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Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium.

Feng Y, Sakamoto N, Wu R, Liu JY, Wiese A, Green ME, Green M, Akyol A, Roy BC, Zhai Y, Cho KR, Fearon ER - PLoS Genet. (2015)

Bottom Line: Ctnnb1 hemizygous dose markedly inhibited increases in β-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key β-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base.A critical threshold for β-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for β-catenin in Ctnnb1 gene expression and CTNNB1 transcription.The active β-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Adenomatous polyposis coli (APC) inactivating mutations are present in most human colorectal cancers and some other cancers. The APC protein regulates the β-catenin protein pool that functions as a co-activator of T cell factor (TCF)-regulated transcription in Wnt pathway signaling. We studied effects of reduced dosage of the Ctnnb1 gene encoding β-catenin in Apc-mutation-induced colon and ovarian mouse tumorigenesis and cell culture models. Concurrent somatic inactivation of one Ctnnb1 allele, dramatically inhibited Apc mutation-induced colon polyposis and greatly extended Apc-mutant mouse survival. Ctnnb1 hemizygous dose markedly inhibited increases in β-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key β-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base. A critical threshold for β-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for β-catenin in Ctnnb1 gene expression and CTNNB1 transcription. The active β-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells. In mouse ovarian endometrioid adenocarcinomas (OEAs) arising from Apc- and Pten-inactivation, while Ctnnb1 hemizygous dose affected β-catenin levels and some β-catenin/TCF target genes, Myc induction was retained and OEAs arose in a fashion akin to that seen with intact Ctnnb1 gene dose. Our findings indicate Ctnnb1 gene dose exerts tissue-specific differences in Apc mutation-instigated tumorigenesis. Differential expression of selected β-catenin/TCF-regulated genes, such as Myc, likely underlies context-dependent effects of Ctnnb1 gene dosage in tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Heterozygous Ctnnb1 inactivation does not prevent mouse ovarian endometrioid adenocarcinoma (OEA) development and the reduction in Ctnnb1 dosage or CTNNB1 transcripts shows differential effects on selected β-catenin/TCF target genes.(A) Representative photomicrographs of H&E stained ovarian cancer tissue sections from Apcfl/flPtenfl/fl, and Apcfl/flPtenfl/flCtnnb1fl/+ mice show similar morphologic characteristics independent of Ctnnb1 gene dosage, with areas of glandular, overtly epithelial differentiation admixed with more poorly differentiated spindle-cell areas. Scale bars, 100 μm. (B) Representative photomicrographs of β-catenin immunohistochemical staining in tumors from Apcfl/flPtenfl/fl mice, showing strong nuclear β-catenin translocation and moderate cytoplasmic staining, whereas tumors from Apcfl/flPtenfl/flCtnnb1fl/+ mice showed weak nuclear and moderate cytoplasmic β-catenin staining. Scale bars, 25 μm. (C) cDNAs were obtained from murine normal ovaries (Cntrl, n = 4) and OEAs from Apcfl/flPtenfl/flCtnnb1fl/+ (n = 6), and Apcfl/flPtenfl/fl (n = 6) mice following AdCre injection. Transcript levels of Ctnnb1, Axin2, Myc and Nkd1 were measured by qRT-PCR analysis and normalized with β-actin. The gene expression levels were set to 1 for control (Cntrl) group. Error bars denote S.D. **P < 0.01 and *P < 0.05. (D) TOV-112D cells (the human OEA-derived cell line harboring β-catenin gain of function mutation), stably transduced with two different doxycycline-inducible shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl), were treated for 4 days with DOX (“+ DOX”) or a solvent control (“- DOX”). CTNNB1 shRNA-mediated effects on CTNNB1 (left) and MYC (right) gene expression are shown. Gene expression was assessed by qRT-PCR and normalized to HPRT expression. Error bars denote S.D. *P < 0.05 in Student's t test.
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pgen.1005638.g008: Heterozygous Ctnnb1 inactivation does not prevent mouse ovarian endometrioid adenocarcinoma (OEA) development and the reduction in Ctnnb1 dosage or CTNNB1 transcripts shows differential effects on selected β-catenin/TCF target genes.(A) Representative photomicrographs of H&E stained ovarian cancer tissue sections from Apcfl/flPtenfl/fl, and Apcfl/flPtenfl/flCtnnb1fl/+ mice show similar morphologic characteristics independent of Ctnnb1 gene dosage, with areas of glandular, overtly epithelial differentiation admixed with more poorly differentiated spindle-cell areas. Scale bars, 100 μm. (B) Representative photomicrographs of β-catenin immunohistochemical staining in tumors from Apcfl/flPtenfl/fl mice, showing strong nuclear β-catenin translocation and moderate cytoplasmic staining, whereas tumors from Apcfl/flPtenfl/flCtnnb1fl/+ mice showed weak nuclear and moderate cytoplasmic β-catenin staining. Scale bars, 25 μm. (C) cDNAs were obtained from murine normal ovaries (Cntrl, n = 4) and OEAs from Apcfl/flPtenfl/flCtnnb1fl/+ (n = 6), and Apcfl/flPtenfl/fl (n = 6) mice following AdCre injection. Transcript levels of Ctnnb1, Axin2, Myc and Nkd1 were measured by qRT-PCR analysis and normalized with β-actin. The gene expression levels were set to 1 for control (Cntrl) group. Error bars denote S.D. **P < 0.01 and *P < 0.05. (D) TOV-112D cells (the human OEA-derived cell line harboring β-catenin gain of function mutation), stably transduced with two different doxycycline-inducible shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl), were treated for 4 days with DOX (“+ DOX”) or a solvent control (“- DOX”). CTNNB1 shRNA-mediated effects on CTNNB1 (left) and MYC (right) gene expression are shown. Gene expression was assessed by qRT-PCR and normalized to HPRT expression. Error bars denote S.D. *P < 0.05 in Student's t test.

Mentions: To assess the role of β-catenin function in another Apc mutation-dependent tumor model, we explored the role of Ctnnb1 gene dosage in a mouse model of ovarian endometrial adenocarcinoma (OEA) arising from bi-allelic inactivation of both the Apc and Pten genes [29]. Prior studies have shown that the Wnt/β-catenin/Tcf signaling pathway is deregulated by mutations in 16%–38% of human OEAs, and PTEN mutations are often seen in the OEAs with Wnt pathway mutations [29–32]. In the mouse OEA model, tumors are initiated by conditional inactivation of the Apc and Pten genes following injection of AdCre into the right ovarian bursa of Apcfl/flPtenfl/fl mice [29]. Interestingly, in both Apcfl/flPtenfl/fl mice and Apcfl/flPtenfl/flCtnnb1fl/+ mice, adenocarcinomas morphologically similar to human OEAs formed following AdCre injection, with 100% penetrance and no difference in tumor latency between mice with two wild type Ctnnb1 alleles or one wild type and one floxed Ctnnb1 allele (Table 1). In addition, no significant differences in survival rates, tumor volumes, and rates of liver metastasis were found between AdCre-injected Apcfl/flPtenfl/fl mice and Apcfl/flPtenfl/flCtnnb1fl/+ littermates (Table 1), and OEAs arising in both lines of mice shared similar histological features and immunohistochemical staining patterns for cytokeratin-8 (CK8), E-cadherin and α-inhibin (Fig 8A and Table 1). Efficient Cre-mediated deletion of Ctnnb1 and Apc was confirmed in tumors from these mice, and no OEAs arose in the AdCre-injected right ovaries in Apcfl/flPtenfl/flCtnnb1fl/fl mice, indicating OEAs could not arise from cells completely lacking β-catenin.


Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium.

Feng Y, Sakamoto N, Wu R, Liu JY, Wiese A, Green ME, Green M, Akyol A, Roy BC, Zhai Y, Cho KR, Fearon ER - PLoS Genet. (2015)

Heterozygous Ctnnb1 inactivation does not prevent mouse ovarian endometrioid adenocarcinoma (OEA) development and the reduction in Ctnnb1 dosage or CTNNB1 transcripts shows differential effects on selected β-catenin/TCF target genes.(A) Representative photomicrographs of H&E stained ovarian cancer tissue sections from Apcfl/flPtenfl/fl, and Apcfl/flPtenfl/flCtnnb1fl/+ mice show similar morphologic characteristics independent of Ctnnb1 gene dosage, with areas of glandular, overtly epithelial differentiation admixed with more poorly differentiated spindle-cell areas. Scale bars, 100 μm. (B) Representative photomicrographs of β-catenin immunohistochemical staining in tumors from Apcfl/flPtenfl/fl mice, showing strong nuclear β-catenin translocation and moderate cytoplasmic staining, whereas tumors from Apcfl/flPtenfl/flCtnnb1fl/+ mice showed weak nuclear and moderate cytoplasmic β-catenin staining. Scale bars, 25 μm. (C) cDNAs were obtained from murine normal ovaries (Cntrl, n = 4) and OEAs from Apcfl/flPtenfl/flCtnnb1fl/+ (n = 6), and Apcfl/flPtenfl/fl (n = 6) mice following AdCre injection. Transcript levels of Ctnnb1, Axin2, Myc and Nkd1 were measured by qRT-PCR analysis and normalized with β-actin. The gene expression levels were set to 1 for control (Cntrl) group. Error bars denote S.D. **P < 0.01 and *P < 0.05. (D) TOV-112D cells (the human OEA-derived cell line harboring β-catenin gain of function mutation), stably transduced with two different doxycycline-inducible shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl), were treated for 4 days with DOX (“+ DOX”) or a solvent control (“- DOX”). CTNNB1 shRNA-mediated effects on CTNNB1 (left) and MYC (right) gene expression are shown. Gene expression was assessed by qRT-PCR and normalized to HPRT expression. Error bars denote S.D. *P < 0.05 in Student's t test.
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pgen.1005638.g008: Heterozygous Ctnnb1 inactivation does not prevent mouse ovarian endometrioid adenocarcinoma (OEA) development and the reduction in Ctnnb1 dosage or CTNNB1 transcripts shows differential effects on selected β-catenin/TCF target genes.(A) Representative photomicrographs of H&E stained ovarian cancer tissue sections from Apcfl/flPtenfl/fl, and Apcfl/flPtenfl/flCtnnb1fl/+ mice show similar morphologic characteristics independent of Ctnnb1 gene dosage, with areas of glandular, overtly epithelial differentiation admixed with more poorly differentiated spindle-cell areas. Scale bars, 100 μm. (B) Representative photomicrographs of β-catenin immunohistochemical staining in tumors from Apcfl/flPtenfl/fl mice, showing strong nuclear β-catenin translocation and moderate cytoplasmic staining, whereas tumors from Apcfl/flPtenfl/flCtnnb1fl/+ mice showed weak nuclear and moderate cytoplasmic β-catenin staining. Scale bars, 25 μm. (C) cDNAs were obtained from murine normal ovaries (Cntrl, n = 4) and OEAs from Apcfl/flPtenfl/flCtnnb1fl/+ (n = 6), and Apcfl/flPtenfl/fl (n = 6) mice following AdCre injection. Transcript levels of Ctnnb1, Axin2, Myc and Nkd1 were measured by qRT-PCR analysis and normalized with β-actin. The gene expression levels were set to 1 for control (Cntrl) group. Error bars denote S.D. **P < 0.01 and *P < 0.05. (D) TOV-112D cells (the human OEA-derived cell line harboring β-catenin gain of function mutation), stably transduced with two different doxycycline-inducible shRNAs targeting CTNNB1 (CTNNB1-1 and CTNNB1-2) or a non-silencing scramble shRNA (Scrmbl), were treated for 4 days with DOX (“+ DOX”) or a solvent control (“- DOX”). CTNNB1 shRNA-mediated effects on CTNNB1 (left) and MYC (right) gene expression are shown. Gene expression was assessed by qRT-PCR and normalized to HPRT expression. Error bars denote S.D. *P < 0.05 in Student's t test.
Mentions: To assess the role of β-catenin function in another Apc mutation-dependent tumor model, we explored the role of Ctnnb1 gene dosage in a mouse model of ovarian endometrial adenocarcinoma (OEA) arising from bi-allelic inactivation of both the Apc and Pten genes [29]. Prior studies have shown that the Wnt/β-catenin/Tcf signaling pathway is deregulated by mutations in 16%–38% of human OEAs, and PTEN mutations are often seen in the OEAs with Wnt pathway mutations [29–32]. In the mouse OEA model, tumors are initiated by conditional inactivation of the Apc and Pten genes following injection of AdCre into the right ovarian bursa of Apcfl/flPtenfl/fl mice [29]. Interestingly, in both Apcfl/flPtenfl/fl mice and Apcfl/flPtenfl/flCtnnb1fl/+ mice, adenocarcinomas morphologically similar to human OEAs formed following AdCre injection, with 100% penetrance and no difference in tumor latency between mice with two wild type Ctnnb1 alleles or one wild type and one floxed Ctnnb1 allele (Table 1). In addition, no significant differences in survival rates, tumor volumes, and rates of liver metastasis were found between AdCre-injected Apcfl/flPtenfl/fl mice and Apcfl/flPtenfl/flCtnnb1fl/+ littermates (Table 1), and OEAs arising in both lines of mice shared similar histological features and immunohistochemical staining patterns for cytokeratin-8 (CK8), E-cadherin and α-inhibin (Fig 8A and Table 1). Efficient Cre-mediated deletion of Ctnnb1 and Apc was confirmed in tumors from these mice, and no OEAs arose in the AdCre-injected right ovaries in Apcfl/flPtenfl/flCtnnb1fl/fl mice, indicating OEAs could not arise from cells completely lacking β-catenin.

Bottom Line: Ctnnb1 hemizygous dose markedly inhibited increases in β-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key β-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base.A critical threshold for β-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for β-catenin in Ctnnb1 gene expression and CTNNB1 transcription.The active β-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Adenomatous polyposis coli (APC) inactivating mutations are present in most human colorectal cancers and some other cancers. The APC protein regulates the β-catenin protein pool that functions as a co-activator of T cell factor (TCF)-regulated transcription in Wnt pathway signaling. We studied effects of reduced dosage of the Ctnnb1 gene encoding β-catenin in Apc-mutation-induced colon and ovarian mouse tumorigenesis and cell culture models. Concurrent somatic inactivation of one Ctnnb1 allele, dramatically inhibited Apc mutation-induced colon polyposis and greatly extended Apc-mutant mouse survival. Ctnnb1 hemizygous dose markedly inhibited increases in β-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key β-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base. A critical threshold for β-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for β-catenin in Ctnnb1 gene expression and CTNNB1 transcription. The active β-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells. In mouse ovarian endometrioid adenocarcinomas (OEAs) arising from Apc- and Pten-inactivation, while Ctnnb1 hemizygous dose affected β-catenin levels and some β-catenin/TCF target genes, Myc induction was retained and OEAs arose in a fashion akin to that seen with intact Ctnnb1 gene dose. Our findings indicate Ctnnb1 gene dose exerts tissue-specific differences in Apc mutation-instigated tumorigenesis. Differential expression of selected β-catenin/TCF-regulated genes, such as Myc, likely underlies context-dependent effects of Ctnnb1 gene dosage in tumorigenesis.

No MeSH data available.


Related in: MedlinePlus