Adenomatous Polyposis Coli Function Research Articles

Outcomes in patients with pancreatic and ampullary cancer whose tumors harbor pathogenic APC mutations.

693 Background: APC (adenomatous polyposis coli) is master regulator of the WNT signaling and beta-catenin pathway. Pathogenic (path) mutations in APC are well known for their connection to familial adenomatous polyposis, but germline (G) mutations in APC can give rise to other cancers, and somatic (S) mutations in APC can be found in multiple cancer subtypes. The clinical relevance for tumors that harbor path APC mutations is unknown. Methods: An established database of patients whose tumors have undergone genetic testing (Next-generation DNA sequencing, NGS) was queried for the presence of an APC mutation. Each APC mutation identified was assessed using the NCI Clinvar data base for determination of a path/likely path APC mutation. Patient/disease characteristics and treatment (tx) information were captured from electronic medical record review. Median overall survival (OS) was defined from the time of diagnosis of advanced/metastatic disease until death. Median time to progression (TTP) for each tx regimen was defined from the initiation of tx until documented disease progression. Results: A database of over 13000 patients entered from 2018 to present included 133 patients (1%) with G- or S-APC mutations, of which 41 were identified as path/likely path. Of the 41 patients, 56% were male, 73% were Caucasian and 24% African Americans, with a median age of 67 (range 34-90). Only 32% of patients had known G-APC mutations, but 34% had a second primary cancer, and 37% had a family history of known G-APC mutation-associated cancers. Only one patient had clinical manifestations of a polyposis syndrome. Of the 13 patients with known G-APC mutations, clinical characteristics were similar to the larger group, except that 100% of the G-APC-mutated patients were Caucasian, and the rate of family history of APC-associated cancers was 54%. Fifty-four percent of the overall group of 41 patients had a pathological diagnosis of pancreatic cancer, while the remaining 46% were ampullary or duodenal cancers. (Only 2 patients [5%] had tail of the pancreas cancers). Of the 37 patients who had somatic tumor NGS, only 70% harbored a KRAS gene mutation. For the OS analysis (n=24), the mOS was 11.7 months (range 5.2 – 56.9). Of the 22 patients evaluable for TTP, the mTTP for 5FU-based tx was 5.8 months, and 6.4 months for gemcitabine-based tx. Conclusions: APC-mutated pancreatic and ampullary cancers are uncommon, but exhibit survival rates, as well as TTP on both 5FU and gemcitabine-based txs that are similar to that seen with most pancreatic adenocarcinomas. However, 30% of APC-mutated cancers are KRAS wild type, and may harbor other driving mutations. As the loss of APC function triggers an accumulation of beta-catenin, targeted therapies that inhibit beta-catenin-regulated transcription and downregulate WNT-related signaling could be a promising tx approach for patients whose tumors harbor path/likely path APC mutations.

Clinical significance of SPOP and APC gene alterations in colorectal cancer in Indian population.

Speckle-Type Poz Protein (SPOP) involved in the regulation of proteasome-mediated degradation of several oncoproteins, resulting in cancer initiation and progression. Mutations in Adenomatous Polyposis Coli (APC) gene is reported in most sporadic and hereditary colorectal cancer (CRC). Identifying the cellular changes involved in carcinogenesis when APC is mutated is an important issue that needs attention. The tumor suppressive function of SPOP and APC has long been a major focus in the research field of colorectal cancer. However, the clinical significance of SPOP and APC gene alteration in CRC has not been established to date. Mutational analysis was performed by single-strand conformational polymorphism followed by Sanger sequencing, methylation status by methylation-specific PCR, and protein expression by immunohistochemistry on 142 tumor tissues along with their adjacent non-cancerous specimens. The overall survival (OS) and recurrence free survival (RFS) were estimated by Kaplan-Meier Curve. Mutation rates of APC and SPOP gene were 2.8% and 11.9% while that of promoter hypermethylation were 37% and 47%, respectively. The grade of differentiation and Lymph node metastasis were significantly correlated with APC methylation pattern (p ≤ 0.05). The down regulation of APC was more often seen in colonic cancer compared to rectal cancer (p = 0.07) and more commonly in T3-4 depth of invasion (p = 0.07) and in patients without lymphovascular and perineural invasion (p = 0.007, p = 0.08 respectively). The median overall survival and recurrence free survival (RFS) was 67 & 36months while 3-yr and 5-yr OS and RFS were 61.1% & 56.4% and 49.2% & 44.8%, respectively. APC promoter methylation had a better overall survival (p = 0.035) while loss of SPOP expression had a worse survival (p = 0.09). Our findings reveal high percentage of SPOP gene mutations in CRC. A significant link is found between promoter hyper methylationand protein expression in all mutant cases of APC and SPOP, suggesting that both genes may be associated in the development of colorectal cancer in people of Indian decent. Hypermethylation of APC gene and loss of SPOP expression have shown an association with disease prognosis and could be further studied looking at its potential role in planning adjuvant treatment in CRC patients.

Altered microbiota caused by disordered gut motility leads to an overactivation of intestinal immune system in APC1638T mice.

Adenomatous polyposis coli (APC) is recognized as an antioncogene related to familial adenomatous polyposis and colorectal cancers. However, APC is a large protein with multiple binding partners, indicating APC has diverse roles besides as a tumor suppressor. We have ever studied the roles of APC by using APC1638T/1638T (APC1638T) mice. Through those studies, we have noticed stools of APC1638T mice were smaller than those of APC+/+ mice and hypothesized there be a disturbance in fecal formation processes in APC1638T mice. The gut motility was morphologically analyzed by immunohistochemical staining of the Auerbach's plexus. Gut microbiota was analyzed by terminal restriction fragment length polymorphism (T-RFLP). IgA concentration in stools was determined by enzyme-linked immunosorbent assay (ELISA). As results, macroscopic findings suggestive of large intestinal dysmotility and microscopic findings of disorganization and inflammation of the plexus were obtained in APC1638T mice. An alteration of microbiota composition, especially increased Bacteroidetes population was observed. Increases in IgA positive cells and dendritic cells in the ileumwith high fecalIgA concentration were also confirmed, suggesting over-activation of gut immunity. Our findings will contribute to our understanding of APC's functions in the gastrointestinal motility, and lead to a development of novel therapies for gut dysmotility-related diseases.

Abstract P014: A novel role of mutant APC and n3-PUFA in reshaping cholesterol-dependent Wnt pathway-associated proteolipid nanocluster organization and signaling in colorectal cancer

Abstract Colorectal cancer (CRC) is the 3rd most common type of cancer in the U.S., and it is estimated it will account for an alarming 151,030 (8%) of new total cancer cases and 52,580 (9%) of total cancer deaths in 2022. Remarkably, dysregulation of the Wnt signaling pathway has been linked to ~90% of all colorectal cancer (CRC) cases. The vast majority (>80%) of sporadic CRC cases display mutations in Adenomatous Polyposis Coli (APC), a central Wnt signaling regulator. Loss of APC function causes aberrant stabilization of β-catenin (βcat), a crucial step in CRC initiation. Notably, attempts towards “drugging” this pathway still poses multiple hurdles due to poor tumor cell targeting, negative side effects associated with required long-term treatments and poorly understood mechanisms of action. Consequently, there is an urgent need to gather additional mechanistic insights associated with the Wnt pathway to develop novel therapeutic approaches. With respect to Wnt biology, two key plasma membrane receptors, LRP5/6 and Frizzled (Fzd) require lipid raft localization and nanoclustering for efficient signaling. Interestingly, various effectors that disrupt raft dynamics, alter LRP5/6-Fzd clustering, leading to reduced βcat stabilization. Thus, we examined the effect of mutant APC (mAPC) on plasma membrane lipid/protein-mediated interactions of Wnt-associated effectors in a CRC mouse model, human mAPC-expressing organoids and cell lines, and Drosophila. We show for the first time that mAPC significantly increased the levels of plasma membrane free cholesterol, a major component of lipid rafts known to selectively activate canonical Wnt signaling, in the intestinal epithelium including Lgr5+ colonic stem cells. Moreover, this change in plasma membrane cholesterol was associated with alterations in colonocyte membrane rigidity and raft organization. We subsequently examined the effect of mAPC on the interactions between Wnt signaling receptors/effectors in mAPC-expressing cell lines. mAPC significantly increased LRP6 and Fzd7 homo-clustering and LRP6-Fzd7 hetero-clustering as well as their interactions with key lipids, e.g., PIP2, resulting in the enhancement of Wnt signaling activation. Interestingly, the effects of mAPC were recapitulated by the addition of exogenous cholesterol to wild type colonocytes. Finally, we demonstrate that n-3 polyunsaturated fatty acids (n3-PUFA), i.e., DHA and EPA, reduces mAPC-driven colonic polyp formation in mice in part by reducing plasma membrane cholesterol, rigidity and LRP6-Fzd7 clustering in colonocytes. Collectively, these findings indicate that mAPC can directly perturb lipid homeostasis in the colon, thereby altering raft stability, LRP6-Fzd7 membrane nanoclusters and downstream Wnt signaling and supports the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane lipid composition, to reduce Wnt signaling and cancer risk. Citation Format: Alfredo Erazo-Oliveras, Mohamed Mlih, Mónica Muñoz-Vega, Eunjoo Kim, Venkat Thiriveedi, Michael L. Salinas, Xiaoli Wang, Jatin Roper, Jason Karpac, Robert S. Chapkin. A novel role of mutant APC and n3-PUFA in reshaping cholesterol-dependent Wnt pathway-associated proteolipid nanocluster organization and signaling in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P014.

Multivalent Interaction of Beta-Catenin With its Intrinsically Disordered Binding Partner Adenomatous Polyposis Coli.

The Wnt signalling pathway plays key roles in cell proliferation, differentiation and fate decisions in embryonic development and maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between β-catenin’s ARM repeat domain and the intrinsically disordered protein adenomatous polyposis coli (APC). APC is a giant multivalent scaffold that brings together the different components of the so-called “β-catenin destruction complex”, which drives β-catenin degradation via the ubiquitin-proteasome pathway. Mutations and truncations in APC, resulting in loss of APC function and hence elevated β-catenin levels and upregulation of Wnt signalling, are associated with numerous cancers including colorectal carcinomas. APC has a long intrinsically disordered region (IDR) that contains a series of 15-residue and 20-residue binding regions for β-catenin. Here we explore the multivalent nature of the interaction of β-catenin with the highest affinity APC repeat, both at equilibrium and under kinetic conditions. We use a combination of single-site substitutions, deletions and insertions to dissect the mechanism of molecular recognition and the roles of the three β-catenin-binding subdomains of APC.

Adenomatous Polyposis Coli (APC) in cell migration

Adenomatous Polyposis Coli (APC) protein is mostly known as a tumor suppressor that regulates Wnt signaling, but is also an important cytoskeletal protein. Mutations in the APC gene are linked to colorectal cancer and various neurological disorders and intellectual disabilities. Cytoskeletal functions of APC appear to have significant contributions to both types of these disorders. As a cytoskeletal protein, APC can regulate both actin and microtubule cytoskeletons, which together form the main machinery for cell migration. As APC is a multifunctional protein with numerous interaction partners, the complete picture of how APC regulates cell motility is still unavailable. However, some molecular mechanisms begin to emerge. Here, we review available information about roles of APC in cell migration and propose a model explaining how microtubules, using APC as an intermediate, can initiate leading edge protrusion in response to external signals by stimulating Arp2/3 complex-dependent nucleation of branched actin filament networks via a series of intermediate events.

EB1 Directly Regulates APC-Mediated Actin Nucleation

EB1 was discovered 25 years ago as a binding partner of the tumor suppressor adenomatous polyposis coli (APC) [1]; however, the significance of EB1-APC interactions has remained poorly understood. EB1 functions at the center of a network of microtubule end-tracking proteins (+TIPs) [2-5], and APC binding to EB1 promotes EB1 association with microtubule ends and microtubule stabilization [6, 7]. Whether EB1 interactions govern functions of APC beyond microtubule regulation has not been explored. The C-terminal basic domain of APC (APC-B) directly nucleates actin assembly, and this activity is required invivo for directed cell migration and for maintaining normal levels of F-actin [8-10]. Here, we show that EB1 binds APC-B and inhibits its actin nucleation function by blocking actin monomer recruitment. Consistent with these biochemical observations, knocking down EB1 increases F-actin levels in cells, and this can be rescued by disrupting APC-mediated actin nucleation. Conversely, overexpressing EB1 decreases F-actin levels and impairs directed cell migration without altering microtubule organization and independent of its direct binding interactions with microtubules. Overall, our results define a new function for EB1 in negatively regulating APC-mediated actin assembly. Combining these findings with other recent studies showing that APC interactions regulate EB1-dependent effects on microtubule dynamics [7], we propose that EB1-APC interactions govern bidirectional cytoskeletal crosstalk by coordinating microtubule and actin dynamics.

Molecular Aspects of Colorectal Adenomas: The Interplay among Microenvironment, Oxidative Stress, and Predisposition.

The development of colorectal cancer (CRC) is a multistep process initiated by a benign polyp that has the potential to evolve into in situ carcinoma through the interactions between environmental and genetic factors. CRC incidence rates are constantly increased for young adult patients presenting an advanced tumor stage. The majority of CRCs arise from colonic adenomas originating from aberrant cell proliferation of colon epithelium. Endoscopic polypectomy represents a tool for early detection and removal of polyps, although the occurrence of cancers after negative colonoscopy shows a significant incidence. It has long been recognized that the aberrant regulation of Wingless/It (Wnt)/β-Catenin signaling in the pathogenesis of colorectal cancer is supported by its critical role in the differentiation of stem cells in intestinal crypts and in the maintenance of intestinal homeostasis. For this review, we will focus on the development of adenomatous polyps through the interplay between renewal signaling in the colon epithelium and reactive oxygen species (ROS) production. The current knowledge of molecular pathology allows us to deepen the relationships between oxidative stress and other risk factors as lifestyle, microbiota, and predisposition. We underline that the chronic inflammation and ROS production in the colon epithelium can impair the Wnt/β-catenin and/or base excision repair (BER) pathways and predispose to polyp development. In fact, the coexistence of oxidative DNA damage and errors in DNA polymerase can foster C>T transitions in various types of cancer and adenomas, leading to a hypermutated phenotype of tumor cells. Moreover, the function of Adenomatous Polyposis Coli (APC) protein in regulating DNA repair is very important as therapeutic implication making DNA damaging chemotherapeutic agents more effective in CRC cells that tend to accumulate mutations. Additional studies will determine whether approaches based on Wnt inhibition would provide long-term therapeutic value in CRC, but it is clear that APC disruption plays a central role in driving and maintaining tumorigenesis.

Colorectal cancer: the APC-lncRNA link.

The adenomatous polyposis coli (APC) gene plays, among other things, a crucial role in the regulation of cell proliferation and survival through its ability to regulate canonical Wnt signaling. In this issue of the JCI, Wang et al. provide an intriguing new mechanism for APC function involving the regulation of a novel long noncoding RNA (lncRNA), leading to changes in exosome production. APC signaling via this novel pathway can regulate cell proliferation and invasion as well as angiogenesis. In addition to enhancing our understanding of APC function, this new mechanism is of particular clinical significance, as it may provide additional targets for the treatment of APC-mutated cancers.

Identification of ICAT as an APC Inhibitor, Revealing Wnt-Dependent Inhibition of APC-Axin Interaction

Adenomatous polyposis coli (APC) and Axin are core components of the β-catenin destruction complex. How APC's function is regulated and whether Wnt signaling influences the direct APC-Axin interaction to inhibit the β-catenin destruction complex is not clear. Through a CRISPR screen of β-catenin stability, we have identified ICAT, a polypeptide previously known to block β-catenin-TCF interaction, as a natural inhibitor of APC. ICAT blocks β-catenin-APC interaction and prevents β-catenin-mediated APC-Axin interaction, enhancing stabilization of β-catenin in cells harboring truncated APC or stimulated with Wnt, but not in cells deprived of a Wnt signal. Using ICAT as a tool to disengage β-catenin-mediated APC-Axin interaction, we demonstrate that Wnt quickly inhibits the direct interaction between APC and Axin. Our study highlights an important scaffolding function of β-catenin in the assembly of the destruction complex and suggests Wnt-inhibited APC-Axin interaction as a mechanism of Wnt-dependent inhibition of the destruction complex.

Validation and application of a novel APC antibody in western blotting, immunoprecipitation, and immunohistochemistry.

Adenomatous polyposis coli (APC) is a large protein with multiple binding partners, suggesting diverse functions besides its well-known role in the destruction of β-catenin. To elucidate these complex functions, it is crucial to evaluate the precise subcellular distribution of APC within a cell and tissue. However, most of the commercially available anti-APC antibodies can only be used for limited applications, resulting in the use of independently generated antibodies. This has led to various discrepancies between studies as a common antibody has not been established. In this study, we generated an antibody against the c-terminal domain of human APC, designated APC-C antibody, and evaluated its specificity and application in various immunological methods. Our data indicate that this novel APC-C antibody is a specific and versatile antibody that can be used in western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. Widespread use of this APC antibody will help enhance our understanding of APC's function in both normal and cancer cell biology.

N-terminal and central domains of APC function to regulate branch number, length and angle in developing optic axonal arbors in vivo

During formation of neuronal circuits, axons navigate long distances to reach their target locations in the brain. When axons arrive at their target tissues, in many cases, they extend collateral branches and/or terminal arbors that serve to increase the number of synaptic connections they make with target neurons. Here, we investigated how Adenomatous Polyposis Coli (APC) regulates terminal arborization of optic axons in living Xenopus laevis tadpoles. The N-terminal and central domains of APC that regulate the microtubule cytoskeleton and stability of β-catenin in the Wnt pathway, were co-expressed with GFP in individual optic axons, and their terminal arbors were then imaged in tectal midbrains of intact tadpoles. Our data show that the APCNTERM and APCβ-cat domains both decreased the mean number, and increased the mean length, of branches in optic axonal arbors relative to control arbors in vivo. Additional analysis demonstrated that expression of the APCNTERM domain increased the average bifurcation angle of branching in optic axonal arbors. However, the APCβ-cat domain did not significantly affect the mean branch angle of arbors in tecta of living tadpoles. These data suggest that APC N-terminal and central domains both modulate number and mean length of branches optic axonal arbors in a compensatory manner, but also define a specific function for the N-terminal domain of APC in regulating branch angle in optic axonal arbors in vivo. Our findings establish novel mechanisms for the multifunctional protein APC in shaping terminal arbors in the visual circuit of the developing vertebrate brain.

APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting β-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wntsignaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased β-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote β-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.

Identification of Eendogenous <i>Adenomatous Polyposis Coli</i> Interaction Partners and &#946-Catenin-Independent Targets by Proteomics

Adenomatous polyposis coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the pro-proliferative Wnt signaling pathway by promoting the degradation of &#946-catenin, but the extent to which APC exerts Wnt/&#946-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and &#946-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed TMT mass spectrometry. Affinity enrichment-mass spectrometry revealed over 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of &#946-catenin. By combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an alternative APC-containing destruction complex and provide evidence for the potential contribution of MINK1 to APC mutant phenotypes. Collectively, our results highlight the extent and importance of Wntindependent APC functions in epithelial biology and disease.

Disruption of the RP-MDM2-p53 pathway accelerates APC loss-induced colorectal tumorigenesis.

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor is frequently found in colorectal cancer. Loss of APC function results in deregulation of the Wnt/β-catenin signaling pathway causing overexpression of the c-MYC oncogene. In lymphoma, both p19ARF and ribosomal proteins RPL11 and RPL5 respond to c-MYC activation to induce p53. Their role in c-MYC-driven colorectal carcinogenesis is unclear, as p19ARF deletion does not accelerate APC loss-triggered intestinal tumorigenesis. To determine the contribution of the RP-MDM2-p53 pathway to APC loss-induced tumorigenesis, we crossed mice bearing MDM2C305F mutation, which disrupts RPL11- and RPL5-MDM2 binding, with Apcmin/+ mice, which are prone to intestinal tumor formation. Interestingly, loss of RP-MDM2 binding significantly accelerated colorectal tumor formation while having no discernable effect on small intestinal tumor formation. Mechanistically, APC loss leads to overexpression of c-MYC, RPL11 and RPL5 in mouse colonic tumor cells irrespective of MDM2C305F mutation. However, notable p53 stabilization and activation were observed only in Apcmin/+;Mdm2+/+ but not Apcmin/+;Mdm2C305F/C305F colon tumors. These data establish that the RP-MDM2-p53 pathway, in contrast to the p19ARF-MDM2-p53 pathway, is a critical mediator of colorectal tumorigenesis following APC loss.

Interaction between APC and Fen1 during breast carcinogenesis.

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER - adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) - promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.

Three novel mutations of APC gene in Chinese patients with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder characterized by the development of hundreds to thousands of colonic adenomas and an increased risk of colorectal cancer. Adenomatous polyposis coli (APC), encoding a large multidomain protein involved in antagonizing the Wnt signaling pathway, has been identified as the main causative gene responsible for FAP. In this study, we identified three novel mutations as well as two recurrent mutations in the APC in five Chinese FAP families by sequencing. Immunohistochemical analysis revealed that among these mutations, a nonsense mutation (c.2510C>G) and two small deletions (c.2016_2047del, c.3180_3184del) led to the truncation of the APC protein and the cytoplasmic and nuclear accumulation of β-catenin in the colorectal samples from affected individuals, respectively. Our study expands the database on mutations of APC and provides evidence to understand the function of APC in FAP.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression throughFxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells

Background: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention.Objective: We investigated howAPC inactivation influences the regulation ofFXR expression in colonic mucosal cells. We hypothesized thatAPC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation.Methods: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J andApc-deficient (ApcMin/+) male mice, respectively. The expression ofFxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation ofFxr in bisulfonated genomic DNA. In vitro, we measured the impact ofAPC inactivation and deoxycholic acid (DCA) treatment onFXR expression in human colon cancer HCT-116 cells transfected with silencing RNA forAPC and HT-29 cells carrying inactivatedAPC.Results: InApcMin/+ mice, constitutive CpG methylation of theFxrα3/4 promoter was linked to reduced (60–90%) baselineFxr,Ibabp, andShp and increasedCox-2 expression in apparently normal adjacent mucosa and colon tumors.Apc knockdown in HCT-116 cells increased cellular myelocytomatosis(c-MYC) and lowered (∼50%)FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA inducedFXR expression and lowered CpG methylation ofFXR.Conclusions: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.

Adenomatous polyposis coli (APC) regulates miR17-92 cluster through β-catenin pathway in colorectal cancer.

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

Epigenetic regulation of APC in the molecular pathogenesis of gallbladder cancer.

Background & objectives:Loss of function of adenomatous polyposis coli (APC) has been reported in cancer. The two promoters of APC, 1A and 1B also have roles in cancer. But, the epigenetic role of APC promoters is not yet clear in gallbladder cancer (GBC) and gallstone diseases (GSD). We undertook this study to determine the epigenetic role of APC in GBC and GSD.Methods:Methylation-specific (MS)-PCR was used to analyze the methylation of APC gene. The expression of APC gene was studied by semi-quantitative PCR, real-time PCR and immunohistochemistry (IHC) in GBC, GSD and adjacent normal tissues.Results:Of the two promoters, APC 1A promoter was found methylated in 96 per cent GBC (P=0.0155) and 80 per cent GSD (P=0.015). Exon 1 was downregulated in grade II (P=0.002) and grade III (P=0.0001) of GBC, while exon 2 was normally expressed. Scoring analysis of IHC revealed 0 or negativity in 34.48 per cent (P=0.057) and 1+ in 24.14 per cent (P=0.005) GBC cases suggesting loss of APC expression.Interpretation & conclusions:The present findings indicate epigenetic silencing of APC in advanced GBC. The methylation pattern, followed by expression analysis of APC may be suggested for diagnostic, prognostic and therapeutic purposes in GBC in future.