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X. L. Xu, Y. Fang, T. C. Lee, S. C. Jhanwar, D. H. Abramson, D. Cobrinik; MDM2, N-myc and Cone-Specific RXR and TRβ2 Signaling Are Essential for Retinoblastoma Cell Proliferation and Survival. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1697.
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The cell type-specific circuitry that predisposes to retinoblastoma (RB) has not been defined. Previously, we showed that RB cells express markers of cone precursors, including RXRγ, TRβ2, and L/M opsin. In contrast, mouse retinal tumors that develop due to the combined loss of Rb and Rb-related proteins express markers of horizontal or amacrine cells. Due to the cone-like features of the tumors, we examined whether cone precursor signaling circuitry might be relevant to RB tumorigenesis.
Immunofluorescent staining was used to examine MDM2 and N-myc expression in human fetal and tumor associated retina, and in E14 to P42 mouse retinas. Lentivirus-transduced shRNAs were used to knockdown MDM2, ARF, RXRγ, N-myc, and TRβ2 in RB cells. Xenografts were performed by injecting Y79 cells into the subretinal space of nude mice. Chromatin immunoprecipitation (ChIP) was performed using two RXRγ antibodies, and luciferase assays performed using MDM2 P2 promoter-luciferase reporter constructs with wild type or mutated RXRγ binding sequences.
Human but not mouse cone precursors prominently express MDM2 and N-Myc, particularly in the central retina of fetal weeks 16 to 21. MDM2 was detected at lower levels in human and mouse horizontal cells and in a subset of mouse amacrine cells. MDM2 and N-Myc were highly expressed, and required for proliferation and survival of RB cells. Knockdown of MDM2 induced apoptosis, which was counteracted by co-knockdown of ARF. An RXRγ recognition element was identified in the human but not mouse MDM2 P2 promoter and contributed to the expression of an MDM2 P2 promoter-luciferase reporter construct. ChIP assays showed that RXRγ associated with the MDM2 P2 promoter in RB cells, and RXRγ knockdown rapidly diminished MDM2 expression and induced RB cell death. TRβ2 knockdown impaired RB cell proliferation in vitro and suppressed tumor growth in vivo.
MDM2, N-Myc and the cone-specific RXRγ and TRβ2 are prominently expressed in cone precursors and RB cells and required for RB cell proliferation and survival. MDM2 is needed to suppress ARF-dependent apoptosis, and its expression depends on the cone-specific RXRγ and an RXRγ consensus element in the human, but not mouse, MDM2 P2 promoter. These findings suggest that high levels of MDM2 could sensitize cone precursors to the oncogenic effects of RB1 mutations, and thus provide further support for a cone precursor origin of retinoblastoma.
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