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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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Cone precursor gene expression response to Rb depletiona–b, Fold change in RNA level relative to day 0 uninfected cells for (a) RB1, or (b) the indicated E2F-responsive genes, or (c) the indicated p53-regulated genes, 3 and 6 days after transduction of each population with a mixture of shRB1-733 and shRB1-737, or with scrambled control. Comparing shRB1 and scrambled control: *, p < 0.01; #, p < 0.05. Data are representative of two sets of qPCR analyses.
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Figure 7: Cone precursor gene expression response to Rb depletiona–b, Fold change in RNA level relative to day 0 uninfected cells for (a) RB1, or (b) the indicated E2F-responsive genes, or (c) the indicated p53-regulated genes, 3 and 6 days after transduction of each population with a mixture of shRB1-733 and shRB1-737, or with scrambled control. Comparing shRB1 and scrambled control: *, p < 0.01; #, p < 0.05. Data are representative of two sets of qPCR analyses.

Mentions: RB1 shRNAs induced similar RB1 knockdown in each retinal cell population (Extended Data Fig. 3a). After two weeks, Ki67 was detected in 80% of cells in the cone-enriched population (Fig. 2c), likely reflecting a high ratio of shRNA-expressing lentivirus to target cells and cone precursor proliferation. After three weeks, cone precursor numbers increased (Fig. 2c). Rb depletion did not induce proliferation in RPCs and glia, but increased the proportion of CC3+ cells entering apoptosis (Fig. 2c). Sorted populations transduced with the scrambled control had higher CC3+ rates than unsorted cultures, potentially reflecting separation of RPCs and glia from neurons13,14. Nevertheless, Rb knockdown induced proliferation and apoptosis in cells with the same immunophenotypes as in unsorted cultures. Notably, Rb depletion induced the cell cycle-related genes CCNE1, SKP2, E2F1, RBL1, CCNB1, and CDK1 in cone precursors and induced p53-responsive genes in sorted RPCs and glia (Extended Data Fig. 3). Cell cycle-related genes were induced several days before Ki67, suggesting that further reprogramming was needed for cell cycle entry.


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)

Cone precursor gene expression response to Rb depletiona–b, Fold change in RNA level relative to day 0 uninfected cells for (a) RB1, or (b) the indicated E2F-responsive genes, or (c) the indicated p53-regulated genes, 3 and 6 days after transduction of each population with a mixture of shRB1-733 and shRB1-737, or with scrambled control. Comparing shRB1 and scrambled control: *, p < 0.01; #, p < 0.05. Data are representative of two sets of qPCR analyses.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Cone precursor gene expression response to Rb depletiona–b, Fold change in RNA level relative to day 0 uninfected cells for (a) RB1, or (b) the indicated E2F-responsive genes, or (c) the indicated p53-regulated genes, 3 and 6 days after transduction of each population with a mixture of shRB1-733 and shRB1-737, or with scrambled control. Comparing shRB1 and scrambled control: *, p < 0.01; #, p < 0.05. Data are representative of two sets of qPCR analyses.
Mentions: RB1 shRNAs induced similar RB1 knockdown in each retinal cell population (Extended Data Fig. 3a). After two weeks, Ki67 was detected in 80% of cells in the cone-enriched population (Fig. 2c), likely reflecting a high ratio of shRNA-expressing lentivirus to target cells and cone precursor proliferation. After three weeks, cone precursor numbers increased (Fig. 2c). Rb depletion did not induce proliferation in RPCs and glia, but increased the proportion of CC3+ cells entering apoptosis (Fig. 2c). Sorted populations transduced with the scrambled control had higher CC3+ rates than unsorted cultures, potentially reflecting separation of RPCs and glia from neurons13,14. Nevertheless, Rb knockdown induced proliferation and apoptosis in cells with the same immunophenotypes as in unsorted cultures. Notably, Rb depletion induced the cell cycle-related genes CCNE1, SKP2, E2F1, RBL1, CCNB1, and CDK1 in cone precursors and induced p53-responsive genes in sorted RPCs and glia (Extended Data Fig. 3). Cell cycle-related genes were induced several days before Ki67, suggesting that further reprogramming was needed for cell cycle entry.

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