Adenomatous Polyposis Coli mRNA Research Articles

APC Loss Prevents Doxorubicin-Induced Cell Death by Increasing Drug Efflux and a Chemoresistant Cell Population in Breast Cancer.

Chemoresistance is a major health concern affecting cancer patients. Resistance is multifactorial, with one mechanism being the increased expression of ABC transporters (such as MDR1 and MRP1), which are drug efflux transporters capable of preventing intracellular accumulation of drugs and cell death. Our lab showed that the loss of Adenomatous Polyposis Coli (APC) caused an intrinsic resistance to doxorubicin (DOX), potentially through an enhanced tumor-initiating cell (TIC) population and the increased activation of STAT3 mediating the expression of MDR1 in the absence of WNT being activated. Here, in primary mouse mammary tumor cells, the loss of APC decreased the accumulation of DOX while increasing the protein levels of MDR1 and MRP1. We demonstrated decreased APC mRNA and protein levels in breast cancer patients compared with normal tissue. Using patient samples and a panel of human breast cancer cell lines, we found no significant trend between APC and either MDR1 or MRP1. Since the protein expression patterns did not show a correlation between the ABC transporters and the expression of APC, we evaluated the drug transporter activity. In mouse mammary tumor cells, the pharmacological inhibition or genetic silencing of MDR1 or MRP1, respectively, decreased the TIC population and increased DOX-induced apoptosis, supporting the use of ABC transporter inhibitors as therapeutic targets in APC-deficient tumors.

METTL3 promotes tumour development by decreasing APC expression mediated by APC mRNA N6-methyladenosine-dependent YTHDF binding

The adenomatous polyposis coli (APC) is a frequently mutated tumour suppressor gene in cancers. However, whether APC is regulated at the epitranscriptomic level remains elusive. In this study, we analysed TCGA data and separated 200 paired oesophageal squamous cell carcinoma (ESCC) specimens and their adjacent normal tissues and demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in tumour tissues. m6A-RNA immunoprecipitation sequencing revealed that METTL3 upregulates the m6A modification of APC, which recruits YTHDF for APC mRNA degradation. Reduced APC expression increases the expression of β-catenin and β-catenin-mediated cyclin D1, c-Myc, and PKM2 expression, thereby leading to enhanced aerobic glycolysis, ESCC cell proliferation, and tumour formation in mice. In addition, downregulated APC expression correlates with upregulated METTL3 expression in human ESCC specimens and poor prognosis in ESCC patients. Our findings reveal a mechanism by which the Wnt/β-catenin pathway is upregulated in ESCC via METTL3/YTHDF-coupled epitranscriptomal downregulation of APC.

Risk Assessment in Hereditary Colorectal Cancer Family by Using APC and MSH2 mRNA Gene Expression and Bayesian Analysis

BACKGROUND: Some of colorectal cancers (CRCs) are familial, however, heterozygote relatives have approximately 80% lifetime risk of cancer. Risk assessment of CRC’s family could be calculated by direct measurement of mRNA gene expression and Bayesian theorem which is modifying initial background of pedigree risk with additional conditional information. This application has not been reported.METHODS: The cross-sectional translational sequential studies were performed: (1) adenomatous polyposis coli (APC) and MutS homolog (MSH)2 mRNA quantitative RT PCR gene expressions in tissue and whole blood CRC patients; (2) gene expression was determined in matched controls; and (3) pedigree and Bayesian analysis was calculated in the patient’s family of Proband.RESULTS: Fourty CRC and 31 control subjects were enrolled. The mean blood APC level control’s group was 13,261±670 fold-change (fc) and blood MSH2 level was 12,219±756 fc. The cut-off points for hereditary APC was 12,195 fc and MSH2 was 11,059 fc. The mean APC blood level in CRC subject was 11,578±2,638 fc and MSH2 blood level was 11,411±2,912 fc. There were significant differences APC and MSH level between tissue and blood level in CRC. Eight of 40 CRC subjects had a history of familial CRC. Four patients and 10 Probands were available for recurrence risk evaluation of pedigree analysis, RNA PCR quantitative and Bayesian calculation.CONCLUSION: There was determined a cut-off point of hereditary mRNA quantitative expression. The APC and MSH2 levels in CRC subjects were significantly lower than controls. Bayesian analysis allowed for the calculation of relative risk in CRC family members and considered in clinical practice.KEYWORDS: hereditary CRC, APC gene, MSH gene, Bayesian analysis

APC Promoter Hypermethylation as a Prognostic Marker in Breast Cancer Patients.

Background:Adenomatous polyposis coli (APC) promoter hypermethylation implicated in breast cancer development through Wnt signaling pathway, hypermethylation may result in inactivation of APC expression. This study aimed to investigated whether hypermethylation of APC promoter, the aggressive behavior of breast cancer cells, and correlated them with clinicopathological parameters and survival. Methods:Sixty-one fresh tissues of breast tumor were evaluated for APC promoter hypermethylation with methylation-specific PCR techniques (MS-PCR) and APC mRNA expression level analysis by quantitative real-time reverse transcription-PCR. Results:Our results show aberrant APC hypermethylation status was founded in 27 of 61 cases (44%), and significantly associated with chemotherapy treatment (OR= 6.9, 95%CI=1.5-31.01, P = 0.01), distant metastasis (OR = 5.52, 95%CI = 1.27-24.08, P = 0.04) as well as APC methylated status also associated with shorter overall survival than those without (8.4 and 11.0 years respectively, P = 0.02). Conclusion:The findings indicated hypermethylation of APC promoter may be used as a useful prognostic biomarker in breast cancer patients.

The Oncogenic Role of miR-BART19-3p in Epstein-Barr Virus-Associated Diseases.

Accumulating evidence so far has shown that EBV's miRNAs have been found to be involved in cancer progression. However, the comprehensive EBV miRNA expression profiles and their biological significance in EBV-associated diseases are not well documented. A comprehensive profiling of EBV-encoded miRNAs expressed in CAEBV, EBV-HLH, and nasopharyngeal carcinoma (NPC) patients was constructed, and the results showed that miR-BART19-3p was upregulated in all these diseases. Ectopic expression of miR-BART19-3p induced EBV-negative cell proliferation and suppressed cell apoptosis. Molecularly, adenomatous polyposis coli (APC) was identified to be a direct target of miR-BART19-3p, and APC mRNA expression was inversely correlated with miR-BART19-3p in CAEBV samples. Our results demonstrated that miR-BART19-3p contributes to the tumorigenesis of EBV-associated diseases and may be a potential therapeutic target.

Promoter epigenetics of APC gene and its implication in sporadic breast cancer patients from South Indian population

BackgroundThe adenomatous polyposis coli (APC) gene is a tumor suppressor gene associated with both familial and sporadic cancer, whose protein plays an important role in the WNT signal transduction pathway. WNT signaling is a key developmental pathway involved in embryonic development, cell differentiation, cell proliferation and tissue maintenance in adults. APC protein acts as an antagonist of the WNT signaling pathway by binding and regulating the β-catenin protein. Impaired APC gene function usually leads to a lack of degradation of β-catenin, which could cause uncontrolled cell growth and lead to tumorigenic transformation. APC gene may be inactivated by aberrant DNA methylation of CpG islands in their promoter regions. ObjectiveHence the present study was designed to determine the role of promoter methylation of APC genes in sporadic breast cancer patients from South Indian population. MethodsDNA methylation analyses of APC gene was performed by methylation-specific polymerase chain reaction (MSP).·Fifty biopsy samples of breast tumor and their corresponding non-malignant portions as controls were studied comparatively. APC mRNA expression analysis was also done using real time PCR. ResultsThirty eight percent (38%) cancerous tissue samples (19/50) showed promoter hypermethylation in APC gene; however, none of normal tissues showed promoter hypermethylation. The difference in methylation frequency between cancerous and normal tissue was statistically significant (p = 0.0001). APC promoter methylation was positively associated with lymph node involvement (p = 0.048), APC Promote hyper methylation and decreased mRNA expression was significantly associated (p = 0.0034). Lower APC-mRNA expression was associated with patient's age above 50 years (p = 0.044). ConclusionIn conclusion, our data showed that promoter hypermethylation of APC gene was a prognostic factor in breast cancer patients from South Indian population. The loss of APC gene function usually leads to an accumulation of β-catenin, which may promote tumorigenesis.

Abstract 2890: MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells

Abstract The canonical WNT signaling plays a critical role in many adult stem cells, including those of the breast and intestine. The fact that the canonical WNT signaling is implicated in both stem cell self-renewal and cancer suggests that normal physiological regulator of stem cell functions might be “hijacked” in cancer. Adenomatous polyposis coli (APC) is a component of the destruction complex that destabilizes β-catenin and suppresses the activity of the canonical WNT signaling. MicroRNAs (miRNAs) are important regulators of stem cell functions. We have previously reported a set of 37 miRNAs that are upregulated or downregulated in human breast cancer stem cells (BCSCs, a CD44+CD24-/lowlineage- population of human breast cancer cells) as compared to non-tumorigenic breast cancer cells (NTCs). Among them, miR-200c targets BMI1 that is a critical regulator of the stem cell maintenance, and strongly impairs the functions of human BCSCs in vivo. In this study, we compared the expression profiles of miRNAs, mRNAs and proteins between BCSCs and NTCs isolated from the patient specimens of human breast cancers and patient-derived tumor xenografts (PDXs) established by their transplantation. Luciferase assays were performed using the plasmid in which the 3’UTR region of candidate mRNA was cloned downstream of a luciferase minigene. The effect of miRNAs on the activity of WNT signaling was evaluated using a TCF reporter plasmid. Finally, the abilities to form organoids and to form tumors in immunodeficient mice were evaluated using the human BCSCs infected with the miRNA inhibitor expressing lentivirus. We found that miR-142 was highly upregulated in BCSCs, but was hardly expressed in NTCs in the patient breast cancer specimens. We confirmed that miR-142 targeted the sequence within the 3’UTR of APC mRNA and suppressed APC protein expression. Accordingly, miR-142 activated the canonical WNT signaling pathway in an APC-suppression dependent manner. The results of mRNA and protein expression profiling of the BCSCs isolated from human breast cancer PDXs suggested that the canonical WNT signaling was activated in BCSCs. Finally, inhibition of miR-142 in the BCSCs suppressed the tumor growth in vivo. These results suggest that the miR-142, a miRNA frequently upregulated in human BCSCs, could provide at least a part of the molecular mechanism for aberrant activation of the canonical WNT signaling in breast cancer in which APC mutations are much less frequent than colon cancer. Citation Format: Yohei Shimono, Taichi Isobe, Andrei Turtoi, Junko Mukohyama, Toru Mukohara, Akira Suzuki, Vincent Castronovo, Hironobu Minami. MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2017-2890

CXCL12/CXCR4 axis induced miR-125b promotes invasion and confers 5-fluorouracil resistance through enhancing autophagy in colorectal cancer

The activation of CXCL12/CXCR4 axis is associated with potential progression of cancer, such as invasion, metastasis and chemoresistance. However, the underlying mechanisms of CXCL12/CXCR4 axis and cancer progression have been poorly explored. We hypothesized that miRNAs might be critical downstream mediators of CXCL12/CXCR4 axis involved in cancer invasion and chemoresistance in CRC. In human CRC cells, we found that the activation of CXCL12/CXCR4 axis promoted epithelial-mesenchymal transition (EMT) and concurrent upregulation of miR-125b. Overexpression of miR-125b robustly triggered EMT and cancer invasion, which in turn enhanced the expression of CXCR4. Importantly, the reciprocal positive feedback loop between CXCR4 and miR-125b further activated the Wnt/β-catenin signaling by targeting Adenomatous polyposis coli (APC) gene. There was a negative correlation of the expression of miR-125b with APC mRNA in paired human colorectal tissue specimens. Further experiments indicated a role of miR-125b in conferring 5-fluorouracil (5-FU) resistance in CRC probably through increasing autophagy both in vitro and in vivo. MiR-125b functions as an important downstream mediator upon the activation of CXCL12/CXCR4 axis that involved in EMT, invasion and 5-FU resistance of CRC. These findings shed a new insight into the role of miR-125b and provide a potential therapeutic target in CRC.

Correlation between mutations and mRNA expression of APC and MUTYH genes: new insight into hereditary colorectal polyposis predisposition.

BackgroundTranscript dosage imbalance may influence the transcriptome. To gain insight into the role of altered gene expression in hereditary colorectal polyposis predisposition, in the present study we analyzed absolute and allele-specific expression (ASE) of adenomatous polyposis coli (APC) and mutY Homolog (MUTYH) genes.MethodsWe analyzed DNA and RNA extracted from peripheral blood mononuclear cells (PBMC) of 49 familial polyposis patients and 42 healthy blood donors selected according similar gender and age. Patients were studied for germline alterations in both genes using dHPLC, MLPA and automated sequencing. APC and MUTYH mRNA expression levels were investigated by quantitative Real-Time PCR (qRT-PCR) analysis using TaqMan assay and by ASE assays using dHPLC-based primer extension.ResultsTwenty out of 49 patients showed germline mutations: 14 in APC gene and six in MUTYH gene. Twenty-nine patients did not show mutations in both genes. Results from qRT-PCR indicated that gene expression of both APC and MUTYH was reduced in patients analyzed. In particular, a significant reduction in APC expression was observed in patients without APC germline mutation vs control group (P < 0.05) while APC expression in the mutation carrier patients, although lower compared to control individuals, did not show statistical significance. On the other hand a significant reduced MUTYH expression was detected in patients with MUTYH mutations vs control group (P < 0.05). Altered ASE of APC was detected in four out of eight APC mutation carriers. In particular one case showed a complete loss of one allele. Among APC mutation negative cases, 4 out of 13 showed a moderate ASE. ASE of MUTYH did not show any altered expression in the cases analyzed. Spearman’s Rho Test analysis showed a positive and significant correlation between APC and MUTYH genes both in cases and in controls (P = 0.020 and P < 0.001).ConclusionsAPC and MUTYH showed a reduced germline expression, not always corresponding to gene mutation. Expression of APC is decreased in mutation negative cases and this appears to be a promising indicator of FAP predisposition, while for MUTYH gene, mutation is associated to reduced mRNA expression. This study could improve the predictive genetic diagnosis of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0244-4) contains supplementary material, which is available to authorized users.

TGF‐β and Wnt Crosstalk Require SMAD 3 for Msi1 Induction in Colon

RNA binding proteins regulate mRNA stability, transport and translation. Musashi 1 (Msi1) binds to the 3'UTR of target mRNAs to block translation. Known Msi1 targets Numb and p21 regulate cellular proliferation, differentiation and apoptosis, events that are critical for maintaining homeostasis in rapidly dividing tissues. One such tissue, the intestine, is lined by a single layer of epithelial cells which form tube-like invaginations called crypts. Stem cells near the crypt base give rise to transit amplifying cells which migrate and differentiate toward the top of the crypt, eventually dying and shedding into the lumen. We previously showed that Msi-1 inhibits translation of the critical intestinal tumor suppressor protein adenomatous polyposis coli (APC). APC destroys proto-oncoprotein β-catenin in cells not encountering Wnt ligand which is secreted from cells below the base of the intestinal crypt. In contrast to Wnt, TGF-β signals originate near the top of the crypt, blocking cell proliferation and promoting differentiation. The coordination of Wnt and TGF-β signaling is critical for intestinal homeostasis, yet the links between these two pathways are not well-defined. Our earlier observation that lung epithelial cells treated with TGF-β showed reduced APC protein but not mRNA levels provided a clue about signal coordination. Here we show in intestinal cells, APC mRNA is stabilized by Msi1 which is induced by TGF-β. Msi1 is also a Wnt target gene and we show that SMAD3 is critical for Wnt / TGF-β crosstalk to control Msi1 expression. Finally, activity of a Msi1 promoter reporter in cells exposed to various TGF-β:Wnt ratios recapitulated Msi1 protein levels seen in the colon crypt.

APC promoter 1B deletion in seven American families with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome caused by mutations in the adenomatous polyposis coli (APC) gene. Clinical genetic testing fails to identify disease causing mutations in up to 20% of clinically apparent FAP cases. Following the inclusion of multiplex ligation-dependent probe amplification (MLPA) probes specific for APC promoter 1B, seven probands were identified with a deletion of promoter 1B. Using haplotype analysis spanning the APC locus, the seven families appear to be identical by descent from a common founder. The clinical phenotype of 19 mutation carriers is classical FAP with colectomy at an average age of 24. The majority of cases had a large number of duodenal and gastric polyps. Measurements of allele-specific expression of APC mRNA using TaqMan assay confirmed that relative expression in the allele containing the promoter 1B deletion was reduced 42-98%, depending on tissue type. This study confirms the importance of APC promoter deletions as a cause of FAP and identifies a founder mutation in FAP patients from the United States.

Abstract 1110: Musashi 1 stabilizes and blocks translation of Adenomatous Polyposis Coli mRNA in intestinal cells.

Abstract Most colorectal cancers are thought to be initiated by mutation of the tumor suppressor Adenomatous polyposis coli gene (APC). APC mutations that result in loss of function lead to deregulation of Wnt signaling and inappropriate proliferation within intestinal crypts. Understanding APC regulation within normal intestinal cells is important for developing methods to restore function in a pathological state. Using cultured human colonocytes, we have previously shown that translation of APC can be blocked by the RNA binding protein, Musashi 1 (MSI1) and that MSI1 is a Wnt target. We propose that a double negative feedback loop between APC and MSI1 functions in maintenance of intestinal cell homeostasis. To explore the MSI1/APC interaction in vivo, we analyzed intestinal tissue from MSI1 knock-out mice (Msi1-/-). We found higher levels of APC and less Wnt signal activation in intestines from the Msi1-/- mice. There were also more enterocytes within the intestinal crypts of Msi1-/-mice, indicating an increase in differentiation. These three observations provide confirmation of the proposed APC/MSI1 double negative feedback loop in vivo. Evidence from cultured human cells and mouse models supports a role for an APC/MSI1 double negative feedback loop in the response of intestinal cells to Wnt signaling. However, the underlying mechanisms remain to be determined. Proteins that bind to a specific motif in the 3’UTR of target mRNA can induce translation inhibition or promotion as well as stabilization or destabilization of the mRNA. We already established that MSI1 blocks APC translation; however, the effect of MSI1 on APC mRNA stability was unknown. Here, we show that MSI1 binding to the 3’ UTR of APC mRNA stabilizes the message. In contrast, Numb, another established MSI1 target, does not appear to be stabilized by MSI1 binding. If MSI1 bound to the 3’UTR of APC mRNA stabilizes the message, but inhibits translation, then something likely leads to MSI1 release when APC translation resumes. One possibility is that MSI1 protein is inherently unstable. In this case, MSI1 binds to APC mRNA and blocks translation but is rapidly degraded and therefore releases APC mRNA for translation unless additional MSI1 is made. Because MSI1 is a Wnt target, a cell stimulated by Wnt ligand should continually replace the MSI1, therefore APC mRNA translation would remain blocked until the Wnt signal is removed. Our data showing that MSI1 has a relatively long half-life does not support this model. Current work is aimed to explore whether MSI1 release from APC mRNA is regulated by post-translational modifications or other protein-protein interactions. In conclusion, by acting as a Wnt antagonist, APC blocks MSI1 transcription. On the other hand, MSI1 binds to and stabilizes APC mRNA while also blocking APC translation. The MSI/APC double negative feedback loop appears important for maintaining homeostasis of intestinal epithelial cells. Citation Format: Andy R. Wolfe, Erick Spears, Amanda Ernlund, Kristi L. Neufeld. Musashi 1 stabilizes and blocks translation of Adenomatous Polyposis Coli mRNA in intestinal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2013-1110

Molecular characterization of parathyroid tumors from two patients with hereditary colorectal cancer syndromes

The tumor suppressor adenomatous polyposis coli (APC) has recently been implicated in parathyroid development. We here report clinical, histopathological and molecular investigations in parathyroid tumors arising in two patients; one familial adenomatous polyposis (FAP) syndrome patient carrying a constitutional APC mutation, and one Lynch syndrome patient demonstrating a germline MLH1 mutation as well as a non-classified, missense alteration of the APC gene. We sequenced the entire APC gene in tumor and constitutional DNA from both cases, assessed the levels of APC promoter 1A and 1B methylation by bisulfite Pyrosequencing analysis and performed immunohistochemistry for APC and parafibromin. In addition, copy number analysis regarding the APC gene on chromosome 5q21-22 was performed using qRT-PCR. Histopathological workup confirmed both tumors as parathyroid adenomas without signs of malignancy or atypia. No somatic mutations or copy number changes for the APC gene were discovered in the tumors; however, in both cases, the APC promoter 1A was hypermethylated while the APC promoter 1B was unmethylated. APC promoter 1B-specific mRNA and total APC mRNA levels were higher than in normal parathyroid samples. Immunohistochemical analyses revealed strong APC protein immunoreactivity and positive parafibromin expression in both parathyroid tumors. Absence of additional somatic APC mutations and copy number changes in addition to the positive APC immunoreactivity obtained suggest that the tumors arose without biallelic inactivation of the APC tumor suppressor gene. The finding of an unmethylated APC promoter 1B and high APC 1B mRNA levels could explain the maintained APC protein expression. Moreover, the findings of positive parafibromin and APC immunoreactivity as well as a low MIB-1 proliferation index and absence of histopathological features of malignancy/atypical adenoma indicate that the parathyroid adenomas arising in these patients did not harbor malignant potential.

Programmed ribosomal frameshifting in the expression of the regulator of intestinal stem cell proliferation, adenomatous polyposis coli (APC)

A programmed ribosomal frameshift (PRF) in the decoding of APC (adenomatous polyposis coli) mRNA has been identified and characterized in Caenorhabditis worms, Drosophila and mosquitoes. The frameshift product lacks the C-terminal approximately one-third of the product of standard decoding and instead has a short sequence encoded by the -1 frame which is just 13 residues in C. elegans, but is 125 in D. melanogaster. The frameshift site is A_AA.A_AA.C in Caenorhabditids, fruit flies and the mosquitoes studied while a variant A_AA.A_AA.A is found in some other nematodes. The predicted secondary RNA structure of the downstream stimulators varies considerably in the species studied. In the twelve sequenced Drosophila genomes, it is a long stem with a four-way junction in its loop. In the five sequenced Caenorhabditis species, it is a short RNA pseudoknot with an additional stem in loop 1. The efficiency of frameshifting varies significantly, depending on the particular stimulator within the frameshift cassette, when tested with reporter constructs in rabbit reticulocyte lysates. Phylogenetic analysis of the distribution of APC programmed ribosomal frameshifting cassettes suggests it has an ancient origin and raises questions about a possibility of synthesis of alternative protein products during expression of APC in other organisms such as humans. The origin of APC as a PRF candidate emerged from a prior study of evolutionary signatures derived from comparative analysis of the 12 fly genomes. Three other proposed PRF candidates (Xbp1, CG32736, CG14047) with switches in conservation of reading frames are likely explained by mechanisms other than PRF.

MK-801 induces schizophrenic behaviors through downregulating Wnt signaling pathways in male mice

Wnt signaling is important in various neuropsychiatric diseases. However, its actions on modulating schizophrenia are largely unknown. Our previous study found that three SNPs in adenomatous polyposis coli (APC), a negative regulator of the Wnt signaling, were associated with schizophrenia, and the mRNA levels of APC in blood leucocytes of patients with schizophrenia were significantly increased. This prompted us to further investigate the effects of Wnt signaling components on the pathogenesis of schizophrenia. In our current study, mouse schizophrenia was induced by i.p. injection of MK-801 for 7days and the brain prefrontal cortex (PFC) and ventral tegmental area (VTA) were isolated to investigate the Wnt signaling pathway. Compared with control, schizophrenic mice had increased inhibitory phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) in PFC and VTA, which is disassociated with augmented beta-catenin phosphorylation. However, APC mRNA and protein in the PFC and VTA isolated from the schizophrenic group were increased and matched with increased beta-catenin phosphorylation. The total dendritic length was significantly increased in both PFC and VTA from MK-801-treated mice compared to control. By using cultured SK-N-SH and PC12 cells with and without transfection of APC siRNA, we found that the APC protein facilitates neurite growth in vitro. Our data suggested that MK-801-induced schizophrenia is associated with attenuated Wnt signaling pathway in the brain, which may be due to augmented APC protein during schizophrenia. APC facilitates neurite growth, potentially contributing to the pathology of schizophrenia.

Novel Double-negative Feedback Loop between Adenomatous Polyposis Coli and Musashi1 in Colon Epithelia

Loss of tumor suppressor adenomatous polyposis coli (APC) is thought to initiate the majority of all colorectal cancers. The predominant theory of colorectal carcinogenesis implicates stem cells as the initiating cells. However, relatively little is known about the function of APC in governing the homeostasis of normal intestinal stem cells. Here, we identify a novel double-negative feedback loop between APC and a translation inhibitor protein, Musashi1 (MSI1), in cultured human colonocytes. We show APC as a key factor in MSI1 regulation through Wnt signaling and identify APC mRNA as a novel target of translational inhibition by MSI1. We propose that APC/MSI1 interactions maintain homeostatic balance in the intestinal epithelium.

Adenomatous Polyposis Coli and Hypoxia-inducible Factor-1α Have an Antagonistic Connection

The tumor suppressor adenomatous polyposis coli (APC) is mutated in the majority of colorectal cancers and is best known for its role as a scaffold in a Wnt-regulated protein complex that determines the availability of β-catenin. Another common feature of solid tumors is the presence of hypoxia as indicated by the up-regulation of hypoxia-inducible factors (HIFs) such as HIF-1α. Here, we demonstrate a novel link between APC and hypoxia and show that APC and HIF-1α antagonize each other. Hypoxia results in reduced levels of APC mRNA and protein via a HIF-1α-dependent mechanism. HIF-1α represses the APC gene via a functional hypoxia-responsive element on the APC promoter. In contrast, APC-mediated repression of HIF-1α requires wild-type APC, low levels of β-catenin, and nuclear factor-κB activity. These results reveal down-regulation of APC as a new mechanism that contributes to the survival advantage induced by hypoxia and also show that loss of APC mutations produces a survival advantage by mimicking hypoxic conditions.

Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype

Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.

Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer

Inactivation of the adenomatous polyposis coli (APC) gene is a major initiating event in colorectal tumorigenesis. Most of the mutations in APC generate premature stop codons leading to truncated proteins that have lost beta-catenin binding sites. APC-free beta-catenin stimulates the Wnt signaling pathway, leading to active transcription of target genes. In the current study, we describe a novel mechanism for APC regulation. We show that miR-135a&b target the 3' untranslated region of APC, suppress its expression, and induce downstream Wnt pathway activity. Interestingly, we find a considerable up-regulation of miR-135a&b in colorectal adenomas and carcinomas, which significantly correlated with low APC mRNA levels. This genetic interaction is also preserved in full-blown cancer cell lines expressing miR-135a&b, regardless of the mutational status of APC. Thus, our results uncover a miRNA-mediated mechanism for the control of APC expression and Wnt pathway activity, and suggest its contribution to colorectal cancer pathogenesis.

Hydralazine inhibits human cervical cancer cell growth in vitro in association with APC demethylation and re-expression

The tumor suppressor adenomatous polyposis coli (APC) is frequently silenced by promoter hypermethylation in human cervical cancer. Clinically, it has been approved that DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (5-Aza-dC), can reverse APC promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. Hydralazine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. The purpose of this study was to evaluate the effects of hydralazine on APC reactivation and the inhibition of human cervical cancer cells in vitro. Expression of APC gene, and methylation status were analyzed by RT-PCR, quantitative real time RT-PCR, and methylation-specific PCR methods. beta-Catenin protein that correlates closely with APC was detected by immunohistochemistry method after treatment with hydralazine. MTT and FCM assays were used to observe the changes of proliferation activity, cell cycle, and apoptosis of the cells. Methylated APC was not expressed in HeLa cell, hemimethylated APC was expressed in CaSki cells, and unmethylated APC was expressed normally in SiHa cells. Hydralazine induces APC expression and promotes demethylation in HeLa and CaSki cells. After treatment with 40 mumol/L hydralazine for 72 h, growth inhibitive rates (%) of HeLa, CaSki, and SiHa cell lines were 52.12 +/- 3.78, 44.31 +/- 2.59, and 47.73 +/- 4.73, respectively. On the contrary, the normal cell ECV304 growth inhibitory rate was only 27.18 +/- 0.79. The expression of APC mRNA in HeLa, CaSki, and SiHa cell lines increased 10.35-, 11.40-, and 0.73-fold, respectively. HeLa and CaSki cells were arrested in S phase of the cell cycle by hydralazine, and the percentage of apoptotic cells in the two cell lines treated with hydralazine was increased significantly compared to the untreated cells (P < 0.01). The expression of beta-catenin protein in the cell membrane was observed after the treatment with hydralazine. Hydralazine, an effective inhibitor of APC methylation and promoter of APC re-expression, can inhibit cell growth in human cervical cancer in vitro and be potentially used for the clinical treatment of human cervical cancer.