Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of Rb- and Rb/p130-depleted cone precursor tumorsa, Intraocular tumor four months after Rb-depleted cone precursor xenograft. b, Summary of subretinal xenograft Groups 1, 2, 3. Sample size was as needed to assess tumor phenotypes. Mice were randomly assigned to different xenograft regimens and the investigator blinded to the assignment until the tumor analyses. Two mice with early death were excluded from the analyses. c, SNP-array analysis of one Rb/p130-depleted (tumor 1) or one Rb-depleted (tumor 2) cone precursor-derived tumors from xenograft Group 3, revealing no megabase-size LOH or CNAs. d, qPCR analysis of pLKO shRNA vector copy number in tumors derived from Rb/p130-depleted cone precursors (m-Cone1, m-Cone2) or from Rb/p130-depleted unsorted retinal cells (m-All3, m-All4), or in mouse ocular tissue (m-Cone-SCR), Y79 cells, or FW19 retina (Normal). All tumors retained shRB1 and/or shp130 vector sequences, confirming their engineered cone precursor origin. e, qPCR analysis of MDM2, MDM4, RB1, and MYCN copy number in three cone-derived tumors and normal retina (n=6). DNA copy number data (d, e) are representative of two analyses.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232224&req=5

Figure 12: Characterization of Rb- and Rb/p130-depleted cone precursor tumorsa, Intraocular tumor four months after Rb-depleted cone precursor xenograft. b, Summary of subretinal xenograft Groups 1, 2, 3. Sample size was as needed to assess tumor phenotypes. Mice were randomly assigned to different xenograft regimens and the investigator blinded to the assignment until the tumor analyses. Two mice with early death were excluded from the analyses. c, SNP-array analysis of one Rb/p130-depleted (tumor 1) or one Rb-depleted (tumor 2) cone precursor-derived tumors from xenograft Group 3, revealing no megabase-size LOH or CNAs. d, qPCR analysis of pLKO shRNA vector copy number in tumors derived from Rb/p130-depleted cone precursors (m-Cone1, m-Cone2) or from Rb/p130-depleted unsorted retinal cells (m-All3, m-All4), or in mouse ocular tissue (m-Cone-SCR), Y79 cells, or FW19 retina (Normal). All tumors retained shRB1 and/or shp130 vector sequences, confirming their engineered cone precursor origin. e, qPCR analysis of MDM2, MDM4, RB1, and MYCN copy number in three cone-derived tumors and normal retina (n=6). DNA copy number data (d, e) are representative of two analyses.

Mentions: After several months, some Rb- or Rb/p130-depleted cone precursor cultures formed suspension aggregates resembling retinoblastoma cells (Extended Data Fig. 7a). Rb/p130-depleted cultures proliferated more robustly and longer than those with Rb depletion alone, consistent with p130 losses in many retinoblastoma cell lines (Extended Data Fig. 6a). The cultures had properties consistent with Rb/p130-depleted cone precursors (Extended Data Fig. 7b–h). When engrafted either 3 months or within one week after knockdown, Rb- or Rb/p130-depleted cone precursors formed retinoblastoma-like tumors in subretinal xenografts (Fig. 4a; Extended Data Fig. 8, 9). For cells engrafted within one week, tumors appeared within 6–14 months (Extended Data Fig. 8b), similar to the time needed to form tumors in children.


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)

Characterization of Rb- and Rb/p130-depleted cone precursor tumorsa, Intraocular tumor four months after Rb-depleted cone precursor xenograft. b, Summary of subretinal xenograft Groups 1, 2, 3. Sample size was as needed to assess tumor phenotypes. Mice were randomly assigned to different xenograft regimens and the investigator blinded to the assignment until the tumor analyses. Two mice with early death were excluded from the analyses. c, SNP-array analysis of one Rb/p130-depleted (tumor 1) or one Rb-depleted (tumor 2) cone precursor-derived tumors from xenograft Group 3, revealing no megabase-size LOH or CNAs. d, qPCR analysis of pLKO shRNA vector copy number in tumors derived from Rb/p130-depleted cone precursors (m-Cone1, m-Cone2) or from Rb/p130-depleted unsorted retinal cells (m-All3, m-All4), or in mouse ocular tissue (m-Cone-SCR), Y79 cells, or FW19 retina (Normal). All tumors retained shRB1 and/or shp130 vector sequences, confirming their engineered cone precursor origin. e, qPCR analysis of MDM2, MDM4, RB1, and MYCN copy number in three cone-derived tumors and normal retina (n=6). DNA copy number data (d, e) are representative of two analyses.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Characterization of Rb- and Rb/p130-depleted cone precursor tumorsa, Intraocular tumor four months after Rb-depleted cone precursor xenograft. b, Summary of subretinal xenograft Groups 1, 2, 3. Sample size was as needed to assess tumor phenotypes. Mice were randomly assigned to different xenograft regimens and the investigator blinded to the assignment until the tumor analyses. Two mice with early death were excluded from the analyses. c, SNP-array analysis of one Rb/p130-depleted (tumor 1) or one Rb-depleted (tumor 2) cone precursor-derived tumors from xenograft Group 3, revealing no megabase-size LOH or CNAs. d, qPCR analysis of pLKO shRNA vector copy number in tumors derived from Rb/p130-depleted cone precursors (m-Cone1, m-Cone2) or from Rb/p130-depleted unsorted retinal cells (m-All3, m-All4), or in mouse ocular tissue (m-Cone-SCR), Y79 cells, or FW19 retina (Normal). All tumors retained shRB1 and/or shp130 vector sequences, confirming their engineered cone precursor origin. e, qPCR analysis of MDM2, MDM4, RB1, and MYCN copy number in three cone-derived tumors and normal retina (n=6). DNA copy number data (d, e) are representative of two analyses.
Mentions: After several months, some Rb- or Rb/p130-depleted cone precursor cultures formed suspension aggregates resembling retinoblastoma cells (Extended Data Fig. 7a). Rb/p130-depleted cultures proliferated more robustly and longer than those with Rb depletion alone, consistent with p130 losses in many retinoblastoma cell lines (Extended Data Fig. 6a). The cultures had properties consistent with Rb/p130-depleted cone precursors (Extended Data Fig. 7b–h). When engrafted either 3 months or within one week after knockdown, Rb- or Rb/p130-depleted cone precursors formed retinoblastoma-like tumors in subretinal xenografts (Fig. 4a; Extended Data Fig. 8, 9). For cells engrafted within one week, tumors appeared within 6–14 months (Extended Data Fig. 8b), similar to the time needed to form tumors in children.

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