Heterozygous APC germline mutations impart predisposition to colorectal cancer

Abstract

Familial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how different APC germline mutations predict disease severity. Three FAP-human embryonic stem cell lines (FAP1/2/3-hESC lines) carrying germline mutations at different locations of the APC gene, and two control hESC lines free of the APC mutation, were differentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP-hESCs carrying a complete loss-of-function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP-hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst-like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical differentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non-pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non-FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufficient to initiate early molecular tumorigenic activity. In addition, the results hint that patient-specific hESC-derived colon organoids can probably predict disease severity among FAP patients.

Figure 1

Characterization of hESC-derived colon organoids. (A) Immunostaining of H9 hESC-derived definitive endoderm (day 4) and hindgut endoderm (day 8). Scale bars: 200 µM. (B) Representative bright-field images of developing hESC-derived colon organoids. Scale bars: 200 µM. (C) Immunostaining of hESC-derived colon organoids. Adult human colon tissue samples and H9 hESCs served as controls. Scale bars: 200 µM. (D) RT-PCR analysis of colonic cell markers in hESC-derived D28 colonic spheroids and D40 colon organoids. Adult human colon tissue samples and H9 hESCs served as positive and negative controls, respectively. Data are presented as relative mean expression (± standard deviation) of triplicate samples from one experiment.

Figure 2

A molecular summary of APC germline mutations in the FAP-hESC lines. (A) Description of the type and location of APC germline mutations in each of the three FAP-hESC lines derived following preimplantation genetic diagnosis (PGD). (B) Schematic structure of the APC protein. Positions of the APC germline mutations in FAP1, FAP2 and FAP3-hESC lines are indicated by arrows. O-oligomerization domain, MCR- mutation cluster region, basic microtubule binding site.

Figure 3

Capability of FAP-hESC lines to form colon organoids. (A) Representative images of 3D colon organoids derived from control hESC lines (H9 and Hues13), and FAP-hESC lines (FAP 1–3), each bearing a different germline APC mutation. Scale bar: 200 µm. (B) Number and complexity of day-48 colon organoids of control (H9 and Hues13) and FAP-hESC lines (FAP 1–3). Presented are the number (#) of experiments, # of Matrigel (MG) droplets created in all experiments performed, average # of organoids developed/MG droplet and average # cysts/MG droplet.

Figure 4

Lineage characterization of cells within colon organoids. (A) Representative images of immunohistochemical staining of colon epithelial markers keratin 20 and CDX2 and the mesodermal marker VIM in control and FAP-colon organoids on day 48 of differentiation. Normal colon tissue served as a positive control. Scale bars: 200 µM. (B) Quantification of VIM⁺ cells by the ImageJ image processing program. Analyses were performed on 10 photos randomly selected from each sample (10 ×). *p < 0 .05, ***p < 0 .005; One-way ANOVA.

Figure 5

Cell proliferation in FAP-hESCs and their derived colon organoids. (A) Representative photos of immunohistochemical staining of the cell proliferation marker Ki67 (right) and the colon epithelial marker CDX2 (left) in control and FAP-colon organoids on day 48 of differentiation. Scale bars: 100 µM. (B) Quantification of Ki67/CDX2-positive cells was performed using the image processing program ImageJ, using ImmunoRatio software, on 5 different fields taken from each sample (magnification: 10 ×). One-way ANOVA *p < 0.05, **p < 0.01. (C) Representative images of the Ki67 immunostaining pattern in normal control colonic mucosa (WT) versus normal-appearing crypts from FAP patients (APC + / −). Scale bars = 100 µM.

Figure 6

Transcriptome of FAP-hESC lines at different stages of differentiation into colon organoids. (A) Heat map of differentially expressed genes (DEGs) during differentiation of FAP-hESCs into colon organoids. Columns represent samples and rows represent genes, categorized in relation to pluripotency and to cell lineage (endoderm, ectoderm, and mesoderm). The heat map illustrates lower (blue) to high (red) gene expression levels with distinct transcriptional profiles across the differentiation process. (B) The volcano plots of DEGs in all three FAP-hESC lines compared to WT, on day 8 of differentiation into colon organoids. (C) Gene ontology biological process analysis of upregulated DEGs in FAP compared to WT cells, on day 8 of colon organoid differentiation. (D) Venn diagram showing the overlap of biological processes at day 8 of differentiation in FAP and WT cells.