Intestinal tumor suppression in ApcMin/+ mice by prostaglandin D2 receptor PTGDR.

Our earlier work showed that knockout of hematopoietic prostaglandin D synthase (HPGDS, an enzyme that produces prostaglandin D2) caused more adenomas in Apc(Min/+) mice. Conversely, highly expressed transgenic HPGDS allowed fewer tumors. Prostaglandin D2 (PGD2) binds to the prostaglandin D2 receptor known as PTGDR (or DP1). PGD2 metabolites bind to peroxisome proliferator-activated receptor γ (PPARG). We hypothesized that Ptgdr or Pparg knockouts may raise numbers of tumors, if these receptors take part in tumor suppression by PGD2. To assess, we produced Apc(Min/+) mice with and without Ptgdr knockouts (147 mice). In separate experiments, we produced Apc(Min/+) mice expressing transgenic lipocalin-type prostaglandin D synthase (PTGDS), with and without heterozygous Pparg knockouts (104 mice). Homozygous Ptgdr knockouts raised total numbers of tumors by 30-40% at 6 and 14 weeks. Colon tumors were not affected. Heterozygous Pparg knockouts alone did not affect tumor numbers in Apc(Min/+) mice. As mentioned above, our Pparg knockout assessment also included mice with highly expressed PTGDS transgenes. Apc(Min/+) mice with transgenic PTGDS had fewer large adenomas (63% of control) and lower levels of v-myc avian myelocytomatosis viral oncogene homolog (MYC) mRNA in the colon. Heterozygous Pparg knockouts appeared to blunt the tumor-suppressing effect of transgenic PTGDS. However, tumor suppression by PGD2 was more clearly mediated by receptor PTGDR in our experiments. The suppression mechanism did not appear to involve changes in microvessel density or slower proliferation of tumor cells. The data support a role for PGD2 signals acting through PTGDR in suppression of intestinal tumors.

Introduction

Prostaglandin studies in intestinal neoplasia usually focus on prostaglandin E2 (PGE2), a pro-tumorigenic compound 1–4. In an opposite effect, knockout of the gene for hematopoietic prostaglandin D synthase (HPGDS) caused more adenomas in ApcMin/+ mice. Moreover, high HPGDS production from transgenes allowed fewer 5. Prostaglandin D2 (PGD2) and PGE2 are both made from PGH2, so Hpgds knockouts could have shunted conversion of PGH2 to PGE2. Likewise, HPGDS transgenes could have drawn prostaglandin synthesis away from PGE2.

Lewis lung cancer cells implanted onto the backs of mice lacking the PGD2 receptor (PTGDR, also known as DP1), grew faster than tumors implanted onto wild-type mice 6. Furthermore, the PTGDR agonist, BW245C, reduced tumor growth. These results support a role for PGD2 itself.

Tumor suppression by PGD2 might also occur through inhibition of inflammatory genes by molecules that bind PGD2 metabolites. For example, PGD2 metabolites bind to peroxisome proliferator-activated receptor γ (PPARG). Such binding can induce conjugation of small ubiquitin-related modifier-1 (SUMO-1) to PPARG. SUMOylation is thought to increase PPARG binding to nuclear receptor corepressor complexes, causing transrepression of inflammatory genes 7. Additionally, 15-deoxy-Δ12,14-PGJ2 may down-regulate inflammatory genes, through covalent binding to nuclear factor-κB or IκB kinase 8.

Here, we show that knockouts of Ptgdr increased tumor numbers in ApcMin/+ mice, indicating that PGD2 and PTGDR act to suppress tumors. PPARG had smaller effects in our experiments.

Material and Methods

Mice

The protocol and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Los Angeles Biomedical Research Institute. C57BL/6, FVB/N, and ApcMin/+ (C57BL/6; no. 002020) mice came from Jackson (Bar Harbor, ME), as did mice carrying the Cre transgene controlled by the adenovirus EIIa promoter [Tg(EIIa-Cre) C5379Lmgd/J; FVB/N strain; no. 003314] 9. Mice in which exon 2 of the Pparg gene is flanked by loxP sites were from F. Gonzalez (Ppargflox/flox FVB/N mice).

To produce ApcMin/+ mice with heterozygous Ptgdr knockouts, we crossed male ApcMin/+ mice with female homozygous Ptgdr knockout mice 10. Male ApcMin/+ mice with heterozygous or homozygous Ptgdr knockouts were then bred with female homozygous Ptgdr knockout mice to produce ApcMin/+ mice with homozygous Ptgdr knockouts (all 100% C57BL/6).

Our PTGDS transgenic mice (line B20; FVB/N) overexpress human PTGDS in all tissues 11. Reported basal brain levels of PGD2 were 1.5-fold higher than wild-type levels and rose fivefold upon stimulation. PGE2 levels did not change. The mice had more eosinophilia in a bronchial asthma model, compared to HPGDS transgenic mice 12.

To generate heterozygotic Pparg knockout mice, we crossed Ppargflox/flox mice with Tg(EIIa-Cre) mice and identified heterozygotes lacking exon 2 of the Pparg gene (Pparg+/− mice; all FVB/N) 13. We then crossed female Pparg+/− FVB/N mice with male ApcMin/+ C57BL/6 mice to produce ApcMin/+ Pparg+/−, ApcMin/+, and Pparg+/− mice, all on an F1 mixed background. Similarly, we bred PTGDS transgenic FVB/N males with C57BL/6 females to produce transgenic mice on an F1 C57BL/6 × FVB/N background. We intercrossed these various offspring to obtain additional mice with desired genotypes. Fifteen of the 104 mice used were C57BL/6 × FVB/N F1 mice, and 89 were from matings of F1 mice or mice in later generations (all 50% C57BL/6).

Intestinal histopathology and definitions of tumor sizes

Adenomas were counted histologically at 6 or 14 weeks, without knowing genotypes 5. We used 24 Swiss roll sections spaced 150 μm apart for PTGDS transgenic mice, Pparg knockout mice, and their controls. We used 10 Swiss roll sections (250 μm apart) for Ptgdr knockout mice and their controls. Tumors sizes were gauged by the number of sections spanned. Small tumors were defined as those seen in only 1 section. Large tumors were those with profiles in multiple sections. Mitotic figures were identified as described 14.

Statistical analyses of tumor data

Tumor data were analyzed by nonparametric methods (Kruskal–Wallis and Mann–Whitney), because numbers of tumors per mouse did not follow a Gaussian distribution. We analyzed total, small, large, and colon tumors. We also calculated ratios of the geometric mean number of tumors in genetically modified mice to the geometric mean number in controls. Ratios were estimated from differences in logarithm-transformed tumor numbers. For the colon, we added 0.5 to all numbers of tumors before taking logarithms, to handle zero values. Data from 6- and 14-week-old mice were analyzed separately. These statistical methods were also used to reanalyze tumor data from ApcMin/+ mice with transgenic HPGDS (and controls) from earlier work 5.

Immunohistochemistry

Antibodies used were: mouse monoclonal anti-human PTGDS 15; rabbit polyclonal anti-human HPGDS; mouse monoclonal anti-rat proliferating cell nuclear antigen (PCNA); and rat monoclonal anti-mouse CD31. Staining for HPGDS, PCNA, and CD31 was done on slides from ApcMin/+ mice with transgenic HPGDS from earlier work 5.

In situ hybridization

Digoxigenin-labeled probes were prepared by in vitro transcription from a linearized plasmid vector containing the mouse PTGDR cDNA (DIG RNA labeling kit; Roche; Indianapolis, IN). T7 RNA polymerase was used to make anti-sense probes. SP6 RNA polymerase was used to prepare control sense probes 16.

mRNA analyses by reverse transcription and real-time PCR (RT-PCR)

Primers, probes, and procedures for preparing RNA and determining copy numbers of RNA transcripts are in Supporting Information. Assays for v-myc avian myelocytomatosis viral oncogene homolog (MYC), GAPDH, and vascular endothelial growth factor A (VEGFA) were performed with kits (Applied Biosystems; Grand Island, NY; Mm00487803_m1, Mm99999915_g1, Mm00437304_m1, respectively).

Results

Tumor scoring

We histologically examined >35,000 tumors in Swiss roll sections (Fig.1A–E), including 9837 tumors from 147 mice in Ptgdr knockout experiments, 21,763 tumors from 104 mice in experiments on PTGDS transgenic and Pparg knockout mice, and 3431 tumors reexamined from 39 HPGDS transgenic mice and controls from earlier work 5.

Figure 1

(A–E) Swiss roll section (14 weeks). (A) The box outlines B. Scale bar, 5 mm. (B) The top, middle, and bottom boxes outline E, C, and D, respectively. Scale bar, 1 mm. (C) An early adenoma abutting against a larger adenoma. Scale bar, 200 μm. (D) An early adenoma expanding the villus base. Scale bar, 200 μm. (E) A large adenoma. Scale bar, 200 μm. (F–G) In situ hybridization for PTGDR (12 weeks). (F) Detection of PTGDR mRNA with antisense probes. PTGDR mRNA appears as blue deposits in stromal cells, in a pattern consistent with lymphocytes or monocytes, or both. (G) Sense probes showed no staining (negative control). Counterstained with neutral red. Scale bar, 100 μm. (H–O) Immunohistochemistry (14 weeks). (H–K) High production of human PTGDS and HPGDS in transgenic mice, shown by immunoperoxidase staining (with rabbit polyclonal anti-human PTGDS [H, I] or HPGDS [J, K] antibodies). Staining (brown) occurred in all cell types of the small bowel and colon (epithelium and stroma). Scale bars, 50 μm. (H) Small bowel villi from a wild-type mouse. Antibody labeling is mostly in the cytosol of some epithelial cells, with occasional stromal cell staining. (I) A small bowel villus from a PTGDS transgenic mouse. Antibody binding is heavy throughout the villus, with a cytoplasmic staining pattern. (J) Small bowel villi from a wild-type mouse. HPGDS staining is mainly within the stroma of villi, not epithelial cells. Earlier studies showed these cells to be macrophages and monocytes. (K) Small bowel villi from an HPGDS transgenic mouse, showing heavy antibody staining in all cells (as in I). (L, M) Staining for PCNA in intravillar tumors from ApcMin/+ mice, with and without HPGDS transgenes. There was no consistent difference in staining between mice with and without transgenic HPGDS. Scale bars, 100 μm. (L) An intravillar adenoma from an ApcMin/+ mouse. (M) An intravillar tumor from an HPGDS transgenic ApcMin/+ mouse. (N, O) Staining for microvessels with anti-CD31 antibodies in tumors from ApcMin/+ mice with and without HPGDS transgenes. There was no consistent difference in staining between mice with and without transgenic HPGDS. Scale bars, 100 μm. (N) A tumor from an ApcMin/+ mouse. (O) A tumor from an HPGDS transgenic ApcMin/+ mouse.

The earliest tumors were uniglandular, intravillar lesions with a simple cystic configuration, or intravillar neoplasms, also known as intravillous microadenomas 17,18, dysplastic crypts 19, and cystic crypts (Fig. S1A and S1D) 20,21. More advanced early tumors may have other dysplastic features, such as extension of dysplastic cells (Fig. S1E), multiple lumina (Fig. S1C and G), loss of epithelial cell polarity (Fig. S1G), pseudo-stratification, or crowding (Fig. S1F).

Intravillar tumors progressed by enlarging, forming adjoining cysts (Figs. S1C and S2), or erupting through the villus surface into the bowel lumen (Figs. S1B and S3). Although early tumors arise from crypts 17,22, we found only a few examples of out-pouching of cysts from crypts (Fig. S4). Serial sections from two tumors (75 sections each) showed that early tumors may have no crypt connection (Fig. S5) 21. Examples of early colon tumors are shown in Fig. S6.

Ptgdr knockouts and intestinal tumors

At 6 weeks (Fig.2A), homozygous Ptgdr knockouts raised total numbers of tumors (medians 64 vs. 49.5; P = 0.0086; Table S1A) and numbers of small tumors (medians 58 vs. 42.5; P = 0.0089; Table S1B). Large tumors and colon tumors were not affected by Ptgdr knockouts at 6 weeks.

Figure 2

Tumors in ApcMin/+ mice, with and without Ptgdr knockouts (total, small, large, and colon). (A) Tumors at 6 weeks. (B) Tumors at 14 weeks. +/+, control ApcMin/+ mice. +/− and −/−, ApcMin/+ mice with heterozygous and homozygous Ptgdr knockouts, respectively. Filled symbols: females. Open symbols: males. Horizontal bars: medians. *P < 0.025. See Tables S1 and S2 for details.

At 14 weeks (Fig.2B), heterozygous Ptgdr knockouts increased the median number of large tumors (33 vs. 24; P = 0.023; Table S2C). Also at 14 weeks, homozygous Ptgdr knockouts raised median numbers of total tumors (100 vs. 71; P = 0.0060; Table S2A) and large tumors (38 vs. 24; P = 0.0040; Table S2C). Ptgdr knockouts did not affect small or colon tumors at 14 weeks (Table S2B and D).

To obtain data on occurrence of the earliest tumors, we also scored tumors in ten 3-week-old mice: six ApcMin/+ mice (3–8 tumors each); three ApcMin/+ mice with heterozygous Ptgdr knockouts (5–11 tumors each); and one ApcMin/+ mouse with homozygous Ptgdr knockouts (11 tumors). However, data from these 10 mice were not included in statistical analyses, because of the age difference.

In situ hybridization for PTGDR mRNA showed consistent, but weak, staining of inflammatory cells in the mucosal stroma (lymphocytes or monocytes, or both; Fig.1F–G). There was no detectable staining in epithelial cells of crypts or villi. Staining with PTGDR antibodies was not conclusive (not shown).

Expression of transgenic PTGDS

Human PTGDS transgenes were highly expressed in the intestines, as measured RT-PCR. Specifically, we found 1.61 × 105 and 8.13 × 105 copies of human PTGDS transcripts per nanogram of total RNA in two transgenic mice (geometric mean, 3.6 × 105 copies). These values were comparable to levels for HPGDS transgenes in earlier work (7.5 × 105 copies—a 375-fold increase in expression of transgenic HPGDS over endogenous mouse Hpgds) 5. Immunohistochemistry showed heavy staining of transgenic PTGDS in all intestinal cells (Fig.1H–I). Endogenous mouse PTGDS mRNA was not detectable in the colon.

Transgenic PTGDS and large tumors

With 104 ApcMin/+ mice, we scored intestinal tumors in relation to transgenic PTGDS, with and without heterozygous Pparg knockouts. Among mice without Pparg knockouts, only large tumors were reduced in number by transgenic PTGDS (medians were 52 vs. 83 for controls; P = 0.011; Fig.3A–D; Table S3). Tumor suppression was also reflected by the ratio of the geometric mean number of large tumors in PTGDS transgenic mice to the geometric mean number in controls (ratio = 0.56 for large tumors; 95% confidence interval 0.34–0.92; Table S3C). Large tumors were >150–300 μm in diameter, based on the spacing between sections.

Figure 3

Tumors in ApcMin/+ mice with PTGDS transgenes, with and without heterozygous Pparg knockouts (all 14 weeks). Data are for total (A), small (B), large (C), and colon tumors (D). There were statistically significant reductions in numbers of large tumors (C) in PTGDS transgenic mice without Pparg knockouts. See Table S3 for details. Pparg +/+ indicates no Pparg knockout. Pparg +/− indicates heterozygous Pparg knockout. Symbols are as in Figure2. *P < 0.025.

We measured colon mRNA levels for VEGFA and MYC, relative to endogenous GAPDH transcript levels. PTGDS transgenes lowered median levels of MYC and VEGFA transcripts by 50% in ApcMin/+ mice (Fig. S7).

Heterozygotic Pparg knockouts and transgenic PTGDS

Without PTGDS transgenes, the numbers of tumors in heterozygotic Pparg knockout mice were comparable to numbers in mice without Pparg knockouts (Fig.3; Table S3; see “Control”). Thus, heterozygous Pparg knockouts alone did not increase tumors in ApcMin/+ mice.

On the other hand, ApcMin/+ mice with both transgenic PTGDS and heterozygotic Pparg knockouts had intermediate numbers of large tumors. Specifically, going by medians, there were 52 large tumors in mice with PTGDS transgenes alone, 88 in mice with heterozygotic Pparg knockouts alone, and 70 in mice with both mutations (Table S3C). Similarly, the ratio of the mean number of large tumors in PTGDS transgenic mice to the mean number in controls was 0.56 for mice without heterozygotic Pparg knockouts (95% confidence interval, 0.34–0.92), compared to 0.78 for mice with heterozygotic Pparg knockouts (95% confidence interval, 0.48–1.26).

PTGDS versus HPGDS

As mentioned above, RT-PCR showed similar expression of transgenic PTGDS, compared to transgenic HPGDS (as measured in our previous work) 5. Also, immunohistochemistry showed high levels of PTGDS and HPGDS (Fig.1H–K). Both experiments scored tumors in the same way (24 Swiss roll sections; 150 μm between sections). Therefore, we reanalyzed slides from HPGDS transgenic mice from our first report 5 to directly compare PTGDS to HPGDS (Fig. S8; Table S4). Ratios of the mean total number of tumors in transgenic mice to the mean total in controls were 0.70 for PTGDS, compared to 0.28 for HPGDS (Tables S3A and S4A). Thus, HPGDS may be two times stronger than PTGDS in suppressing tumors.

We assessed tumor cell proliferation in relation to HPGDS transgenes, by use of immunohistochemistry with anti-PCNA antibodies. Again, we used slides from our earlier work on ApcMin/+ mice with HPGDS transgenes 5. We focused on intravillar tumors, because they are fairly uniform in size. There was no difference in PCNA staining in intravillar tumors in HPGDS transgenic versus nontransgenic ApcMin/+ mice (Fig.1L–M). We also counted mitotic figures in all intravillar tumors of 24 HPGDS transgenic mice and 15 ApcMin/+ controls. There were 0.18 mitoses per tumor in HPGDS transgenic ApcMin/+ mice (33 mitotic figures in 182 intravillar tumors), compared to 0.11 mitoses per tumor in non-transgenic ApcMin/+ mice (64 mitotic figures in 587 intravillar tumors). Thus, transgenic HPGDS did not reduce tumor cell proliferation.

Immunohistochemistry with anti-CD31 antibodies showed no consistent difference in microvessel staining between HPGDS transgenic and nontransgenic tumors (Fig.1N–O). Thus, microvessel growth does not appear to explain occurrence of fewer tumors with PGD2.

Tumors in eight mutants with altered PGD2 synthesis or binding

We have now analyzed tumors in eight different ApcMin/+ mouse mutants that have altered PGD2 production or binding, due to knockouts or transgenes. Some experiments used different procedures for cutting sections. For example, we used up to 24 Swiss roll sections for scoring tumors in our first report 5 and in the PTGDS and PPARG experiments shown here. Alternatively, we used 10 sections per Swiss roll in the PTGDR experiments, because reanalysis of earlier data showed that the same conclusions can be reached with 8–10 sections.

To compare data across experiments, we converted the total number of tumors for each mouse to a “multiple of the median” value. Specifically, we divided the total number of tumors for each mouse by the median number of tumors for that mouse's controls. By this analysis, the most tumor-promoting mutations were Hpgds knockouts and homozygous Ptgdr knockouts—raising tumor numbers 40% above control values (Fig.4; all mice were analyzed at 14 weeks). In contrast, HPGDS transgenes were the most tumor-suppressing mutations—reducing tumor numbers to 20–30% of the control value.

Figure 4

Tumors in ApcMin/+ mice with various mutations that affect PGD2 production or binding (all at 14 weeks). Total numbers of tumors scored for each mouse were divided by the median total number of tumors scored among that mouse's controls. HPGDS transgenes are the most tumor-suppressive mutations, whereas homozygous Ptgdr knockouts and homozygous and heterozygous Hpgds knockouts are the most tumorigenic. The dotted line represents median control values (defined as 1.0). TG, transgenic; +/−, heterozygous knockouts; −/−, homozygous knockouts. Horizontal bars, median values: HPGDS TG, 0.23; PTGDS TG, 0.67; Ptgds −/−, 0.85; Pparg +/−, 1.03; Ptgdr +/−, 1.18; Hpgds −/−, 1.34; Ptgdr −/−, 1.40; Hpgds +/−, 1.47. *P < 0.05.

Female versus male ApcMin/+ mice

To assess female–male differences in tumor numbers at 14 weeks, we used current data and two earlier reports 5,23 (for a total of 61 female and 75 male ApcMin/+ mice; Fig. S9). Males and females had similar numbers of intestinal tumors (ratio of tumors in males vs. females, 0.82; P = 0.069). However, males had more colon tumors (ratio of colon tumors in males vs. females, 1.6; P = 0.0002). Results are consistent with McAlpine et al. 24.

Discussion

PTGDR and intestinal tumors

Homozygous deletion of the gene for PGD2 receptor PTGDR led to 30–40% more intestinal tumors in ApcMin/+ mice. The result supports an interpretation that PTGDR mediates tumor inhibition by PGD2 in these mice. We now have data on eight different ApcMin/+ mouse mutants, each with a different alteration in PGD2 production or binding. Homozygous Ptgdr and homozygous or heterozygous Hpgds knockout mutations are the most pro-tumorigenic. On the other hand, HPGDS transgenes are the most tumor-suppressive mutations—lowering numbers of tumors by 70-80% (Fig.4).

There was no detectable staining of PTGDR mRNA in the epithelium of intestinal crypts or villi. However, PTGDR mRNA was consistently detected in inflammatory cells in the mucosal stroma (Fig.1F–G). Tissue-specific gene knockouts will be needed to more conclusively identify the cells that respond to PGD2.

Mutoh et al. 25 treated homozygous Ptgdr knockout mice with azoxymethane starting at 7 weeks and examined colons at 12 weeks. They did not find more aberrant crypt foci in the colons of knockout mice, compared to controls. Our results are consistent with Mutoh et al., because we did not observe more colon tumors at 6 or 14 weeks with Ptgdr knockouts (Fig.2A–B). However, a role for PTGDR in colon tumor growth is supported by human data. Gustafsson et al. 26 found fivefold lower expression of PTGDR in colorectal cancers, compared to normal tissues (62 tumors and 43 normal tissues, from 62 patients). Galamb et al. 27 showed a trend toward decreased PTGDR expression going from normal tissues, to adenomas, to early cancers, and to advanced cancers.

Comparison of PTGDS and HPGDS effects

Transgenic PTGDS in ApcMin/+ mice reduced numbers of large adenomas (>150–300 μm; Fig.3C; Table S3C). In this way, PTGDS had a tumor blocking effect. However, transgenic PTGDS was less effective than transgenic HPGDS in suppressing tumors (Fig.4). Reasons are unknown. A difference between PTGDS and HPGDS is secretion of PTGDS into body fluids, whereas HPGDS stays in the cytosol 28. We recognize that our comparison between PTGDS and HPGDS is based on only one transgenic mouse line for each mutant. But these lines had comparable numbers of PTGDS or HPGDS mRNA transcripts in the intestines (3.6 × 105 and 7.5 × 105 copies, respectively).

Transgenic PTGDS was associated with lower colon expression of MYC (Fig. S7). MYC is a major part of WNT signaling following Apc loss 29. Moreover, disruption of Myc restores the normal appearance of intestinal crypts in mice with intestine-specific Apc knockouts 30. Thus, lower intestinal levels of MYC may be part of the tumor preventive mechanism of PGD2.

Reduced levels of VEGFA mRNA were also seen in PTGDS transgenic mice (Fig. S7). The finding is consistent with VEGFA effects in Apc mice 31. However, we did not see a decrease in microvessel density in large tumors in ApcMin/+ mice with transgenic HPGDS (Fig.1N–O). Thus, tumor suppression by PGD2 did not appear to involve antiangiogenesis in our experiments 32, at a level detectable by anti-CD31 immunohistochemistry.

Transgenic HPGDS did not reduce PCNA immunostaining in early tumors in ApcMin/+ mice (Fig.1L–M). PCNA is a marker of intestinal cell proliferation and belongs to the family of sliding DNA clamps that bind factors at replication forks 33. Similarly, transgenic HPGDS did not lower numbers of mitotic figures in early tumors. Thus, PGD2 does not appear to suppress tumors by lowering rates of tumor cell division.

A possible explanation is increased tumor cell death with PGD2 and PTGDR, as shown by Lewis lung cancer cells implanted onto the backs of mice 6. Alternatively, PGD2 may prevent tumors by slowing initiation.

PPARG and intestinal tumors

Heterozygous Pparg knockouts alone did not increase the numbers of tumors in our ApcMin/+ mice. The result is consistent with earlier reports 24,34. However, McAlpine et al. 24 found ∼30% more tumors in male ApcMin/+ mice with heterozygous or homozygous intestine-specific Pparg deletions.

In our transgenic mice with PTGDS overproduction and reduced adenoma occurrence, the decrease in numbers of large tumors caused by PTGDS appeared blunted in heterozygous Pparg knockout mice (Fig.3C and Table S3C). Such blunting could be compatible with tumor suppression by PGD2 metabolites bound to PPARG 35, when PGD2 production is increased. A limitation in our experiments with heterozygous Pparg knockouts and PTGDS transgenes was the use of mice with mixed C57BL/6-FVB/N backgrounds (all 50% C57BL/6, but not all F1). However, fairly large numbers of mice were used in the Pparg experiments (104 in total). The 147 mice in the Ptgdr knockout experiments were all 100% C57BL/6.

PGD2 and inflammation

Mechanisms for tumor suppression by PGD2 in the intestines have not been proven, but useful information is available. For example, in the skin 36 and lung 37, PGD2 delays migration of dendritic cells to draining lymph nodes, where T cells are primed. PGD2 also reduces the ability of dendritic cells to stimulate naïve T cells 38,39. In the intestinal mucosa, dendritic cells produce IL-23, to stimulate release of IL-22 by immune cells (innate lymphoid cells 40,41, TH17 cells 42, and TH22 cells 43). In turn, IL-22 induces proliferation of epithelial cells, production of inflammatory mediators, and release of antimicrobial proteins, to guard against invaders 44. This cytokine can be neutralized by IL-22-binding protein, a soluble receptor also made by dendritic cells in the colon. Huber et al. 45 showed that IL-22 gene knockouts allowed fewer tumors in ApcMin/+ mice, whereas knockouts of IL-22-binding protein caused more (in the colon). Further work is needed to determine if these functions of dendritic cells explain tumor suppression by PGD2. Identification of mechanisms involving PGD2 and PTGDR may suggest molecular targets for tumor prevention studies.

Conclusions

By scoring tumors in ApcMin/+ mice histologically at 6 and 14 weeks, we showed that homozygous knockouts of the gene for the PGD2 receptor, PTGDR, raised median numbers of tumors by 30–40%. The results support an interpretation that PGD2 is a tumor-suppressing molecule, acting through PTGDR. Heterozygous Pparg knockouts had smaller effects in our experiments. The observation that PGD2 and PTGDR can affect tumorigenesis may have impact for prevention.