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Objective: Colorectal cancer is a common malignancy and a common cause of tumor-related death. Long non-coding RNAs (lncRNAs) have become an important regulatory factor and tissue specific biomarker for a variety of cancers, including colorectal cancer. Recent evidence indicates that the novel lncRNA SLC30A10 plays an important role in tumor progression and metastasis. However, its role and molecular mechanisms in colorectal cancer are unclear., Patients and Methods: SLC30A10 expression was detected in 12 colorectal cancer and adjacent normal tissues by quantitative reverse transcription PCR. Insights into the underlying mechanisms of competitive endogenous RNAs (ceRNAs) were determined by transwell assay, CCK8 assay, and luciferase assay., Results: SLC30A10 was down-regulated in colorectal cancer tissues and cell lines, and its low expression was positively correlated with colorectal cancer progression and metastasis. Functionally, SLC30A10 depletion promotes cell proliferation and migration in colorectal cancer cells, while SLC30A10 overexpression has the opposite effect. Bioinformatics prediction and luciferase assay indicated that miR-21c is a direct target of SLC30A10, which plays the role of ceRNA in regulating colorectal cancer metastasis. In addition, miR-21c specifically targets APC gene., Conclusions: Our findings suggest that reduced expression of SLC30A10 is associated with aggressive tumor phenotypes and poor patient outcomes in colorectal cancer. SLC30A10 inhibits colorectal cancer progression and metastasis by acting as a ceRNA for miR-21c to regulate APC expression, suggesting that SLC30A10 may serve as a potential prognostic biomarker and anti-metastatic therapeutic target for colorectal cancer.

We explored the role of APC-mediated MDR-1/CLCX-1 signaling pathway in ovarian tumors. In this study, ovarian tumor cell lines SKOV-3, ES-2 and MCV-152 were used to conduct the research. Fluorescence quantitative PCR and Western-blotting were used to investigate the effects of MDR-1/CLCX-1 signaling pathway in ovarian tumors. The effects of the APC gene silencing and overexpression on proliferation and apoptosis of ovarian tumor cells were detected by flow cytometry. Compared to normal cells, the expression of APC gene mRNA in ovarian tumor cells was significantly decreased (p less than 0.05). Western-blotting results showed that the level of APC protein in ovarian tumor cells was significantly lower than that in normal tissue, while MDR-1/CLCX-1 related proteins levels were significantly increased (p less than 0.05). In the APC gene silenced ovarian tumor cell lines, the expression of MDR-1/CLCX-1 was significantly higher than that of the untreated group (p less than 0.05), and apoptosis of ovarian tumor cells decreased. However, in ovarian tumor cell lines that over-expressed APC gene, the expression of MDR-1/CLCX-1 was significantly lower than that of the untreated group (p less than 0.05), and apoptosis of ovarian tumor cells was increased. There is a certain correlation between the APC gene and ovarian tumors, and the APC gene mediates the apoptosis of tumor cells through the MDR-1/CLCX-1 signaling pathway.

Background: Gastric cancer is the fifth most frequent cancer and the third most common cause of cancer-related deaths worldwide.It has been reported that Wnt/ betacatenin pathway is activated in 30-50% of these tumors. However,the deregulation of this pathway has not been fully elucidated., Aim: To determine the expression of E-cadherin, betacatenin, APC, TCF-4 and survivin proteins in gastric adenocarcinoma tissues and correlate with clinical and pathological parameters., Method: Seventy-one patients with gastric adenocarcinoma undergoing gastrectomy were enrolled. The expression of E-cadherin, betacatenin, APC, TCF-4 and survivin proteins was detected by immunohistochemistryand related to the clinical and pathological parameters., Results: The expression rates of E-cadherin in the membrane was 3%; betacatenin in the cytoplasm and nucleus were 23,4% and 3,1% respectively; APC in the cytoplasm was 94,6%; TCF-4 in the nucleus was 19,4%; and survivin in the nucleus 93,9%. The expression rate of E-cadherin was correlated with older patients (p=0,007), while betacatenin with tumors <5 cm (p=0,041) and APC with proximal tumors (p=0,047). Moreover, the expression of TCF-4 was significantly higher in the diffuse type (p=0,017) and T4 tumors (p=0,002)., Conclusion: The Wnt/betacatenin is not involved in gastric carcinogenesis. However, the high frequency of survivin allows to suggest that other signaling pathways must be involved in the transformation of gastric tissue., Competing Interests: none

Adenomatous polyposis coli (APC) mutation is the most common genetic change in sporadic colorectal cancer (CRC). Although deregulations of miRNAs have been frequently reported in this malignancy, APC-regulated miRNAs have not been extensively documented. Here, by using an APC-inducible cell line and array analysis, we identified a total of 26 deregulated miRNAs. Among them, members of miR-17-92 cluster were dramatically inhibited by APC and induced by enforced expression of β-catenin. Furthermore, we demonstrate that activated β-catenin resulted from APC loss binds to and activates the miR-17-92 promoter. Notably, enforced expression of miR-19a overrides APC tumor suppressor activity, and knockdown of miR-19a in cancer cells with compromised APC function reduced their aggressive features in vitro. Finally, we observed that expression of miR-19a significantly correlates with β-catenin levels in colorectal cancer specimens, and it is associated to the aggressive stage of tumor progression. Thus, our study reveals that miR-17-92 cluster is directly regulated by APC/β-catenin pathway and could be a potential therapeutic target in colon cancers with aberrant APC/β-catenin signaling.

Purpose and Experimental Design: The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer cells, and we identified a novel EGFR-independent oncogenic signal of EGF that works under coexpressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contribute to further progression of intestinal adenoma following APC loss., Results: The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven KITENIN expression induces increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes compared with those of nontransgenic mice. Among the four ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells coexpressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of the ErB4-CYT-2 mRNA as well as increased EGFR expression were observed in intestinal adenoma of APC(min/+) mice, which makes the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APC(min/+) mice, intestinal tumor tissues in the crossed mice showed the characteristics of early-stage invading adenocarcinoma. In patients with colorectal cancer, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no significant differences in tumor tissue expression were found between different colorectal cancer stages. Furthermore, the mRNA expression of KITENIN and that of ErbB4-CYT-2 were positively correlated in human colorectal cancer tissue., Conclusions: Elevated coexpression of KITENIN and ErbB4-CYT-2 promotes the transition of colon adenoma to adenocarcinoma within an APC loss-associated tumor microenvironment., (©2015 American Association for Cancer Research.)

Objective: To validate our earlier observation that 11 chemoresistance-associated mRNAs are molecular markers of poor overall survival in ovarian serous carcinoma., Methods: Ovarian serous carcinomas (n=112) and solid metastases (n=63; total=175) were analyzed for mRNA expression of APC, BAG3, EGFR, S100A10, ITGAE, MAPK3, TAP1, BNIP3, MMP9, FASLG and GPX3 using quantitative real-time PCR. mRNA expression was studied for association with clinicopathologic parameters and survival. Tumor heterogeneity was assessed in 20 cases with >1 specimen per patient. APC, BAG3, S100A10 and ERK1 protein expression by immunohistochemistry was analyzed in 58 specimens (38 primary carcinomas, 20 metastases)., Results: BAG3 (p=0.013), TAP1 (p=0.014), BNIP3 (p<0.001) and MMP9 (p=0.036) were overexpressed in primary tumors, whereas S100A10 (p=0.027) and FASLG (p=0.006) were overexpressed in metastases. Analysis of patient-matched primary carcinomas and metastases showed overexpression of APC (p=0.022), MAPK3 (p=0.002) and BNIP3 (p=0.004) in the former. In primary carcinomas, higher APC (p=0.003) and MAPK3 (p=0.005) levels were related to less favorable chemoresponse. Higher S100A10 (p=0.029) and MAPK3 (p=0.041) levels were related to primary chemoresistance. Higher BAG3 (p=0.026) and APC (p=0.046) levels in primary carcinomas were significantly related to poor overall survival in univariate, though not in multivariate survival analysis. S100A10 protein expression was related to poor chemoresponse (p=0.002) and shorter overall (p=0.005) and progression-free (p<0.001) survival, the latter finding retained in multivariate analysis (p=0.035)., Conclusions: Our data provide evidence of heterogeneity in ovarian serous carcinoma and identify APC, MAPK3, BAG3 and S100A10 as potential biomarkers of poor chemotherapy response and/or poor outcome in this cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rheumatoid arthritis (RA) is a symmetrical polyarticular autoimmune disease of unknown etiology. In this present study, we observed that the adenomatous polyposis coli (APC) expression is down-regulated and the expression of microRNA (miR)-663 increased significantly in synovium from RA patients compared with control. Target gene prediction for miR-663 revealed that the mRNA of APC gene, which is a member of the canonical Wnt signaling pathway, has a miR-663 binding site in its 3'-untranslated region (3'UTR). The result showed that increased miR-663 suppressed the APC expression significantly, and this down-regulation of APC expression triggered the activation of canonical Wnt signaling through accumulation of β-catenin in fibroblast-like synoviocytes (FLS). In addition, increased miR-663 induced the FLS proliferation and the expression MMP3 and fibronectin during disease development. Therefore, miR-663 can be considered as a critical regulator of RA pathogenesis and can be utilized for developing miRNA-based therapeutic agents for RA patients., (Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

The aim of the present study was to investigate the correlation between the expression of DNA (cytosine‑5)‑methyltransferase 1 (DNMT1), glutathione S‑transferase‑P1 (GSTP1) and adenomatous polyposis coli (APC), and the methylation status of GSTP1 and APC in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to examine its clinical significance. Immunohistochemistry and reverse transcription‑polymerase chain reaction (RT‑PCR) was used to detect the expression of DNMT1, GSTP1 and APC in 56 samples of PCa tissue and 10 samples of BPH tissue. Methylation‑specific‑PCR was used to detect the methylation status of the CpG island promoters of GSTP1 and APC. The positive rate of expression of DNMT1 in poorly‑differentiated PCa, moderately‑differentiated PCa, well‑differentiated PCa and BPH was 86.7%, 70.6%, 55.6% and 30.0%, respectively (P<0.05); for GSTP1, the positive rate was 13.3%, 29.4%, 44.4% and 90.0%, respectively (P<0.05); and for APC, the positive rate was 23.3%, 47.6%, 55.6% and 70.0%, respectively (P<0.05). The correlation coefficient for the association between the expression of DNMT1 and GSTP1 was ‑0.891 (P<0.05). Between the expression of DNMT1 and APC, the correlation coefficient was ‑0.721 (P<0.05). GSTP1 and APC were hypermethylated in the majority of PCa tissue samples. The positive rate of methylation of these genes in poorly‑differentiated PCa was 83.3% and 73.3%, respectively. By contrast, hypomethylation (or demethylation) was observed in BPH samples, in which the positive rate of methylation was 10.0% and 20.0%, respectively (P<0.05). The increased expression of DNMT1, and the reduced expression of GSTP1 and APC have an important role in the development of PCa. Due to the high expression of DNMT1 mRNA, it is likely that the hypermethylation of CpG islands contributed to the silencing of GSTP1 and APC in PCa tissues.

Background: The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in up to 70% of sporadic breast cancers depending on subtype; however, the effects of APC mutation on tumorigenic properties remain unexplored. Using the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we identified enhanced breast tumorigenesis and alterations in genes critical in therapeutic resistance independent of Wnt/β-catenin signaling. Apc mutation changed the tumor histopathology from solid to squamous adenocarcinomas, resembling the highly aggressive human metaplastic breast cancer. Mechanistic studies in tumor-derived cell lines demonstrated that focal adhesion kinase (FAK)/Src/JNK signaling regulated the enhanced proliferation downstream of Apc mutation. Despite this mechanistic information, the role of APC in mediating breast cancer chemotherapeutic resistance is currently unknown., Methods: We have examined the effect of Apc loss in MMTV-PyMT mouse breast cancer cells on gene expression changes of ATP-binding cassette transporters and immunofluorescence to determine proliferative and apoptotic response of cells to cisplatin, doxorubicin and paclitaxel. Furthermore we determined the added effect of Src or JNK inhibition by PP2 and SP600125, respectively, on chemotherapeutic response. We also used the Aldefluor assay to measure the population of tumor initiating cells. Lastly, we measured the apoptotic and proliferative response to APC knockdown in MDA-MB-157 human breast cancer cells after chemotherapeutic treatment., Results: Cells obtained from MMTV-PyMT;ApcMin/+ tumors express increased MDR1 (multidrug resistance protein 1), which is augmented by treatment with paclitaxel or doxorubicin. Furthermore MMTV-PyMT;ApcMin/+ cells are more resistant to cisplatin and doxorubicin-induced apoptosis, and show a larger population of ALDH positive cells. In the human metaplastic breast cancer cell line MDA-MB-157, APC knockdown led to paclitaxel and cisplatin resistance., Conclusions: APC loss-of-function significantly increases resistance to cisplatin-mediated apoptosis in both MDA-MB-157 and the PyMT derived cells. We also demonstrated that cisplatin in combination with PP2 or SP600125 could be clinically beneficial, as inhibition of Src or JNK in an APC-mutant breast cancer patient may alleviate the resistance induced by mutant APC.

Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1(-/-) and Mlh1(+/+) mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1(+/+) mice. Colon tumors developed after DSS treatment alone in Mlh1(-/-) mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and β-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Background and Aims: β-Catenin and adenomatous polyposis coli (APC) are major components of the Wnt pathway. This study aimed to investigate the expression of β-catenin and APC in tumors and lymph nodes in colorectal cancer (CRC) patients and the mutational spectrum of the genes coding these proteins., Methods: Expression of APC and β-catenin was examined in 124 tumors and 41 lymph nodes. Exon 3 of CTNNB1 and the mutation cluster region (MCR) in exon 15 of the APC gene were screened for mutation by PCR-sequencing., Results: Nuclear/cytoplasmic immunostaining of β-catenin was detected in 58.1 and 48.8% in tumors and lymph nodes, respectively. In tumors, abnormal expression of β-catenin correlated with tumor size and with those in lymph nodes. Membranous β-catenin expression occurred in 41.9 and 14.6% of tumors and lymph nodes, respectively. In tumors, lack of membranous β-catenin correlated with high invasiveness and metastatic potential. Positive immunostaining for APC was observed in 2 and 14% of tumors and lymph nodes, respectively. Overexpression in nucleus/cytoplasm and lack of membranous β-catenin significantly correlated with a reduced overall survival. Among 25 tumors, four harbour mutation in Ser33 and Ser47 and overexpress the β-catenin in the nucleus/cytoplasm. Mutations were identified in the APC gene in 13 tumors and six mutations were novel., Conclusions: Positive association between aberrant expression of β-catenin in the nucleus/cytoplasm of tumors and lymph nodes was observed. Nucleus/cytoplasmic accumulation of β-catenin and loss of membranous expression are related to reduced survival and could serve as a candidate prognostic predictor., (Copyright © 2015 IMSS. Published by Elsevier Inc. All rights reserved.)

Small chemical compound sulindac has been approved as a preventive approach against colon cancer for its effectiveness in treatment of precancerous adenoma. Due to its severe toxicities in the cardiovascular, gastrointestinal and renal systems, however, a combination of low-dose sulindac with other chemopreventive agents has been sought after as an alternative therapeutic strategy that could increase its effectiveness, while minimizing its adverse effects. To identify the promising alternative approach, we investigated the therapeutic potential of targeting the interleukin (IL)-8/CXCR2 pathway in colon cancer treatment using both loss-of-function (CXCR2 knockout) and gain-of-function (IL-8 overexpression) mouse models, as the IL-8/CXCR2 pathway has been shown to be activated in intestinal tumors of both human and experimental animals. We found that deletion of CXCR2 gene and ectopic expression of IL-8 suppresses and enhances, respectively, intestinal tumor development caused by a mutation in the APC gene. Moreover, a single copy deletion of CXCR2 gene resulted in abrogation of COX-2 and Gro-α upregulation in intestinal tumors caused by the APC mutation. Moreover, a single copy (heterozygote) deletion of CXCR2 gene was sufficient to synergize with a low-dose sulindac treatment in suppressing APCmin-induced intestinal polyposis. Together, our study provides a therapeutic justification of combined inhibition of CXCR2 and sulindac treatment in colon cancer prevention., (© 2013 UICC.)

Deregulation of the Wnt/APC/β-catenin signaling pathway is an important consequence of tumor suppressor APC dysfunction. Genetic and molecular data have established that disruption of this pathway contributes to the development of colorectal cancer. Here, we demonstrate that the transcriptional coregulator RIP140 regulates intestinal homeostasis and tumorigenesis. Using Rip140-null mice and mice overexpressing human RIP140, we found that RIP140 inhibited intestinal epithelial cell proliferation and apoptosis. Interestingly, following whole-body irradiation, mice lacking RIP140 exhibited improved regenerative capacity in the intestine, while mice overexpressing RIP140 displayed reduced recovery. Enhanced RIP140 expression strongly repressed human colon cancer cell proliferation in vitro and after grafting onto nude mice. Moreover, in murine tissues and human cancer cells, RIP140 stimulated APC transcription and inhibited β-catenin activation and target gene expression. Finally, RIP140 mRNA and RIP140 protein levels were decreased in human colon cancers compared with those in normal mucosal tissue, and low levels of RIP140 expression in adenocarcinomas from patients correlated with poor prognosis. Together, these results support a tumor suppressor role for RIP140 in colon cancer.

Aberrant activation of the Wnt signalling pathway is a key feature of many cancers. β-Catenin, adenomatous polyposis coli (APC) and E-cadherin are major players in this pathway. The aim of this study is to examine the expression of β-catenin, APC and E-cadherin in tumour tissues of 80 Tunisian patients with gastric carcinoma and to determine the methylation status of the APC promoter in tumour tissues. Associations between protein expression and clinico-pathological parameters, including prognosis, were performed. Positive expression of β-catenin, APC and E-cadherin was observed in 77.5, 68.7 and 60% of cases, respectively. Tumours lacking membranous expression of β-catenin had greater extent of lymph node metastasis, poor differentiation and advanced T-stage. The expression of E-cadherin correlated with poor differentiation (P = 0.05) and β-catenin expression (P = 0.004). With regards to prognosis, the overall survival time was significantly prolonged for patients showing normal β-catenin expression (exclusively or predominantly membranous staining) alone or combined with positive APC expression (P log rank = 0.008 and 0.003, respectively). The methylated pattern of APC promoter 1A was detected in 43.8% of cases and correlated with T-stage (P = 0.046) and distant metastasis (P = 0.037). No correlation was found between the methylated profile of APC promoter 1A and the expression of APC protein in tumour tissues. Our findings suggest that deregulation of the Wnt pathway via abnormal expression of β-catenin and E-cadherin occurred frequently in gastric carcinoma and correlated with worse clinical behaviour.

Epigenetic modulators, particularly histone deacetylases (HDACs), are valid targets for cancer prevention and therapy. Recent studies report that HDAC2 overexpression is associated with colon tumor progression and is a potential target for colon cancer prevention. This study tested chemopreventive and dose-response effects of Ohio State University HDAC42 (OSU-HDAC42), a selective HDAC2 inhibitor, using a rat colon carcinogenesis model to assess aberrant crypt foci inhibition and a familial adenomatous polyposis model to assess intestinal tumor inhibition. Colonic aberrant crypt foci were induced by azoxymethane (AOM) (15 mg/kg body weight, once-weekly subcutaneous injections at 8 and 9 weeks age). One week after AOM treatment, groups of rats were fed an AIN-76A diet containing 0, 75, 150, and 300 ppm OSU-HDAC42 for 8 weeks, and colonic aberrant crypt foci were evaluated. To assess the inhibitory effect of OSU-HDAC42 on small-intestinal polyps and colon tumor growth, 6-week-old male C57Bl/6J-APC(min/+)mice were fed an AIN-76A diet containing 150 ppm OSU-HADC42 or 300 ppm pan-HDAC inhibitor suberoylanilide hydroxyamic acid (SAHA) for 80 days. Our results demonstrate that dietary OSU-HDAC42 produced dose-dependent inhibition of AOM-induced colonic aberrant crypt foci formation (13-50%; P < 0.01 to < 0.0001) and reduced multiple crypts with ≥ 4 crypts per focus (25-57%; P < 0.01 to < 0.0001) in F344 rats. Our findings show that 150 ppm OSU-HDAC42 significantly inhibited small-intestinal polyps (>46%; P < 0.001), with polyp size measuring >1 mm (P < 0.001), and colon tumors (>26%) in APC(min/+)mice, whereas 300 ppm SAHA showed nonsignificant inhibition. Mice fed 150 ppm OSU-HDAC42 had significantly decreased HDAC2, proliferating cell nuclear antigen, B cell lymphoma 2, cyclin-dependent kinase 2, and cell division cycle homolog 25C expression levels and increased p53 expression levels. These observations demonstrate the chemopreventive efficacy of OSU-HDAC42 against chemically induced and polyposis models of intestinal tumorigenesis.

Wnt signaling is often constitutively active in colorectal cancer cells. The expression of the intestinal specific transcription factor CDX2 is found to be transiently decreased in invasive cells at the tumor/stroma interface. A recent ChIP-Seq study has indicated that several Wnt signaling-related genes are regulated by CDX2. The aim was to investigate the role of decreased CDX2 level on the expression of APC, AXIN2 and GSK3β in migrating colon cancer cells at the invasive front. CDX2-bound promoter and enhancer regions from APC, AXIN2 and GSK3β were analyzed for gene regulatory activity and the expression pattern of APC and GSK3β at the invasive front was evaluated by immunohistochemical procedures. Transfection of intestinal and non-intestinal cell lines demonstrated that CDX2 activated APC and AXIN2 promoter activities via intestinal cell-specific enhancer elements. Suppressed CDX2 expression was associated with endogenous downregulation of APC and AXIN2 expression in Caco-2 cells but did not affect GSK3β expression. Furthermore, elevated levels of nuclear β-catenin and reduced levels of cytoplasmic APC were correlated to a low CDX2 expression in migrating colon cancer cells in vivo. These results suggest that a low CDX2 level has influence on the Wnt signaling in invasive colon cancer cells possibly promoting cellular migration.

ApcMin/+ mice spontaneously develop multiple intestinal adenomas along the length of the small intestine and colon. Currently little is known about the role of the immune system in regulating intestinal tumorigenesis in these animals. This study characterised small intestinal intraepithelial lympho-- cyte (IEL) populations in C56BL/6J ApcMin/+ mice and wild-type (Apc+/+) mice. We also determined the effect that T cells expressing either γδ or αβ encoded T cell receptors (TcR) exert on intestinal tumorigenesis. ApcMin/+ mice had significantly lower numbers of CD3+ IELs compared with Apc+/+ littermates and displayed reduced cytotoxicity against tumour target cells. Further analysis of IEL cytotoxicity revealed differences in the cytotoxic pathways utilised by IELs in ApcMin/+ and Apc+/+ mice with ApcMin/+ IELs displaying an absence of perforin/granzyme-mediated killing and increased levels of Fas-FasL-mediated cytotoxicity compared with wild-type IELs. Analysis of ApcMin/+ mice crossed with αβ T-cell deficient (TcRβ-/-) or γδ T-cell deficient (TcRδ-/-) mice on the same genetic background revealed decreased tumour multiplicity in the absence of both αβ and γδ T-cells. This study demonstrates that altered T-cell subsets play important roles in promoting tumorigenesis in ApcMin/+ mice and forms the basis for future mechanistic studies.

Familial adenomatous polyposis (FAP) is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Two promoters, 1A and 1B, have been recognized in APC, and 1B is thought to have a minor role in the regulation of the gene. We have identified a novel deletion encompassing half of this promoter in the largest family (Family 1) of the Swedish Polyposis Registry. The mutation leads to an imbalance in allele-specific expression of APC, and transcription from promoter 1B was highly impaired in both normal colorectal mucosa and blood from mutation carriers. To establish the significance of promoter 1B in normal colorectal mucosa (from controls), expression levels of specific transcripts from each of the promoters, 1A and 1B, were examined, and the expression from 1B was significantly higher compared with 1A. Significant amounts of transcripts generated from promoter 1B were also determined in a panel of 20 various normal tissues examined. In FAP-related tumors, the APC germline mutation is proposed to dictate the second hit. Mutations leaving two or three out of seven 20-amino-acid repeats in the central domain of APC intact seem to be required for tumorigenesis. We examined adenomas from mutation carriers in Family 1 for second hits in the entire gene without any findings, however, loss of the residual expression of the deleterious allele was observed. Three major conclusions of significant importance in relation to the function of APC can be drawn from this study; (i) germline inactivation of promoter 1B is disease causing in FAP; (ii) expression of transcripts from promoter 1B is generated at considerable higher levels compared with 1A, demonstrating a hitherto unknown importance of 1B; (iii) adenoma formation in FAP, caused by impaired function of promoter 1B, does not require homozygous inactivation of APC allowing for alternative genetic models as basis for adenoma formation.

Background: Because of clinicopathologic and genetic differences between left-sided colorectal cancer (LSCRC) and right-sided colon cancer (RSCC), cyclooxygenase-2(COX-2) and adenomatous polyposis coli (APC) expression may be of clinical relevance., Materials and Methods: Clinicopathologic information for 72 primary colon tumors, 44 left and 28 right, from 72 patients (34 F, 38 M) were analyzed. COX-2 and wild-type APC (W-APC) immunohistochemical expressions were determined for each case. The data were analyzed using the Chi-square test and exact binomial confidence intervals., Results: Overall, 31 out of 44 (70%) LSCRC were W-COX-2 positive vs. 13 out of 28 (46%) RSCC (p-value=0.042). When evaluated independently of the anatomic location, COX-2 expression showed a borderline statistical correlation with the lack of W-APC protein (p-value=0.054). When considering location of tumors, the inverse correlation between COX-2 and W-APC expression became statistically significant (p-value=0.024)., Conclusion: We report a strong inverse correlation between COX-2 and W-APC expression, with COX-2 being more frequently as expressed in LSCRC. These data may be useful to stratify colorectal cancer patients into right- and left-sided and COX-2 expressor and non-expressor subsets, when evaluating COX-2 inhibitor and other targeted therapies in colon cancer.

This research is motivated by a pilot colorectal adenoma study, where the outcome of interest is the presence of colorectal adenoma representing risk for colorectal cancer, and the predictors of interest are protein biomarkers that are repeatedly measured with errors along the length of a microscopic structure in the human colon, the colon crypt. Biomarkers of this type are referred to as functional biomarkers. The investigators are interested in identifying features of functional biomarkers that are associated with risk for colorectal cancer. In this paper, we investigate a joint modeling approach, where the binary clinical outcome is modeled using a logistic regression model with the unobserved true functional biomarkers as the predictors. Most existing methods are developed either for linear models or for functional biomarkers measured without errors and cannot be directly applied to our data. The applicable methods include a two-step method and a maximum likelihood method, which have some limitations. We propose a robust semiparametric method to overcome the limitations of the existing methods. We study the properties of the proposed method, and show in simulations that it compares favorably with other methods and also offers significant savings in CPU time. We analyze the pilot colorectal adenoma data and show that expression levels of AFC, a tumor suppressor gene, in the transitional area from the proliferation zone to the differentiation zone of colon crypts are likely to be associated with risk for colorectal cancer. Given the relatively small sample size in the pilot study, our results need to be validated in the future full-scale studies., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The identification of parathyroid carcinomas is based upon histopathological criteria in which an invasive growth pattern or distant metastasis is demonstrated. A dilemma arises when tumours present with atypical histopathological features but lack direct evidence of malignancy. Recently, reduced expression or loss of the tumour suppressor proteins parafibromin and adenomatous polyposis coli (APC) has been associated with parathyroid malignancy. We report results from APC and parafibromin expression analyses by immunohistochemistry and Western blot in five cases of atypical adenoma, a single case of carcinoma and 54 adenomas without atypical features. Complete loss of APC immunoreactivity and reduced expression of parafibromin was evident in two of the atypical adenomas and in the parathyroid carcinoma. By contrast, all adenomas displayed APC expression, including two cases with hyperparathyroidism 2 gene (HRPT2) mutations and loss of parafibromin expression. We conclude that loss of APC is a frequent molecular event in atypical adenomas and carcinomas, but not in adenomas. Following verification in an independent material, APC could become a valuable tool when assessing parathyroid tumours in the clinical setting. Furthermore, the molecular resemblance of atypical adenomas with carcinoma concerning parafibromin and APC expression indicates that atypical adenomas should be subjects to watchful follow-up.

Recent studies have suggested that APC loss alone may be insufficient to promote aberrant Wnt/beta-catenin signalling. Our aim was to comprehensively characterize Wnt signalling components in a set of APC-associated familial adenomatous polyposis (FAP) tumours. Sixty adenomas from six FAP patients with known pathogenic APC mutations were included. Somatic APC and KRAS mutations, beta-catenin immunostaining, and qRT-PCR of APC, MYC, AXIN2 and SFRP1 were analysed. Array-comparative genomic hybridization (aCGH) was also assessed in 26 FAP adenomas and 24 paired adenoma-carcinoma samples. A somatic APC alteration was present in 15 adenomas (LOH in 11 and four point mutations). KRAS mutations were detected in 10% of the cases. APC mRNA was overexpressed in adenomas. MYC and AXIN2 were also overexpressed, with significant intra-case heterogeneity. Increased cytoplasmic and/or nuclear beta-catenin staining was seen in 94% and 80% of the adenomas. beta-Catenin nuclear staining was strongly associated with MYC levels (p value 0.03) but not with KRAS mutations. Copy number aberrations were rare. However, the recurrent chromosome changes observed more frequently contained Wnt pathway genes (p value 0.012). Based on beta-catenin staining and Wnt pathway target genes alterations the Wnt pathway appears to be constitutively activated in all APC-FAP tumours, with alterations occurring both upstream and downstream of APC. Wnt aberrations are present at both the DNA and the RNA level. Somatic profiling of APC-FAP tumours provides new insights into the role of APC in tumourigenesis., (Copyright (c) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Objective: To explore the effect of recombinant pEGFP-N3-APC vectors carrying various APC functional domains on the expression of beta-catenin in human colorectal cancer cells HT-29., Methods: The recombinant plasmids were transfected into HT-29 cells mediated by lipofectamine(TM) 2000, and detected by green fluorescence and RT-PCR. Western blot was applied to detect beta-catenin expression level in HT-29 cells after transfection, and gray scales of electrophoresis strips were analyzed by SPSS 13.0., Results: Green fluorescence and RT-PCR made clear that all 5 recombinant plasmids were successfully expressed in HT-29 cells. Western blot showed that beta-catenin expression level in HT-29 cells was not affected after being transfected with pEGFP-N3-APC1, pEGFP-N3-APC2 and pEGFP-N3-APC3, and was distinctly affected after being transfected with pEGFP-N3-APC4 and pEGFP-N3-APC5, especially the later one., Conclusion: The selected APC5 gene fragment with 15-amino acid repeats and SAMP repeats, which is relatively short, can degrade beta-catenin level in HT-29 cells and may be applied in the gene therapy.

Previous findings have suggested that methylation of the APC gene may be associated with some tumors including lung cancer. To explore the pattern of APC methylation and the effect of APC gene methylation on its protein expression in lung cancer cell lines, we investigated APC promotor methylation by methylation specific PCR (MSP) and bisulfite sequencing and analyzed the APC protein levels by western blot in three lung cancer cell lines. Monoallelic methylation and 20 methylated CpGs in CpG island near the open reading frame (ORF) of the APC gene were found in the NCI-H460 cell line, and were stablely inherited within 10 generations of the cell line in culture. Our results showed that two special CpG sites (794, 797) might be binding sites for proteins that regulate APC expression. Protein expression of the APC gene in the NCI-H460 cell line declined, but was enhanced after the treatment with 5-aza-2-deoxycytidine (5-aza-dC). Inherited monoallelic methylation of the APC gene may play an important role in lung cancer. Demethylation of the APC gene by 5-aza-dC may be useful for the treatment of lung cancer.

The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor kappaB (NF-kappaB) and has recently been shown to be a target of the Wnt/beta-catenin pathway. In this study, we tested the hypothesis that Wnt/beta-catenin signaling might regulate cytokine- or tumor necrosis factor alpha (TNFalpha)-induced hiNOS expression through interaction with NF-kappaB. A cytokine mixture of TNFalpha + interleukin (IL)-1beta + IFNgamma induced a 2- to 3-fold increase in hiNOS promoter activity in HCT116 and DLD1 colon cells, but produced a 2-fold decrease in SW480 colon cancer cells. A similar differential activity was seen in liver cancer cells (HepG2, Huh7, and Hep3B). Overexpression of beta-catenin produced a dose-dependent decrease in NF-kappaB reporter activity and decreased cytokine mixture-induced hiNOS promoter activity. Gel shift for TNFalpha-induced hiNOS NF-kappaB activation showed decreased p50 binding and decreased NF-kappaB reporter activity in the beta-catenin-mutant HAbeta18 cells. Conversely, enhanced p50 binding and increased NF-kappaB reporter activity were seen in HAbeta85 cells, which lack beta-catenin signaling. Coimmunoprecipitation confirmed that beta-catenin complexed with both p65 and p50 NF-kappaB proteins. NF-kappaB-dependent Traf1 protein expression also inversely correlated with the level of beta-catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased beta-catenin protein and increased TNFalpha-induced p65 NF-kappaB binding as well as iNOS and Traf1 expression. Finally, beta-catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that beta-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-kappaB-dependent gene expression. These findings underscore the complex role of Wnt/beta-catenin, NF-kappaB, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis.

The aims of this study were to analyze the cadherin/catenin adhesion complex in cells from abdominal and extra-abdominal aggressive fibromatosis tumors, and to estimate the correlation between the expression of the tested proteins and the clinical data of the desmoid patients. Immunohistochemistry was used to examine the expression of the cadherin/catenin adhesion complex: APC protein, alpha-, beta-catenin, and N-cadherin in archival material derived from 15 cases of extra-abdominal desmoid tumor (E-AD) and 20 cases of abdominal (AD) desmoid tumor. The tested proteins demonstrated cytoplasmic (c) staining. Furthermore, nuclear (n) or cytoplasmic and nuclear (c+n) staining was observed for beta-catenin. The mean values of the percentage of positive cells for the tested proteins between E-AD vs. AD did not demonstrate any statistically significant difference except for alpha-catenin. In the E-AD group, in both cases of recurrent tumors, no alpha-catenin expression was observed but the expression of this protein was detected in primary tumors. In the groups investigated, no statistically significant correlation was found between alpha-catenin, beta-catenin (c), (n) and (c+n) expression, and tumor size (p>0.1). The results regarding beta-catenin expression obtained in our study confirm the previous findings that nuclear accumulation of this protein plays a crucial role in the pathogenesis of aggressive fibromatosis.

Purpose: We investigated the impact of promoter methylation on APC protein expression in patients with hepatocellular carcinoma (HCC)., Materials and Methods: 50 patients [HCC (n=19), liver metastasis (n=19), cholangiocellular cancer (n=7), and benign liver tumors (n=5)] were studied for methylation using Methylight analysis. APC mutation was investigated by protein truncation test and direct sequencing of genomic DNA. The protein expression was evaluated by immunohistochemistry and Western blot analysis., Results: The APC promoter was hypermethylated in 81.8% of non-cancerous liver tissue samples. All HCC samples and ten patients with liver metastasis (52.6%) exhibited APC promoter methylation. The degree of methylation was significantly higher in samples from HCC compared to the non-cancerous liver tissue samples (63.1% vs. 24.98%; p=0.001). The level of APC protein expression was significantly reduced in HCC samples compared to that of the corresponding non-tumor liver tissue (p<0.05)., Conclusions: Promoter methylation of the APC gene seems to be of significance in hepatocarcinogenesis and results in reduced protein expression in HCC. Interestingly, APC promoter methylation is also present in the vast majority of non-cancerous liver tissue whose (patho)physiological function remains unresolved.

Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of E-cadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and beta-catenin.

Background: Mammary tumors are a very common neoplasm in the dog and show histological features and biological behaviour similar to human mammary carcinomas. Recently, a pathway named Wnt-1, involving beta-catenin and APC protein, has emerged as an important player in many human tumor types, including mammary neoplasms., Materials and Methods: Thirty-five samples of canine mammary tumors (10 benign and 25 malignant) were studied in order to evaluate the co-expression of beta-catenin, APC protein and E cadherin with confocal laser microscopical observation, by western blot analysis and by correlating data obtained with the histological grade of tumours., Results: A progressive decrease of E-cadherin together with disruption of beta-catenin expression was observed in less differentiated malignant tumors. In addition, a loss of beta-catenin membranous distribution and a cytoplasmic accumulation was often coexpressed with disrupted expression of APC protein. Western blot analysis showed a progressive increase of beta-catenin in malignant tumors, which could be the expression of disrupted /-catenin catabolism leading to cytoplasmic accumulation. In some less differentiated malignant tumors, a marked beta-catenin decrease was also observed. This feature could be linked to mutations in beta-catenin gene coding for a truncated and lighter protein., Conclusion: These results may indicate the multifunctional role played by beta-catenin in canine mammary oncogenesis.

To understand the role of the colonic extracellular calcium-sensing receptor (CaSR) in calcium chemoprotection against colon cancer, we activated the CaSR with 5 mM Ca(2+) on HT-29 cells, an adenocarcinoma cell line. High Ca(2+) stimulated the upregulation (as assessed by RT-PCR) and the secretion of Wnt5a (assessed by Western blot), a noncanonical Wnt family member. Inhibiting CaSR activity with a short interfering RNA (siRNA) duplex against the CaSR reduced CaSR protein and prevented the secretion of Wnt5a. Dominant negative CaSR (R185Q) or siRNA blocked the high Ca(2+)-mediated inhibition of the beta-catenin reporter TOPflash. The CaSR/Wnt5a inhibition of beta-catenin reporter was prevented by dominant negative ubiquitin ligase seven in absentia homolog 2 (Siah2). In low-calcium medium, overexpressing Wnt5a increased Siah2 amplicons and protein. Inducing the expression of full-length adenomatous polyposis coli (APC) prevented CaSRmediated increases of Siah2 and Wnt5a. Overexpressing the receptor tyrosine kinase-like orphan receptor 2 (Ror2) increased Wnt5a and CaSR-mediated inhibition of TOPflash. Conditioned medium from Wnt5a-transfected cells added to HT-29 cells in low-Ca(2+) medium inhibited the beta-catenin reporter. This inhibition was blocked dose responsively by Frizzled-8/Fc chimeric antibody. Overexpression of Ror2 in HT-29 cells in low-Ca(2+) medium increased the inhibition of beta-catenin reporter caused by recombinant Wnt5a protein compared with addition of Wnt5a protein alone. Our findings demonstrate that APC status plays a key role as a determinant of Wnt5a secretion and suggest that CaSR-mediated secretion of Wnt5a will inhibit defective Wnt signaling in APC-truncated cells in an autocrine manner.

Little is known about the molecular pathogenesis of neuroendocrine tumors (NET) of the gastro-entero-pancreatic (GEP) system. We analyzed genetic and epigenetic alterations as well as the CpG island methylator phenotype (CIMP). The study comprised 118 well-differentiated fore- and mid-gut GEP-NET from 71 patients. In addition to loss of heterozygosity (LOH), microsatellite instability (MSI) and the methylation status of various tumor associated genes were examined. The expression profile of p16, APC and MENIN was investigated by immunohistochemistry. None of the tumors was highly microsatellite unstable, LOH was found in 22.2%. Significant differences in promoter hypermethylation were identified in the RUNX3 and the O(6)-MGMT genes. We found a significant loss of p16 expression in insulinomas (p = 0.05) and functional NET (p = 0.01), respectively. APC was expressed less in gastrinomas (p = 0.01) and functional GEP-NET (p = 0.05) vs. nonfunctional tumors. MENIN expression was reduced in pancreatic vs. extrapancreatic NET (p = 0.008) and in insulinomas vs. nonfunctional GEP-NET (p = 0.019) and NET associated with the carcinoid syndrome (p = 0.029). Further CIMP and a Ki-67 index >10% showed a close correlation. Outcome analysis of 19 patients showed a better survival for CIMP-negative patients. The analyses identified significant genetic and epigenetic alterations in well-differentiated fore- and mid-gut NET. CIMP, similar to Ki-67, might turn out to be of prognostic relevance., ((c) 2007 Wiley-Liss, Inc.)

Aims: To investigate dysregulation of the wnt signalling pathway by assessing beta-catenin expression/increasing expression and loss of cytoplasmic adenomatous polyposis coli (APC) and membranous E-cadherin in colorectal cancer (CRC) and determining the prognostic significance of these variables., Methods and Results: Unselected, non-consecutive CRC resections (n = 1420) were subdivided into three groups: mismatch repair (MMR)-proficient, MLH1- and presumed hereditary non-polyposis colonic cancer (HNPCC). Immunohistochemical analysis of beta-catenin expression (0% versus > 0%) and increasing expression (increasing percentage-positivity) and loss of APC and E-cadherin was performed using the tissue microarray technique. In MMR-proficient CRC, increased nuclear beta-catenin expression and loss of membranous E-cadherin were independently associated with higher N stage (P = 0.03 and < 0.0001), vascular invasion (P < 0.01 and < 0.001) and worse survival (P < 0.01 and < 0.001). Additionally, there was an association between loss of membranous E-cadherin and higher T stage (P = 0.03). In MLH1- CRC, loss of membranous E-cadherin was associated with higher N stage (P = 0.05) and worse survival (P = 0.03). In presumed HNPCC CRC nuclear beta-catenin and membranous E-cadherin were not associated with tumour progression or worse survival. In all CRC subsets loss of cytoplasmic APC was not associated with clinicopathological features., Conclusions: Increasing nuclear beta-catenin expression and loss of membranous E-cadherin are independent, adverse prognostic factors in MMR-proficient and MLH1- CRC.

Inhibitors of histone deacetylases (HDAC) inhibit malignant cell growth and induce apoptosis through unknown mechanisms. Here, we report that the expression status of adenomatous polyposis coli (APC) protein determines the relative sensitivity of colon cancer cells to HDAC inhibitor-induced apoptosis. HCA-7 cells (expressing wild-type beta-catenin and APC proteins) are more sensitive to apoptosis induced by HDAC inhibitors valproic acid (VPA) and suberoylanilide hydroxamic acid than SW620 or HT-29 cells (both expressing mutant APC). When wild-type APC protein was expressed using an inducible expression system, HT-29 cells became sensitive to apoptosis in response to VPA. Conversely, knocking down of endogenous APC protein by small interfering RNA (siRNA) blocked VPA-induced apoptosis in HCA-7 cells. APC mediated VPA-induced apoptosis through down-regulation of survivin. The level of survivin protein decreased in HCA-7 and HT-29/APC cells, but not in SW620 and HT-29/beta-Gal cells after VPA treatment. Whereas knocking down of survivin by siRNA sensitized SW620 cells to VPA-induced apoptosis, overexpression of survivin blocked VPA-induced apoptosis in HCA-7 cells. Down-regulation of survivin transcription occurred through changes in GSK-3beta/beta-catenin/Tcf-4 signaling molecules. VPA also induced proteasome-mediated degradation of survivin protein in HCA-7 cells. Furthermore, we have shown that APC mutation-mediated resistance to apoptosis can be overcome by cotreatment with Flavopiridol, which promotes survivin degradation. These results suggest that APC is a critical determinant of HDAC inhibitor-induced apoptosis in colon cancer cells and survivin is a potential target to enhance apoptotic response to HDAC inhibitors.

Familial adenomatous polyposis (FAP) is an autosomal dominant familial cancer syndrome caused by germline mutations of the tumor suppressor adenomatous polyposis coli (APC) gene. Heterozygous apc mutations have been identified in the majority of classical FAP patients who develop more than 100 colorectal adenomas. However, classical FAP patients often fail to display germline APC mutations detectable by routine mutation analysis. These apparently mutation-negative cases may be caused by heterozygous large genomic deletions. In the present study, FAP patients who showed no APC germline mutation detectable by the protein truncation assay and direct sequencing of protein coding exons were screened for APC gene deletion by a gene dose assay based on double competitive polymerase chain reaction. Gene dosage measurements within exon 15 of the APC gene identified two patients with gene deletion and one with possible gene duplication among 41 apparently mutation-negative patients. The deleted sequences in the two patients were determined by fine gene dose mapping around the APC gene and nucleotide sequencing of the deletion breakpoints. They were approximately 435-kilobase pair (kb) and 737-kb regions including the whole APC gene and flanked by a 4-base pair repeat and LINE-1 repetitive sequences, respectively. The chimeric LINE-1 element created at the breakpoint in the latter case also contained a short sequence derived from another LINE-1 element, suggesting a complex unequal homologous recombination event. These findings indicate that this gene dose assay is a useful technique to detect large gene deletions of the APC gene and to determine their genomic breakpoints.

Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo- (binds KAP3A, B56alpha, and ASEF) and beta-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of beta-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of beta-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of beta-catenin reduced APC at membrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with beta-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only beta-catenin stimulated APC anchorage at the membrane. Our findings identify beta-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.

In the present investigation, we have examined the transcriptional regulation of adenomatous polyposis coli (APC) gene expression in the spontaneously immortalized human normal breast epithelial cell line, MCF10A, in response to carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The APC mRNA levels and the APC gene's promoter (pAPCP) activity were increased in MCF10A cells after treatment with DMBA. A sequential deletion analysis and site-directed mutagenesis of the pAPCP promoter revealed that the DMBA response is mediated through a GC-box element. Also, the GC-box binding agent mithramycin A, which prevents binding of proteins to the GC-box region, abolished DMBA-mediated increase of the pAPCP promoter activity. The specificity of the proteins binding to the GC-box region was characterized by gel-shift analysis. An increased binding of the GC-box binding proteins was observed in the gel-shift analysis with nuclear extracts from DMBA-treated MCF10A cells, which corresponded to the increased levels of Sp1 and Sp3 proteins. However, a super-shift of the DNA-protein complexes was observed with only anti-Sp3 antibody. Based on the chromatin-immunoprecipitation assay results, the Sp3 appeared to be a genuine protein binding to the GC-box site of the pAPCP promoter. In RNA interference experiments, in which the Sp3 expression was knocked down, the DMBA response on the pAPCP promoter activity was reduced, suggesting that the binding of Sp3 to the GC-box site is critical for DMBA-induced pAPCP promoter activity. From these results we conclude that the increased pAPCP promoter activity in the MCF10A cell line in response to DMBA treatment is mediated by Sp3.

Objective: To investigate the promoter methylation status and mRNA expression of APC gene in MCF10 model of breast cancer progression., Methods: Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect the methylation status of APC promoter 1A in normal breast tissues, conventional breast cancer cell line MCF-7 and MCF10 model cell lines including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell lines, producing slowly progressing hyperplastic and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in-situ cell line, producing ductal carcinoma in-situ), MCF10CA1a, MCF10CA1d, MCF10CA1h cell lines (invasive breast carcinoma cell line, forming aggressive tumors of different morphology and metastatic potential). In addition, mRNA expression of APC was determined by reverse transcriptase PCR and real-time PCR assays., Results: Hypomethylation of APC promoter 1A was identified in hyperplastic cell line MCF10A, pre-malignant cell line MCF10AT, ductal carcinoma in-situ cell line MCF10DCIS.com, invasive carcinoma cell lines MCF10CA1a, MCF10CA1d, MCF10CA1h and normal breast tissue. MCF-7 showed partial methylation at the promoter. Statistically significant reduction of APC mRNA expression was not found in all MCF10 cell lines and MCF-7, compared with that of normal breast tissue (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h and MCF10DCIS.com showed reduced mRNA expressions of APC at 0.27, 0.96, 1.78, 2.70, and 2.03 times respectively. MCF10A and MCF-7 even showed an increase of APC mRNA expression at 0.02 and 0.33 times, respectively)., Conclusion: The aberrant promoter methylation of APC is not related to the breast cancer progression, at least in the MCF10 model system.

Mucin core proteins are expressed in a tissue and cell type specific manner in the normal gastrointestinal tract. Aberrant expression of mucin core proteins have been reported in colorectal neoplasms. To examine the relationship between subsets of colorectal polyps and non-mucinous and mucinous adenocarcinomas of the colorectum, we evaluated the frequency of the expression of cell lineage associated mucin core proteins (MUC5AC and MUC2), trefoil factors (TFF1 and TFF3), and APC and p21 in these tissues. An immunohistochemical study was performed in 10 normal rectal mucosa samples (NM) 21 hyperplastic polyps (HP), 20 serrated adenomas (SA), 25 tubular adenomas (TA), 13 tubulovillous adenomas (TVA), 7 villous adenomas (VA), 42 non-mucinous colorectal cancers (NMC), and 19 mucinous colorectal cancers (MC). A higher frequency of ectopic expression of gastric foveolar mucin, MUC5AC, and the expression of intestinal goblet cell mucins, MUC2, was observed respectively in HP (100%, 100%), SA (85%, 85%), TVA (85%, 85%), and VA (100%, 100%), compared to TA (32%, p<0.002; 36%, p<0.01). MC (68%, 100%) also showed a higher frequency of the expression of MUC5AC and MUC2 compared to NMC (31%, p=0.001; 38%, p<0.001), and TFF1 showed similar patterns of expression. APC protein and p21 were also expressed at a higher frequency in HP (100%, 100%), and SA (67%, 83%), than in TA (29%, p<0.03; 46%, p<0.05). MC (68%, 100%) showed a higher frequency of expression of APC protein and p21 than NMC (19%, p<0.001; 45%, p<0.01). Our results showed that MUC2 expression and de novo ectopic expression of MUC5AC and TFF1 are more frequent in HP, SA, TVA, VA, and MC than in TA and NMC. These results suggest that simultaneous activation of differentiation pathways of goblet cells and gastric foveolar cells may occur predominantly in the pathogenesis of HP, SA, TVA, VA, and MC, while the pathogenesis of TA and NMC are less likely to involve these processes.

Mutations of Apc and p53 have both been implicated in human and murine mammary neoplasia. To investigate potential interactions between Apc and p53, we conditionally inactivated Apc in both the presence and the absence of functional p53. Apc deficiency on its own leads to the development of metaplasia but not neoplasia. We show here that these areas of metaplasia are characterized by elevated levels of both p53 and p21. In the additional absence of p53,there is rapid progression to neoplasia, with 44.4% of lymphoma-free mice developing a mammary tumor with earliest observed onset at pregnancy. To investigate the mechanism by which p53 deficiency accelerates neoplasia, we used the Rosa26R reporter strain as a marker of Cre-mediated recombination and show a role for p53 in the loss of Apc-deficient cells. This role seems limited to pregnancy and subsequent time points. We therefore show clear synergy between these two mutations in mammary gland neoplasia and present data to suggest that at least one mechanism for this acceleration is the p53-dependent loss of Apc-deficient cells.

In the two conditions juvenile polyps (JPs) and juvenile polyposis coli (JPC), colonic polyps may have overlapping histologic and phenotypic appearance, but JPC confers a significant risk for colon adenocarcinoma. Although not thought to contain adenomatous polyposis coli (APC) mutations, the status of beta-catenin and full-length APC protein expression in JPs is not known. We evaluated beta-catenin and full-length APC protein expression in JPs from children with JPs and JPC. Cases were identified through endoscopic procedure records. Immunohistochemistry was performed for beta-catenin and full-length APC protein. Loss of heterozygosity at the APC gene locus on chromosome 5 was assessed using two APC-linked microsatellite markers. Polyp and normal colonic tissue were analyzed from 36 children with JPs and 9 with JPC. Both APC and beta-catenin immunoreactivity were present in epithelial cells from all samples but in different patterns. In all normal colon and polyp samples, APC expression was cytoplasmic with maximal immunoreactivity in the goblet cells. In contrast, beta-catenin immunoreactivity in epithelial cells was limited to the plasma membrane in normal colon but was both cytoplasmic and nuclear in all 45 JPs. No evidence of APC gene loss of heterozygosity was found. In polyps from children with JPs and JPC, nuclear beta-catenin accumulation is a consistent feature, and it is not due to APC gene mutation or loss of full-length APC protein expression. Thus, beta-catenin accumulation may be intrinsic to the formation of juvenile-type polyps through an as-yet-undefined mechanism.

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.

Clusterin is a widely expressed glycoprotein that has been paradoxically observed to have both pro- and antiapoptotic functions. Recent reports suggest this apparent dichotomy of function may be related to two different isoforms, one secreted and cytoplasmic, the other nuclear. To clarify the functional role of clusterin in regulating apoptosis, we examined its expression in human colon cancer tissues and in human colon cancer cell lines. We additionally explored its expression and activity using models of adenomatous polyposis coli (APC)- and chemotherapy-induced apoptosis. Clusterin RNA and protein levels were decreased in colon cancer tissues largely devoid of wild-type APC when compared with matched normal tissue controls, suggesting a means for invasive cancers to avoid apoptosis. Conversely, induction of apoptosis by expression of wild-type APC or by treatment with chemotherapy led to increased clusterin RNA and protein levels localizing to apoptotic nuclei. We found that transient transfection of clusterin to colon cancer cell lines directly enhanced basal and chemotherapy-induced apoptosis. Clusterin-induced apoptosis was inhibited by antisense clusterin and was found to be highly dependent on p21 but not p53 expression, yet a deficit in p21 can be subverted by clusterin transfection. Collectively, these data support the hypothesis that nuclear clusterin function is proapoptotic when induced by APC or chemotherapy in the context of p21 expression. Absent of p21, clusterin in not induced, and apoptosis is significantly inhibited. These data support a potential therapeutic role for clusterin in enhancing chemotherapy-induced apoptosis and in promoting apoptosis in cells deficient in p21.

Adenomatous polyposis coli (APC) promoter hypermethylation has been reported frequently in normal gastric mucosa, but it remained to be clarified whether this occurs in every individual. In this study, methylation of the APC promoter was analyzed in histologically normal-appearing gastric mucosa samples by methylation-sensitive single-strand conformation analysis and by a methylation-sensitive dot blot assay. Epithelial cell samples were collected by microdissection from tissue sections. Equal amounts of methylated and unmethylated APC alleles were found in all gastric mucosa samples from patients without any gastric lesions (20 samples). Allele-specific methylation analysis showed that the methylation of the APC promoter was monoallelic; however, which allele was methylated depended on the cell type. Increased or decreased methylation was found in 10 of 36 (28%) normal gastric mucosa samples adjacent to a gastric or esophageal adenocarcinoma. No allelic loss was found at the APC locus. Modification of the methylation status was also found in 3 of 21 (14%) normal-appearing gastric mucosa samples adjacent to intestinal metaplasia. In contrast, all normal mucosa samples in cases with chronic gastritis but without metaplasia or dysplasia showed a monoallelic methylation pattern. Our results indicate the following: (a) In normal gastric mucosa, the APC promoter shows monoallelic methylation, which is not due to imprinting but most likely due to allelic exclusion; (b) the excluded allele differs between foveolar and glandular epithelial cells; (c) the APC methylation pattern is frequently altered in normal-appearing gastric mucosa of gastric or esophageal adenocarcinoma patients; and (d) such alterations also occur in normal gastric mucosa adjacent to intestinal metaplasia.

We identified the mouse homolog of human DRAPC1 (APCDD1) gene, shown to be a target of Wnt/beta-catenin signaling pathway in cancer cell lines. Analysis of its spatiotemporal expression in mouse embryos from E7.5 to E14 showed that Drapc1 is expressed during development of the extraembryonic structures, nervous system, vascular system and inner ear. In addition, Drapc1 is expressed in the mesenchyme of several developing organs at sites of epithelio-mesenchymal interactions. Drapc1 expression was also found in the hair follicles of the adult mouse skin. Similarity of Drapc1 expression pattern to location of active beta-catenin in developing mouse embryo further suggests that mouse Drapc1 is a novel in vivo target gene of Wnt/beta-catenin signaling pathway.

As an important regulator in Wnt-signaling pathway, the APC gene is involved in apoptosis and cell cycle arrest. The loss of APC function is observed in most familial adenomatous polyposis-associated and sporadic colorectal cancer. APC gene is frequently inactivated by DNA mutations. However, hypermethylation in APC gene promoter was also observed in different cancers. In this study, by analyzing the methylation status of APC promoter in 22 colorectal cancer cell lines with different APC expression levels, we identified Regions A and B in the promoter, where the methylation of CpG sites was invariably correlated with the loss of gene expression. By nuclease accessibility assay, we also observed a correlation between the closed chromatin conformation in APC promoter and loss of gene expression. When the nonexpressing cell lines were treated with a DNA methyltransferase inhibitor, 5-Aza-2'-Deoxycytidine, the APC expression in these cells was induced, CpG sites were demethylated, and closed chromatin conformation was opened. However, when these cell lines were treated with a histone deacetylase inhibitor, Trichostatin A, no significant changes in APC expression, methylation status, and chromatin conformation were observed. Using transient transfection assay, a CCAAT box located in Region B was identified, which was involved in up-regulation of APC expression. Methylation of CpG sites around the CCAAT box resulted in a significant inhibition in the gene expression. The specific binding of a transcription factor CCAAT-binding factor (CBF) to the CCAAT box was determined by electrophoretic mobility shift analysis. The binding was inhibited after CpG sites close to the CCAAT box were methylated, indicating that DNA methylation can silence gene expression through interfering with the binding of transcription factors to the promoter. The biological function of CBF in APC gene regulation was further indicated by the decrease of luciferase activities in cells cotransfected with a plasmid carrying APC promoter/luciferase gene and a plasmid expressing dominant negative CBF mutant. In summary, methylation of CpG sites around CCAAT box in APC promoter inhibits the gene expression by changing the chromatin conformation and interfering with the binding of transcription factor CBF to CCAAT box.

The Apc1638N/+ mouse has a chain-terminating mutation in one allele of the adenomatous polyposis coli (Apc) gene that is similar to most mutations observed in the human familial adenomatous polyposis syndrome. Aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon, are morphologically abnormal structures that are identifiedmicroscopically in the grossly normal colonic mucosas of rodents treated with colon carcinogens and of human patients. The colons and cecums of 62 Apc1638N/+ mice were evaluated for the spontaneous occurrence of ACF and tumors. Both male and female mice were killed at different times between 5 and 28 weeks of age. Wild-type littermates, ie, Apc(+/+) mice, at 22 to 26 weeks of age served as controls. ACF were identified in 97% of the Apc1638N/+ mice starting at 5 weeks of age and not in any wild-type littermates. Although the number of ACF increased with age (P < 0.0001), the average number of crypts per focus of the ACF did not increase significantly. In addition, wild-type Apc protein was detected by immunohistochemistry in all 22 ACF evaluated. Together these data suggest that heterozygous loss of Apc may be sufficient to initiate ACF in these mice and that these mice may be suitable models to study the interaction of environmental factors with an inherited mutation of the Apc gene that is associated with colon cancer.

The adenomatous polyposis coli (APC) gene, a member of the WNT pathway, has been shown to assign intestinal epithelial cells to a program of proliferation or differentiation through regulation of the beta-catenin/TCF-4 complex. Wild-type APC, in certain cellular contexts, appears to induce differentiation and apoptosis, although mutant forms of APC, known to produce polyps and ultimately cancers, may suppress these events. Here, we show that mutant forms of APC can induce repression of select terminal caspases as a potential means of attenuating responses to apoptotic stimuli. Using gene expression profiling to interrogate the intact intestines of Apc(+/min) mice harboring numerous polyps, we identified a reduction in the mRNA expression of both caspases 3 and 7. We additionally identified a reduction in protein levels of caspase-3, caspase-7, and caspase-9 in human colon cancer specimens known to harbor APC mutations. A reduction in caspase protein levels resulted in resistance to apoptotic-inducing agents and restoration of caspase levels reinstated apoptotic capacities. Consistent with Wnt pathway involvement, dominant negative TCF/LEF induced caspase protein expression. These data provide support for the hypothesis that one of the functions of APC is the regulation of caspase activity and other apoptotic proteins by controlling their expression levels in the cell.

Mutations in the adenomatous polyposis coli (APC) gene and K-ras occur in the majority of human colorectal cancers. Loss of functional APC protein activates the Wnt signal transduction pathway, allowing the nuclear accumulation of beta-catenin, which then binds to T-cell factor-4 (Tcf-4), causing increased transcriptional activation of downstream target genes. We investigated the hypothesis that the activation of the WNT pathway regulates cyclooxygenase-2 (COX-2). COX-2 was down-regulated after the induction of full-length APC in the HT29-APC cell line. We identified a Tcf-4-binding element (TBE) in the COX-2 promoter that specifically bound to Tcf-4 in an electrophoretic mobility shift assay. COX-2 promoter luciferase activity is down-regulated by APC in a promoter reporter construct containing the, TBE but not with mutant TBE. Mutant beta-catenin expression up-regulated the COX-2 promoter activity and the endogenous COX-2 mRNA expression in HuH7, hepatocellular carcinoma cell line, which is partially abrogated by cotransfection with a dominant-negative Tcf-4 expression vector. Although beta-catenin alone did not increase COX-2 protein to detectable levels in HuH7 cells, coexpression of both mutant beta-catenin and mutant K-ras increased COX-2 protein expression, which is consistent with the previous reports that K-ras can stabilize COX-2 mRNA. Taken together, our data support the hypothesis that COX-2 is down-regulated by APC and up-regulated by nuclear beta-catenin accumulation, and additionally implicate the Wnt signal transduction pathway in colon and liver carcinogenesis.

Germline mutations within the adenomatous polyposis coli (APC) gene, a key member of the Wnt signalling pathway, have been shown to cause adenoma development in familial adenomatous polyposis (FAP), a dominantly inherited predisposition to colorectal cancer. Although it has been suggested for several years that alterations within the Wnt pathway are the underlying events for the development of colorectal adenomas in FAP patients, no detailed analysis of the gene expressions of Wnt pathway members has been available in fresh colorectal tissue of FAP patients, so far. Thus, we investigated potential differences in the expressions of APC and its Wnt partners conductin, beta-catenin, cyclin D1, and c-myc in normal colorectal mucosa and matched adenoma tissue of 14 FAP patients using real-time quantitative PCR. The expressions of both Wnt target genes, cyclin D1 and c-myc, were significantly increased in adenoma compared to matched normal mucosa. Furthermore, the overexpressions of these two genes showed a highly significant positive correlation. Our data suggest that the concomitant overexpression of the Wnt targets and cell cycle regulators cyclin D1 and c-myc plays an important role in the neoplastic proliferation of adenomas in FAP patients.

The wnt pathway plays an important role in embryonal patterning and cell fate determination, involving stabilization of nuclear and cytoplasmic beta-catenin (CTNNB1) mediated by APC, axin, and other proteins. Uncomplexed beta-catenin binds to TCF/LEF transcription factors and activates the expression of growth regulatory target genes such as c-myc or cyclin D1. In colorectal and other cancers, constitutive wnt signaling results frequently from mutations in one or more pathway components, e.g. APC and beta-catenin, resulting in nuclear and/or cytoplasmic accumulation of beta-catenin. In the present study, the most frequent alterations in the CTNNB1 and APC genes were investigated in primary urothelial bladder tumors and cell lines. Snap-frozen bladder tumors (n=99) of different stages and grades and 4 cell lines (RT4, RT112, J82, UROtsa) were investigated for APC allelic deletions by loss of heterozygosity (LOH) analysis. The most frequent mutated regions of CTNNB1 (degradation box in the third exon) and APC (mutation cluster region) were directly sequenced. Beta-catenin expression was analyzed by immunofluorescence in the cell lines. LOH at the APC gene locus on chromosome 5q21 was found in 7 of 72 (10%) of the informative cases. No mutations were found in either CTNNB1 or APC. A previously described polymorphism at codon 1493 of the APC gene was detected in 8 tumors and 3 cell lines. All cell lines showed normal membranous beta-catenin staining without evidence for nuclear or cytoplasmic accumulation. Alteration of APC and beta-catenin, which are the most frequent wnt pathway alterations in many tumor types, are rare events in urothelial carcinomas. Other wnt pathway members, such as axin, may play an important role in urothelial carcinogenesis.