us to carry out GSEA on the unfiltered data generated from the microarray protocols (34, 35). All microarray data were subjected to ANOVA to examine the effect of time, treatment, and genotype on gene expression, and Biocarta pathways were mapped onto the gene expression data (Supplementary Tables S3 and S5). Because we were primarily interested in the role of Gstp in the process of skin tumorigenesis, we focused on the effect of genotype in the pathway and Gene Ontology analysis. As shown in Supplementary Table S8, the upregulated biological processes and molecular functions statistically enriched in Gstp−/−/Tg.AC mice relative to Gstp+/+/Tg.AC were highly similar, with the exception of a more antiapoptotic and proinflammatory environment at the early time point, and alterations to lipid and sterol metabolism, and the Wnt receptor signaling pathway, at 4 weeks. The latter is interesting because transient activation of β-catenin signaling has previously been shown to be required for the initiation of hair follicle development (36), but higher and more sustained, or continuous, activation is required to maintain tumors derived from hair follicles such as those generated in the model used in this study (37).
Although the changes observed at 4 weeks could reflect changes in the constitution of the skin, those at 6 hours must reflect changes in short-term regulatory control and not be related to cell type. It is important to note that the high number of papillomas seen in these studies suggest that at the 4-week time point, the entire skin is highly predisposed to tumor formation, reflected in the altered pathways of hair follicle morphogenesis and development, and epidermal morphogenesis observed, and suggesting a more dedifferentiated phenotype in the skin of Gstp−/−/Tg.AC mice.
We also carried out analysis of patterns of gene expression in papillomas from Gstp−/−/Tg.AC versus Gstp+/+/Tg.AC animals. Although there were some differences in patterns of gene expression between the 2 genotypes, we were unable to identify regulatory networks from which these gene expression changes were derived. Relative to the gene expression differences in normal skin, remarkably few changes were observed in the papillomas of the different genotypes, further strengthening the evidence that Gstp affects tumor promotion rather than tumor genetics. However, some of the differences observed are worthy of note. Galectin-4 and galectin-6 were elevated in Gstp−/−/Tg.AC mice; galectins have been implicated in a number of biological processes, including inflammation, innate and adaptive immunity, and cancer, indeed galectin-4 has been proposed as a marker for breast cancer (38). Mug1 expression was induced both in papillomas (3.2-fold) and skin (11.3-fold) from Gstp−/−/Tg.AC mice. Mug1 is a novel protease inhibitor and a member of the α2-macroglobulin family whose members act as binding proteins for growth factors and cytokines including TNFα, TGFβ, and interleukins; Mug1 knockout mice are more susceptible to diet-induced acute pancreatitis (39). The elevated expression of Mug1 in both skin and papillomas from Gstp−/−/Tg.AC mice may reflect the presence of a significantly enhanced inflammatory environment, both basally and following TPA treatment. CHi3l3 and Chi3l4 mRNA were both significantly lower in papillomas from Gstp−/−/Tg.AC mice; chitinase-like proteins have been associated with infection, T-cell–mediated inflammation, and allergy (40). Interestingly, Chi3l3 was induced in the skin of both Gstp+/+/Tg.AC and Gstp−/−/Tg.AC mice 6 hours after TPA treatment (Supplementary Table S2) but to a significantly greater extent in the latter (6- vs. 84-fold). These data show that the absence of Gstp can cause marked changes in gene expression profiles in tumors containing dominant oncogenes. Such changes could explain why GSTP is methylated in human tumors of prostate, liver, and breast (12, 41, 42).
A number of studies have reported a reduction in papilloma multiplicity following crossing of the Tg.AC mouse with different transgenic mouse strains (43–45), whereas others have shown the transgene to initiate earlier onset of papillomagenesis (46) or an increase in tumor size (47). However, as reported in this study, the effects of the absence of Gstp on the Tg.AC background appear to be particularly marked with regard to both the incidence and multiplicity of papillomas.
Ras-related signaling has been show to be activated by mutation in a significant number of skin cancers in man, for example, B-Raf mutations are commonly found in melanoma (48, 49). The finding that Gstp can alter tumors induced by Ras signaling raises the interesting possibility that it may affect susceptibility to skin cancer in humans. In support of this possibility, polymorphisms in GSTP have been reported to increase susceptibility to basal cell and squamous cell carcinomas (50, 51). In future work, it will be interesting to establish whether GSTP also alters tumor incidence in other models of skin cancer such as the conditional B-Raf mutant mouse and whether tumorigenicity in these models can be inhibited by exogenous agents.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work was funded by a Programme Grant awarded to C.R. Wolf by Cancer Research UK (C4639/A5661).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The authors thank Catherine Hughes, Susanne van Schelven, and Jennifer Kennedy for assistance with mouse work; Dr. Shaun Walsh (Pathology, NHS Tayside) for expert analysis of histology sections; and P. Chakravarty (Cancer Research UK London Research Institute) for bioinformatic analyses.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
C.J. Henderson and K.J. Ritchie are joint first authors.
- Received March 30, 2011.
- Revision received July 28, 2011.
- Accepted August 16, 2011.
- ©2011 American Association for Cancer Research.