Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells.

ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/ß-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling.

PIGA Mutations and Glycosylphosphatidylinositol Anchor Dysregulation in Polyposis-Associated Duodenal Tumorigenesis.

The pathogenesis of duodenal tumors in the inherited tumor syndromes familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumors and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyzes the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA. IMPLICATIONS: PIGA somatic mutation in duodenal tumors from patients with FAP and MAP and loss of membrane GPI-anchors may present new opportunities for understanding and intervention in duodenal tumorigenesis.

Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth.

ZRANB1 (human Trabid) missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterized these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organization and neural development. Zranb1 knock-in mice harboring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalized causing impaired organization of the cytoskeleton that underlie the neuronal and developmental phenotypes.

The adenomatous polyposis coli protein 30 years on.

Mutations in the gene encoding the Adenomatous polyposis coli protein (APC) were discovered as driver mutations in colorectal cancers almost 30 years ago. Since then, the importance of APC in normal tissue homeostasis has been confirmed in a plethora of other (model) organisms spanning a large evolutionary space. APC is a multifunctional protein, with roles as a key scaffold protein in complexes involved in diverse signalling pathways, most prominently the Wnt signalling pathway. APC is also a cytoskeletal regulator with direct and indirect links to and impacts on all three major cytoskeletal networks. Correspondingly, a wide range of APC binding partners have been identified. Mutations in APC are extremely strongly associated with colorectal cancers, particularly those that result in the production of truncated proteins and the loss of significant regions from the remaining protein. Understanding the complement of its role in health and disease requires knowing the relationship between and regulation of its diverse functions and interactions. This in turn requires understanding its structural and biochemical features. Here we set out to provide a brief overview of the roles and function of APC and then explore its conservation and structure using the extensive sequence data, which is now available, and spans a broad range of taxonomy. This revealed conservation of APC across taxonomy and new relationships between different APC protein families.

Tumor Suppressor Adenomatous Polyposis Coli Sustains Dendritic Cell Tolerance through IL-10 in a ß-Catenin-Dependent Manner.

Dendritic cells (DC) play important roles in balancing immunity and tolerance, in which ß-catenin signaling plays an important role, yet the underlying mechanisms remain elusive. In this study, we investigated the functions of the tumor suppressor adenomatous polyposis coli (APC), also a key component of the ß-catenin upstream destruction complex in DC. APC depletion in DC does not alter DC and T cell homeostasis under resting conditions. However, APC deficiency in DC leads to attenuated antitumor immunity in mice, which exhibit fewer CD8+ T cells and more Foxp3+ regulatory T cells in tumor and draining lymph nodes. Loss of APC in DC does not affect the expression levels of costimulatory molecules. However, APC-deficient DC produce more IL-10 and exhibit a higher ability of inducing regulatory T cells but a lower ability of priming CD8+ T cells, both of which can be reversed by IL-10 inhibition. Lastly, ß-catenin depletion in APC-deficient DC rescues their antitumor immunity and reverses elevated IL-10 production. Taken together, our results identify that APC drives DC tolerance via the ß-catenin/IL-10 axis.

Cortical Parvalbumin-Positive Interneuron Development and Function Are Altered in the APC Conditional Knockout Mouse Model of Infantile and Epileptic Spasms Syndrome.

Infantile and epileptic spasms syndrome (IESS) is a childhood epilepsy syndrome characterized by infantile or late-onset spasms, abnormal neonatal EEG, and epilepsy. Few treatments exist for IESS, clinical outcomes are poor, and the molecular and circuit-level etiologies of IESS are not well understood. Multiple human IESS risk genes are linked to Wnt/ß-catenin signaling, a pathway that controls developmental transcriptional programs and promotes glutamatergic excitation via ß-catenin's role as a synaptic scaffold. We previously showed that deleting adenomatous polyposis coli (APC), a component of the ß-catenin destruction complex, in excitatory neurons (APC cKO mice, APCfl/fl x CaMKIIαCre) increased ß-catenin levels in developing glutamatergic neurons and led to infantile behavioral spasms, abnormal neonatal EEG, and adult epilepsy. Here, we tested the hypothesis that the development of GABAergic interneurons (INs) is disrupted in APC cKO male and female mice. IN dysfunction is implicated in human IESS, is a feature of other rodent models of IESS, and may contribute to the manifestation of spasms and seizures. We found that parvalbumin-positive INs (PV+ INs), an important source of cortical inhibition, were decreased in number, underwent disproportionate developmental apoptosis, and had altered dendrite morphology at P9, the peak of behavioral spasms. PV+ INs received excessive excitatory input, and their intrinsic ability to fire action potentials was reduced at all time points examined (P9, P14, P60). Subsequently, GABAergic transmission onto pyramidal neurons was uniquely altered in the somatosensory cortex of APC cKO mice at all ages, with both decreased IPSC input at P14 and enhanced IPSC input at P9 and P60. These results indicate that inhibitory circuit dysfunction occurs in APC cKOs and, along with known changes in excitation, may contribute to IESS-related phenotypes.SIGNIFICANCE STATEMENT Infantile and epileptic spasms syndrome (IESS) is a devastating epilepsy with limited treatment options and poor clinical outcomes. The molecular, cellular, and circuit disruptions that cause infantile spasms and seizures are largely unknown, but inhibitory GABAergic interneuron dysfunction has been implicated in rodent models of IESS and may contribute to human IESS. Here, we use a rodent model of IESS, the APC cKO mouse, in which ß-catenin signaling is increased in excitatory neurons. This results in altered parvalbumin-positive GABAergic interneuron development and GABAergic synaptic dysfunction throughout life, showing that pathology arising in excitatory neurons can initiate long-term interneuron dysfunction. Our findings further implicate GABAergic dysfunction in IESS, even when pathology is initiated in other neuronal types.

The mitotic role of adenomatous polyposis coli requires its bilateral interaction with tubulin and microtubules.

Adenomatous polyposis coli (APC) is a scaffold protein with tumour suppressor properties. Mutations causing the loss of its C-terminal domain (APC-C), which bears cytoskeleton-regulating sequences, correlate with colorectal cancer. The cellular roles of APC in mitosis are widely studied, but the molecular mechanisms of its interaction with the cytoskeleton are poorly understood. Here, we investigated how APC-C regulates microtubule properties, and found that it promotes both microtubule growth and shrinkage. Strikingly, APC-C accumulates at shrinking microtubule extremities, a common characteristic of depolymerases. Cryo-electron microscopy revealed that APC-C adopts an extended conformation along the protofilament crest and showed the presence of ring-like tubulin oligomers around the microtubule wall, which required the presence of two APC-C sub-domains. A mutant of APC-C that was incapable of decorating microtubules with ring-like tubulin oligomers exhibited a reduced effect on microtubule dynamics. Finally, whereas native APC-C rescued defective chromosome alignment in metaphase cells silenced for APC, the ring-incompetent mutant failed to correct mitotic defects. Thus, the bilateral interaction of APC-C with tubulin and microtubules likely contributes to its mitotic functions.

Bacterial translocation and barrier dysfunction enhance colonic tumorigenesis.

In the development of colon cancer, the intestinal dysbiosis and disruption of barrier function are common manifestations. In the current study, we hypothesized that host factors, e.g., vitamin D receptor deficiency or adenomatous polyposis coli (APC) mutation, contribute to the enhanced dysbiosis and disrupted barrier in the pathogenesis of colorectal cancer (CRC). Using the human CRC database, we found enhanced tumor-invading bacteria and reduced colonic VDR expression, which was correlated with a reduction of Claudin-10 mRNA and protein. In the colon of VDRΔIEC mice, deletion of intestinal epithelial VDR led to lower protein of tight junction protein Claudin-10. Lacking VDR and a reduction of Claudin-10 are associated with an increased number of tumors in the mice without myeloid VDR. Intestinal permeability was significantly increased in the mice with myeloid VDR conditional deletion. Further, mice with conditional colonic APC mutation showed reduced mucus layer, enhanced bacteria in tumors, and loss of Claudin-10. Our data from human samples and colon cancer models provided solid evidence- on the host factor regulation of bacterial translocation and dysfunction on barriers in colonic tumorigenesis. Studies on the host factor regulation of microbiome and barriers could be potentially applied to risk assessment, early detection, and prevention of colon cancer.

Rational design of a sensitivity-enhanced tracer for discovering efficient APC-Asef inhibitors.

The adenomatous polyposis coli (APC)-Rho guanine nucleotide exchange factor 4 (Asef) protein-protein interaction (PPI) is essential for colorectal cancer metastasis, making it a promising drug target. Herein, we obtain a sensitivity-enhanced tracer (tracer 7) with a high binding affinity (Kd = 0.078 µM) and wide signal dynamic range (span = 251 mp). By using tracer 7 in fluorescence-polarization assays for APC-Asef inhibitor screening, we discover a best-in-class inhibitor, MAI-516, with an IC50 of 0.041 ± 0.004 µM and a conjugated transcriptional transactivating sequence for generating cell-permeable MAIT-516. MAIT-516 inhibits CRC cell migration by specifically hindering the APC-Asef PPI. Furthermore, MAIT-516 exhibits no cytotoxic effects on normal intestinal epithelial cell and colorectal cancer cell growth. Overall, we develop a sensitivity-enhanced tracer for fluorescence polarization assays, which is used for the precise quantification of high-activity APC-Asef inhibitors, thereby providing insight into PPI drug development.

Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer.

Inactivation of adenomatous polyposis coli (APC) is common across many cancer types and serves as a critical initiating event in most sporadic colorectal cancers. APC deficiency activates WNT signaling, which remains an elusive target for cancer therapy, prompting us to apply the synthetic essentiality framework to identify druggable vulnerabilities for APC-deficient cancers. Tryptophan 2,3-dioxygenase 2 (TDO2) was identified as a synthetic essential effector of APC-deficient colorectal cancer. Mechanistically, APC deficiency results in the TCF4/ß-catenin-mediated upregulation of TDO2 gene transcription. TDO2 in turn activates the Kyn-AhR pathway, which increases glycolysis to drive anabolic cancer cell growth and CXCL5 secretion to recruit macrophages into the tumor microenvironment. Therapeutically, APC-deficient colorectal cancer models were susceptible to TDO2 depletion or pharmacologic inhibition, which impaired cancer cell proliferation and enhanced antitumor immune profiles. Thus, APC deficiency activates a TCF4-TDO2-AhR-CXCL5 circuit that affects multiple cancer hallmarks via autonomous and nonautonomous mechanisms and illuminates a genotype-specific vulnerability in colorectal cancer. SIGNIFICANCE: This study identifies critical effectors in the maintenance of APC-deficient colorectal cancer and demonstrates the relationship between APC/WNT pathway and kynurenine pathway signaling. It further determines the tumor-associated macrophage biology in APC-deficient colorectal cancer, informing genotype-specific therapeutic targets and the use of TDO2 inhibitors. This article is highlighted in the In This Issue feature, p. 1599.

Neddylation is essential for ß-catenin degradation in Wnt signaling pathway.

ß-Catenin is a central component in the Wnt signaling pathway; its degradation has been tightly connected to ubiquitylation, but it is rarely examined by loss-of-function assays. Here we observe that endogenous ß-catenin is not stabilized upon ubiquitylation depletion by a ubiquitylation inhibitor, TAK-243. We demonstrate that N-terminal phosphorylated ß-catenin is quickly and strongly stabilized by a specific neddylation inhibitor, MLN4924, in all examined cell types, and that ß-catenin and TCF4 interaction is strongly enhanced by inhibition of neddylation but not ubiquitylation. We also confirm that the E3 ligase ß-TrCP2, but not ß-TrCP1, is associated with neddylation and destruction of ß-catenin. GSK3ß and adenomatous polyposis coli (APC) are not required for ß-catenin neddylation but essential for its subsequent degradation. Our findings not only clarify the process of ß-catenin modification and degradation in the Wnt signaling pathway but also highlight the importance of reassessing previously identified ubiquitylation substrates.

A pan-cancer analysis on the carcinogenic effect of human adenomatous polyposis coli.

Adenomatous polyposis coli (APC) is the most commonly mutated gene in colon cancer and can cause familial adenomatous polyposis (FAP). Hypermethylation of the APC promoter can also promote the development of breast cancer, indicating that APC is not limited to association with colorectal neoplasms. However, no pan-cancer analysis has been conducted. We studied the location and structure of APC and the expression and potential role of APC in a variety of tumors by using The Cancer Genome Atlas and Gene Expression Omnibus databases and online bioinformatics analysis tools. The APC is located at 5q22.2, and its protein structure is conserved among H. sapiens, M. musculus with C. elaphus hippelaphus. The APC identity similarity between homo sapiens and mus musculus reaches 90.1%. Moreover, APC is highly specifically expressed in brain tissues and bipolar cells but has low expression in most cancers. APC is mainly expressed on the cell membrane and is not detected in plasma by mass spectrometry. APC is low expressed in most tumor tissues, and there is a significant correlation between the expressed level of APC and the main pathological stages as well as the survival and prognosis of tumor patients. In most tumors, APC gene has mutation and methylation and an enhanced phosphorylation level of some phosphorylation sites, such as T1438 and S2260. The expressed level of APC is also involved in the level of CD8+ T-cell infiltration, Tregs infiltration, and cancer-associated fibroblast infiltration. We conducted a gene correlation study, but the findings seemed to contradict the previous analysis results of the low expression of the APC gene in most cancers. Our research provides a comparative wholesale understanding of the carcinogenic effects of APC in various cancers, which will help anti-cancer research.

The prognostic and clinicopathological roles of microsatellite instability, PD-L1 expression and tumor-infiltrating leukocytes in familial adenomatous polyposis.

BACKGROUND: Microsatellite instability, programmed death-ligand 1 and tumor-infiltrating leukocytes are prognostic biomarkers in colorectal cancer but unknown toward familial adenomatous polyposis. AIM: To investigate the prognostic and clinicopathological roles of microsatellite instability, programmed death-ligand 1 and tumor-infiltrating leukocytes in familial adenomatous polyposis. METHODS: Clinical data and paraffin embedded tissues from 45 familial adenomatous polyposis patients were collected. Microsatellite instability was detected by immunohistochemistry and polymerase chain reaction. Programmed death-ligand 1 was detected by immunohistochemistry. Tumor-infiltrating leukocytes comprising CD8+ T cells, M1 and M2 tumor associated macrophages, CD56bright and CD56dim natural killer cells were analyzed using multiple fluorescence immunohistochemistry. RESULTS: Microsatellite instability high was noted in 6 samples but not associated with overall survival or progression-free survival. Programmed death-ligand 1 is negative on tumor cells but positive on tumor-infiltrating leukocytes, and positive programmed death-ligand 1 expression on tumor-infiltrating leucocytes is associated with overall survival. Low CD56bright natural killer cell infiltration was associated with longer progression-free survival and was an independent prognostic factor in FAP. CONCLUSION: For familial adenomatous polyposis, microsatellite instability high can be found but has no correlation with prognosis; programmed death-ligand 1 on tumor-infiltrating leukocytes is related with overall survival; CD56bright natural killer cell is an independent prognostic factor associating with longer progression-free survival.

Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome.

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.

The Second Allele: A Key to Understanding the Timing of Sporadic and Hereditary Colorectal Tumorigenesis.

Our understanding of the molecular basis of colorectal neoplasia is derived from Mendelian genetics, with tumor suppressor genes contributing more to the deregulation of growth than oncogenes. In patients with hereditary syndromes, expression of one allele of a key tumor suppressor gene is absent at birth. The loss of the expression of the second allele precipitates tumorigenesis. However, there are multiple ways in which the expression of the second allele of a tumor suppressor gene is lost. Here, we review these ways and their possible effect on phenotype.

Negative regulation of TGFß-induced apoptosis by RAC1B enhances intestinal tumourigenesis.

RAC1B is a tumour-related alternative splice isoform of the small GTPase RAC1, found overexpressed in a large number of tumour types. Building evidence suggests it promotes tumour progression but compelling in vivo evidence, demonstrating a role in driving tumour invasion, is currently lacking. In the present study, we have overexpressed RAC1B in a colorectal cancer mouse model with potential invasive properties. Interestingly, RAC1B overexpression did not trigger tumour invasion, rather it led to an acceleration of tumour initiation and reduced mouse survival. By modelling early stages of adenoma initiation we observed a reduced apoptotic rate in RAC1B overexpressing tumours, suggesting protection from apoptosis as a mediator of this phenotype. RAC1B overexpressing tumours displayed attenuated TGFß signalling and functional analysis in ex vivo organoid cultures demonstrated that RAC1B negatively modulates TGFß signalling and confers resistance to TGFß-driven cell death. This work defines a novel mechanism by which early adenoma cells can overcome the cytostatic and cytotoxic effects of TGFß signalling and characterises a new oncogenic function of RAC1B in vivo.

A genome-wide library of MADM mice for single-cell genetic mosaic analysis.

Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.

Microencapsulated Bifidobacterium bifidum and Lactobacillus gasseri in Combination with Quercetin Inhibit Colorectal Cancer Development in ApcMin/+ Mice.

Recent studies have suggested that flavonoids such as quercetin and probiotics such as Bifidobacterium bifidum (Bf) and Lactobacillus gasseri (Lg) could play a relevant role in inhibiting colon cancer cell growth. Our study investigated the role of dietary supplementation with microencapsulated probiotics (Bf and Lg) along with quercetin in the development of mouse colorectal cancer (CRC). Methods: Adenomatous polyposis coli/multiple intestinal neoplasia (ApcMin/+) mice were fed a standard diet or the same diet supplemented with microencapsulated probiotics (Bf and Lg strains, 107 CFU/100 g food) or both probiotics strains plus microencapsulated quercetin (15 mg/100 g food) for 73 days. Changes in body and organ weights, energy metabolism, intestinal microbiota, and colon tissue were determined. The expression of genes related to the Wnt pathway was also analyzed in colon samples. Results: Dietary supplementation with microencapsulated probiotics or microencapsulated probiotics plus quercetin reduced body weight loss and intestinal bleeding in ApcMin/+ mice. An improvement in energy expenditure was observed after 8 weeks but not after 10 weeks of treatment. A supplemented diet with microencapsulated Bf and Lg reduced the number of aberrant crypt foci (ACF) and adenomas by 45% and 60%, respectively, whereas the supplementation with Bf, Lg and quercetin decreased the number of ACF and adenomas by 57% and 80%, respectively. Microencapsulated Bf and Lg in combination with quercetin could exert inhibition of the canonical Wnt/ß-catenin signaling pathway in the colon of ApcMin/+ mice Conclusions: The administration of microencapsulated Bf and Lg, individually or in combination with quercetin, inhibits the CRC development in ApcMin/+ mice.

Role of adenomatous polyposis coli in proliferation and differentiation of colon epithelial cells in organoid culture.

Adenomatous polyposis coli (APC) is a tumor-suppressing protein whose inactivation triggers the formation of colorectal polyps. Numerous studies using cell lines or genetically engineered mice have revealed its role in suppressing Wnt/ß-catenin signaling pathway and regulating cell proliferation and differentiation. Here, we performed genetic analyses of APC using a three-dimensional organoid culture of mouse colon epithelia, which enables the detailed examination of epithelial properties. Analyses of Apc-knockout colon organoids not only confirmed the importance of APC in suppressing Wnt/ß-catenin signaling and regulating cell differentiation, but also revealed several novel features: a significant decrease in proliferating speed and an increase in cross-sectional area of cells. Moreover, we found a significant number of lysozyme-positive Paneth-like cells, which were never observed in wild-type colon tissues or organoids, but have been reported to emerge in colon cancers. Therefore, APC autonomously suppresses ectopic differentiation into lysozyme-positive cells, specifically in the colon epithelia. Colon organoids would be an ideal material to investigate the molecular mechanism and biological importance of the ectopic differentiation associated with cancer development.

Expression of apoptosis repressor with caspase recruitment domain (ARC) in familial adenomatous polyposis (FAP) adenomas and its correlation with DNA mismatch repair proteins, p53, Bcl-2, COX-2 and beta-catenin.

BACKGROUND: Colorectal familial adenomatous polyposis (FAP) adenomas exhibit a uniform pathogenetic basis caused by a germline mutation in the adenomatous polyposis gene (APC), but the molecular changes leading to their development are incompletely understood. However, dysregulated apoptosis is known to substantially affect the development of colonic adenomas. One of the key regulatory proteins involved in apoptosis is apoptosis repressor with caspase recruitment domain (ARC). METHODS: The expression of nuclear and cytoplasmic ARC in 212 adenomas from 80 patients was analyzed by immunohistochemistry. We also compared expression levels of ARC with the expression levels of p53, Bcl-2, COX-2, and MMR proteins. Statistical analyses were performed by Spearman's rank correlation and linear regression test. RESULTS: ARC was overexpressed in the nuclei and cytoplasm of most FAP adenomas investigated. Cytoplasmic ARC staining was moderately stronger (score 2) in 49.1% (n = 104/212) and substantially stronger (score 3) in 32.5% (n = 69/212) of adenomas compared to non-tumorous colorectal mucosa. In 18.4% (n = 39/212) of adenomas, cytoplasmic ARC staining was equivalent to that in non-tumorous mucosa. Nuclear expression of ARC in over 75% of cells was present in 30.7% (n = 65/212) of investigated adenomas, and nuclear expression in 10-75% of cells was detected in 62.7% (n = 133/212). ARC expression in under 10% of nuclei was found in 6.6% (n = 14/212) of adenomas. The correlation between nuclear ARC expression and cytoplasmic ARC expression was highly significant (p = 0.001). Moreover, nuclear ARC expression correlated positively with overexpression of Bcl-2, COX-2 p53 and ß-catenin. Cytoplasmic ARC also correlated with overexpression of Bcl-2. Sporadic MMR deficiency was detected in very few FAP adenomas and showed no correlation with nuclear or cytoplasmic ARC. CONCLUSIONS: Our results demonstrated that both cytoplasmic and nuclear ARC are overexpressed in FAP adenomas, thus in a homogenous collective. The highly significant correlation between nuclear ARC and nuclear ß-catenin suggested that ARC might be regulated by ß-catenin in FAP adenomas. Because of its further correlations with p53, Bcl-2, and COX-2, nuclear ARC might play a substantial role not only in carcinomas but also in precursor lesions. Video Abstract.