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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Mol Cancer Res. 2013 Jun 5;11(9):1040–1050. doi: 10.1158/1541-7786.MCR-13-0084-T

Epithelial 11β-hydroxysteroid dehydrogenase type II deletion inhibits Apc+/min mouse tumorigenesis via COX-2 pathway inhibition and induction of G1 cell cycle arrest

Li Jiang 1,4, Shilin Yang 1, Huiyong Yin 1,3, Xiaofeng Fan 1, Suwan Wang, Bing Yao, Ambra Pozzi 1, Xiaoping Chen 4, Raymond C Harris 1, Ming-Zhi Zhang 1,2
PMCID: PMC3778073  NIHMSID: NIHMS487224  PMID: 23741059

Abstract

Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) promotes colorectal tumorigenesis. Glucocorticoids are endogenous and potent COX-2 inhibitors, and their local actions are down-regulated by 11β–hydroxysteroid dehydrogenase type II (11ßHSD2)-mediated metabolism. We previously reported that 11ßHSD2 increased in human colonic and Apc+/min mouse intestinal adenomas and correlated with increased COX-2 expression and activity, and 11ßHSD2 inhibition suppressed the COX-2 pathway and decreased tumorigenesis. 11ßHSD2 is expressed in Apc+/min mouse intestinal adenoma stromal and epithelial cells. We generated Apc+/min mice with selective deletion of 11ßHSD2 in intestinal epithelial cells (Vil-HSD2-/- Apc+/min). 11ßHSD2 deletion in intestinal epithelia led to marked inhibition of Apc+/min mouse intestinal tumorigenesis. Immunostaining indicated decreased 11ßHSD2 and COX-2 expression in adenoma epithelia, while stromal COX-2 expression was intact in Vil-HSD2-/- Apc+/min mice. In Vil-HSD2-/- Apc+/min mouse intestinal adenomas, both p53 and p21 mRNA and protein levels were increased, with concomitant decrease in phosphorylation of retinoblastoma protein, indicating glucocorticoid-mediated G1 cell cycle arrest. Regulated in development and DNA damage responses 1 (REDD1), a novel stress-induced gene that inhibits mammalian target of rapamycin (mTOR) signaling pathway, was increased, while the mTOR signaling pathway was inhibited. Therefore, in Vil-HSD2-/- Apc+/min mice, epithelial cell 11ßHSD2 deficiency leads to inhibition of adenoma initiation and growth by attenuation of COX-2 expression, increased G1 cell cycle arrest and inhibition of mTOR signaling as a result of increased tumor intracellular active glucocorticoids. 11ßHSD2 inhibition may represent a novel approach for colorectal cancer chemoprevention by increasing tumor glucocorticoid activity, which in turn inhibits tumor growth by multiple pathways.

Keywords: adenoma, epithelia, cyclooxygenase-2, glucocorticoid-metabolism, mice

Introduction

Colorectal cancer (CRC) is a leading cause of cancer death. Most patients present with advanced disease, and metastatic disease remains largely incurable. Therefore, prevention remains the best approach to reduce the overall morbidity and mortality of CRC. There is a clear link between cyclooxygenase-2 (COX-2) derived PGE2 and CRC progression (1,2) Clinical evidence indicates a 40-50% reduction in CRC in individuals who take NSAIDs regularly, either in the context of sporadic CRC or in patients of familial adenomatous polyposis (FAP) (3-6). NSAIDs reduce the relative risk of CRC primarily due to their ability to inhibit COX-2-mediated prostaglandin production (7). However, long-term use of traditional NSAIDs increases gastrointestinal side-effects due primarily to COX-1 inhibition (8,9) Selective COX-2 inhibitors have shown similar efficacy to traditional NSAIDs in treating acute and chronic inflammatory conditions and in reducing the number and size of adenomas in FAP patients and in Apc+/min mice with fewer gastrointestinal side-effects (10,11), and were touted as promising agents for chemoprevention and chemotherapy of CRC. However, long-term use of selective COX-2 inhibitors has been found to be associated with an increased incidence of cardiovascular events (12,13), thought to be due to inhibition of endothelial cell-derived COX-2 activity, with selective inhibition of COX-2 derived PGI2 production but without inhibition of COX-1 mediated prothrombotic platelet thromboxane A2 production (14).

Glucocorticoids (GCs) are the most potent, endogenous, specific COX-2 inhibitors, acting to suppress COX-2, but not COX-1 expression, through stimulating glucocorticoid receptors (15-17). In addition to inhibiting COX-2 expression, GCs also reduce prostaglandin production through inhibition of cytosolic phospholipase A2 activity, which prevents the release of arachidonic acid from membrane phospholipids (18), and through inhibition of microsomal prostaglandin E synthetase (mPGES-1) expression, a major terminal synthetase in PGE2 biosynthesis (19). In addition to treatment of hematologic malignancies, GCs can inhibit solid tumor growth, regress tumor mass, and prevent metastasis by blocking angiogenesis (20,21). However, the undesirable side-effects of immune suppression limit their application in cancer chemoprevention and chemotherapy.

In tissues, GC effects are regulated by a “pre-receptor” regulatory mechanism involving 11ß–hydroxysteroid dehydrogenase type I (11ßHSD1) and 11ßHSD2 (12). 11ßHSD1 produces active GCs from inactive metabolites, while 11ßHSD2 converts GCs to their inactive keto-forms. We recently reported that 11ßHSD2 expression was increased in human colonic and Apc+/min mouse intestinal adenomas and correlated with increased COX-2 expression and activity (23). 11ßHSD2 inhibition reduces tumor COX-2-mediated PGE2 production and tumor growth by increasing the tonic glucocorticoid-mediated suppression of the COX-2 signaling pathway, suggesting that 11ßHSD2 inhibition may be a potential therapeutic option to prevent and/or treat colorectal cancer. To exclude the possible off-targets of glycyrrhizic acid, the 11ßHSD2 inhibitor we used in our previous report (23), and to investigate the role of intestinal epithelial 11ßHSD2 in colorectal tumorigenesis, we generated Apc+/min mouse with selective deletion of 11ßHSD2 in intestinal epithelial cells and determined that 11ßHSD2 in intestinal epithelial cells plays an important role in adenoma development and growth.

Materials and Methods

Generation of a mouse line with selective deletion of 11βHSD2 in intestinal epithelial cells

We utilized a BAC engineering strategy to produce an Hsd11b2 conditional gene targeting vector, in which a Neo cassette was flanked by two Flrt sites (Neoflrt), and the Hsd11b2 gene was flanked by two loxP sites (Hsd11b2flox). The detailed procedures and information regarding the generation of Neoflrt;Hsd11b2flox mice are illustrated in Supplemental Figure 1. The targeting construct was linearized with Not I and electroporated into 129 ES cells. The targeting events were found in 6 out of 237 colonies resistant to G418 as demonstrated by an expected 3.0-kb product amplified by PCR and confirmed by Southern blot. Two positive ES clones were used for implantation. Three male chimeras with targeted events (with Neoflrt/Hsd11b2flox allele) were obtained. These chimeras were crossed with female congeneic C57BL/6 mice. Germline transmission of the targeted events in the F1 mice was screened by PCR and further confirmed by Southern blot as illustrated in Supplemental Figure 2. The Neo cassette was effectively removed by crossing with ACTB:FLPe mice. The mice bearing the Hsd11b2flox allele were backcrossed to C57BL/6 background for 10 generations, and then crossed with C57BL/6 Vil-Cre mice (Jackson Lab, Stock # 004586), in which the Cre recombinase is controlled by the villin promoter, which is active as early as E12.5 in the entire intestinal epithelium (24).

Animals

All animal experiments were performed according to animal care guidelines and were approved by the Vanderbilt University IACUC. Germline mutations in the adenomatous polyposis coli (APC) gene lead to familial adenomatous polyposis, and inactivation of APC is also found in the majority of sporadic CRC. The Apc+/min mouse generated by random ethylnitrosourea mutagenesis carries a T to A transversion at nucleotide 2549 of the Apc gene. The resulting point mutation in codon 850 produces a premature stop codon and a truncated polypeptide of approximately 95 kD (25). The autosomal dominant heterozygous nonsense mutation of the Apc gene in the Apc+/min mouse is homologous to human germline and somatic APC mutations. Apc+/min mice develop grossly detectable adenomas within a few months. Vil-Cre/Hsd11b2flox mice were crossed with Apc+/min (C56BL/6, Jackson Laboratory) to generate Apc+/min mice in which the Hsd11b2 gene in intestinal epithelial cells has been deleted selectively (Vil-HSD2-/- Apc+/min mice). At 20 weeks of age, 10 wild type Apc+/min mice and 8 Vil-HSD2-/- Apc+/min mice were sacrificed for analysis. Under anesthesia with Nembutal (60 mg/kg i.p., Abbot Laboratories), the entire intestine was dissected, flushed thoroughly with ice-cold PBS (pH 7.4), and then filled with fixative (26). The intestine was transferred to 70% ethanol for 24 h, opened longitudinally, and examined using a dissecting microscope to count polyps in a blinded fashion. The tumor diameter was measured with a digital caliper. After tumors were counted, intestinal tissues were processed for paraffin embedding. A subset of mice (6 wild type Apc+/min mice and 6 Vil-HSD2-/- Apc+/min mice) was sacrificed for collection of adenomas and intestine tissue for quantitative real-time PCR, Western blot analysis and determination of tissue levels of corticosterone and 11-keto-corticosterone. All animals were genotyped twice (after weaning and before sacrifice). Probes 0IMR0033, 0IMR0034 and 0IMR0758 were used for Apc+/min genotyping and probes 0IMR0015 and 0IMR0016 for villin-Cre genotyping.

Wild type Apc+/min mice (16-18 weeks of age) were treated with the selective COX-2 inhibitor SC-58236 (2mg/kg/day, daily gastric gavage, a gift from Searle Monsanto) for 7 days and tumors with similar size were collected for Western analysis.

Cell culture

Mouse colon adenocarcinoma MC38 cells (C57BL/6) were obtained from Dr. Bixiang Zhang (Huazhong University of Science & Technology, PR. China) in March, 2012. MC38 cells were authenticated by DNA short-tandem repeat analysis in February, 2012 and used for experiments within 5 months. MC-38 cells were grown in RPMI1640 supplemented with 4,500 mg/l glucose, 2 mM l-glutamine, 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 and 95% air at 37°C (27). MC-38 cells were cultured in medium with 1% fetal bovine serum overnight and then treated with 1μM dexamethasone or dexamethasone plus 10 μM glucocorticoid receptor antagonist, RU486 in normal culture medium for 6 hours. RU486 was added 30 min before addition of dexamethasone.

RNA isolation and quantitative real time PCR (qRT-PCR)

Total RNA was isolated from tumors using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions. qRT-PCR was performed using TaqMan real time PCR machine (7900HT, Applied Biosystems). The Master Mix and all gene probes were also purchased from Applied Biosystems. The probes used in the experiments included mouse S18 (Mm02601778), p53 (Trp53, Mm01731290), p21 (Cdkn1a, 04205640), p27 (Dctn6, 00495994), and REDD1 (Ddit4, Mm00512504).

Measurement of corticosterone and 11-keto-corticosterone

Colonic tissues were collected and stored at −80°C. Tissue levels of corticosterone (active form) and 11-keto-corticosterone (inactive form) were measured using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (23). Corticosterone was from ICN Biomedicals, and 11-keto-corticosterone was from Steraloids.

Antibodies

Affinity-purified rabbit anti-mouse 11βHSD2 (catalog no. BHSD22-A) was purchased from Alpha Diagnostic International; rabbit anti-murine COX-2 (catalog no. 160106) was from Cayman Chemicals; rabbit anti-c-Myc was from Santa Cruz Biotechnology, rabbit anti-REDD1 was from Lifespan (LC-53286); rabbit anti- p21waf1/cip1(29470), rabbit anti-p-Rb (9308), rabbit anti-p-mTOR (2971, Ser2448), rabbit anti-p-p70 S6K (9234), rabbit anti-p-4E-BP1 (9451), rabbit anti-p-S6 ribosomal protein (p-rpS6) (4858, Ser235/236), rabbit anti-cleaved caspase-3 (9661) and mouse anti-cyclin D1 (2926) were from Cell Signaling.

Immunohistochemistry and Western analysis

Immunostaining and western blot analysis were carried out as previously reported (28).

Quantitative image analysis

Immunostaining of β-catenin, cleaved caspase-3 and p21waf1/cip1 was quantified using the BIOQUANT image analysis system (R & M Biometrics, Nashville, TN) (23). Bright-field images from a Leitz Orthoplan microscope with DVC digital RGB video camera were digitized and saved as computer files. Contrast and color level adjustment (Adobe Photoshop) were performed for the entire image; i.e., no region- or object-specific editing or enhancements were performed.

Statistical analysis

Values are presented as means ± S.E.M. ANOVA and Bonferroni t-test were used for statistical analysis, and differences were considered significant when P < 0.05.

Results

Since 11ßHSD2 is primarily localized to epithelial cells in both human colonic and Apc+/min mouse intestinal adenomas (23), we generated Apc+/min mouse line with selective deletion of 11ßHSD2 in intestinal epithelia by crossing Apc+/min mouse with Hsd11b2flox/flox mice and villin Cre mice (Supplementary Figures 1&2). Apc+/min mice are normally on the C57BL/6 background, and Apc+/min mouse adenoma development and growth are sensitive to genetic background (29). We therefore backcrossed Hsd11b2flox/+ mouse onto C57BL/6 background for 10 generations, then crossed with Vil-Cre mouse (C57/BL/6) and Apc+/min mouse to get Apc+/min mice with Hsd11b2flox/flox alleles (wild type Apc+/min mice) or with Hsd11bΔ/Δ alleles (Vil-Cre/Hsd11b2flox/flox/Apc+/min mice, designated as Vil-HSD2-/- Apc+/min mice).

To determine whether colonic 11ßHSD2 expression was decreased in Vil-HSD2-/- Apc+/min mice, colonic tissues were collected for Western analysis. As indicated in Figure 1A, colonic 11ßHSD2 levels were markedly decreased in Vil-HSD2-/- Apc+/min mice compared to wild type Apc+/min mice. As expected, colonic COX-2 levels were also markedly decreased in Vil-HSD2-/- Apc+/min mice compared to wild type Apc+/min mice (Figure 1B). 11ßHSD2 and COX-2 levels in small intestine were also significantly lower in Vil-HSD2-/- Apc+/min mice than wild type Apc+/min mice (data not shown).

Figure 1. Intestinal 11ßHSD2 and COX-2 expression decreased in Vil-HSD2-/- Apc+/min mice.

Figure 1

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(A) Immunoblotting indicated decreased intestinal 11ßHSD2 expression in Vil-HSD2-/- Apc+/min mice compared to wild type Apc+/min mice. (B) Immunoblotting demonstrated decreased intestinal COX-2 expression in Vil-HSD2-/- Apc+/min mice (5 wild type mice and 6 Vil-HSD2-/- mice). (C) 11ßHSD2 deletion in intestinal epithelia significantly increased corticosterone levels and decreased 11-keto-corticosterone levels in intestines. n = 6. *P < 0.05 vs. wild type Apc+/min mice.

To investigate whether selective deletion of 11ßHSD2 in intestinal epithelia is associated with functional alterations in glucocorticoid metabolism, intestinal tissues were collected and glucocorticoid levels were measured. As indicated in Figure 1C, intestinal corticosterone levels (active glucocorticoid) were > 10-folds higher in Vil-HSD2-/- Apc+/min mice than wild type Apc+/min mice (corticosterone: 30.34 ± 12.35 vs. 2.73 ± 0.57 ng/g of wild type Apc+/min mice, p< 0.05, n = 6 in each group). In contrast, intestinal 11-keto-corticosterone levels (inactive glucocorticoid) were significantly lower in Vil-HSD2-/- Apc+/min mice than wild type Apc+/min mice (11-keto-corticosterone: 1.16 ± 0.45 vs. 4.56 ± 1.15 ng/g of wild type Apc+/min mice, p< 0.05). Therefore, selective deletion of 11ßHSD2 in intestinal epithelia was associated with increases in active glucocorticoid levels and decreases in inactive glucocorticoid levels in intestine of Vil-HSD2-/- Apc+/min mouse.

To collect enough adenoma tissue for analysis, animals were sacrificed at 20 weeks of age. Immunohistochemistry demonstrated that adenoma epithelial 11ßHSD2 immunostaining was evident in wild type Apc+/min mice but diminished in Vil-HSD2-/- Apc+/min mice (Figure 2A). In Vil-HSD2-/- Apc+/min mouse adenoma epithelia, decreased 11ßHSD2 expression was associated with decreased COX-2 expression. In contrast, adenoma stromal COX-2 expression was comparable between wild type Apc+/min mice and Vil-HSD2-/- Apc+/min mice (Figure 2B). Immunoblotting confirmed decreased adenoma COX-2 expression in Vil-HSD2-/- Apc+/min mice (Figure 2C). Therefore, 11ßHSD2 deletion in adenoma epithelia led to selective COX-2 inhibition in epithelia in Vil-HSD2-/- Apc+/min mouse.

Figure 2. Adenoma epithelial cell 11ßHSD2 and COX-2 expression decreased in Vil-HSD2-/- Apc+/min mice.

Figure 2

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(A) Immunostaining indicated that 11ßHSD2 expression in adenoma epithelial cells disappeared in Vil-HSD2-/- Apc+/min mice (arrow). Original magnification: Left lanes: × 100; right lanes: × 250. (B) Adenoma COX-2 expression disappeared in epithelial cells (arrow), but was still apparent in stromal cells (arrow head) in Vil-HSD2-/- Apc+/min mice. Original magnification: Left lanes: × 100; right lanes: × 250. (C) Western blot analysis demonstrated decreased adenoma COX-2 expression in Vil-HSD2-/- Apc+/min mice. n = 4. *P < 0.01 vs. wild type Apc+/min mice.

At 20 weeks of age, total intestinal adenoma number was markedly lower in Vil-HSD2-/- Apc+/min mice than wild type Apc+/min mice (44.3 ± 5.3 vs. 83.9 ± 4.9 in wild type, P < 0.001, n = 10 in wild type group and n = 8 Vil-HSD2-/- group) (Figure 3). Adenoma size is an independent risk factor for the progression of colorectal cancer (30). Adenomas were stratified by size to determine whether selective deletion of 11ßHSD2 in intestinal epithelia affected size. Vil-HSD2-/- Apc+/min mice showed significant decreases in polyps smaller than 2 mm, but had no difference in polyps larger than 2 mm (Figure 3), suggesting that inhibiting intestinal epithelial 11ßHSD2 primarily prevented the initiation of polyp formation.

Figure 3. Selective deletion of 11ßHSD2 in intestinal epithelia inhibited tumorigenesis in Apc+/min mice.

Figure 3

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(A) 11ßHSD2 deletion in intestinal epithelia led to markedly reduced intestinal adenoma multiplicity (*P < 0.001 vs. wild type Apc+/min mice, n = 10 in wild type group, n = 8 in Vil-HSD2-/- group). (B) Adenomas smaller than 2 mm decreased significantly in Vil-HSD2-/- Apc+/min mice (*P < 0.001, ** P < 0.01 vs. wild type Apc+/min mice).

To determine whether inhibition of tumorigenesis in Vil-HSD2-/- Apc+/min mice was related to decreased cell proliferation and/or increased apoptosis, the expression levels of cyclin D1, c-Myc, β-catenin and cleaved caspase-3 were investigated. Immunostaining indicated that cyclin D1 was primarily localized to the nuclei of adenoma epithelial cells. Both cyclin D1 immunostaining density and the number of cyclin D1 positive epithelial cells were reduced in Vil-HSD2-/- Apc+/min mice compared to wild type Apc+/min mice (Figure 4A). Western blot analysis confirmed the decreased adenoma cyclin D1 expression in Vil-HSD2-/- Apc+/min mice. Similarly, the expression levels of adenoma c-Myc and β-catenin were also reduced in Vil-HSD2-/- Apc+/min mice (Figure 4 B&C). In contrast, the number of adenoma cells that were positive for cleaved caspase-3, a specific marker of apoptosis, was significantly higher in Vil-HSD2-/- Apc+/min mice compared to wild type Apc+/min mice (Figure 4 D). Therefore, there was decreased cell proliferation and increased apoptosis in adenomas of Vil-HSD2-/- Apc+/min mice.

Figure 4. Selective deletion of 11ßHSD2 in intestinal epithelia decreased tumor cell proliferation and increased tumor cell apoptosis.

Figure 4

Figure 4

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(A) Western blot showed decreased cyclin D1 expression in adenomas from Vil-HSD2-/- Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 4). Immunostaining demonstrated that cyclin D1 was primarily localized to adenoma epithelial cell nuclei, and its expression decreased in Vil-HSD2-/- Apc+/min mice. (B) Western blot indicated decreased c-Myc expression in adenomas from Vil-HSD2-/- Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 4). Immunostaining demonstrated that c-Myc was expressed in nuclei and cytosols in adenoma epithelial cells and its expression decreased in Vil-HSD2-/- Apc+/min mice. (C) Immunostaining demonstrated that adenoma ß-catenin expression was markedly inhibited in Vil-HSD2-/- Apc+/min mice compared to wild type Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 4). (D) Adenoma apoptosis increased in Vil-HSD2-/- Apc+/min mice as indicated by increase in cleaved-caspase-3 positive cells, a specific marker of apoptosis (*P < 0.01 vs. wild type Apc+/min mice, n = 4). Original magnification: × 100 in left panels of A, B, and C; × 250 in right panels of A, B, and C as well as in D.

The retinoblastoma protein (Rb), a tumor suppressor, inhibits cell proliferation through its interaction with the E2F family of transcription factors and the resultant regression of genes that are essential for DNA synthesis (31). The inactivation of retinoblastoma protein is a prerequisite for cell proliferation. Inactivation of Rb is achieved through cyclin-dependent protein kinase-mediated phosphorylation during cell cycle progression. Glucocorticoids activate Rb through p53-mediated induction of p21waf1/cip1, an inhibitor of cyclin-dependent kinase (28, 31-35). qRT-PCR determined that both adenoma p53 and p21 mRNA levels increased markedly in Vil-HSD2-/- Apc+/min mice compared to wild type Apc+/min mice (p53 mRNA: 27.8 ± 3.5 vs. 14.4 ± 2.8 of wild type Apc+/min mice, P < 0.05; p21waf1/cip1 mRNA: 94.1 ± 5.6 vs. 51.7 ± 5.6 of wild type Apc+/min mice, P < 0.001, n = 6 in each group) (Figure 5A). The expression of adenoma p27, another inhibitor of cyclin-dependent protein kinase, was not affected. Immunostaining demonstrated that p21waf1/cip1 in adenomas was predominantly localized to nuclei, and the density of p21waf1/cip1 positive cells was markedly higher in Vil-HSD2-/- Apc+/min mice than in wild type Apc+/min mice (p21waf1/cip1 cells/hpf: 41. 3 ± 4.8 vs. 11.8 ± 1.6 of wild type Apc+/min mice, P < 0.001, n = 4) (Figure 5B). We further found that the phosphorylation levels of retinoblastoma protein in adenomas were dramatically reduced in Vil-HSD2-/- Apc+/min mice (Figure 5C). Immunostaining determined that phosphorylated retinoblastoma protein in adenomas was present in nuclei as well as in the cytosol and its expression was reduced in Vil-HSD2-/- Apc+/min mice (Figure 5C). Therefore, in Vil-HSD2-/- Apc+/min mice, increased active glucocorticoids in adenoma epithelial cells stimulated the signaling pathway regulating retinoblastoma protein mediated cell cycle arrest.

Figure 5. Selective deletion of 11ßHSD2 in intestinal epithelia activated adenoma retinoblastoma protein pathway in Apc+/min mice.

Figure 5

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(A) Adenoma mRNA levels of p53 and p21waf1/cip1 were increased in Vil-HSD2-/- Apc+/min mice (*P < 0.01, **P < 0.05 versus wild type Apc+/min mice, n = 6). (B) The number of adenoma p21waf1/cip1 positive cells markedly increased in Vil-HSD2-/- Apc+/min mice (*P < 0.01, vs. wild type Apc+/min mice, n = 4). (C) The levels of the active form of the tumor suppressor, retinoblastoma protein (Rb) increased in adenomas from Vil-HSD2-/- Apc+/min mice as indicated by decreased phosphorylation (*P < 0.01 vs. wild type Apc+/min mice, n = 4). Immunostaining confirmed decreased number of phosphorylated Rb positive cells in adenomas from Vil-HSD2-/- Apc+/min mice. Original magnification: × 250.

Glucocorticoids may also inhibit tumor growth through suppression of the mTOR signal pathway (36, 37). REDD1 (RTP801, an mTOR complex 1, mTORC1, repressor) is a novel stress-induced gene linked to regression of mTOR signaling and is induced by glucocorticoids (38-40). Therefore, we investigated whether REDD1 expression and activity of mTOR pathway were altered in adenomas from Vil-HSD2-/- Apc+/min mice. Adenoma REDD1 mRNA levels were markedly higher in Vil-HSD2-/- Apc+/min mice than in wild type Apc+/min mice (32.8 ± 2.9 vs. 18.8 ± 2.2 of wild type Apc+/min, P < 0.01, n = 6) (Figure 6A) and adenoma REDD1 protein level was more than 2-folds higher in Vil-HSD2-/- Apc+/min mice than in wild type Apc+/min mice (Figure 6B). In contrast, the activity of adenoma mTOR pathway was suppressed in Vil-HSD2-/- Apc+/min mice as indicated by decreased phosphorylated mTOR (Ser2448) levels (Figure 6C) as well as decreased levels of phosphorylated p70 S6K (Figure 6D) and phosphorylated 4E-BP1 (Figure 6E), two major downstream targets of mTOR signaling. In addition, levels of adenoma phosphorylated S6 ribosomal protein (rpS6, Ser235/236), a downstream target of p70 S6K, were decreased in Vil-HSD2-/- Apc+/min mice (Figure 6F). The levels of adenoma phosphorylated eEF2K, another downstream target of p70 S6K, were also reduced in Vil-HSD2-/- Apc+/min mice (data not shown).

Figure 6. Selective deletion of 11ßHSD2 in intestinal epithelia inhibited adenoma activity of the mTOR pathway in Apc+/min mice.

Figure 6

Figure 6

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(A) Adenoma REDD1 mRNA levels were increased in Vil-HSD2-/- Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 6). (B) Adenoma REDD1 protein levels were increased in Vil-HSD2-/- Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 4). (C) Adenoma mTOR activity was markedly inhibited in Vil-HSD2-/- Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 4). (D&E) Adenoma p-70 S6K and 4E-BP1 activities were markedly inhibited in Vil-HSD2-/- Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 4). (F) Adenoma levels of phosphorylated S6 ribosomal protein (p-rpS6) were markedly inhibited in Vil-HSD2-/- Apc+/min mice (*P < 0.01 vs. wild type Apc+/min mice, n = 4).

To investigate whether inhibition of the COX-2 pathway contributes to cell cycle arrest and suppression of mTOR pathway seen in Vil-HSD2-/- Apc+/min mice, wild type Apc+/min mice were treated with a selective COX-2 inhibitor and activity of retinoblastoma protein and mTOR was determined. As indicated in Supplementary Figure 3, inhibition of COX-2 pathway had no effect on activity of mTOR (Ser2448) and retinoblastoma protein. In cultured mouse colon adenocarcinoma MC-38 cells, treatment with dexamethasone, a synthetic glucocorticoid, led to induction of p53, p21 and REDD1 (supplemental Figure 4A) but inhibition of the phosphorylation of retinoblastoma protein, mTOR (Ser2448), and p70 S6K, all of which were reversed by the glucocorticoid receptor inhibitor, RU486 (Supplementary Figure 4B), indicating that glucocorticoids may inhibit colon cancer cell proliferation through activating the tumor suppressor retinoblastoma protein pathway and inhibiting the mTOR pathway. In addition, inhibition of mTOR activity or inhibition of COX-2 activity did not affect the expression levels of p53, p21 and REDD1 in MC38 cells (Supplemental Figure 5).

Discussion

Our present results demonstrate that intestinal epithelial 11ßHSD2 activity contributes to increased COX-2 expression in Apc+/min mouse intestinal adenomas and that 11ßHSD2 deficiency in intestinal epithelial cells suppresses adenoma development and growth due to inhibition of the COX-2 pathway, induction of G1 cell cycle arrest, and inhibition of the mTOR pathway as a result of increased intracellular active glucocorticoids in tumor epithelial cells (Figure 7). Recent studies have demonstrated a clear molecular link between COX-2-derived PGE2 and colorectal cancer progression (1), and inhibition of COX-2-derived PGE2 production by traditional NSAIDs or selective COX-2 inhibitors reduces the number and size of adenomas in Apc+/min mice and in FAP patients (3-6),(10,11,28). However, increased gastrointestinal side effects of traditional NSAIDs and increased cardiovascular risks of selective COX-2 inhibitors limit their use in chemoprevention of CRC. That most patients present with advanced disease and that metastatic disease is largely incurable underscore that prevention remains the best approach to reduce the overall morbidity and mortality of CRC.

Figure 7. Proposed mechanism underlying epithelial cell 11ßHSD2 activity and colorectal tumorigenesis.

Figure 7

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In tumor epithelial cells, glucocorticoids are converted to inactive 11-keto-forms, reducing glucocorticoid receptor activity, while 11ßHSD2 deletion leads to increased levels of epithelial intracellular active glucocorticoid. The subsequent inhibition of the COX-2 pathway and induction of G1 cell cycle arrest through activation of retinoblastoma protein (Rb, a tumor suppressor) due to induction of p53 and p21 and inhibition of the mTOR pathway due to induction of REDD1 lead to inhibition of colorectal tumorigenesis.

GCs and NSAIDs inhibit prostaglandin biosynthesis through different mechanisms. NSAIDs inhibit prostaglandin biosynthesis by non-competitive inhibition of both COX-1 and COX-2 enzymatic activity. GCs are the most potent, endogenous, specific COX-2 inhibitors. GCs suppress prostaglandin production through inhibiting cytosolic phospholipase A2 activity and suppressing COX-2 and mPGES-1 expression. The actions of GCs are regulated systemically and locally (7). Circulating glucocorticoid levels are regulated by the hypothalamo-pituitary-adrenal axis. Within target tissues, the actions of GCs are down-regulated by 11ßHSD2-mediated metabolism. 11ßHSD2 is expressed predominantly in mineralocorticoid responsive tissues such as the kidney and colon to convert GCs to their inactive keto-forms, thus providing mineralocorticoid receptor selectivity to aldosterone.

GCs are known to inhibit cell proliferation and induce cell differentiation through activation of glucocorticoid receptors. 11ßHSD2 has been thought to be pro-proliferative due to its ability to inactivate glucocorticoids (41,44). We recently reported that 11ßHSD2 expression is increased in epithelial cells and stromal cells in human colonic and Apc+/min mouse intestinal adenomas and is correlated with increased COX-2 expression and activity, and that inhibition of 11ßHSD2 activity with glycyrrhizic acid suppresses tumor COX-2 pathway and prevents adenoma formation, tumor growth, and metastasis as a result of increased tumor intracellular active glucocorticoids (23). One limitation of our previous study is that glycyrrhizic acid is not a specific 11ßHSD2 inhibitor, and its potential off-target effects could not be excluded. The other limitation is that pharmacologic inhibition of 11ßHSD2 activity cannot distinguish the role of epithelial vs. stromal 11ßHSD2 in tumorigenesis.

In the current studies, we generated Apc+/min mice with selective deletion of 11ßHSD2 in intestinal epithelia, Vil-HSD2-/- Apc+/min mice, which had reduced 11ßHSD2 and COX-2 expression with concomitant increased active corticosterone levels and decreased inactive 11-keto-corticosterone levels in the intestine (Figure 1). In adenomas from Vil-HSD2-/- Apc+/min mouse, both epithelial 11ßHSD2 and COX-2 expression decreased, while stromal COX-2 expression was not affected. Therefore, in Vil-HSD2-/- Apc+/min mouse adenomas, selective deletion of 11ßHSD2 in epithelia was associated with selective inhibition of COX-2 that was confined to the epithelia. In Vil-HSD2-/- Apc+/min mouse, epithelial 11ßHSD2 and COX-2 deficiency was associated with a marked decrease in total adenoma number while the number of adenomas larger than 2 mm was not affected, suggesting that epithelial 11ßHSD2 and/or COX-2 may primarily promote the initiation and formation of adenomas or that in large adenomas 11ßHSD2 was not sufficiently deleted. Since COX-2 inhibitors and COX-2 deletion dramatically decrease both adenoma formation and size (10, 11, 28, 45), the present results suggested that the stroma may be the major source of prostaglandins that mediate adenoma growth (46). Treatment with a peroxisome proliferator-activated receptor-δ agonist primarily increases intestinal adenoma growth in Apc+/min mice (30), further suggesting that Apc+/min mouse intestinal adenoma development and growth may be regulated by different signal pathways.

A recent study with overexpression of COX-2 in intestinal epithelia suggests that increased intestinal epithelial COX-2 is not itself sufficient to initiate tumorigenesis (47). In Vil-HSD2-/- Apc+/min mouse, tumorigenesis was markedly inhibited, although only epithelial COX-2 expression was decreased, suggesting involvement of further signaling pathways. Indeed, increased intracellular active glucocorticoids in epithelia after 11ßHSD2 deletion could inhibit cell proliferation and induce differentiation through different mechanisms such as induction of G1 cell cycle arrest and inhibition of the mTOR pathway (36-38, 48).

Glucocorticoids induce G1 cell cycle arrest through p-53 mediated induction of p21waf1/cip1, known as a cyclin-dependent kinase inhibitor, which directly binds to and inhibits the activity of cyclin E/CDK2 and cylin D/CDK4/6 complexes, leading to hypophosphorylation of its major downstream target, the retinoblastoma protein, a tumor suppressor. Activated Rb due to dephosphorylation interacts with the E2F family of transcription factors and inhibits cell cycle progression (31). In adenomas from Vil-HSD2-/- Apc+/min mouse, p53 and p21waf1/cip1 levels were markedly increased while the levels of phosphorylated Rb were decreased, suggesting G1 cell cycle arrest contributes to tumor inhibition due to 11ßHSD2 deletion in epithelia as a result of increased intracellular active glucocorticoids. mTOR signaling is critical in cell growth (49). REDD1 is an mTOR inhibitor and has been found to be upregulated in several colon cancer cell lines when their growth is inhibited (50). Glucocorticoids have been reported to inhibit the mTOR pathway through induction of REDD1 (38-40). In adenomas from Vil-HSD2-/- Apc+/min mouse, both REDD1 mRNA and protein levels increased, while activity of the mTOR pathway was inhibited as indicated by decreased phosphorylation of mTOR (Ser2448), p70-S6K, 4E-BP1, and S6 ribosomal protein (Figure 6).

The current investigation supports our previous studies that suggested that inhibition of 11ßHSD2 activity with glycyrrhizic acid and its analogs may represent a novel approach for CRC chemoprevention with the following advantages by increasing tumor active glucocorticoids: 1) GCs selectively inhibit the COX-2 but not COX-1 pathway, therefore having the beneficial effects of traditional NSAIDs to prevent and regress colorectal cancers without the gastrointestinal side-effects associated with COX-1 inhibition; 2) Physiologic 11ßHSD2 expression is largely restricted to colon and kidney. Therefore, inhibition of 11ßHSD2 activity is not expected to incur the cardiovascular risk posed by COX-2 inhibitors that suppress COX-2-derived PGI2 production in vascular endothelial cells; 3) Intracellular active GCs are only increased in tissues with elevated 11ßHSD2 expression. 11ßHSD2 inhibition will not produce immunosuppression or other systemic side-effects of conventional glucocorticoid therapy; 4) In addition to inhibiting the COX-2 pathway, increased tumor active GCs also inhibit tumor development and growth through induction of G1 cell cycle arrest and inhibition of the mTOR pathway (Figure 7). Although 11ßHSD2 inhibition may result in salt-dependent hypertension due to activation of mineralocorticoid receptors by GCs, development of locally acting enteric 11ßHSD2 inhibitors that are not systemically absorbed would be a potential therapeutic means to prevent colorectal tumorigenesis (7).

Supplementary Material

1
2

Acknowledgments

Grant Support

These studies were supported by grants from the National Institutes of Health (CA122620, DK38226, DK51265, DK62794, GM15431, ES13125, NCI P50 95103 Special Program of Research Excellence), by the Vanderbilt O’Brien Center (DK79341), by funds from the Veterans Administration, by grant from National Natural Science Foundation of China (31170809), by National Key Basic Research Program of China (973 Program, #2012CB524900) and by Hundred Talents Program from CAS (2012OHTP07).

Footnotes

Disclosure of Potential Conflicts of Interest

The authors have no potential conflicts of interest

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