Introduction-Most cases of colorectal cancer initiate as a result of inactivation of APC gene, a negative regularor of the Wnt-β-catenin pathway. Familial Adenomatous Polyposis (FAP) patients inherit a germline mutation in one APC allele and loss of the second allele leads to cancer initiation. However, it is not currently understood which molecular mechanisms are activated by APC loss that leads to cancer.
Aim-Explore the earliest molecular events leading to malignant transformation using human embryonic stem cell (hESCs) lines derived from APC mutated embryos following PGD for FAP.
Methods–The FAP-hESCs were cultured and the timing and location of the 'second hit' was evaluated by four different methodologies:1) DNA sequencing of the APC gene; 2) Luciferase assay for examining Wnt-β-catenin mediated transcription 3) β-catenin expression by Western bloting; 4) β-catenin localization by immunofluoresence.
Results -Extended culturing of FAP1-hESCs resulted in activation of Wnt pathway. Interestingly, theses cultures showed changes in the subcellular localization of β-catenin from membrane to perinuclear distribution in 91% of FAP1-hESCs colonies at late passage, as compare to normal APC hESCs lines in which β-catenin is localized solely in the membrane. No mutation was found in the sequences of the APC mutation cluster region and hot spots in FAP-hESCs by the DNA sequences examined thus far.
Conclusions–Our novel FAP-hESCs suggest the acquisition of the somatic APC mutation in-vitro, as demonstrated by Wnt/β-catenin activation. They may shed light on the primary molecular changes occuring during malignant transformation and demonstrate the importance of hESCs in modeling cancer development.
