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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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Effect of cone- and Rb-related circuitry on cone precursor response to Rb depletiona, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cells (a1), among RXRγ+, CRX+ cells (a2), or among cone arrestin+, CRX+ cells (a3); and percentage of L/M-opsin+, CRX+ cells among all cells with DAPI+ nuclei (a4) after transduction of dissociated FW18 retina with shRB1-733 and shRNAs against p130, p107, TRβ2, SKP2, MDM2, and MYCN. b, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cone-like cells (top) and proliferative response (bottom) after transduction of dissociated FW18 retina with shRB1-733 and with shRNAs against RXRγ and p27 (shRNAs 856+930), or with overexpression of p27 and p27-T187A. c, High-level Thr187 phosphorylated p27 (p27-T187-Ph, top) coinciding with down-regulation of total p27 (bottom) and prominent Rb during cone precursor maturation. c1, Perifoveal region of FW18 retina. c2, Enlarged view of boxed regions in c1. Arrows, cone precursors identified by large, strongly Rb+ nuclei and lack of p27 signal in characteristic outer nuclear layer (ONL) position7,16. d, Effect of two RBL1-p107 or two RBL2-p130 shRNAs on proliferation of Rb-depleted isolated cone precursors. e, Knockdown efficacy of two RBL1-p107 or two RBL2-p130 shRNAs in Y79 and RB177 retinoblastoma cells. f, Impaired proliferation of Weri-RB1 retinoblastoma cells after transduction with BN-p130 compared to vector control. g, Impaired proliferation of RB177 retinoblastoma cells following transduction with two p107 shRNAs. h, i, Impaired proliferation and MYCN expression in Y79 cells following p107 knockdown with two p107-directed shRNAs, and rescue by shRNA-resistant BN-p107 constructs. j, p27 accumulation and growth suppression following p107 knockdown with shp107-2 rescued by BN-p107-2r in RB1-wild type SKN-BE(2) neuroblastoma cells. p107 overexpression impaired SKN-BE(2) growth, contrary to its effects in Y79. Compared to SCR or Vector control: *, P < 0.01. #, P < 0.05. Compared to RB1-KD+SCR or RB1-KD+BN-Vector: $, P < 0.01; &, P < 0.05. Compared to shp107-2+BN-Vector: $, P < 0.01; &, P<0.05 (h and i). Data are representative of more than two independent experiments except for SKN-BE(2) analyses.
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Figure 9: Effect of cone- and Rb-related circuitry on cone precursor response to Rb depletiona, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cells (a1), among RXRγ+, CRX+ cells (a2), or among cone arrestin+, CRX+ cells (a3); and percentage of L/M-opsin+, CRX+ cells among all cells with DAPI+ nuclei (a4) after transduction of dissociated FW18 retina with shRB1-733 and shRNAs against p130, p107, TRβ2, SKP2, MDM2, and MYCN. b, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cone-like cells (top) and proliferative response (bottom) after transduction of dissociated FW18 retina with shRB1-733 and with shRNAs against RXRγ and p27 (shRNAs 856+930), or with overexpression of p27 and p27-T187A. c, High-level Thr187 phosphorylated p27 (p27-T187-Ph, top) coinciding with down-regulation of total p27 (bottom) and prominent Rb during cone precursor maturation. c1, Perifoveal region of FW18 retina. c2, Enlarged view of boxed regions in c1. Arrows, cone precursors identified by large, strongly Rb+ nuclei and lack of p27 signal in characteristic outer nuclear layer (ONL) position7,16. d, Effect of two RBL1-p107 or two RBL2-p130 shRNAs on proliferation of Rb-depleted isolated cone precursors. e, Knockdown efficacy of two RBL1-p107 or two RBL2-p130 shRNAs in Y79 and RB177 retinoblastoma cells. f, Impaired proliferation of Weri-RB1 retinoblastoma cells after transduction with BN-p130 compared to vector control. g, Impaired proliferation of RB177 retinoblastoma cells following transduction with two p107 shRNAs. h, i, Impaired proliferation and MYCN expression in Y79 cells following p107 knockdown with two p107-directed shRNAs, and rescue by shRNA-resistant BN-p107 constructs. j, p27 accumulation and growth suppression following p107 knockdown with shp107-2 rescued by BN-p107-2r in RB1-wild type SKN-BE(2) neuroblastoma cells. p107 overexpression impaired SKN-BE(2) growth, contrary to its effects in Y79. Compared to SCR or Vector control: *, P < 0.01. #, P < 0.05. Compared to RB1-KD+SCR or RB1-KD+BN-Vector: $, P < 0.01; &, P < 0.05. Compared to shp107-2+BN-Vector: $, P < 0.01; &, P<0.05 (h and i). Data are representative of more than two independent experiments except for SKN-BE(2) analyses.

Mentions: We next determined whether Rb-depleted cone precursors and retinoblastoma cells depend upon similar signaling circuitry. Retinoblastoma cell proliferation requires several proteins that are prominent in cone precursors, including TRβ2, RXRγ, MYCN, and MDM27. Depletion of these factors suppressed Ki67 expression and cone precursor proliferation both in dissociated retinal cultures (Extended Data Fig. 5a, b) and in isolated populations (Fig. 3a). Retinoblastoma cell proliferation also requires SKP2-mediated degradation of Thr187-phosphorylated p27 (Ref. 15). Concordantly, SKP2 depletion suppressed cone precursor proliferation and increased CC3 (Fig. 3a, Extended Data Fig. 5a). Notably, maturing cone precursors had exceptionally high Thr187-phosphorylated p27 (Extended Data Fig. 5c), coincident with a maturation-associated decrease in total p27 (Ref. 16), suggesting that SKP2-mediated p27 degradation might enable cone precursor proliferation. Consistent with this view, cone precursor proliferation was suppressed by ectopic p27 and enhanced by ectopic SKP2 or p27 knockdown (Fig. 3b, Extended Data Fig. 5b), as in retinoblastoma cells15. Thus, Rb-depleted cone precursors and retinoblastoma cells had similar signaling requirements.


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)

Effect of cone- and Rb-related circuitry on cone precursor response to Rb depletiona, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cells (a1), among RXRγ+, CRX+ cells (a2), or among cone arrestin+, CRX+ cells (a3); and percentage of L/M-opsin+, CRX+ cells among all cells with DAPI+ nuclei (a4) after transduction of dissociated FW18 retina with shRB1-733 and shRNAs against p130, p107, TRβ2, SKP2, MDM2, and MYCN. b, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cone-like cells (top) and proliferative response (bottom) after transduction of dissociated FW18 retina with shRB1-733 and with shRNAs against RXRγ and p27 (shRNAs 856+930), or with overexpression of p27 and p27-T187A. c, High-level Thr187 phosphorylated p27 (p27-T187-Ph, top) coinciding with down-regulation of total p27 (bottom) and prominent Rb during cone precursor maturation. c1, Perifoveal region of FW18 retina. c2, Enlarged view of boxed regions in c1. Arrows, cone precursors identified by large, strongly Rb+ nuclei and lack of p27 signal in characteristic outer nuclear layer (ONL) position7,16. d, Effect of two RBL1-p107 or two RBL2-p130 shRNAs on proliferation of Rb-depleted isolated cone precursors. e, Knockdown efficacy of two RBL1-p107 or two RBL2-p130 shRNAs in Y79 and RB177 retinoblastoma cells. f, Impaired proliferation of Weri-RB1 retinoblastoma cells after transduction with BN-p130 compared to vector control. g, Impaired proliferation of RB177 retinoblastoma cells following transduction with two p107 shRNAs. h, i, Impaired proliferation and MYCN expression in Y79 cells following p107 knockdown with two p107-directed shRNAs, and rescue by shRNA-resistant BN-p107 constructs. j, p27 accumulation and growth suppression following p107 knockdown with shp107-2 rescued by BN-p107-2r in RB1-wild type SKN-BE(2) neuroblastoma cells. p107 overexpression impaired SKN-BE(2) growth, contrary to its effects in Y79. Compared to SCR or Vector control: *, P < 0.01. #, P < 0.05. Compared to RB1-KD+SCR or RB1-KD+BN-Vector: $, P < 0.01; &, P < 0.05. Compared to shp107-2+BN-Vector: $, P < 0.01; &, P<0.05 (h and i). Data are representative of more than two independent experiments except for SKN-BE(2) analyses.
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Figure 9: Effect of cone- and Rb-related circuitry on cone precursor response to Rb depletiona, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cells (a1), among RXRγ+, CRX+ cells (a2), or among cone arrestin+, CRX+ cells (a3); and percentage of L/M-opsin+, CRX+ cells among all cells with DAPI+ nuclei (a4) after transduction of dissociated FW18 retina with shRB1-733 and shRNAs against p130, p107, TRβ2, SKP2, MDM2, and MYCN. b, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cone-like cells (top) and proliferative response (bottom) after transduction of dissociated FW18 retina with shRB1-733 and with shRNAs against RXRγ and p27 (shRNAs 856+930), or with overexpression of p27 and p27-T187A. c, High-level Thr187 phosphorylated p27 (p27-T187-Ph, top) coinciding with down-regulation of total p27 (bottom) and prominent Rb during cone precursor maturation. c1, Perifoveal region of FW18 retina. c2, Enlarged view of boxed regions in c1. Arrows, cone precursors identified by large, strongly Rb+ nuclei and lack of p27 signal in characteristic outer nuclear layer (ONL) position7,16. d, Effect of two RBL1-p107 or two RBL2-p130 shRNAs on proliferation of Rb-depleted isolated cone precursors. e, Knockdown efficacy of two RBL1-p107 or two RBL2-p130 shRNAs in Y79 and RB177 retinoblastoma cells. f, Impaired proliferation of Weri-RB1 retinoblastoma cells after transduction with BN-p130 compared to vector control. g, Impaired proliferation of RB177 retinoblastoma cells following transduction with two p107 shRNAs. h, i, Impaired proliferation and MYCN expression in Y79 cells following p107 knockdown with two p107-directed shRNAs, and rescue by shRNA-resistant BN-p107 constructs. j, p27 accumulation and growth suppression following p107 knockdown with shp107-2 rescued by BN-p107-2r in RB1-wild type SKN-BE(2) neuroblastoma cells. p107 overexpression impaired SKN-BE(2) growth, contrary to its effects in Y79. Compared to SCR or Vector control: *, P < 0.01. #, P < 0.05. Compared to RB1-KD+SCR or RB1-KD+BN-Vector: $, P < 0.01; &, P < 0.05. Compared to shp107-2+BN-Vector: $, P < 0.01; &, P<0.05 (h and i). Data are representative of more than two independent experiments except for SKN-BE(2) analyses.
Mentions: We next determined whether Rb-depleted cone precursors and retinoblastoma cells depend upon similar signaling circuitry. Retinoblastoma cell proliferation requires several proteins that are prominent in cone precursors, including TRβ2, RXRγ, MYCN, and MDM27. Depletion of these factors suppressed Ki67 expression and cone precursor proliferation both in dissociated retinal cultures (Extended Data Fig. 5a, b) and in isolated populations (Fig. 3a). Retinoblastoma cell proliferation also requires SKP2-mediated degradation of Thr187-phosphorylated p27 (Ref. 15). Concordantly, SKP2 depletion suppressed cone precursor proliferation and increased CC3 (Fig. 3a, Extended Data Fig. 5a). Notably, maturing cone precursors had exceptionally high Thr187-phosphorylated p27 (Extended Data Fig. 5c), coincident with a maturation-associated decrease in total p27 (Ref. 16), suggesting that SKP2-mediated p27 degradation might enable cone precursor proliferation. Consistent with this view, cone precursor proliferation was suppressed by ectopic p27 and enhanced by ectopic SKP2 or p27 knockdown (Fig. 3b, Extended Data Fig. 5b), as in retinoblastoma cells15. Thus, Rb-depleted cone precursors and retinoblastoma cells had similar signaling requirements.

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