Human Cone Precursor Program Underlying a Proliferative Response to pRB Loss

Abstract

Purpose : RB1 inactivation collaborates with cone precursor circuitry, including intrinsically high MDM2, high MYCN, and low p27, to initiate retinoblastoma tumorigenesis. In contrast, mouse cone precursors lack prominent MDM2 and MYCN and fail to proliferate in response to pRb loss. This study aims to 1) define the spatio-temporal expression of human cone precursor proliferation-related proteins that could contribute to retinoblastoma, 2) determine whether these features appear in mouse cone precursors, and 3) test whether ectopic high-level MDM2 and MYCN can sensitize mouse cone precursors to the loss of pRb function.

Methods : Human fetal week 17-18 retinas and mouse post-natal day (P) 6, P10, and P20 retinas were immunostained and protein expression quantitated using ImageJ. Transgenic Red-Green-opsin Promoter (RGP)-MDM2 mice were mated with RGP-Cre and Rb1^l/l mice to generate mice with cone-directed pRb loss and MDM2 expression. Intact retinas of various genotypes were infected with lentiviruses expressing RB1 shRNA and/or overexpressing MYCN. Cell cycle entry was evaluated at day 14 post-infection by EdU staining.

Results : Cyclin E and MYCN were prominently expressed throughout human cone precursor development and increased with maturation. E2F3, pRB, and p-p27(T187) were induced with similar timing and increased with maturation. MDM2 and cone arrestin were co-induced only in the most mature cone precursors. p27 declined with cone precursor maturation, with lowest levels in the fovea. In mouse cone precursors, cyclin E, N-Myc, E2f3, pRb, and p-p27(T187) were low or not detected. RGP-MDM2 mice expressed high MDM2 in cones beginning at P8, yet RGP-MDM2 RGP-Cre, Rb1^l/l mice showed no cone cell proliferation at P8-P20 and no tumors when followed for up to 18 months. Whereas RB1 shRNA induced cone precursor proliferation in cultured human retina, Rb1 shRNA either with or without MDM2 and MYCN overexpression did not induce cone precursor cell cycle entry in cultured mouse retina.

Conclusions : The temporal sequence of human cone precursor proliferation-related circuitry is: Cyclin E, MYCN, p27 followed by E2F3, pRB and p-p27 (T187) followed by MDM2 onset and p27 downregulation. Mouse cone precursors fail to induce cyclin E, N-Myc, pRb, E2F3 or Mdm2. Cone-directed MDM2 and MYCN overexpression is not sufficient to sensitize pRb deficient mouse cones to retinoblastoma.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.