APC Expression Research Articles

Modulation of the Wnt/β-catenin pathway is associated with the basal-like breast cancer phenotype.

Abstract Abstract #4066 Background: Basal-like breast tumors are aggressive breast cancers that are distinct from other types of breast tumors by gene expression signatures. The Wnt/β-catenin signal transduction pathway is deregulated in numerous tumor types, including breast cancer, and is implicated in promoting the mesenchymal phenotype characteristic of basal-like breast tumor cells. Nuclear β-catenin has been associated with poor survival in some breast cancer patients, and analysis of human sporadic breast cancers indicate that as many as 44% of sporadic breast cancers or cell lines demonstrate inactivation of the APC tumor suppressor, a Wnt/β-catenin regulator. However, it is unclear whether deregulation of this pathway occurs in a specific subclass of breast cancers and whether therapeutically targeting this pathway in patients might be warranted. We hypothesized that the Wnt/β-catenin pathway is activated specifically in basal-like breast tumors and is causally involved in the morphology and behavior of basal-like tumor cells.
 Materials and Methods: To test our hypothesis, we utilized three complementary approaches. 1) In EpH4 mammary epithelial cells, β-catenin was stabilized by treatment with a proteasome (ALLN) or GSK3β (SB216763) inhibitor or by overexpression of a non-degradable mutant (S37A β-catenin). Specificity of the drug effects was tested by transfection of β-catenin-specific siRNAs. 2) APC knockdown was achieved in 4T07 mammary tumor cells using transient transfection of APC-specific siRNAs compared to non-specific control siRNAs. Cell morphology and cell-cell interactions were analyzed using phase-contrast microscopy and immunofluorescence. 3) Using tissue microarrays containing 32 normal breast, 7 DCIS, 41 invasive breast cancers and 23 metastases specimens, β-catenin immunohistochemistry was performed and scored for nuclear, membranous and cystosolic localization. A colon cancer tissue microarray was used as positive control for nuclear β-catenin localization.
 Results: In mammary epithelial cells, stabilization of β-catenin was sufficient to induce a morphology that resembles mesenchymal cells, including flattening and extension of robust cell protrusions, an effect that was rescued by β-catenin knockdown. Metastatic murine mammary tumor cells with a robust mesenchymal phenotype were converted to an epithelial phenotype when APC expression was suppressed. Furthermore, we found that nuclear localization of β-catenin, a read-out of Wnt/β-catenin pathway activity, was found exclusively in a subset of basal-like breast tumors (7 of 18) but not in normal breast or other tumor subtypes.
 Discussion: Our data indicate that alterations in Wnt/β-catenin pathway components can dramatically modify the phenotype of both normal and transformed breast cells. Further, these results indicate that activation of the Wnt/β-catenin pathway is specifically associated with basal-like breast tumors. These findings suggest that pharmacologic modulation of this pathway might be useful for basal-like tumors in further pre-clinical studies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4066.

Analysis of APC, α-, β-catenins, and N-cadherin protein expression in aggressive fibromatosis (desmoid tumor)

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, α-, β-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 β-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 α-catenin. In the E-AD group, in both cases of recurrent tumors, no α-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 α-catenin, β-catenin (c), (n) and (c+n) expression, and tumor size ( p>0.1). The results regarding β-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.

The property of methylated APC gene promotor and its influence on lung cancer cell line

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.

APC ain't always necessary for axons

Like many neuroscientists, Rusan et al. considered that the mechanism controlling axonal positioning and outgrowth might be the same in all neurons. As they now show, however, one neuron's must-have axon-promoting protein is virtually dispensable in other neurons. Figure 1 A central brain neuron grows a perfectly good axon even in the absence of APC. In a young neuron, multiple mini neurites protrude until one is chosen to become the axon. A microtubule-associated protein called APC was thought to be required for axon outgrowth, as dominant-negative APC expression in cultured neurons reduced axon growth dramatically. Other reports had suggested that APC delivers a polarity protein called Par3, necessary for axon growth, to the chosen neurite. Despite the strong evidence for APC's axon-promoting role, definitive knockout experiments in neurons were limited to one study, which showed that medulla neurons behaved much as expected—no APC, no axon extension. Now, the same lab have found that neurons from the central brain, mushroom body, and lobular plug will happily grow axons without APC. Cells from these parts of the brain were not entirely normal: precursor neuroblasts had slightly longer cell cycles and some had bent spindles. Though, neither problem prevented mitosis. The need for APC in some neurons but not others seems to reflect a requirement for Wnt signaling. As well as its cytoskeletal role, APC is a downstream regulator of Wnt, and while medulla neurons need Wnt for axon growth, the other types of neurons did not. It will be of interest to check whether hippocampal neurons, where the dominant-negative experiments were carried out, are also Wnt responsive. Rusan, N.M., et al. 2008. J. Cell Biol. .10.1083/jcb.200807079 [PubMed] [Cross Ref]

Promoter hypermethylation leads to decreased APC mRNA expression in familial polyposis and sporadic colorectal tumours, but does not substitute for truncating mutations

Germline mutations in the tumour suppressor APC cause familial adenomatous polyposis (FAP), and somatic mutations are common in sporadic colorectal cancers (CRCs). Hypermethylation of APC promoter 1A has been reported in a substantial proportion of sporadic CRCs and may cause transcriptional silencing. Methylation has been proposed as an alternative to mutation or loss of heterozygosity as a mechanism of gene inactivation. However, the significance of APC methylation has remained unclear, because it has not previously been related to the presence of mono- or bi-allelic mutations at APC. We examined 103 FAP adenomas, 11 attenuated FAP adenomas, 31 sporadic CRCs and 30 CRC cell lines, all with known germline and/or somatic APC mutations. Overall, APC promoter 1A methylation was detected in 27–45% of colorectal tumours and cell lines, but generally not in histologically normal colorectum. In contrast to previous reports, methylation was detected in similar proportions of FAP/AFAP and sporadic CRCs. Importantly, methylation was independent of the presence, number and positions of APC mutations and was not associated with the CpG island methylator phenotype. Methylation resulted in a decrease or loss of 1A isoform mRNA and reduced total APC transcript levels, although expression was retained from promoter 1B. However, neither APC protein levels, nor transcription of a panel of Wnt target genes was associated with methylation status. Our data suggest that APC promoter 1A hypermethylation may influence APC expression levels in a subtle fashion, but methylation does not result in complete gene inactivation or act as a ‘second hit’.

Switch of transforming growth factor β function from tumor suppression to stimulation in adenomatous polyposis coli (APC) knocked-down human colon carcinoma cells

TGFβ exerts a potent tumor-suppressive effect in the human colon carcinoma CBS and Moser cells. However, TGFβ can also function as a tumor promoter. The mechanisms underlying the tumor promoting effect of TGFβ are not understood. Both the CBS and Moser cells were found to express mutant (truncated) APC. Expression of this form of APC did not interfere with the tumor-suppressive function of TGFβ. However, when APC expression was knocked down in these cells, TGFβ function switched from that of tumor suppression to that of tumor promotion. TGFβ stimulated cellular invasion and anchorage-independent growth in APC knocked-down cells. Knocking down APC expression abrogated the ability of TGFβ to induce the expression of the tumor suppressor E-cadherin and the cyclin dependent kinase inhibitor p21/Waf1. TGFβ now stimulated the constitutive TCF transcriptional activation activity associated with the β-catenin/Wnt pathway in the APC knocked-down cells. Thus, the level of APC expression determined the type of TGFβ function in these human colon carcinoma cells.

Clinical characterization and the mutation spectrum in Swedish adenomatous polyposis families

BackgroundThe dominantly inherited condition familial adenomatous polyposis (FAP) is caused by germline mutations in the APC gene. Finding the causative mutations has great implications for the families. Correlating the genotypes to the phenotypes could help to improve the diagnosis and follow-up of patients.MethodsMutation screening of APC and the clinical characterization of 96 unrelated FAP patients from the Swedish Polyposis Registry was performed. In addition to generally used mutation screening methods, analyses of splicing-affecting mutations and investigations of the presence of low-frequency mutation alleles, indicating mosaics, have been performed, as well as quantitative real-time polymerase chain reaction to detect lowered expression of APC.ResultsSixty-one different APC mutations in 81 of the 96 families were identified and 27 of those are novel. We have previously shown that 6 of the 96 patients carried biallelic MUTYH mutations. The 9 mutation-negative cases all display an attenuated or atypical phenotype. Probands with a genotype (codon 1250–1464) predicting a severe phenotype had a median age at diagnosis of 21.8 (range, 11–49) years compared with 34.4 (range, 14–57) years among those with mutations outside this region (P < 0.017). Dense polyposis (> 1000) occurred in 75% of the probands with a severe phenotype compared with 30% in those with mutations outside this region. The morbidity in colorectal cancer among probands was 25% at a mean age of 37.5 years and 29% at a mean age of 46.6 years.ConclusionUsing a variety of mutation-detection techniques, we have achieved a 100% detection frequency in classical FAP. Probands with APC mutations outside codon 1250–1464, although exhibiting a less-severe phenotype, are at high risk of having a colorectal cancer at diagnosis indicating that age at diagnosis is as important as the severity of the disease for colorectal cancer morbidity.

The APC Tumor Suppressor Inhibits DNA Replication by Directly Binding to DNA via Its Carboxyl Terminus

The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of beta-catenin levels in the cell. Transient over expression of the tumor suppressor gene APC in colon cancer cells prevents entry into S phase of the cell cycle, a phenotype only partially restored by cotransfection of a transcriptionally active form of beta-catenin. In an attempt to define its transcription-independent tumor suppressor functions, we tested whether APC directly affects DNA replication. A transcriptionally quiescent in vitro DNA replication system, the polymerase chain reaction, DNA binding assays, and transient transfections in colon cancer cell lines were used to determine the effects of APC on DNA replication and the mechanism by which it works. We report that exogenous full-length APC inhibits replication of template DNA through a function that maps to amino acids 2140-2421, a region of the protein commonly lost by somatic or germline mutation. This segment of APC directly interacts with DNA, while mutation of the DNA-binding S(T)PXX motifs within it abolishes DNA binding and reduces inhibition of DNA replication. Phosphorylation of this segment by cyclin-dependent kinases also reduces inhibition of DNA replication. Furthermore, transient transfection of an APC segment encoding amino acids 2140-2421 into a colon cancer cell line with mutant APC prevents cell cycle progression into or through S phase. Our results suggest that APC can negatively regulate cell cycle progression through inhibition of DNA replication by direct interaction with DNA.

Characterization of the specific role and activation of the High Mobility Group A1 (HMGA1) in colon cancer

The High Mobility Group A1 (HMGA1) proteins are a class of oncogenes whose specific role in colon and other cancers is currently emerging. Our analysis of the HMGA1 promoter region revealed several putative sites for beta‐catenin/tcf binding suggesting that HMGA1 may be activated by the adenomatous polyposis coli (Apc)/beta‐catenin pathway in colon cancer. To better understand the specific role of HMGA1 in colon carcinoma formation, we evaluated the expression of HMGA1 in the Apc/min colon cancer mouse model. Results of quantitative real‐time RT‐PCR (qRT‐PCR) show a 9‐fold increase over normal intestine in HMGA1 mRNA where Apc is truncated and in tumors. Consistent with qRT‐PCR data, HMGA1 protein levels were also significantly higher in Apc‐min intestinal tumors than those from normal mouse tissues. To determine if Apc/beta catenin signaling directly impacts HMGA1 expression, we tested the effect of Apc recovery on HMGA1 expression. Preliminary results indicate that zinc‐induced full‐length Apc expression suppresses HMGA1 levels and that truncations in Apc may activate HMGA1 to promote cancer initiation and progression. Collectively, these results provide the first line of evidence supporting the cooperation of truncated Apc and HMGA1 in colon cancer.This project was supported by NIH grant P20 RR‐16461 in support of SC INBRE.

Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung

Atypical adenomatous hyperplasia (AAH) is now recognized as a precursor lesion from which lung adenocarcinomas arise and thus represents an ideal target for studying the early genetic and epigenetic alterations associated with lung tumorigenesis such as alterations of the Wnt pathway. We assessed the level of Wnt signaling activity in lung cancer cell lines by determining the level of active beta-catenin and determined the level of expression of Wnt antagonists APC, DKK1, DKK3, LKB1, SFRP1, 2, 4, 5, WIF1 and RUNX3 using reverse transcription-polymerase chain reaction. Using multiplex nested methylation-specific polymerase chain reaction, we analyzed promoter region methylation of these genes in resected lung tissue in the histopathologic sequence of glandular neoplasia (normal lung parenchyma, low-grade and high-grade AAH, adenocarcinoma). The majority of non-small cell lung cancer cell lines (11 of 16, 69%) have evidence of active Wnt signaling and silencing of Wnt antagonists correlated with promoter hypermethylation. Promoter region methylation of Wnt antagonists was common in primary lung adenocarcinoma and there was a significant increase in the frequency of methylation for Wnt antagonist genes and the number of genes methylated with each stage of tumorigenesis (test for rend P <or= 0.01). Additionally, odds ratios for promoter hypermethylation of individual or multiple Wnt antagonist genes and adenocarcinomas were statistically significantly elevated and ranged between 3.64 and 48.17. These results show that gene silencing of Wnt antagonists by promoter hypermethylation occurs during the earliest stages of glandular neoplasia of the lung and accumulates with progression toward malignancy.

Sporadic childhood hepatoblastomas show activation of β-catenin, mismatch repair defects and p53 mutations

Hepatoblastoma, a rare embryonic tumor that may arise sporadically or in the context of hereditary syndromes (familial adenomatous polyposis and Beckwith–Wiedemann's) is the most frequent liver cancer of childhood. Deregulation of the APC/β-catenin pathway occurs in a consistent fraction of hepatoblastomas, with mutations in the APC and β-catenin genes implicated in familial adenomatous polyposis-associated and sporadic hepatoblastomas, respectively. Alterations in other cancer-related molecular pathways have not been reported. We investigated a series of 21 sporadic paraffin-embedded hepatoblastoma cases for mutations in the p53 (exons 5–8) and β-catenin (exon 3) genes, loss of heterozygosity at APC, microsatellite instability and immunohistochemical expression of β-catenin and of the two main mismatch repair proteins, MLH1 and MSH2. No loss of heterozygosity at APC was detected. We found mutations in β-catenin and p53 in 4/21 (19%) and 5/21 (24%) cases respectively, β-catenin protein accumulation in 14/21 cases (67%), microsatellite instability in 17/21 cases (81%), of which eight resulted positive for high-level of microsatellite instability (in four cases associated with loss of MLH1/MSH2 immunostaining). No correlations between involved molecular pathway(s) and hepatoblastoma histotype(s) emerged. This study confirms that β-catenin deregulation is involved in sporadic hepatoblastoma and also suggests that mismatch repair defects and p53 mutations contribute to this rare liver cancer. Sporadic hepatoblastoma appears to be molecularly and phenotypically heterogeneous and may reflect different pathways of liver carcinogenesis.

Mild Depletion of Dietary Folate Combined with Other B Vitamins Alters Multiple Components of the Wnt Pathway in Mouse Colon ,

Preclinical and clinical studies suggest that diminished folate status increases the risk of colorectal carcinogenesis. However, many biochemical functions of folate are dependent on the adequate availability of other 1-carbon nutrients, including riboflavin, vitamin B-6, and vitamin B-12. Aberrations in the Wnt pathway are thought to play an important role in human colorectal cancers. This study therefore investigated if mild depletion of folate combined with depletion of riboflavin, vitamin B-6, and vitamin B-12 could induce alterations in the Wnt pathway in the colonic mucosa. Ninety-six mice were pair-fed diets with different combinations of B vitamin depletion for 10 wk. Genomic DNA methylation and uracil misincorporation were measured by LC/MS and GC/MS. Gene-specific methylation, strand breaks, and expressions were measured by real-time PCR and immunoblotting. Proliferation and apoptosis were determined by immunohistochemistry. DNA strand breaks within the Apc mutation cluster region were induced by folate depletion combined with inadequacies of riboflavin, vitamin B-6, and vitamin B-12 (P < 0.05), but such effects were not induced by folate depletion alone. Similarly, minor changes in the expression of Apc, β-catenin, and cyclin D1 produced by mild folate depletion were significantly magnified by multiple vitamin depletion. Apoptosis, which can be suppressed by increased Wnt-signaling, was attenuated by the combined deficiency state (P < 0.05) but not by singlet or doublet deficiencies. These findings indicate that a mild depletion of folate that is of insufficient magnitude by itself to induce alterations in components of the Wnt pathway may produce such effects when present in conjunction with mild inadequacies of other 1-carbon nutrients.

Polymeric black tea polyphenols inhibit 1,2-dimethylhydrazine induced colorectal carcinogenesis by inhibiting cell proliferation via Wnt/β-catenin pathway

Tea polyphenols like epigallocatechin gallate and theaflavins are established chemopreventive agents for colorectal carcinogenesis. However, studies on evaluating similar chemopreventive properties of thearubigins or polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, are limited. Hence, in the present study we aim to investigate chemopreventive effects along with probable mechanisms of action of PBP extract employing 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis in Sprague–Dawley rats as experimental model. The present study suggests that PBPs, like other tea polyphenols, also inhibit DMH-induced colorectal tumorigenesis by decreasing tumor volume and multiplicity. This study also shows that although the pretreatment with PBP extract could induce detoxifying enzymes in hepatic and colorectal tissue, it did not show any additional chemopreventive effects when compared to treatments with PBP extract after initiation with DMH. Mechanistically, PBP extract may inhibit colorectal carcinogenesis by decreasing DMH-induced cell proliferation via Wnt/β-catenin pathway. Treatments with PBP extract showed decreased levels of COX-2, c-MYC and cyclin D1 proteins which aid cell proliferation probably by regulating β-catenin by maintaining expression of APC and decreasing inactivation of GSK3β. DMH-induced activation of MAP kinases such as ERK and JNK was also found to be inhibited by treatments with PBP extract. In conclusion, the protective effects of PBP extract could be attributed to inhibition of DMH-induced cellular proliferation probably through β-catenin regulation.

Target gene activation of the Wnt-signalling pathway in the adamantinomatous subtype of craniopharyngiomas

Objectives: Craniopharyngiomas (CPs) are rare, benign, epithelial tumours of the sellar region, histologically classified into adamantinomatous and papillary subtypes. The Wnt signalling pathway is involved in the pathogenesis of adamantinomatous CP, indicated by activating mutations within exon 3 of the β-catenin gene as well as nuclear accumulation of ß-catenin protein in 95% of respective tumours. However, several issues remain to be addressed, e.g. whether activating mutations in exon 3 induce nuclear β-catenin accumulation per se, or are other active and passive nuclear transport mechanisms also be involved (i.e. APC)? In addition, there is no proven evidence that nuclear ß-catenin activates target genes of the Wnt-signalling cascade in affected tumour cells. To answer these intriguing questions we performed a laser-microdissection based study comparing accumulating vs. non-accumulating tumour cells in adamantinomatous CPs. Mutation analysis as well as gene expression profiling with real-time PCR were conducted. Methods: Laser-microdissected cell clusters with or without β-catenin accumulation were subjected to mutational subsequent SSCP analysis of exon 3 of the β-catenin gene, in order to explore a potential genotypic heterogeneity (n=3). Furthermore, mutational analysis was extended towards exons 8 to 12 encoding armadillo repeats, which are crucial for passive nuclear transport of ß-catenin. Since active nuclear transport of ß-catenin is operated by APC, we have included an immunohistochemical analysis in 71 adamantinomatous and 16 papillary craniopharyngiomas using APC – specific antibodies. To adress the issue whether nuclear ß-catenin activates the Wnt-signaling cascade in adamantinomatous CPs, real-time RT-PCR analysis of conductin, a well recognised target gene and inhibitor of β-catenin, was examined in snap frozen tumour samples as well as in a laser – microdissected formalin fixed paraffin embedded tumour specimen. Results: Mutations in exon 3 of the β-catenin gene were identified in both, accumulating and non-accumulating cell populations. However, analysis of exons 8 to 12 did not reveal additional alterations in a series of 44 craniopharyngiomas. Whereas APC-mutations were not detectable in CP, our immunohistochemical analysis showed nuclear APC accumulation in 60 out of 71 (85%) adamantinomatous, and 6 out of 16 (38%) papillary CPs. A distinct overlap of nuclear β-catenin and APC expression was identified only in adamantinomatous CP. In addition, the β-catenin accumulating cell fraction revealed a 20 fold upregulation of conductin compared to non – accumulating adamantinomatous or papillary CPs. Conclusion: Our data confirm activating β-catenin mutations in exon 3 in adamantinomatous CPs. However, these mutations may not associate with nuclear β-catenin transfer per se, but rather point to the nuclear APC export machinery of β-catenin to be activated, but not genetically compromised. Immunohistochemical colocalisation of APC and ß-catenin within cell nuclei support this notion. Furthermore, nuclear β-catenin accumulation results in target gene activation and overexpression of inhibitors of the Wnt-signalling pathway i.e. conductin. The present results further highlight the Wnt-signaling pathway as molecular basis of the distinct and challenging clinical and morphological phenotype of adamantinomatous CPs.

Distinct Endosomal Trafficking Requirements for Presentation of Autoantigens and Exogenous Lipids by Human CD1d Molecules

CD1d molecules present both self Ags and microbial lipids to NKT cells. Previous studies have established that CD1d lysosomal trafficking is required for presentation of autoantigens to murine invariant NKT cells. We show in this study that this is not necessary for autoantigen presentation by human CD1d, but significantly affects the presentation of exogenous Ags. Wild-type and tail-deleted CD1d molecules stimulated similar autoreactive responses by human NKT clones, whereas presentation of exogenous lipids by tail-deleted CD1d was highly inefficient. Chloroquine treatment markedly inhibited exogenous Ag presentation by wild-type CD1d transfectants, but did not affect NKT autoreactive responses. Conversely, APC expression of HLA-DRalphabeta and the invariant chain (Ii) was associated with faster internalization of CD1d into the endocytic system and enhanced CD1d-mediated presentation of exogenous Ags, but did not appear to augment NKT autoreactivity. Knockdown of the Ii by small interfering RNA resulted in reduced CD1d surface expression and slower internalization in HLA-DR+ APCs, but not HLA-DR- APCs, demonstrating a direct effect of MHC/Ii complexes on CD1d trafficking. CD1d-mediated presentation of exogenous Ags was much more efficient in immature dendritic cells, which actively recycle MHC class II molecules through the endocytic system, than in mature dendritic cells that have stabilized MHC class II expression at the cell surface, suggesting a physiological role for MHC/Ii complexes in modulating CD1d function. These results indicate that autoantigens and exogenous lipids are acquired by human CD1d at distinct cellular locations, and that Ii trafficking selectively regulates CD1d-mediated presentation of extracellular Ags.

APC Inactivation Associates With Abnormal Mitosis Completion and Concomitant BUB1B/MAD2L1 Up-Regulation

Chromosomal instability, a hallmark of most colorectal cancers, has been related to altered chromosome segregation and the consequent deficit in genetic integrity. A role for the tumor suppressor gene APC has been proposed in colorectal cancer that leads to compromised chromosome segregation even though the molecular mechanism is not yet understood. Here, we tackled the genetic basis for the contribution of APC to chromosomal instability in familial adenomatous polyposis and sporadic colorectal cancer. We have used video-microscopy of primary cultures and molecular genetic methods to address these issues in human samples and in genetically defined mouse models that either recapitulate the familial adenomatous polyposis syndrome (Apc(1638N)), or develop tumors in the absence of APC mutations (pvillin-KRASV12G). Mutations in APC were associated with an increased incidence in cell cycle defects during the completion of cytokinesis. Transcriptome analysis performed on mouse models indicated a significant up-regulation of genes that regulate accurate mitosis. Notably, we identified up-regulated expression of BUB1B and MAD2L1, 2 genes that are involved in the mitotic checkpoint, but have so far not been implicated in chromosomal instability induced by APC loss of function. In vitro modulation of APC expression suggested a causal association for this upregulation, which was consistently found in sporadic and familial adenomatous polyposis lesions, as an early event in colorectal tumorigenesis. In addition to the known function of APC during correct spindle assembly and positioning, we propose a concomitant involvement of APC in the surveillance mechanism of accurate mitosis.

Different Activation of WNT Signaling Pathway in B-Cell and T-Cell Acute Leukemias.

WNT signaling pathway proteins function as hematopoietic growth factors and regulate proliferation in normal T-cell and B-cell development. Recent experimental evidence demonstrated that oncogenic transformation in leukemias of both lymphoid and myeloid lineages is dependent on WNT signaling. Not much is known about activation of WNT signaling pathway, its ligands and receptors in hematopoiesis and leukemia pathogenesis. To define its role in leukemia, we aimed to determine mRNA levels of the critical members of WNT pathway (WNT5A, WNT10B, FZ5, β catenin, APC, TCF-1 and LEF-1) by using quantitative real time PCR in acute lymphoblastic leukemia (ALL) patients (T-cell n=42, B-cell n=46 and pre B-cell n=30) and normal hematopoietic cells (bone marrow n=6, peripheral blood n=10, and CD19+ cells from peripheral blood). These genes expressed varying levels in B-cells, preB-cells and T-cells. In the B-cell leukemia patients, WNT5A was expressed notably (OR=58.05 CI 95% 1.63–1219.55, p>0,001). WNT5A directs Ca++ dependent signaling by PKC and a G protein dependent manner which is an alternative pathway for beta-catenin mediated signaling. Also LEF-1 levels were higher in B-ALL patients and APC expression was down regulated when compared to normal tissue (OR=18.81 CI 95% 0.34–5703, p>0.001 and OR=0.212 CI 95% 0.006–8.816, p=0.001, respectively). It is known that LEF-1 blocks APC mediated β catenin nuclear export and activates transcription of various transforming genes, including cyclin, D1, c-myc, MMP7, and LEF-1 itself. WNT5A or WNT10B proteins were not found to be up regulated in preB-ALL whereas APC and LEF-1 gene expressions were increased compared to normal hematopoietic cells (OR=32.97 CI 95% 0.27–1281, 38 p>0.001 and OR=5.57 CI 95% 0.28–89.51, p=0.01, respectively). We found increased TCF-1 expression (7.4 fold) without any β catenin accumulation in T-ALL patients. It is known that TCF-1 in absence of β catenin functions as a tumor suppressor gene. WNT5A, APC and LEF-1 gene expression levels were also different between T-cell, B-cell and preB cell ALL cases. WNT5A expression had the highest levels in B-ALL compared to T-ALL cases, whereas the highest APC expression levels were observed in preB and T-ALL patients. Also LEF-1 expression levels were significantly different between preB and T-cell ALL patients. Taken together these results indicate that WNT signaling genes have abnormal expression and are active in acute lymphoblastic leukemia. This data suggests different WNT activation mechanisms exist in the leukemic transformation in different hematopoietic cells.

The CpG Island Methylator Phenotype and Chromosomal Instability Are Inversely Correlated in Sporadic Colorectal Cancer

The CpG island methylator phenotype (CIMP) is one of the mechanisms involved in colorectal carcinogenesis (CRC). Although CIMP is probably the cause of high-frequency microsatellite instability (MSI-H) sporadic CRCs, its role in microsatellite stable (MSS) tumors is debated. The majority of MSS CRCs demonstrate chromosomal instability (CIN) with frequent loss of heterozygosity (LOH) at key tumor suppressor genes. We hypothesized that the majority of sporadic CRCs without CIN would be associated with CIMP. We tested 126 sporadic CRCs for MSI and LOH and categorized tumors into MSI, LOH, or MSI-/LOH- subgroups. Methylation status was evaluated using 6 CIMP-related markers (MINT1, MINT2, MINT31, p16(INK4alpha), p14(ARF), and hMLH1) and 6 tumor suppressor genes (PTEN, TIMP3, RUNX3, HIC1, APC, and RARbeta2). BRAF V600E mutation analysis was performed using allele-specific polymerase chain reaction and DNA sequencing. We observed frequent methylation at all 12 loci in all CRCs. BRAF V600E mutations correlated with the MSI (P < .0001) and MSI-/LOH- (P = .03) subgroups. MSI and MSI-/LOH- tumors exhibited more promoter methylation than CRCs with LOH (P < .0001). We also found an inverse correlation between the frequencies of methylation and LOH (rho = -0.36; P < .0001). The associations between methylation frequencies at CIMP-related markers and MSI or MSI-/LOH- sporadic CRCs suggest that the majority of these tumors evolve through CIMP. These findings suggest that CIN and CIMP represent 2 independent and inversely related mechanisms of genetic and epigenetic instability in sporadic CRCs and confirm that MSI cancers arise as a consequence of CIMP.

Expressions and clinical implications of kruppel-like factor 6 and APC in human colorectal carcinoma

To investigate the expressions and clinical implications of kruppel-like factor 6(KLF-6) and APC in human colorectal carcinoma. The expressions of KLF-6 and APC in tumor and normal tissues from 32 patients with colorectal carcinoma were investigated by RT-PCR and immunohistochemical technique. The expression rates of KLF-6 and APC mRNA were 37.5% and 34.3% in tumor tissue, 96.9% and 93.8% respectively in normal tissues (both P< 0.05). The expression rates of KLF-6 and APC protein were 28.1% and 25.0% in colorectal carcinomas, 81.3% and 84.43% respectively in normal tissues (both P< 0.05). There was a significant correlation between the expressions of KLF-6 and APC in colorectal carcinomas (P < 0.05). The expressions of KLF-6 and APC were significantly correlated with tumor differentiation, depth of infiltration, lymph node metastasis and clinical stage (P< 0.05). Down-regulations of KLF-6 and APC might play an important role in the carcinogenesis, development, metastasis of human colorectal carcinoma.

Tumor suppressor and genetic instability during development of esophageal adenocarcinoma

Genomic instability (GIN) occurs in majority of human solid tumors, particularly in GI cancers. The significance and molecular regulators of GIN remain poorly understood. The roles of p53 and APC in protecting the genomic integrity have been well investigated, largely from studies in vitro. Precancerous Barrett's esophagus (BE) provides an important in vivo model to investigate GIN in human. This study was undertaken to determine GIN in relation to changes in p53 and APC in BE. We analyzed specimens from endoscopic biopsies or esophagectomies in patients with BE (10 cases) or with BE-associated esophageal adenocarcinoma (10 cases), with normal gastro-esophageal junction (5 cases) as controls. Chromosomal enumeration probe Cep 7, 11, 12, 17 and 18 were detected by fluorescence in situ hybridization (FISH). Expression of tumor suppressor p53 and APC were determined by the sensitive avidin-biotin immunohistochemistry. Up-regulation of p53, an indicator of p53 mutations, was observed in BE with high grade dysplasia (HGD), which increased further in BE associated esophageal cancer (EC). The expression of wildtype APC was decreased in BE with HGD and in advanced EC. Chromosomal abnormalities of all probes tested were found in each BE associated EC (100%). Numeric changes of chromosome 7, 11 and 12 were observed in BE stage in 14%, 64% and 43% of cases, respectively. Interestingly, aneusomy of chromosome 11 and 12 were found in BE without pathological dysplasia, in the presence of normal expression of p53 and APC. Our results suggest that GIN is an early event that occurs at precancerous stage prior to changes in tumor suppressor p53 and APC in BE-associated cancer progression.