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Proc Natl Acad Sci U S A. Nov 16, 2010; 107(46): 20027–20032.
Published online Nov 1, 2010. doi:  10.1073/pnas.1010430107
PMCID: PMC2993406
Medical Sciences

Chemoprevention by nonsteroidal anti-inflammatory drugs eliminates oncogenic intestinal stem cells via SMAC-dependent apoptosis

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac effectively prevent colon cancer in humans and rodent models. However, their cellular targets and underlying mechanisms have remained elusive. We found that dietary sulindac induced apoptosis to remove the intestinal stem cells with nuclear or phosphorylated β-catenin in APCMin/+ mice. NSAIDs also induced apoptosis in human colonic polyps and effectively removed cells with aberrant Wnt signaling. Furthermore, deficiency in SMAC, a mitochondrial apoptogenic protein, attenuated the tumor-suppressive effect of sulindac in APCMin/+ mice by blocking apoptosis and removal of stem cells with nuclear or phosphorylated β-catenin. These results suggest that effective chemoprevention of colon cancer by NSAIDs lies in the elimination of stem cells that are inappropriately activated by oncogenic events through induction of apoptosis.

Prevention of human cancers by using chemical agents or dietary manipulation represents a promising anticancer strategy (1, 2). Widely used nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac and aspirin effectively prevent colon cancer in humans and rodent models (3, 4). However, their cellular targets and underlying mechanisms have remained elusive. Colorectal tumorigenesis is initiated by genetic alterations in the APC tumor suppressor pathway through Wnt signaling, leading to accumulation of β-catenin and its subsequent nuclear translocation (5). This process has been largely recapitulated in animal models such as APCMin/+ mice, which contain an APC mutation and exhibit intestinal adenoma formation (6). Emerging evidence suggests that initial neoplastic proliferation in APCMin/+ mice impinges upon loss of APC in intestinal stem cells (7, 8), including crypt base columnar (CBC) cells near the crypt bottom, as well as those located in position 4–6 (+4) counting from the crypt bottom (9). Several intestinal stem cell markers have been identified, such as Lgr5 (10), Bmi1 (8), and OLFM4 (11).

Substantial evidence indicates that the chemopreventive effects of NSAIDs are mediated by induction of apoptosis, a safeguard mechanism protecting against neoplastic transformation (12, 13). Our previous work established that NSAIDs induce mitochondria- and Bax-dependent apoptosis in colon cancer cells (14), and that SMAC (second mitochondria-derived activator of caspase), a mitochondrial apoptogenic protein (15), is an essential downstream mediator of Bax in NSAID-induced apoptosis (16, 17). In this study, we investigated the role of intestinal stem cell apoptosis in chemoprevention by NSAIDs. Our data suggest a critical role of SMAC-mediated apoptosis in removing early neoplastic stem cells in cancer chemoprevention by NSAIDs.

Results

Sulindac Treatment Induced Apoptosis in Intestinal Stem Cells of APCMin/+ Mice.

Dietary supplementation with NSAIDs such as sulindac for several months prevents adenoma formation in the small intestine of APCMin/+ mice (18). To study the role of apoptosis in chemoprevention by NSAIDs, we first determined the time window for analyzing sulindac-induced apoptosis in APCMin/+ mice because of the rapid and transient nature of apoptotic events. We found that sulindac given for only 1 wk markedly induced apoptosis detected by TUNEL staining in the small intestinal crypts of APCMin/+ mice, with 22.1% of crypts containing at least one TUNEL-positive cell, compared with only 4.0% in mice receiving control diet (Fig. 1A). Importantly, this short exposure reduced the number of macroadenomas by 66.7% (Fig. 1B), consistent with observations made by others (19). Sulindac treatment for 2 wk or longer further decreased polyp numbers (Fig. S1A). However, TUNEL staining detected little apoptosis at 2 wk or later after treatment (Fig. 1A and Fig. S1B), suggesting that most of the apoptosis had occurred earlier. As previously shown (20), sulindac treatment did not significantly affect polyp formation in the colon of APCMin/+ mice. These observations indicate that sulindac rapidly induces apoptosis in the small intestine of APCMin/+ mice, and this early apoptosis may be responsible for effective chemoprevention. Therefore, 1-wk sulindac treatment was chosen for most of the subsequent experiments.

Fig. 1.
Short-term sulindac administration induced apoptosis in the intestinal stem cells and suppressed adenoma formation in APCMin/+ mice. Ten-week-old APCMin/+ mice were fed with control or sulindac-containing (20 mg/kg/d) AIN93G diet for 1 or 2 wk and killed ...

In light of recent reports that APC loss in intestinal stem cells efficiently promotes adenoma formation (7, 8), we further determined the types of cells undergoing apoptosis in APCMin/+ mice following 1 wk of sulindac treatment. Remarkably, a majority of TUNEL-positive cells were the wedge-shaped CBC cells (62.7%) and +4 cells (27.5%), whereas apoptotic cells were rare (<10%) at higher positions in the crypts (Fig. 1 C and D and Fig. S2). Upon introducing the Lgr5-EGFP lineage marking allele (10) into APCMin/+ mice, we found that sulindac treatment induced apoptosis in Lgr5-expressing cells of Lgr5-EGFP/APCMin/+ mice, but not WT mice (Fig. 1 C and E and Fig. S3). The fraction of Lgr5-positive crypts containing one or more TUNEL-positive cells increased from 4.32% in the control mice to 17.60% in the sulindac-treated mice (Fig. 1E). We confirmed that the Lgr5-marked CBC cells and apoptotic cells at the crypt base were interspersed between MMP7-positive Paneth cells (Fig. 1C and Figs. S3B and S4) (21). Active caspase 3 staining verified the induction of apoptosis in these cells (Fig. 1F and Fig. S3C). Interestingly, apoptotic CBC cells were found to be clustered in several neighboring Lgr5-positive crypts (Fig. 1 C and F and Fig. S3A), probably reflecting clonal expansion of the intestinal stem cells in which an early oncogenic event(s) occurred. These data demonstrate that intestinal stem cells are targeted for apoptosis induction following NSAID treatment.

Sulindac Treatment Removed Intestinal Stem Cells with β-Catenin Accumulation and Suppressed β-Catenin Phosphorylation.

Intestinal polyp formation in APCMin/+ mice is always accompanied by loss of the remaining WT APC allele (22), leading to deregulation of Wnt signaling and nuclear translocation of β-catenin (23). We therefore reasoned that sulindac may preferentially induce apoptosis in stem cells with nuclear β-catenin. Indeed, nuclear β-catenin was found in 1.92% of intestinal crypts in the control mice, including both the CBC and +4 cells, but rarely (<0.01% crypts) in other areas of the intestinal epithelium, or in the crypts of WT mice (Fig. 2A). Sulindac treatment for only 1 wk reduced the number of crypts containing cells with nuclear β-catenin by 75% (Fig. 2A). Interestingly, a vast majority (98%, 0.47%/0.48%) of identifiable CBC and +4 cells with nuclear β-catenin in sulindac-treated APCMin/+ mice were TUNEL-positive at this time point (Fig. 2 A and B).

Fig. 2.
Sulindac treatment removed the intestinal cells with nuclear or phospho-β-catenin via apoptosis. WT and APCMin/+ mice were fed with control or sulindac-containing (20 mg/kg/d) diet for 1 or 2 wk and killed immediately after treatment. Small intestinal ...

It has been shown that β-catenin nuclear translocation can be promoted by phosphorylation at Ser552 in the +4 cells (24). We found that the number of cells positive for β-catenin Ser552 phosphorylation (p-β-catenin), including mostly +4 and above +4 cells that did not express Lgr5 and some (11.4%) Lgr5-expressing cells (Fig. S5), was ninefold higher in APCMin/+ mice compared with that in WT mice. Sulindac treatment significantly reduced cells with p-β-catenin (Fig. 2C), and induced rapid and significant apoptosis in these cells (Fig. 2D). These results suggest that sulindac treatment rapidly removes intestinal stem cells or progenitors with aberrant activation of Wnt signaling through induction of apoptosis.

NSAID Treatment Induced Apoptosis in Human Colonic Polyps and Removed Cells with Aberrant Wnt Signaling.

To test the relevance of these observations in human patients, we analyzed colonic polyps in patients taking NSAIDs. The percentage of colonic crypts containing TUNEL-positive apoptotic cells increased by more than 10-fold (from 5.04% to 51.9%) in the patients taking NSAIDs compared with those not taking NSAIDs (Fig. 3 A and B and Fig. S6). TUNEL-positive cells could be detected among those stained positive for OLFM4, a Wnt target and a CBC cell marker (11, 25) (Fig. 3C). Interestingly, we found that the number of p-β-catenin-positive cells decreased drastically (by more than sixfold) in patients taking NSAIDs (Fig. 3D). These data suggest that NSAIDs selectively induce apoptosis in human intestinal polyps with aberrant Wnt signaling.

Fig. 3.
NSAIDs induced apoptosis in human colonic polyps and removed cells with activated Wnt signaling. (A) TUNEL staining (brown) of intestinal polyps from patients taking or not taking NSAIDs. Arrows indicate TUNEL-positive apoptotic cells. (B) Quantification ...

SMAC Deficiency Attenuated the Chemopreventive Effect of Sulindac.

Our previous work revealed that SMAC, a mitochondrial apoptogenic protein released into cytosol during apoptosis execution (15), is essential for NSAID-induced apoptosis in colon cancer cells (16, 17). To determine whether such a mechanism operates in vivo, age- and sex-matched cohorts of APCMin/+ mice with WT SMAC (APCMin/+) or SMAC-KO (SMAC−/−/APCMin/+) were generated and subjected to sulindac treatment for 1 wk. SMAC deficiency significantly attenuated the chemopreventive effect of sulindac in APCMin/+ mice (50.2% vs. 69.6%; P < 0.01; Fig. 4A and Fig. S7A). A slight increase in polyp number in SMAC-deficient APCMin/+ mice was observed, and taken into the consideration. Anatomic stratification revealed that the differences were mainly in the middle and distal regions, but not in the proximal region of small intestine (Fig. 4B). No significant difference in polyp size was found.

Fig. 4.
SMAC deficiency attenuated the chemopreventive effect of sulindac in APCMin/+ mice by blocking apoptosis in the intestinal stem cells. Age- and sex-matched parental (APCMin/+) and SMAC-deficient APCMin/+ mice (SMAC−/−/APCMin/+) were fed ...

SMAC Deficiency Impaired Sulindac-Induced Apoptosis and Suppression of Nuclear β-Catenin Accumulation.

Following 1 wk of sulindac treatment, the number of crypts with TUNEL-positive CBC/+4 cells was significantly lower in the SMAC−/−/APCMin/+ mice than in APCMin/+ mice (9.9% vs. 22.1%; P < 0.005; Fig. 4C and Fig. S7B). Apoptosis in the crypts decreased significantly in both strains following 2 wk of sulindac treatment (Fig. 4C and Fig. S7C). Similarly, the number of cells or crypts with nuclear β-catenin was significantly higher in SMAC−/−/APCMin/+ mice compared with that in APCMin/+ mice (1.33% vs. 0.48%; P < 0.05; Fig. 4D), which was correlated with a significant decrease of apoptosis in the CBC/+4 cells with nuclear β-catenin (0.22% vs. 0.47%; P < 0.05; Fig. 4E and Fig. S7B). Furthermore, SMAC deficiency significantly impaired apoptosis and removal of p-β-catenin–positive cells in the crypts (Fig. 4 F and G). In addition, sulindac treatment did not affect SMAC expression in the mucosa of APCMin/+ mice (Fig. S8A) and in colon cancer cells that undergo SMAC-dependent apoptosis (17) (Fig. S8B). These results demonstrate that SMAC-mediated apoptosis in the intestinal stem cells with aberrant activation of Wnt signaling directly contributes to chemoprevention.

Discussion

Neoplastic transformation appears to be driven by accumulation of genetic and epigenetic alterations in tissue stem cells or progenitors with pluripotency and regenerative potential (26, 27). Our results indicate that CBC and +4 intestinal stem cells accumulating nuclear or p-β-catenin are selectively removed by NSAIDs in APCMin/+ mice through apoptosis induction, which translates into effective tumor prevention. Apoptosis in intestinal epithelial cells proceeds rapidly, typically within days (28), which may explain why we could detect sulindac-induced apoptosis only at an early time point. The partial effect of SMAC deficiency on sulindac-mediated chemoprevention is consistent with incomplete block of sulindac-induced apoptosis in SMAC-KO mice (Fig. 4C) and cells (17), and involvement of additional mechanisms including COX inhibition (29). The upstream events that activate Bax to trigger SMAC release following sulindac treatment remain to be delineated, and may involve death receptor signaling as suggested by several recent studies (30, 31).

Several characteristics of stem cells may explain the preferential killing of oncogenic stem cells by sulindac. Stem cells express high levels of “stemness” factors including the oncoprotein c-Myc (32), a well known apoptosis inducer (33). Therefore, stem cells with oncogenic alterations, such as loss of APC, may be more sensitive to NSAID-induced apoptosis, relative to differentiated cells with such alterations. It is also possible that stem cells with oncogenic alterations are simply more prevalent than differentiated cells with such alterations, because stem cells can regenerate and permanently keep acquired genetic changes, whereas differentiated cells with these changes may quickly disappear because of their rapid turnover.

Long-term use of NSAIDs, in particular COX2-specific inhibitors, is associated with side effects, which has stimulated active pursuit of new targets and combination strategies for cancer chemoprevention (34). Induction of apoptosis in oncogenic stem cells is likely to be a useful marker for successful cancer prevention, and may hold the promise for identifying novel and improved cancer chemopreventive agents. Small-molecule SMAC mimetics, which are in clinical development and can sensitize colon cancer cells to NSAID-induced apoptosis (16), may be useful as sensitizers of NSAIDs for safer and more effective cancer chemoprevention.

Methods

Mice and Treatment.

All animal experiments were approved by the Institutional Animal Care and Use Committee at University of Pittsburgh. The SMAC-KO mice on a mixed background (129/C57BL/6) (35) were backcrossed to C57BL/6 background for 10 generations. Female SMAC+/− mice were crossed with APCMin/+ mice (Jackson Laboratory) to generate SMAC+/−/APCMin/+ male mice, which were crossed to SMAC+/− mice to generate APCMin/+ littermates with homozygous WT (+/+) or null (i.e., KO; −/−) SMAC alleles. The previously described Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) mice (10) were crossed with APCMin/+ mice to generate Lgr5-EGFP/APCMin/+ mice. All mice were housed in micro isolator cages in a room illuminated from 7:00 AM to 7:00 PM (i.e., 12-h/12-h light-dark cycle), and allowed access to water and chow ad libitum. Genotyping was performed as previously described for SMAC (35) and for Lgr5 (10). APC genotyping was according to the Jackson Laboratory protocol.

Treatment and Tumor Analysis.

Ten-week-old and sex-matched APCMin/+ mice with different SMAC and Lgr5 genotypes were fed with control or experimental AIN93G diet (Dyets) containing 200 ppm (approximately 20 mg/kg/d) of sulindac (Sigma) for 1, 2, or 22 wk. Mice were killed immediately after treatment. Dissection of small intestine and histological analysis of adenomas (polyps; >0.5 mm in diameter) were performed as previously described (36). The adenoma counts were performed under a dissection microscope at various times following sulindac treatment.

Immunostaining.

Tissue sections (5 μm) were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide, followed by antigen retrieval in boiling 0.1 M citrate (pH 6.0) buffer for 10 min twice. The sections were then blocked by 20% goat/rabbit serum for 30 min. TUNEL staining was performed by using an ApopTag Kit (Chemicon International) according to the manufacturer's protocol. Immunostaining was performed as previously described for MMP7 (21), active caspase 3 (37), and OLFM4 (25). EGFP staining was performed at 4 °C overnight using a mouse anti-EGFP antibody (Santa Cruz Biotechnology), with Alexa 594 (Invitrogen) for signal detection. β-Catenin staining was done at 4 °C overnight using a mouse anti-β-catenin antibody (BD Biosciences), with Alexa 488 (Invitrogen) for signal detection. Staining of p-β-catenin Ser552 was performed as described (24). For double staining, TUNEL staining was performed following EGFP, β-catenin, p-β-catenin, OLFM4, or MMP-7 staining. EGFP staining was performed before MMP7, p-β-catenin, or active caspase 3 staining. Cells with positive staining were scored in at least 500 crypt sections and reported as mean ± SD.

Clinical Samples.

Frozen specimens of polyps from four patients taking NSAIDs and four patients not taking NSAIDs were obtained from the Digestive Disease Tissue Resource of the University of Pittsburgh. Acquisition of tissue samples was approved by the institutional review board at the University of Pittsburgh and written informed consent was received from each patient. Paraffin blocks and sections were prepared as previously described (21) and analyzed by immunostaining.

Two male and two female subjects were represented in each category, with ages ranging from 50 to 65 y in the NSAID group and 58 to 75 y in the non-NSAID group. Subjects taking NSAIDs reported use ranging from one to three tablets per week to greater than seven tablets per week during the preceding year. The specific NSAIDs in use were not recorded. All patients had advanced adenomas by virtue of having polyps at least 1 cm in size. Four patients had tubulovillous histology and four had tubular adenomas.

Statistical Analysis.

Statistical analyses were carried out using GraphPad Prism IV software. P values were calculated by the Student's t test. P < 0.05 was considered to be significant. The means ± 1 SD are displayed in the figures where applicable.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Dr. Monica E. Buchanan and other members of the L.Z. and J.Y. laboratories for helpful discussion and advice, Dr. Ronald A. DePinho at the Dana-Farber Cancer Institute (Boston, MA) for providing rederived Lgr5-EGFP mice, and Dr. Linheng Li at the Stowers Institute for Medical Research (Kansas City, MO) for providing the phospho-β-catenin antibody. This work was supported in part by National Institutes of Health Grants CA121105 (to L.Z.), CA106348 (to L.Z.), CA129829 (to J.Y.), and U01-DK085570 (to J.Y.), the latter as part of the Intestinal Stem Cell Consortium, a collaborative research project funded by the National Institute of Diabetes and Digestive and Kidney Diseases. This work was also supported by American Cancer Society Grants RSG-07-156-01-CNE (to L.Z.) and RGS-10-124-01-CCE (to J.Y.) and a grant from the Flight Attendant Medical Research Institute (to J.Y.).

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1010430107/-/DCSupplemental.

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