Exploration of the resistance’s mechanisms to oncogenesis induced by APC mutation in retinal pigment epithelium cells

Abstract

Purpose : Familial Adenomatous Polyposis (FAP) is characterized by colonic adenomas but also by various extra-intestinal manifestations. Among these manifestations, pigmented ocular fundus lesion (POFL) is the most common. Clinically these lesions are similar to congenital hypertrophy of the retinal pigment epithelium (CHRPE) but postmortem studies in FAP patients demonstrated RPE hyperplasia and RPE hamartomata’s growth in addition to CHRPE. Although the RPE cells seem sensitive to oncogenesis induce by APC, these lesions never derived into Adenocarcinoma. It suggests that APC mutation in RPE cells activates first an oncogenic process and, in a second time, a mechanism of tumor suppression that stops lesion progression. We hypothesize that APC mutation results into oncogene induced senescence in RPE cells and mechanisms underlying this process are under investigation.

Methods : WT (N=2 lines) and APC mutant (N=2 lines) human embryonic stem cells (hESCs) are differentiated into RPE cells to establish the cellular model. In addition, an isogenic APC mutant hESC line has been generated using the CrispR/Cas9 technology. These cells are then compared to identify pathological phenotypes and markers. Cell-cell junctions, epithelial organization, proliferation and senescence markers were explored by immunostaining and immunoblot at day 7, 14, 21 and 35. Moreover pigmentation levels of WT and mutant cells were compared by macroscopic observation and by the quantification of the melanin and the expression of proteins involved in the melanin synthesis pathway.

Results : Preliminary results indicate that APC mutated RPE cells express high level of KI67 at day 21 compare to WT cells, suggesting an over-proliferation and a loss of inhibition contact. APC Mutated RPE cells also exhibit local epithelial disorganization with the loss of the monolayer aspect of the epithelium. Moreover APC mutation induced a hyperpigmentation of the RPE associated with an overexpression of MITF and Tyrosinase.

Conclusions : hESCs derived RPE cells harboring APC mutation recapitulate the main phenotypes associated to POFLs observed in FAP patients. Consequently these cells represent an interesting model to study the mechanisms that protect RPE against oncogenesis induced by APC mutation. We will now determine if APC mutation induces specific senescence in long term cultures.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.