Heterozygous APC germline mutations impart predisposition to colorectal cancer

Livia Preisler,Liron Kuznitsov-Yanovsky,Dalit Ben-Yosef,Yoav Mayshar,Mira Malcov,Ilana Carmel-Gross,Dov Hershkovitz,Guy Shapira,Aline Habib,Foad Azem,Noam Shomron,Revital Kariv

doi:10.1038/s41598-021-84564-4

Livia Preisler, Liron Kuznitsov-Yanovsky + Show 10 more

Open Access

https://doi.org/10.1038/s41598-021-84564-4

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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.

Highlights

Left untreated[10,11,12]
In order to mimic the natural niche of Colorectal cancer (CRC) development, hESCs were differentiated into colon organoids
The current understanding of CRC stems either from the examination of patient samples and cell lines, which are highly variable and provide only a post hoc view of disease progression, or from investigation of animal models, which often fall short in faithfully recapitulating the human c ondition19–24. hESCs carrying specific mutations characterizing human genetic disorders provide a valuable tool for studying the pathophysiology of these diseases in h umans[36,37]

Summary

Introduction

Left untreated[10,11,12]. The site of the ’first hit’ in the APC tumor suppressor gene has an impact on the type of the ’second hit’ that will either cause APC loss of heterozygosity or a truncated p rotein[13,14,15,16,17]. Three FAP-human embryonic stem cell (hESCs) lines carrying various human APC germline mutations that were derived in our lab from donated e mbryos[25,26], were exploited to study whether a mutation in a single allele of APC is sufficient to alter the molecular and cellular phenotype of colon epithelial cells in the in vitro derived colon organoids. This unique research model enabled the study of the correlation of specific mutations to FAP manifestations in vivo in the donor parents

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