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Rb suppresses human cone-precursor-derived retinoblastoma tumours.

Xu XL, Singh HP, Wang L, Qi DL, Poulos BK, Abramson DH, Jhanwar SC, Cobrinik D - Nature (2014)

Bottom Line: This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear.Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion.More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA [2] Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA.

ABSTRACT
Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

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Proliferation status of retinal cells other than cones 15 days after shRB1 transduction of intact FW19 retinasa–c, Combined transduction with pLKO-shRB1-733 and -737. a, Ki67 not detected in NRL+, or rhodopsin+ rod photoreceptors or in calbindin+ horizontal cells. b, Ki67 detected in PAX6lo, nestin+ RPCs (white arrows) but not in PAX6hi, nestin(−) horizontal, amacrine, or ganglion cells (yellow arrows). c, Ki67 detected in CHX10+, CRX(−) RPCs (white arrows) but not in CHX10+, CRX+ bipolar cells (yellow arrows). d, Percentage of cells co-expressing Ki67 and retinal cell markers. e–h, Transduction with yellow fluorescent protein-marked pLKO-YFP-shRB1-733. e, Ki67 detected in YFP+, L/M-opsin+ or YFP+, cone arrestin+ cone precursors (white arrows) and in undefined YFP(−) cell (yellow arrow). f, Ki67 not detected in YFP+, calbindin+ horizontal cells, YFP+, syntaxin+ or YFP+, PAX6+ amacrine cells, or in YFP+, NRL+ rod precursors. g, Ki67 detected (white arrows) or not detected (yellow arrows) in YFP+, nestin+ RPCs or glia, or in YFP+, CHX10+ RPCs or bipolar cells. h, Proportion of Ki67+ cells co-expressing YFP and retinal markers after transduction with pLKO-YFP-shRB1-733 or scrambled control. Scale bars, 20 μm. Analyses in a–d and in e–h represent two independent experiments. All Immunostaining was performed at least twice.
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Figure 8: Proliferation status of retinal cells other than cones 15 days after shRB1 transduction of intact FW19 retinasa–c, Combined transduction with pLKO-shRB1-733 and -737. a, Ki67 not detected in NRL+, or rhodopsin+ rod photoreceptors or in calbindin+ horizontal cells. b, Ki67 detected in PAX6lo, nestin+ RPCs (white arrows) but not in PAX6hi, nestin(−) horizontal, amacrine, or ganglion cells (yellow arrows). c, Ki67 detected in CHX10+, CRX(−) RPCs (white arrows) but not in CHX10+, CRX+ bipolar cells (yellow arrows). d, Percentage of cells co-expressing Ki67 and retinal cell markers. e–h, Transduction with yellow fluorescent protein-marked pLKO-YFP-shRB1-733. e, Ki67 detected in YFP+, L/M-opsin+ or YFP+, cone arrestin+ cone precursors (white arrows) and in undefined YFP(−) cell (yellow arrow). f, Ki67 not detected in YFP+, calbindin+ horizontal cells, YFP+, syntaxin+ or YFP+, PAX6+ amacrine cells, or in YFP+, NRL+ rod precursors. g, Ki67 detected (white arrows) or not detected (yellow arrows) in YFP+, nestin+ RPCs or glia, or in YFP+, CHX10+ RPCs or bipolar cells. h, Proportion of Ki67+ cells co-expressing YFP and retinal markers after transduction with pLKO-YFP-shRB1-733 or scrambled control. Scale bars, 20 μm. Analyses in a–d and in e–h represent two independent experiments. All Immunostaining was performed at least twice.

Mentions: RB1 shRNAs also induced cone precursor proliferation in intact retinas. Ki67 was detected in L/M-opsin+ cone precursors in the fovea, demarcated by cones but not rods, 15 days after transduction (Fig. 2d). Ki67 was not detected in cells expressing rod, amacrine, horizontal, or ganglion cell markers (Extended Data Fig. 4a, d). Ki67 was detected in RPCs and glia marked by PAX6+, nestin+ or by CHX10+, CRX(−), yet in similar proportions after shRB1 and control shRNAs (Extended Data Fig. 4b–d). Moreover, a yellow fluorescent protein (YFP) -expressing shRB1 vector selectively induced Ki67 in YFP+ cones, although all cell types were transduced (Extended Data Fig. 4e–h).


Rb suppresses human cone-precursor-derived retinoblastoma tumours.

Xu XL, Singh HP, Wang L, Qi DL, Poulos BK, Abramson DH, Jhanwar SC, Cobrinik D - Nature (2014)

Proliferation status of retinal cells other than cones 15 days after shRB1 transduction of intact FW19 retinasa–c, Combined transduction with pLKO-shRB1-733 and -737. a, Ki67 not detected in NRL+, or rhodopsin+ rod photoreceptors or in calbindin+ horizontal cells. b, Ki67 detected in PAX6lo, nestin+ RPCs (white arrows) but not in PAX6hi, nestin(−) horizontal, amacrine, or ganglion cells (yellow arrows). c, Ki67 detected in CHX10+, CRX(−) RPCs (white arrows) but not in CHX10+, CRX+ bipolar cells (yellow arrows). d, Percentage of cells co-expressing Ki67 and retinal cell markers. e–h, Transduction with yellow fluorescent protein-marked pLKO-YFP-shRB1-733. e, Ki67 detected in YFP+, L/M-opsin+ or YFP+, cone arrestin+ cone precursors (white arrows) and in undefined YFP(−) cell (yellow arrow). f, Ki67 not detected in YFP+, calbindin+ horizontal cells, YFP+, syntaxin+ or YFP+, PAX6+ amacrine cells, or in YFP+, NRL+ rod precursors. g, Ki67 detected (white arrows) or not detected (yellow arrows) in YFP+, nestin+ RPCs or glia, or in YFP+, CHX10+ RPCs or bipolar cells. h, Proportion of Ki67+ cells co-expressing YFP and retinal markers after transduction with pLKO-YFP-shRB1-733 or scrambled control. Scale bars, 20 μm. Analyses in a–d and in e–h represent two independent experiments. All Immunostaining was performed at least twice.
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Figure 8: Proliferation status of retinal cells other than cones 15 days after shRB1 transduction of intact FW19 retinasa–c, Combined transduction with pLKO-shRB1-733 and -737. a, Ki67 not detected in NRL+, or rhodopsin+ rod photoreceptors or in calbindin+ horizontal cells. b, Ki67 detected in PAX6lo, nestin+ RPCs (white arrows) but not in PAX6hi, nestin(−) horizontal, amacrine, or ganglion cells (yellow arrows). c, Ki67 detected in CHX10+, CRX(−) RPCs (white arrows) but not in CHX10+, CRX+ bipolar cells (yellow arrows). d, Percentage of cells co-expressing Ki67 and retinal cell markers. e–h, Transduction with yellow fluorescent protein-marked pLKO-YFP-shRB1-733. e, Ki67 detected in YFP+, L/M-opsin+ or YFP+, cone arrestin+ cone precursors (white arrows) and in undefined YFP(−) cell (yellow arrow). f, Ki67 not detected in YFP+, calbindin+ horizontal cells, YFP+, syntaxin+ or YFP+, PAX6+ amacrine cells, or in YFP+, NRL+ rod precursors. g, Ki67 detected (white arrows) or not detected (yellow arrows) in YFP+, nestin+ RPCs or glia, or in YFP+, CHX10+ RPCs or bipolar cells. h, Proportion of Ki67+ cells co-expressing YFP and retinal markers after transduction with pLKO-YFP-shRB1-733 or scrambled control. Scale bars, 20 μm. Analyses in a–d and in e–h represent two independent experiments. All Immunostaining was performed at least twice.
Mentions: RB1 shRNAs also induced cone precursor proliferation in intact retinas. Ki67 was detected in L/M-opsin+ cone precursors in the fovea, demarcated by cones but not rods, 15 days after transduction (Fig. 2d). Ki67 was not detected in cells expressing rod, amacrine, horizontal, or ganglion cell markers (Extended Data Fig. 4a, d). Ki67 was detected in RPCs and glia marked by PAX6+, nestin+ or by CHX10+, CRX(−), yet in similar proportions after shRB1 and control shRNAs (Extended Data Fig. 4b–d). Moreover, a yellow fluorescent protein (YFP) -expressing shRB1 vector selectively induced Ki67 in YFP+ cones, although all cell types were transduced (Extended Data Fig. 4e–h).

Bottom Line: This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear.Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion.More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA [2] Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA.

ABSTRACT
Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

Show MeSH
Related in: MedlinePlus