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The Wnt/β-catenin signaling pathway is a growth control pathway involved in various biological processes as well as the development and progression of cancer. Colorectal cancer (CRC) is one of the most common malignancies in the world. The hyperactivation of Wnt signaling is observed in almost all CRC and plays a crucial role in cancer-related processes such as cancer stem cell (CSC) propagation, angiogenesis, epithelial-mesenchymal transition (EMT), chemoresistance, and metastasis. This review will discuss how the Wnt/β-catenin signaling pathway is involved in the carcinogenesis and progression of CRC and related therapeutic approaches.

The acquisition of biallelic mutations in the APC gene is a rate-limiting step in the development of most colorectal cancers and occurs in the earliest lesions. APC encodes a 312-kDa protein that localizes to multiple subcellular compartments and performs diverse functions. APC participates in a cytoplasmic complex that promotes the destruction of the transcriptional licensing factor β-catenin; APC mutations that abolish this function trigger constitutive activation of the canonical WNT signaling pathway, a characteristic found in almost all colorectal cancers. By negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis, and suppresses invasion and tumor progression. APC further antagonizes canonical WNT signaling by interacting with and counteracting β-catenin in the nucleus. APC also suppresses tumor initiation and progression in the colorectal epithelium through functions that are independent of canonical WNT signaling. APC regulates the mitotic spindle to facilitate proper chromosome segregation, localizes to the cell periphery and cell protrusions to establish cell polarity and appropriate directional migration, and inhibits DNA replication by interacting directly with DNA. Mutations in APC are often frameshifts, insertions, or deletions that introduce premature stop codons and lead to the production of truncated APC proteins that lack its normal functions and possess tumorigenic properties. Therapeutic approaches in development for the treatment of APC-deficient tumors are focused on the inhibition of canonical WNT signaling, especially through targets downstream of APC in the pathway, or on the restoration of wild-type APC expression.

The Wnt/β-catenin pathway is critical to maintaining cell fate decisions. Recent study showed that liquid–liquid-phase separation (LLPS) of Axin organized the β-catenin destruction complex condensates in a normal cellular state. Mutations inactivating the APC gene are found in approximately 80% of all human colorectal cancer (CRC). However, the molecular mechanism of the formation of β-catenin destruction complex condensates organized by Axin phase separation and how APC mutations impact the condensates are still unclear. Here, we report that the β-catenin destruction complex, which is constructed by Axin, was assembled condensates via a phase separation process in CRC cells. The key role of wild-type APC is to stabilize destruction complex condensates. Surprisingly, truncated APC did not affect the formation of condensates, and GSK 3β and CK1α were unsuccessfully recruited, preventing β-catenin phosphorylation and resulting in accumulation in the cytoplasm of CRCs. Besides, we propose that the phase separation ability of Axin participates in the nucleus translocation of β-catenin and be incorporated and concentrated into transcriptional condensates, affecting the transcriptional activity of Wnt signaling pathway.

Unravelling the origin and evolution of precancerous lesions is crucial for effectively preventing malignant transformation, yet our current knowledge remains limited 1 , 2 – 3 . Here we used a base editor-enabled DNA barcoding system 4 to comprehensively map single-cell phylogenies in mouse models of intestinal tumorigenesis induced by inflammation or loss of the Apc gene. Through quantitative analysis of high-resolution phylogenies including 260,922 single cells from normal, inflamed and neoplastic intestinal tissues, we identified tens of independent cell lineages undergoing parallel clonal expansions within each lesion. We also found polyclonal origins of human sporadic colorectal polyps through bulk whole-exome sequencing and single-gland whole-genome sequencing. Genomic and clinical data support a model of polyclonal-to-monoclonal transition, with monoclonal lesions representing a more advanced stage. Single-cell RNA sequencing revealed extensive intercellular interactions in early polyclonal lesions, but there was significant loss of interactions during monoclonal transition. Therefore, our data suggest that colorectal precancer is often founded by many different lineages and highlight their cooperative interactions in the earliest stages of cancer formation. These findings provide insights into opportunities for earlier intervention in colorectal cancer. Experiments using DNA barcoding for lineage tracing in mouse models of colorectal cancer reveal polyclonal origins of premalignant lesions, with a transition to monoclonality as they progress to the advanced tumour.

Background/Objectives: Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by pathogenic variants in the adenomatous polyposis coli (APC) gene. Its attenuated form (AFAP) is characterized by fewer colorectal polyps and later onset of colorectal cancer. We aimed to characterize the molecular effects of a novel APC gene variant (NM_000038.6: c.1620_1624delinsT) identified in a patient with AFAP. Methods: A 56-year-old man with the AFAP phenotype underwent germline testing via a multigene NGS panel, which identified a novel APC gene variant (NM_000038.6: c.1620_1624delinsT). In silico analyses predicted disruption of the canonical donor splice site and a frameshift followed by the introduction of a premature stop codon. The transcriptional impact of the identified APC gene variant was investigated by mRNA analysis. Results: mRNA analysis revealed two distinct APC transcripts: the first transcript led to a truncated protein (p.Leu540PhefsTer8), and the second transcript lacked exon 12, resulting in an in-frame 26 amino acid deletion of APC protein (p.Ala517_Gly542del). The transcript lacking exon 12 was more abundant than the transcript with a premature stop codon, likely due to degradation through nonsense-mediated decay. Conclusions: The APC gene variant (NM_000038.6: c.1620_1624delinsT) exhibits a dual transcriptional effect, revealing its pathogenic role in AFAP. This study highlights the diagnostic value of combined DNA–RNA germline testing for improving the clinical classification of novel APC gene variants and their genotype–phenotype correlations in FAP.

BackgroundFamilial adenomatous polyposis (FAP), an autosomal dominant disorder characterized by multiple colonic polyps, is caused by a germline pathogenic variant of the APC gene. However, this variant is not detected in up to 30% of patients with the adenomatous polyposis phenotype.MethodsWe performed next-generation sequencing (NGS) to identify the causative genes in FAP patients with 10 or more polyps. For patients in whom the APC germline variant was not able to be identified, we screened for APC mosaicism using high-coverage NGS of APC with DNA from leucocytes and/or frozen tissue.ResultsThe pathogenic APC germline variant was found in 93.3%, 71.6%, and 17.1% of patients with profuse-type polyposis, sparse-type polyposis, and oligo-polyposis, respectively. The APC germline variant detection rate in patients with FAP-related diseases was 69.7% for fundic gland polyposis, 79.7% for duodenal adenoma, 94.7% for desmoid tumor, and 71.4% for thyroid cancer, with increasing numbers of extracolonic lesions associated with an increasing APC germline variant detection rate. A mosaic test detected nine patients with APC mosaicism. A comparison of APC-associated polyposis with APC mosaicism showed that patients with APC mosaicism had a low frequency of duodenal adenoma and a family history of colonic polyposis.ConclusionsWe determined the detection rate of the APC germline variant by phenotype and identified APC mosaicism. Genetic testing of FAP patients is important because it can help with surgical decision-making, monitoring, and genetic counseling. Furthermore, genetic testing by NGS proved to be an effective method of detecting APC germline variants.

While constitutional pathogenic variants in the APC gene cause familial adenomatous polyposis, APC c.3920T>A; p.Ile1307Lys (I1307K) has been associated with a moderate increased risk of colorectal cancer (CRC), particularly in individuals of Ashkenazi Jewish descent. However, published data include relatively small sample sizes, generating inconclusive results regarding cancer risk, particularly in non-Ashkenazi populations. This has led to different country/continental-specific guidelines regarding genetic testing, clinical management and surveillance recommendations for I1307K. A multidisciplinary international expert group endorsed by the International Society for Gastrointestinal Hereditary Tumours (InSiGHT), has generated a position statement on the APC I1307K allele and its association with cancer predisposition. Based on a systematic review and meta-analysis of the evidence published, the aim of this document is to summarise the prevalence of the APC I1307K allele and analysed the evidence of the associated cancer risk in different populations. Here we provide recommendations on the laboratory classification of the variant, define the role of predictive testing for I1307K, suggest recommendations for cancer screening in I1307K heterozygous and homozygous individuals and identify knowledge gaps to be addressed in future research studies. Briefly, I1307K, classified as pathogenic, low penetrance, is a risk factor for CRC in individuals of Ashkenazi Jewish origin and should be tested in this population, offering carriers specific clinical surveillance. There is not enough evidence to support an increased risk of cancer in other populations/subpopulations. Therefore, until/unless future evidence indicates otherwise, individuals of non-Ashkenazi Jewish descent harbouring I1307K should be enrolled in national CRC screening programmes for average-risk individuals.

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.

Age-period-cohort (APC) analysis has a long, controversial history in sociology and related fields. Despite the existence of hundreds, if not thousands, of articles and dozens of books, there is little agreement on how to adequately analyze APC data. This article begins with a brief overview of APC analysis, discussing how one can interpret APC effects in a causal way. Next, we review methods that obtain point identification of APC effects, such as the equality constraints model, Moore-Penrose estimators, and multilevel models. We then outline techniques that entail point identification using measured causes, such as the proxy variables approach and mechanism-based models. Next, we discuss a general framework for APC analysis grounded in partial identification using bounds and sensitivity analyses. We conclude by outlining a general step-by-step procedure for conducting APC analyses, presenting an empirical example examining temporal shifts in verbal ability.

Background Several genetic and epigenetic alterations are related to the development and progression of Gastric Cancer (GC), one of those being the deregulated microRNA (miRNA) expression profile. miRNAs are small noncoding RNAs that negatively regulate the expression of thousands of genes, including oncogenes and tumor suppressor genes. Our group identified, in previous studies, some miRNAs that are differentially expressed in GC when compared to the gastric mucosa without cancer, including hsa-miR-29c and hsa-miR-135b. The aim of the study was to modulate the expression of the miRNAs hsa-miR-29c-5p and hsa-miR-135b-5p and evaluate the expression of their target genes in 2D and 3D cell cultures. Methods hsa-miR-29c-5p and hsa-miR-135b-5p expression profiles were modulated by transfecting mimics and antimiRs, respectively, in 2D and 3D cell cultures. The expression of the proteins coded by the genes CDC42, DNMT3A (target genes of hsa-miR-29c-5p ) and APC (target gene of hsa-miR-135b-5p ) were measured by Western Blot. Results Results showed that mimics and antimiRs transfection significantly altered the expression of both miRNAs, increasing the expression of hsa-miR-29c-5p and reducing the expression of hsa-miR-135b-5p , especially in the 3D culture of the cell lines. When analyzing the proteins expression, we observed that AGP01 and AGP03 cell lines transfected with mimics had a reduction in the levels of CDC42 and DNMT3A and all three cell lines transfected with antimiRs had an increase in the expression of the protein APC. Conclusion We concluded that three-dimensional culture can be a more representative in vitro model that resembles better the in vivo reality. Our results also showed that hsa-miR-29c-5p is an important regulator of CDC42 and DNMT3A genes in the intestinal subtype gastric cancer and hsa-miR-135b-5p regulates the APC gene in both intestinal and diffuse subtypes of GC. Dysregulation in their expression, and consequently in their respectively signaling pathways, shows how these miRNAs can influence the carcinogenesis of different histological subtypes of gastric cancer.