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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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FACS isolation of retinal cell populationsRetinal cells were isolated according to size, CD133, and CD44 staining. In Study 1, cell type compositions in each fraction (a) were determined by immunostaining with cone arrestin/CRX (b, e), NRL (c), and nestin/PAX6 (d, f). In study 2, cell type compositions (i) were determined by immunostaining with RXRγ/CRX, nestin/CHX10, nestin/PAX6, and CRALBP (j, k). The percentages of the predominant cell types in each population (a, i) and marker specificities (g) are indicated. h, Cone-specific co-staining of cone arrestin and GNAT2 (top) and cone-specific co-staining of RXRγ and CRX (bottom) in FW19 retina. l, Co-staining of cells for EdU and cone arrestin/CRX or RXRγ/CRX 14 days after transduction of the cone-enriched medium plus large CD133hi, CD44(−) population isolated as in (i–k) with two RB1 shRNAs (yellow arrows) but not with the scrambled control (white arrows). In both studies, CD133hi, CD44(−) medium and large size populations mainly consisted of cells expressing cone markers (CRX/cone arrestin or CRX/RXRγ). The CD133hi, CD44(−)small population mainly consisted of cells expressing a rod marker (NRL) with a variable proportion expressing cone markers. All CD133lo, CD44+ populations mainly consisted of cells co-expressing RPC and glial markers (nestin/PAX6, nestin/CHX10, or CRALBP). The CD133lo, CD44(−) small size population consisted of cells with diverse immunophenotypes. Scale bars, 30 μm.
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Figure 6: FACS isolation of retinal cell populationsRetinal cells were isolated according to size, CD133, and CD44 staining. In Study 1, cell type compositions in each fraction (a) were determined by immunostaining with cone arrestin/CRX (b, e), NRL (c), and nestin/PAX6 (d, f). In study 2, cell type compositions (i) were determined by immunostaining with RXRγ/CRX, nestin/CHX10, nestin/PAX6, and CRALBP (j, k). The percentages of the predominant cell types in each population (a, i) and marker specificities (g) are indicated. h, Cone-specific co-staining of cone arrestin and GNAT2 (top) and cone-specific co-staining of RXRγ and CRX (bottom) in FW19 retina. l, Co-staining of cells for EdU and cone arrestin/CRX or RXRγ/CRX 14 days after transduction of the cone-enriched medium plus large CD133hi, CD44(−) population isolated as in (i–k) with two RB1 shRNAs (yellow arrows) but not with the scrambled control (white arrows). In both studies, CD133hi, CD44(−) medium and large size populations mainly consisted of cells expressing cone markers (CRX/cone arrestin or CRX/RXRγ). The CD133hi, CD44(−)small population mainly consisted of cells expressing a rod marker (NRL) with a variable proportion expressing cone markers. All CD133lo, CD44+ populations mainly consisted of cells co-expressing RPC and glial markers (nestin/PAX6, nestin/CHX10, or CRALBP). The CD133lo, CD44(−) small size population consisted of cells with diverse immunophenotypes. Scale bars, 30 μm.

Mentions: To assess whether the Rb-deficient proliferating cone-like cells derived from post-mitotic cone precursors, we examined effects of Rb knockdown in prospectively isolated retinal cell populations. Populations were isolated by sorting for size, for CD133, which is expressed strongly in maturing photoreceptors and weakly in RPCs11, and for a CD44 epitope expressed by Müller glia and RPCs12 (Fig. 2a). Staining for cell type-specific markers revealed populations enriched for cone precursors, for rod plus cone precursors, for RPCs plus glia, and for a mixture of rod, ganglion, bipolar, amacrine, and horizontal cells (Fig. 2b, Extended Data Fig. 2a–g). In medium and large CD133hi, CD44(−) populations, 96–98% of cells co-stained for CRX and cone arrestin, which is cone-specific at FW 19 (Extended Data Fig. 2h). A similar enrichment was observed when cone precursors were identified using CRX and RXRγ (Extended Data Fig. 2h–k).


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)

FACS isolation of retinal cell populationsRetinal cells were isolated according to size, CD133, and CD44 staining. In Study 1, cell type compositions in each fraction (a) were determined by immunostaining with cone arrestin/CRX (b, e), NRL (c), and nestin/PAX6 (d, f). In study 2, cell type compositions (i) were determined by immunostaining with RXRγ/CRX, nestin/CHX10, nestin/PAX6, and CRALBP (j, k). The percentages of the predominant cell types in each population (a, i) and marker specificities (g) are indicated. h, Cone-specific co-staining of cone arrestin and GNAT2 (top) and cone-specific co-staining of RXRγ and CRX (bottom) in FW19 retina. l, Co-staining of cells for EdU and cone arrestin/CRX or RXRγ/CRX 14 days after transduction of the cone-enriched medium plus large CD133hi, CD44(−) population isolated as in (i–k) with two RB1 shRNAs (yellow arrows) but not with the scrambled control (white arrows). In both studies, CD133hi, CD44(−) medium and large size populations mainly consisted of cells expressing cone markers (CRX/cone arrestin or CRX/RXRγ). The CD133hi, CD44(−)small population mainly consisted of cells expressing a rod marker (NRL) with a variable proportion expressing cone markers. All CD133lo, CD44+ populations mainly consisted of cells co-expressing RPC and glial markers (nestin/PAX6, nestin/CHX10, or CRALBP). The CD133lo, CD44(−) small size population consisted of cells with diverse immunophenotypes. Scale bars, 30 μm.
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Related In: Results  -  Collection

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Figure 6: FACS isolation of retinal cell populationsRetinal cells were isolated according to size, CD133, and CD44 staining. In Study 1, cell type compositions in each fraction (a) were determined by immunostaining with cone arrestin/CRX (b, e), NRL (c), and nestin/PAX6 (d, f). In study 2, cell type compositions (i) were determined by immunostaining with RXRγ/CRX, nestin/CHX10, nestin/PAX6, and CRALBP (j, k). The percentages of the predominant cell types in each population (a, i) and marker specificities (g) are indicated. h, Cone-specific co-staining of cone arrestin and GNAT2 (top) and cone-specific co-staining of RXRγ and CRX (bottom) in FW19 retina. l, Co-staining of cells for EdU and cone arrestin/CRX or RXRγ/CRX 14 days after transduction of the cone-enriched medium plus large CD133hi, CD44(−) population isolated as in (i–k) with two RB1 shRNAs (yellow arrows) but not with the scrambled control (white arrows). In both studies, CD133hi, CD44(−) medium and large size populations mainly consisted of cells expressing cone markers (CRX/cone arrestin or CRX/RXRγ). The CD133hi, CD44(−)small population mainly consisted of cells expressing a rod marker (NRL) with a variable proportion expressing cone markers. All CD133lo, CD44+ populations mainly consisted of cells co-expressing RPC and glial markers (nestin/PAX6, nestin/CHX10, or CRALBP). The CD133lo, CD44(−) small size population consisted of cells with diverse immunophenotypes. Scale bars, 30 μm.
Mentions: To assess whether the Rb-deficient proliferating cone-like cells derived from post-mitotic cone precursors, we examined effects of Rb knockdown in prospectively isolated retinal cell populations. Populations were isolated by sorting for size, for CD133, which is expressed strongly in maturing photoreceptors and weakly in RPCs11, and for a CD44 epitope expressed by Müller glia and RPCs12 (Fig. 2a). Staining for cell type-specific markers revealed populations enriched for cone precursors, for rod plus cone precursors, for RPCs plus glia, and for a mixture of rod, ganglion, bipolar, amacrine, and horizontal cells (Fig. 2b, Extended Data Fig. 2a–g). In medium and large CD133hi, CD44(−) populations, 96–98% of cells co-stained for CRX and cone arrestin, which is cone-specific at FW 19 (Extended Data Fig. 2h). A similar enrichment was observed when cone precursors were identified using CRX and RXRγ (Extended Data Fig. 2h–k).

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