Targeted Suppression of β-Catenin Blocks Intestinal Adenoma Formation in APC Min Mice
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- Foley, P.J., Scheri, R.P., Smolock, C.J. et al. J Gastrointest Surg (2008) 12: 1452. doi:10.1007/s11605-008-0519-6
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Mutations involving the adenomatous polyposis coli (APC) tumor suppressor gene leading to activation of β-catenin have been identified in the majority of sporadic colonic adenocarcinomas and in essentially all colonic tumors from patients with Familial Adenomatous Polyposis. The C57BL/6J-APCmin (Min) mouse, which carries a germ line mutation in the murine homolog of the APC gene is a useful model for intestinal adenoma formation linked to loss of APC activity. One of the critical downstream molecules regulated by APC is β-catenin; molecular targeting of β-catenin is, thus, an attractive chemopreventative strategy in colon cancer. Antisense oligodeoxynucleotides (AODNs) capable of downregulating murine β-catenin have been identified.
Analysis of β-catenin Protein Expression in Liver Tissue and Intestinal Adenomas
Adenomas harvested from mice treated for 7 days with β-catenin AODNs demonstrated clear downregulation of β-catenin expression, which was accompanied by a significant reduction in proliferation. There was no effect on proliferation in normal intestinal epithelium. Min mice treated systemically with β-catenin AODNs over a 6-week period had a statistically significant reduction in the number of intestinal adenomas. These studies provide direct evidence that targeted suppression of β-catenin inhibits the formation of intestinal adenomas in APC-mutant mice. Furthermore, these studies suggest that molecular targeting of β-catenin holds significant promise as a chemopreventative strategy in colon cancer.
Approximately 150,000 people will develop colorectal cancer in the United States in 2007, and it is estimated that over 50,000 people will die from it.1 While surgery and chemotherapy are useful modalities for treating patients with colon cancer, additional strategies in the treatment of such patients are needed. It has become apparent that the accumulation of genetic mutations in a clonal cell population results in the transition of normal colonic epithelium to carcinoma.2 Defining the molecular abnormalities involved in the development of neoplasia offers hope for specific molecular targeting in the treatment of established tumors as well as for chemopreventative interventions.
The wnt or β-catenin signaling pathway is important in developmental processes and carcinogenesis.3 In normal cells, β-catenin is tightly regulated by a multiprotein destruction complex that includes GSK-3β, APC, axin, and protein phosphatase 2A.4 In the presence of wnt glycoproteins, β-catenin is stabilized by the inhibition of GSK-3β. Free cytoplasmic β-catenin translocates into the nucleus where it binds to T-cell factor proteins and serves as a coactivator to stimulate transcription of multiple target genes that regulate cellular proliferation, survival, and migration including c-myc, cyclin D1, AF-17, MMP-7, and PPARδ among others.5–11
The adenomatous polyposis coli (APC) gene product is a critical regulatory component of the wnt signaling pathway and is involved in downregulating β-catenin within the cell. Dysregulation of this pathway, leading to the aberrant accumulation of β-catenin and subsequent stimulation of target gene transcription, has been described as a critical step in carcinogenesis.12,13 Mutations involving APC or β-catenin have been described in the vast majority of sporadic colonic adenocarcinomas.14,15 Additionally, inherited germ line mutations in the APC gene result in Familial Adenomatous Polyposis, an autosomal dominant disorder.16,17 Affected individuals develop numerous colonic adenomas at an early age with malignant degeneration occurring in most by age 50.18 C57BL/6J-APCmin (Min) mice carry a germ line mutation in the murine homolog of the APC gene.19 These mice develop multiple intestinal adenomas which make them an excellent model for studying chemopreventative agents for neoplasia related to inactivation of the APC gene.
Targeted therapeutics directed at downregulating β-catenin expression represent an attractive approach to modifying aberrant downstream signaling in colon cancer. Studies utilizing antisense oligodeoxynucleotides (AODNs) and small interfering RNA to selectively inhibit β-catenin expression have demonstrated efficacy in the inhibition of APC-mutant colon cancer cell growth both in vitro and in vivo.20–22 These studies provide a direct link between the disruption of abnormal β-catenin or Tcf signaling and growth inhibition of established tumors. Numerous chemoprevention studies using a variety of agents have demonstrated efficacy in the suppression of adenoma formation in Min mice.23 Many of these agents have been shown to downregulate β-catenin among multiple other targets, but specific targeting of β-catenin signaling in intestinal adenoma formation has not been described.24–31
To directly examine the role of β-catenin in the development of intestinal adenomas and the role of targeted suppression of β-catenin in the prevention of intestinal adenomas, our laboratory employed a strategy utilizing AODNs in Min mice. A novel 20-base phosphorothioate AODN sequence capable of specifically and potently suppressing the expression of murine β-catenin in a dose-dependant manner was utilized. The studies presented here demonstrate a role for aberrant β-catenin signaling in the development of intestinal adenomas and supports chemopreventative strategies aimed at the targeted suppression of β-catenin.
Materials and Methods
Female C57BL/6J-APCmin (Min) mice, at 7 weeks of age, were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Balb/c nude mice, 4- to 8-week-old female, were purchased form the National Cancer Institute (NCI, Frederick, MD, USA). Animals were housed in the University of Pennsylvania, School of Medicine Animal Research Facility with a 12-h light–dark cycle under constant temperature and humidity. Animals were given a standard mouse chow diet and drinking water ad libitum. Guidelines established by the University of Pennsylvania regarding the humane care and use of laboratory animals were followed.
ODNs and Antisense Treatment Protocol
Murine β-catenin Antisense 20mer: 5′-GCT TTT CTG TCC GGC TCC AT-3′
Murine β-catenin Scrambled 20mer: 5′-TGC CTG CCT ACT GTT CTG TC-3′
It should be noted that the scrambled control ODN contains the same nucleotides but differs from the β-catenin AODN at 16 of 20 bases. Lyophilized AODNs were reconstituted in sterile phosphate-buffered saline (PBS) to 20 mg/ml and stored in aliquots at −20°C. Stock β-catenin and scrambled AODNs were diluted in sterile Hanks Balanced Salt Solution (Mediatech, Herndon, VA, USA) to concentrations of 10 and 20 mg/kg in a volume of 0.5 ml per mouse. Following treatment the mice were sacrificed by CO2 asphyxiation. The intestinal tracts were removed from each animal from distal esophagus to rectum, opened longitudinally, flushed with saline, and flattened for tumor counting. Tumors were counted under a 10× dissecting microscope by an observer blinded to the treatment group and genotype of the animal. In some experiments, mice were pulsed with bromodeoxyuridine prior to sacrifice, and the tissue was processed for immunohistochemistry.
Analysis of β-catenin Protein Expression in Liver Tissue and Intestinal Adenomas
Following the 7-day intraperitoneal (i.p.) injection course consisting of either ODN or control, the Balb/c nude mice were sacrificed, and liver specimens were surgically removed. Following sacrifice of the Min mice, representative adenomas were surgically excised under the dissecting microscope after counting was completed. Tissue was homogenized in 10 volumes (w/v) homogenization buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), 50 mM NaCl, 1 mM ethylene diamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, and 1% aprotinin. Tissue debris was removed by centrifugation at 2,000×g for 10 min. The supernatant fraction was transferred to a fresh tube and lysed by the addition of Triton X-100 to a final concentration of 1%. The sample was centrifuged at 30,000×g for an additional 30 min to remove insoluble material. Protein concentration was measured by Bradford colorimetric assay (Bio-Rad).
Balb/c nude mice liver tissue was fixed with Bouin’s solution, embedded in paraffin, and sectioned at 3 μm. Tissue sections were deparaffinized and stained as described below. Min mice were given an intraperitoneal injection of 5-bromo-2-deoxyuridine (120 mg/kg) and 5-fluoro-2′-deoxyuridine (12 mg/kg; Sigma Chemical Co., St. Louis, MO, USA) 90 min prior to sacrifice. Following sacrifice, the intestinal tract was removed, flushed with cold PBS, fixed in Bouin’s solution for 6–12 h, and washed with 70% ethanol. The intestinal tract was opened with an incision along its cephalocaudal axis on the antimesenteric border and, then, rolled from its proximal to distal end. Each of these resulting “swiss rolls” was, then, cut in half parallel to the duodenal ileal axis and placed on a tissue cassette with the cut edge of one half facing down and the cut half of the other half facing up. The tissue was embedded in paraffin, and serial sections were prepared. Sections were stained with hematoxylin and eosin to inspect mucosal histology.
Tissue was deparaffinized in xylene and isopropanol and subjected to an antigen unmasking procedure consisting of a 15-min incubation at 37°C in type II bovine pancreas chymotrypsin (Sigma Chemical Co., St. Louis, MO, USA). Endogenous peroxidases were inactivated by immersing the slides in 2% H2O2 and 100% methanol for 5 min at room temperature. The treated sections were, then, washed in PBS, placed in blocking buffer (1% BSA, 0.3% Triton X-100, and 0.2% powdered milk in PBS), and stained overnight at 4°C with either a monoclonal antibody specific for β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a monoclonal antibody specific for BrdU (Calbiochem, San Diego, CA, USA). Antigen-antibody complexes were detected by using a peroxidase-labeled secondary antibody. BrdU-positive cells were quantified, and the proliferative index was determined by calculating the percentage of BrdU-positive cells to the total number of columnar epithelial cells in a given field. Statistical analysis was performed using the Student’s t-test.
Representative adenomas from each of the treatment groups were surgically excised and snap frozen in liquid nitrogen. Tissue was homogenized in 1 mL of TRIzol® (Invitrogen, Carlsbad, CA, USA), and total RNA was isolated according to the supplier’s instructions. Total RNA was, then, subjected to the Oligotex® mRNA spin column (Qiagen, Valencia, CA, USA) to yield purified mRNA. Equal amounts of mRNA was, then, electrophoresed on a formaldehyde containing 1% agarose gel, transferred onto a nylon membrane (Boehringer Mannheim, Indianapolis, IN, USA), and hybridized with a digoxigenin-labeled murine β-catenin cDNA probe. A digoxigenin-labeled Glyceraldehyde 3-phosphate dehydrogenase cDNA probe (CLONTECH, Palo Alto, CA, USA) was used as a loading and transfer control.
Equal amounts of cell lysate from tissue specimens in each treatment group were subjected to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane (Immobilon-P, Milipore, Bedford, MA, USA). β-Catenin-specific antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An actin-specific monoclonal antibody was obtained from Chemi-Con (Temecula, CA, USA) and was used to control for equal protein loading. All antibodies were used according to the supplier’s recommendations.
Inhibition of β-Catenin Expression in Intestinal Adenomas using Antisense ODNs
A 20-base pair ODN capable of potent suppression of human β-catenin expression has been previously described.20,21 The murine and human β-catenin genes are highly homologous; therefore, the design of the murine β-catenin antisense ODN involved targeting a similar region of the murine β-catenin mRNA sequence as in the human β-catenin antisense sequence. A novel 20-base phosphorothioate antisense oligodeoxynucleotide sequence capable of specifically and potently suppressing the expression of murine β-catenin in a dose-dependant manner was identified.
β-Catenin Antisense Inhibits Cellular Proliferation in Intestinal Adenomas
β-Catenin Antisense Inhibits Intestinal Adenoma Formation in Min Mice
There have been numerous published studies in Min mice using a variety of agents which demonstrate inhibition of intestinal adenomas.23 A number of these studies examined effects on β-catenin signaling, particularly with the use of nonsteroidal anti-inflammatory drugs (NSAIDs). However, NSAIDs have multiple targets including cox-2, NF-κB, PPARγ, PDK-1/Akt, and RSK-2/MAPKs, thereby making specific conclusions regarding the contribution of aberrant β-catenin expression to adenoma formation difficult.32 None of the previously published chemoprevention studies have utilized direct targeting of β-catenin to evaluate its contribution to adenoma formation. The studies presented above have used AODNs to specifically downregulate β-catenin expression in APC-mutant mice and have shown that systemic administration of β-catenin AODNs results in specific and potent suppression of both β-catenin mRNA and protein levels in intestinal adenomas. Downregulation of β-catenin expression was accompanied by a significant decrease in cellular proliferation in intestinal adenomas as well as a significant reduction in the total number of intestinal adenomas. Collectively, these experiments provide evidence for the critical role of altered β-catenin signaling in the development of intestinal adenomas.
Previous studies utilizing β-catenin AODNs to inhibit the growth of APC-mutant colon cancer cells demonstrated these molecules to be sequence- and target-specific, providing evidence that the mechanism for growth inhibition in these cell lines is directly related to suppression of β-catenin signaling.20,21 Further direct evidence of the importance of β-catenin in the growth and survival of APC and β-catenin-mutant colon cancer cells was provided through studies demonstrating growth inhibition of colon cancer cells utilizing siRNA directed against β-catenin.22 Together, these studies have provided a direct link between aberrant β-catenin expression and neoplastic growth.
The experiments presented above have demonstrated that target-specific suppression of β-catenin using AODNs results in significant inhibition of adenoma formation. It is likely that chemoprevention studies demonstrating greater inhibition of adenoma formation are, in part, due to a particular agent’s ability to affect a diverse range of targets involved in cell growth and proliferation. It is also possible that AODNs may reflect less potency in downregulating β-catenin than other agents, particularly NSAIDs. Agents utilized clinically as chemopreventative agents may have toxicities linked to effects on targets other than β-catenin. Our data would support further development of potent, specific agents that target β-catenin for the chemoprevention of colon cancer.
It has become clear that altered β-catenin expression plays a significant role in the enhanced growth and survival of neoplastic cells. The results above indicate that agents designed to specifically target and potently antagonize activation of β-catenin signaling have efficacy in chemoprevention of intestinal adenoma formation. The diversity of genetic disruption involved in the initiation and progression of neoplasia suggests that the most efficacious therapies will be those that target multiple defective pathways. Further study of agents which have the ability to inhibit β-catenin signaling is warranted to determine both the molecular mechanism and advance the design of targeted therapeutics.
Targeted suppression of β-catenin inhibits intestinal adenoma formation in APC-mutant mice. Molecular targeting of β-catenin holds significant promise as a chemopreventative strategy in colon cancer.
This work was supported by National Institutes of Health grant RO1 CA100189 and a Clinician Scientist Award in Translational Research from the Burroughs Wellcome Fund (to JAD).