Dataset Information

The effect of a germline mutation in the APC gene on ?-catenin in human embryonic stem cells.

ABSTRACT: Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-?-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs.Two FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) ?-catenin expression was analyzed by Western blot analysis; 2) Wnt-?-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of ?-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed.We have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced ?-catenin/TCF-mediated activity. Additionally, ?-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in ?-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population.Our results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation.

SUBMITTER: Yedid N

PROVIDER: S-EPMC5180406 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells.

Yedid Nofar N Kalma Yael Y Malcov Mira M Amit Ami A Kariv Revital R Caspi Michal M Rosin-Arbesfeld Rina R Ben-Yosef Dalit D

BMC cancer 20161223 1

<h4>Background</h4>Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele ...[more]

PMID: 28010732

Similar Datasets

Project description:Familial adenomatous polyposis (FAP) is an inherited condition arising from genetic defects in the Adenomatous polyposis coli (APC) gene. Carriers with mutations in the APC gene develop polyps in the colon and rectum which if not managed, transition into colon cancer. In this study, we identified a novel germline mutation in the APC gene in members of an FAP-affected (Familial adenomatous polyposis) family. This unique heterozygous variant (c.735_736insT; p.Ser246PhefsTer6) was identified in ten out of twenty six family members, ranging in age from 6 to 60 years. Polyps were detected in six of the ten individuals (35-60 years) carrying this mutation. The remaining four members (6-23 years) remain polyp free. A significant fraction of FAP affected individuals eventually develop colon cancer and therapeutic interventions to prevent cancer progression remain elusive. To address this issue, we sought to determine if peptides derived from the novel APC mutation could induce a cytotoxic T cell response, thereby qualifying them as vaccine candidates. Peptides harboring the variant amino acids were first interrogated in silico for their immunogenicity using a proprietary neoepitope prioritization pipeline, OncoPeptVAC. A single 9-mer peptide was predicted to be immunogenic. Remarkably, CD8+ T cells isolated from either an FAP+/ APCmut individual, or from a FAP-/ APCmut individual, failed to respond to the peptide, whereas those from either an unaffected family member (FAP-/ APCwt) or from healthy unrelated donors, showed a robust response, suggesting that CD8+ T cells from individuals carrying this germline APC mutation have been tolerized to the mutation. Furthermore, experimental testing of six additional reported APC gene mutation-derived peptides revealed one of the six to be immunogenic. While not all APC mutant peptides are inmmunogenic, a few qualify as vaccine candidates offering novel treatment opportunities to patients with somatic APC gene mutations to delay/treat colorectal cancer.

Dataset Information

The effect of a germline mutation in the APC gene on ?-catenin in human embryonic stem cells.

Publications

The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets