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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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Rb- or Rb/p130-depleted cone precursor tumorsa, Hematoxylin and eosin-stained Rb- and Rb/p130-depleted cone xenograft tumors and human retinoblastoma (n=4). Dashed lines, Flexner-Wintersteiner rosettes. Solid lines, fleurettes. *, rosette cavity. b, Cone and cell cycle related protein expression in human retinoblastoma and cone xenografts (n=6). Scale bars, 40 μm (a,b). c, Transmission electron microscopy of Flexner-Wintersteiner rosettes in human retinoblastoma and a cone-derived tumor, with mitochondria (arrows) between nuclei and rosette cavity (n=2). 25,000X image from boxed area (top). Results are representative of at least two experiments. d, Model of cone-precursor retinoblastoma origin highlighting proteins that suppressed (blue) or promoted (red) the proliferative response.
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Figure 4: Rb- or Rb/p130-depleted cone precursor tumorsa, Hematoxylin and eosin-stained Rb- and Rb/p130-depleted cone xenograft tumors and human retinoblastoma (n=4). Dashed lines, Flexner-Wintersteiner rosettes. Solid lines, fleurettes. *, rosette cavity. b, Cone and cell cycle related protein expression in human retinoblastoma and cone xenografts (n=6). Scale bars, 40 μm (a,b). c, Transmission electron microscopy of Flexner-Wintersteiner rosettes in human retinoblastoma and a cone-derived tumor, with mitochondria (arrows) between nuclei and rosette cavity (n=2). 25,000X image from boxed area (top). Results are representative of at least two experiments. d, Model of cone-precursor retinoblastoma origin highlighting proteins that suppressed (blue) or promoted (red) the proliferative response.

Mentions: We also assessed roles of the Rb-related p107 and p130. In mouse models, retinal tumorigenesis required loss of Rb combined with loss of p107, p130, or p27 (Refs. 10, 17). However, in human retinoblastomas, p130/RBL2 losses are common, whereas p107/RBL1 losses are rare18 (Extended Data Fig. 6a). Moreover, whereas maturing cone precursors had abundant p130 and minimal p107, retinoblastomas had barely detectable p130 yet prominent p107 (Fig. 4b, Extended Data Fig. 6b), implicating p130 but not p107 in retinoblastoma suppression. Concordantly, co-knockdown of p130 with Rb increased cone precursor proliferation (Fig. 3a, Extended Data Fig. 5a, d) and p130 overexpression suppressed cone precursor and retinoblastoma cell proliferation (Fig. 3b, c, e). Meanwhile, p107 knockdown suppressed proliferation both in Rb-depleted cone precursors (Fig. 3a; Extended Data Fig. 5a, d) and in retinoblastoma cells (Fig. 3d–e; Extended Data Fig. 5e,g). In Y79, p107 knockdown decreased expression of MYCN and SKP2, while increasing the SKP2 target, p27 (Fig. 3e). These effects were seen with two shRNAs and were rescued by p107 restoration (Fig 3d,e, Extended Data Fig. 5d–i). Furthermore, p107 overexpression enhanced proliferation of retinoblastoma cells while suppressing that of neuroblastoma cells (Extended Data Fig. 5h–j). Thus, both in Rb-depleted cone precursors and in retinoblastoma cells, p130 suppressed proliferation whereas p107 had a proliferative role distinct from its function in mouse models.


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)

Rb- or Rb/p130-depleted cone precursor tumorsa, Hematoxylin and eosin-stained Rb- and Rb/p130-depleted cone xenograft tumors and human retinoblastoma (n=4). Dashed lines, Flexner-Wintersteiner rosettes. Solid lines, fleurettes. *, rosette cavity. b, Cone and cell cycle related protein expression in human retinoblastoma and cone xenografts (n=6). Scale bars, 40 μm (a,b). c, Transmission electron microscopy of Flexner-Wintersteiner rosettes in human retinoblastoma and a cone-derived tumor, with mitochondria (arrows) between nuclei and rosette cavity (n=2). 25,000X image from boxed area (top). Results are representative of at least two experiments. d, Model of cone-precursor retinoblastoma origin highlighting proteins that suppressed (blue) or promoted (red) the proliferative response.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232224&req=5

Figure 4: Rb- or Rb/p130-depleted cone precursor tumorsa, Hematoxylin and eosin-stained Rb- and Rb/p130-depleted cone xenograft tumors and human retinoblastoma (n=4). Dashed lines, Flexner-Wintersteiner rosettes. Solid lines, fleurettes. *, rosette cavity. b, Cone and cell cycle related protein expression in human retinoblastoma and cone xenografts (n=6). Scale bars, 40 μm (a,b). c, Transmission electron microscopy of Flexner-Wintersteiner rosettes in human retinoblastoma and a cone-derived tumor, with mitochondria (arrows) between nuclei and rosette cavity (n=2). 25,000X image from boxed area (top). Results are representative of at least two experiments. d, Model of cone-precursor retinoblastoma origin highlighting proteins that suppressed (blue) or promoted (red) the proliferative response.
Mentions: We also assessed roles of the Rb-related p107 and p130. In mouse models, retinal tumorigenesis required loss of Rb combined with loss of p107, p130, or p27 (Refs. 10, 17). However, in human retinoblastomas, p130/RBL2 losses are common, whereas p107/RBL1 losses are rare18 (Extended Data Fig. 6a). Moreover, whereas maturing cone precursors had abundant p130 and minimal p107, retinoblastomas had barely detectable p130 yet prominent p107 (Fig. 4b, Extended Data Fig. 6b), implicating p130 but not p107 in retinoblastoma suppression. Concordantly, co-knockdown of p130 with Rb increased cone precursor proliferation (Fig. 3a, Extended Data Fig. 5a, d) and p130 overexpression suppressed cone precursor and retinoblastoma cell proliferation (Fig. 3b, c, e). Meanwhile, p107 knockdown suppressed proliferation both in Rb-depleted cone precursors (Fig. 3a; Extended Data Fig. 5a, d) and in retinoblastoma cells (Fig. 3d–e; Extended Data Fig. 5e,g). In Y79, p107 knockdown decreased expression of MYCN and SKP2, while increasing the SKP2 target, p27 (Fig. 3e). These effects were seen with two shRNAs and were rescued by p107 restoration (Fig 3d,e, Extended Data Fig. 5d–i). Furthermore, p107 overexpression enhanced proliferation of retinoblastoma cells while suppressing that of neuroblastoma cells (Extended Data Fig. 5h–j). Thus, both in Rb-depleted cone precursors and in retinoblastoma cells, p130 suppressed proliferation whereas p107 had a proliferative role distinct from its function in mouse models.

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