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 cancers1, 2, 3, 4, 5, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies6. 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 unclear7, 8. 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.
At a glance
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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Proliferation of cone-like cells after Rb depletion in dissociated FW19 retina. (716 KB)
a, Decreased Rb protein in L/M-opsin+ or TRβ2+ cells (arrows) on days 5 or 23, and decreased RB1 RNA or Rb protein on day 4 after shRB1-733 transduction. b, Cone arrestin+, CRX+ cells (arrows) with or without Ki67 co-expression. c, Ki67+ and cone arrestin+ cells first detected 9 or 14 days after transduction in two experiments. d–f, Co-staining of Ki67 with RXRγ and CRX at 14 days (d), with cone arrestin and CRX at 14 days (e), or with L/M-opsin and CRX at 23 days (f) after transduction with shRB1-733 or a scrambled control. g, Percentage of cells co-expressing Ki67 with L/M-opsin and CRX, RXRγ and CRX, or cone arrestin and CRX, 23 days after transduction. h, Prevalence of cells co-staining for L/M-opsin and CRX, RXRγ and CRX, or cone arrestin and CRX, 23 days after transduction. i, Ki67 not detected in cells expressing markers of rods (NRL), ganglion cells (BRN-3), bipolar cells (strong CHX10), or horizontal cells (PROX1) 14 days after transduction. j, Co-expression of Ki67 with markers of RPCs (nestin, white arrows) or Müller glia (CRALBP or SOX2), but not in PAX6+, nestin− ganglion, amacrine or horizontal cells (yellow arrows) 14 days after transduction. k, l, EdU incorporation in cells expressing markers of cones (cone arrestin and CRX or RXRγ and CRX, yellow arrows in l) but not in cells expressing markers of rods (CNGA1, CNGB1), bipolar cells (CHX10, CRX), or ganglion, horizontal or amacrine cells (syntaxin) (white arrows in l) 14 days after transduction. Black lines above labels demarcate distinct fields. m, Co-staining of phosphohistone H3 (PH3) with cone arrestin and CRX 23 days after transduction. n, Apoptosis marker CC3 in cells expressing RPC and glial marker nestin 14 days after transduction with RB1-directed shRNAs (yellow arrow) but not with scrambled control (white arrow). Values and error bars are mean and s.d. of triplicate assays. Scale bars, 20 μm. Data are representative of at least two independent experiments.
- Extended Data Figure 2: FACS isolation of retinal cell populations. (548 KB)
Retinal 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 and CRX (b, e), NRL (c), and nestin and PAX6 (d, f). In study 2, cell type compositions (i) were determined by immunostaining with RXRγ and CRX, nestin and CHX10, nestin and 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. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. l, Co-staining of cells for EdU with cone arrestin and CRX or with RXRγ and 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 and cone arrestin, or CRX and 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 and PAX6, nestin and CHX10, or CRALBP). The CD133lo CD44− small size population consisted of cells with diverse immunophenotypes. Values and error bars are mean and s.d. of triplicate assays. Scale bars, 30 μm.
- Extended Data Figure 3: Cone precursor gene expression response to Rb depletion. (258 KB)
a–c, Fold change in RNA level relative to day 0 uninfected cells for RB1 (a), or the indicated E2F-responsive genes (b), or the indicated p53-regulated genes (c), 3 and 6 days after transduction of each population with a mixture of shRB1-733 and shRB1-737, or with scrambled control. *P < 0.05, **P < 0.01 (comparing shRB1 and scrambled control). Data are representative of two sets of qPCR analyses. Values and error bars are mean and s.d. of triplicate assays.
- Extended Data Figure 4: Proliferation status of retinal cells other than cones 15 days after shRB1 transduction of intact FW19 retinas. (983 KB)
a–d, Combined transduction with pLKO-shRB1-733 and -737. a, Ki67 not detected in NRL+ or rhodopsin+ rod photoreceptors or in calbindin+ horizontal cells. b, Ki67 detected in PAX6lo, nestin+ RPCs (white arrows) but not in PAX6hi, nestin− horizontal, amacrine or ganglion cells (yellow arrows). c, Ki67 detected in CHX10+, CRX− RPCs (white arrows) but not in CHX10+, CRX+ bipolar cells (yellow arrows). d, Percentage of cells co-expressing Ki67 and retinal cell markers. e–h, Transduction with YFP-marked pLKO-YFP-shRB1-733. e, Ki67 detected in YFP+, L/M-opsin+ or YFP+, cone arrestin+ cone precursors (white arrows) and in an undefined YFP− cell (yellow arrow). f, Ki67 not detected in YFP+, calbindin+ horizontal cells, YFP+, syntaxin+ or YFP+, PAX6+ amacrine cells, or in YFP+, NRL+ rod precursors. g, Ki67 detected (white arrows) or not detected (yellow arrows) in YFP+, nestin+ RPCs or glia, or in YFP+, CHX10+ RPCs or bipolar cells. h, Proportion of Ki67+ cells co-expressing YFP and retinal markers after transduction with pLKO-YFP-shRB1-733 or scrambled control. Values and error bars are mean and s.d. of triplicate assays. Scale bars, 20 μm. Analyses in a–d and in e–h represent two independent experiments. All immunostaining was performed at least twice.
- Extended Data Figure 5: Effect of cone- and Rb-related circuitry on cone precursor response to Rb depletion. (611 KB)
A, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cells (a), among RXRγ+, CRX+ cells (b), or among cone arrestin+, CRX+cells (c); and percentage of L/M-opsin+, CRX+ cells among all cells with DAPI+ nuclei (d) 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 Thr 187 phosphorylated p27 (p-p27(T187), top) coinciding with downregulation of total p27 (bottom) and prominent Rb during cone precursor maturation. C, a, Perifoveal region of FW18 retina. C, b, Enlarged view of boxed regions in C, a. Arrows, cone precursors identified by large, strongly Rb+ nuclei and lack of p27 signal in characteristic outer nuclear layer 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 after 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. *P < 0.05, **P < 0.01 (compared to SCR or vector control); †P < 0.05, ††P < 0.01 (compared to RB1-KD plus SCR or RB1-KD plus BN vector); ‡P < 0.05, ‡‡P < 0.01 (compared to shp107-2 plus BN vector) (H–J). Data are representative of more than two independent experiments except for SKN-BE(2) analyses. Values and error bars are mean and s.d. of triplicate assays.
- Extended Data Figure 6: p130 copy number in retinoblastomas and cone precursor expression. (502 KB)
a, DNA copy number of p130, other 16q genes implicated in retinoblastoma (CDH11, CDH13), and p107 determined by qPCR (n = 6). The percentage of retinoblastomas with copy number (CN) < 1.5 was higher for p130 than for other 16q genes (summarized at right; P values relative to p130 using Fisher’s exact test). b, p130 in peripheral, lateral and central FW19 retina. Boxed region in maturing central retina (top) and enlarged view (bottom) show prominent p130 in weakly DAPI-stained cone precursor nuclei (arrows). Scale bars, 40 μm. Data are representative of at least two independent experiments. Values and error bars are mean and s.d. of triplicate assays.
- Extended Data Figure 7: Characterization of Rb/p130-depleted retinoblastoma-like cells. (422 KB)
a, Similar appearance of Rb/p130-depleted cones and early passage retinoblastoma cells. Scale bar, 40 μm. b, c, DNA copy number of shRNA vectors (b) or selected genes (c) in cell lines derived from Rb/p130-depleted cone precursors (Cone1, Cone2, Cone5) or from Rb/p130-depleted unsorted retinal cells (All3, All4), in Rb-depleted unsorted retinal cells 4 days after transduction (All-RB1-KD-d4), in Y79 cells, or in FW21 retina (normal) (n = 6). All cell lines retained RB1 and p130 shRNA vectors and lacked RB1 or p130 copy number alterations. The Y79 MYCN copy number (~78) is not shown (asterisk). d–g, qPCR gene expression analyses in the indicated cell lines relative to cones transduced with scrambled control or FW21 retina (n = 6). d, All cell lines had diminished RB1 and p130 expression. e–g, Altered expression of cell-cycle-related (e), cone-related (f) and apoptosis-related (g) genes. h, SNP-array analysis of two Rb/p130-depleted cone precursor cell lines (1, 2), revealing no megabase-size loss of heterozygosity (LOH) or copy number alterations (CNA). Data are representative of at least two analyses (b–g) or analyses of two cell lines (h). Values and error bars are mean and s.d. of triplicate assays.
- Extended Data Figure 8: Characterization of Rb- and Rb/p130-depleted cone precursor tumours. (340 KB)
a, Intraocular tumour 4 months after Rb-depleted cone precursor xenograft. b, Summary of subretinal xenograft groups 1, 2 and 3. Sample size was as needed to assess tumour phenotypes. Mice were randomly assigned to different xenograft regimens and the investigator blinded to the assignment until the tumour analyses. Two mice with early death were excluded from the analyses. c, SNP-array analysis of one Rb/p130-depleted (tumour 1) or one Rb-depleted (tumour 2) cone-precursor-derived tumours from xenograft group 3, revealing no megabase-size loss of heterozygosity or copy number alterations . d, qPCR analysis of pLKO shRNA vector copy number in tumours 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 tumours retained RB1 and/or p130 shRNA vector sequences, confirming their engineered cone precursor origin. e, qPCR analysis of MDM2, MDM4, RB1 and MYCN copy number in three cone-derived tumours and normal retina (n = 6). DNA copy number data (d, e) are representative of two analyses. Values and error bars are mean and s.d. of triplicate assays.
- Extended Data Figure 9: Cone and cell-cycle-related proteins in Rb- or Rb/p130-depleted cone precursor tumours engrafted 3 days after transduction. (1,506 KB)
Most tumour cells expressed human nuclear antigen (HuNU), confirming their xenograft origin. They also expressed cone-related proteins (CRX, cone arrestin, L/M-opsin, RXRγ, CD133 and IRBP) and proliferation-related proteins (Ki67, SKP2, p107 and cytoplasmic p27) but lacked Rb. Tumours had elements resembling Flexner–Wintersteiner rosettes (asterisks) and fleurettes (daggers). Scale bars, 40 μm. Data are representative of three independent experiments.
- Extended Data Figure 10: Analysis of non-cone cell markers in cone-precursor-derived tumours and retinoblastomas. (1,167 KB)
a, Proteins detected in normal retina but not in cone-derived tumour or human retinoblastoma cells included markers of rods (rhodopsin and CNGB1), RPCs and Müller glia (nestin, GFAP and PAX6), bipolar cells (CHX10), ganglion, amacrine and horizontal cells (calbindin and PAX6), and ganglion cells (nuclear BRN-3, thin arrows in mouse retina). PAX6+, nestin+ cells detected in human retinoblastoma were previously found to be Rb+ non-tumour cells from tumour-associated retina7. An uncharacterized cytoplasmic BRN-3 signal (bold arrows) was detected in mouse photoreceptor outer segments and in cone-derived tumour and retinoblastoma rosettes. b, L/M-opsin was detected in most cone-derived tumour cells. However, rare cells co-expressed S-opsin and L/M-opsin (arrows), as in immature L/M-cone precursors and human retinoblastomas7. c, One tumour had rare rhodopsin+, Ki67− cells but no detected rhodopsin+, Ki67+ cells, as in a previously characterized retinoma-like regions7. Scale bars, 40 μm. Data are representative of three independent xenograft experiments.
- Supplementary Tables (385 KB)
This file contains Supplementary Tables 1-5.