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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 of cone-like cells after Rb depletion in dissociated FW19 retinaa, Decreased Rb protein in L/M-opsin+ or TRβ2+ cells (arrows) on days 5 or 23, and decreased RB1 RNA or Rb protein on day 4 after shRB1-733 transduction. b, Cone arrestin+, CRX+ cells (arrows) with or without Ki67 co-expression. c, Ki67+, cone arrestin+ cells first detected 9 or 14 days post-transduction in two experiments. d–f Co-staining of Ki67 with RXRγ/CRX at 14 days, (d) with cone arrestin/CRX at 14 days (e), or with L/M-opsin/CRX at 23 days (f) after transduction with shRB1-733 or a scrambled control. g, Percentage of cells co-expressing Ki67 with L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX, 23 days post-transduction. h, Prevalence of cells co-staining for L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX 23 days post-transduction. i, Ki67 not detected in cells expressing markers of rods (NRL), ganglion cells (BRN-3), bipolar cells (strong CHX10), or horizontal cells (PROX1) 14 days post-transduction. j, Co-expression of Ki67 with markers of RPCs (nestin, white arrows) or Müller glia (CRALBP or SOX2), but not in PAX6+, nestin(−) ganglion, amacrine, or horizontal cells (yellow arrows) 14 days post-transduction. k–l, EdU incorporation in cells expressing markers of cones (cone arrestin/CRX or RXRγ/CRX, yellow arrows in l) but not in cells expressing markers of rods (CNGA1, CNGB1), bipolar cells (CHX10/CRX), or ganglion, horizontal, or amacrine cells (syntaxin) (white arrows in l) 14 days after transduction. Black lines above labels demarcate distinct fields. m, Co-staining of phosphohistone H3 (PH3) with cone arrestin/CRX 23 days post-transduction. n, Apoptosis marker CC3 in cells expressing RPC and glial marker nestin 14 days after transduction with RB1-directed shRNAs (yellow arrow) but not with scrambled control (white arrow). Values and error bars are means and standard deviation of triplicate assays for all Extended Data figures. Scale bars, 20 μm. Data are representative of at least two independent experiments.
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Figure 5: Proliferation of cone-like cells after Rb depletion in dissociated FW19 retinaa, Decreased Rb protein in L/M-opsin+ or TRβ2+ cells (arrows) on days 5 or 23, and decreased RB1 RNA or Rb protein on day 4 after shRB1-733 transduction. b, Cone arrestin+, CRX+ cells (arrows) with or without Ki67 co-expression. c, Ki67+, cone arrestin+ cells first detected 9 or 14 days post-transduction in two experiments. d–f Co-staining of Ki67 with RXRγ/CRX at 14 days, (d) with cone arrestin/CRX at 14 days (e), or with L/M-opsin/CRX at 23 days (f) after transduction with shRB1-733 or a scrambled control. g, Percentage of cells co-expressing Ki67 with L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX, 23 days post-transduction. h, Prevalence of cells co-staining for L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX 23 days post-transduction. i, Ki67 not detected in cells expressing markers of rods (NRL), ganglion cells (BRN-3), bipolar cells (strong CHX10), or horizontal cells (PROX1) 14 days post-transduction. j, Co-expression of Ki67 with markers of RPCs (nestin, white arrows) or Müller glia (CRALBP or SOX2), but not in PAX6+, nestin(−) ganglion, amacrine, or horizontal cells (yellow arrows) 14 days post-transduction. k–l, EdU incorporation in cells expressing markers of cones (cone arrestin/CRX or RXRγ/CRX, yellow arrows in l) but not in cells expressing markers of rods (CNGA1, CNGB1), bipolar cells (CHX10/CRX), or ganglion, horizontal, or amacrine cells (syntaxin) (white arrows in l) 14 days after transduction. Black lines above labels demarcate distinct fields. m, Co-staining of phosphohistone H3 (PH3) with cone arrestin/CRX 23 days post-transduction. n, Apoptosis marker CC3 in cells expressing RPC and glial marker nestin 14 days after transduction with RB1-directed shRNAs (yellow arrow) but not with scrambled control (white arrow). Values and error bars are means and standard deviation of triplicate assays for all Extended Data figures. Scale bars, 20 μm. Data are representative of at least two independent experiments.

Mentions: Dissociated retinal cells were transduced with RB1-directed or control short hairpin RNAs (shRNAs), followed by co-staining for the proliferation-associated Ki67 and cell type-specific markers. RB1 shRNAs abrogated Rb expression in long or medium wavelength (L/M)-opsin+ and thyroid hormone receptor β2 (TRβ2)+ cone precursors as well as in other cell types (Extended Data Fig. 1a). After two weeks, Ki67 was detected in cone precursor-like cells co-expressing the photoreceptor marker CRX and the cone markers L/M-opsin, cone arrestin, and RXRγ (Fig. 1a, Extended Data Fig. 1b–h). Ki67+ cone marker+ cells were first detected 9 days after transduction whereas clusters were routinely detected by day 23. Ki67 was not detected in cells expressing markers of rods (NRL), bipolar cells (strong CHX10), ganglion cells (BRN-3), or amacrine or horizontal cells (PROX1+ or PAX6+, nestin(−)) (Fig. 1a, Extended Data Fig. 1i, j). Ki67 was detected in cells expressing markers of RPCs or Müller glia (nestin or CRALBP, SOX2), yet in similar proportions after shRB1 or control shRNA (Fig. 1a, Extended Data Fig. 1j). RB1 shRNAs also induced incorporation of 5-ethynyl-2′-deoxyuridine (EdU), an indicator of S phase entry, increased expression of the mitosis marker phosphohistone H3, suppressed expression of the apoptosis marker cleaved caspase 3 (CC3), and induced proliferation in cells expressing cone but not other retinal cell markers (Fig. 1c, d; Extended Data Fig. 1k–n). In contrast, RB1 shRNAs induced CC3 and decreased the number of cells expressing markers of RPCs and glia (Fig. 1b, d; Extended Data Fig. 1n).


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 of cone-like cells after Rb depletion in dissociated FW19 retinaa, Decreased Rb protein in L/M-opsin+ or TRβ2+ cells (arrows) on days 5 or 23, and decreased RB1 RNA or Rb protein on day 4 after shRB1-733 transduction. b, Cone arrestin+, CRX+ cells (arrows) with or without Ki67 co-expression. c, Ki67+, cone arrestin+ cells first detected 9 or 14 days post-transduction in two experiments. d–f Co-staining of Ki67 with RXRγ/CRX at 14 days, (d) with cone arrestin/CRX at 14 days (e), or with L/M-opsin/CRX at 23 days (f) after transduction with shRB1-733 or a scrambled control. g, Percentage of cells co-expressing Ki67 with L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX, 23 days post-transduction. h, Prevalence of cells co-staining for L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX 23 days post-transduction. i, Ki67 not detected in cells expressing markers of rods (NRL), ganglion cells (BRN-3), bipolar cells (strong CHX10), or horizontal cells (PROX1) 14 days post-transduction. j, Co-expression of Ki67 with markers of RPCs (nestin, white arrows) or Müller glia (CRALBP or SOX2), but not in PAX6+, nestin(−) ganglion, amacrine, or horizontal cells (yellow arrows) 14 days post-transduction. k–l, EdU incorporation in cells expressing markers of cones (cone arrestin/CRX or RXRγ/CRX, yellow arrows in l) but not in cells expressing markers of rods (CNGA1, CNGB1), bipolar cells (CHX10/CRX), or ganglion, horizontal, or amacrine cells (syntaxin) (white arrows in l) 14 days after transduction. Black lines above labels demarcate distinct fields. m, Co-staining of phosphohistone H3 (PH3) with cone arrestin/CRX 23 days post-transduction. n, Apoptosis marker CC3 in cells expressing RPC and glial marker nestin 14 days after transduction with RB1-directed shRNAs (yellow arrow) but not with scrambled control (white arrow). Values and error bars are means and standard deviation of triplicate assays for all Extended Data figures. Scale bars, 20 μm. Data are representative of at least two independent experiments.
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Figure 5: Proliferation of cone-like cells after Rb depletion in dissociated FW19 retinaa, Decreased Rb protein in L/M-opsin+ or TRβ2+ cells (arrows) on days 5 or 23, and decreased RB1 RNA or Rb protein on day 4 after shRB1-733 transduction. b, Cone arrestin+, CRX+ cells (arrows) with or without Ki67 co-expression. c, Ki67+, cone arrestin+ cells first detected 9 or 14 days post-transduction in two experiments. d–f Co-staining of Ki67 with RXRγ/CRX at 14 days, (d) with cone arrestin/CRX at 14 days (e), or with L/M-opsin/CRX at 23 days (f) after transduction with shRB1-733 or a scrambled control. g, Percentage of cells co-expressing Ki67 with L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX, 23 days post-transduction. h, Prevalence of cells co-staining for L/M-opsin/CRX, RXRγ/CRX, or cone arrestin/CRX 23 days post-transduction. i, Ki67 not detected in cells expressing markers of rods (NRL), ganglion cells (BRN-3), bipolar cells (strong CHX10), or horizontal cells (PROX1) 14 days post-transduction. j, Co-expression of Ki67 with markers of RPCs (nestin, white arrows) or Müller glia (CRALBP or SOX2), but not in PAX6+, nestin(−) ganglion, amacrine, or horizontal cells (yellow arrows) 14 days post-transduction. k–l, EdU incorporation in cells expressing markers of cones (cone arrestin/CRX or RXRγ/CRX, yellow arrows in l) but not in cells expressing markers of rods (CNGA1, CNGB1), bipolar cells (CHX10/CRX), or ganglion, horizontal, or amacrine cells (syntaxin) (white arrows in l) 14 days after transduction. Black lines above labels demarcate distinct fields. m, Co-staining of phosphohistone H3 (PH3) with cone arrestin/CRX 23 days post-transduction. n, Apoptosis marker CC3 in cells expressing RPC and glial marker nestin 14 days after transduction with RB1-directed shRNAs (yellow arrow) but not with scrambled control (white arrow). Values and error bars are means and standard deviation of triplicate assays for all Extended Data figures. Scale bars, 20 μm. Data are representative of at least two independent experiments.
Mentions: Dissociated retinal cells were transduced with RB1-directed or control short hairpin RNAs (shRNAs), followed by co-staining for the proliferation-associated Ki67 and cell type-specific markers. RB1 shRNAs abrogated Rb expression in long or medium wavelength (L/M)-opsin+ and thyroid hormone receptor β2 (TRβ2)+ cone precursors as well as in other cell types (Extended Data Fig. 1a). After two weeks, Ki67 was detected in cone precursor-like cells co-expressing the photoreceptor marker CRX and the cone markers L/M-opsin, cone arrestin, and RXRγ (Fig. 1a, Extended Data Fig. 1b–h). Ki67+ cone marker+ cells were first detected 9 days after transduction whereas clusters were routinely detected by day 23. Ki67 was not detected in cells expressing markers of rods (NRL), bipolar cells (strong CHX10), ganglion cells (BRN-3), or amacrine or horizontal cells (PROX1+ or PAX6+, nestin(−)) (Fig. 1a, Extended Data Fig. 1i, j). Ki67 was detected in cells expressing markers of RPCs or Müller glia (nestin or CRALBP, SOX2), yet in similar proportions after shRB1 or control shRNA (Fig. 1a, Extended Data Fig. 1j). RB1 shRNAs also induced incorporation of 5-ethynyl-2′-deoxyuridine (EdU), an indicator of S phase entry, increased expression of the mitosis marker phosphohistone H3, suppressed expression of the apoptosis marker cleaved caspase 3 (CC3), and induced proliferation in cells expressing cone but not other retinal cell markers (Fig. 1c, d; Extended Data Fig. 1k–n). In contrast, RB1 shRNAs induced CC3 and decreased the number of cells expressing markers of RPCs and glia (Fig. 1b, d; Extended Data Fig. 1n).

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