A tumor suppressor activity of Drosophila Polycomb genes mediated by JAK-STAT signaling

Abstract

A prevailing paradigm posits that Polycomb Group (PcG) proteins maintain stem cell identity by repressing differentiation genes, and abundant evidence points to an oncogenic role for PcG proteins in human cancer. Here we show using Drosophila melanogaster that a conventional PcG complex can also have a potent tumor suppressor activity. Mutations in any core PRC1 component cause pronounced hyperproliferation of eye imaginal tissue, accompanied by deregulation of epithelial architecture. The mitogenic JAK-STAT pathway is strongly and specifically activated in mutant tissue; activation is driven by transcriptional upregulation of Unpaired (Upd, also known as Outstretched, Os) family ligands. We show here that upd genes are direct targets of PcG-mediated repression in imaginal discs. Ectopic JAK-STAT activity is sufficient to induce overproliferation, whereas reduction of JAK-STAT activity suppresses the PRC1 mutant tumor phenotype. These findings show that PcG proteins can restrict growth directly by silencing mitogenic signaling pathways, shedding light on an epigenetic mechanism underlying tumor suppression.

Figure 1. PRC1 components are fly tumor suppressors

(a–j). Phenotype of Psc-Su(z)2 mutant eye imaginal discs generated with the eyFlp/cell lethal system. Heterozygous tissue is marked by expression of GFP (green). WT (a) and Psc-Su(z)2 discs (b) stained for Actin to reveal size difference. (c) WT and (d) Psc-Su(z)2 discs stained for Elav (red) and Actin (blue) showing impaired differentiation. Psc-Su(z)2 mutant eye imaginal discs stained for Actin (e, red in f) and E-cadherin (g, red in h) revealing defective epithelial organization. Psc-Su(z)2 mutant eye imaginal discs stained for MMP1 (i, red in j) and Actin (blue in j). (k–t) Phenotype of Pc mutant mosaic eye imaginal discs generated with eyFLP. WT or heterozygous tissue is marked by expression of GFP (green). (k) Wild type and (l) Pc mosaic discs stained for Actin to reveal size difference. (m) Wild type and (n) Pc mosaic discs stained for Elav (red) and Actin (blue) showing impaired differentiation. Pc mosaic discs stained for Actin (o, red in p) and E-cadherin (q, red in r) revealing defective epithelial organization. Pc mosaic discs stained for MMP-1 (s, red in t) and Actin (blue in t). (u) Eye discs mutant for core members of PRC1 (Psc-Su(z)2, ph, Pc and Sce) stained for Actin; strong overgrowth is seen in all genotypes. See Supplementary Table 1 for detailed genotypes.

Figure 2. JAK/STAT signaling is ectopically activated in PRC1 mutant tissue

(a–h) Immunofluorescent stains for reporters in Pc eye mosaic clones; WT cells are marked by GFP (green in b,d,f,h). Fibrillarin (a, red in b), Capicua (c, red in d). expanded-LacZ (e, red in f) and mβ-LacZ (g, red in h) are not consistently upregulated in mutant cells. (i–l) Expression of a JAK/STAT reporter (cyan) in WT or PRC1 mutant eye imaginal discs; actin staining is red. Compared to WT (i), the reporter is strongly and consistently elevated in Psc-Su(z)2 (j), ph (k) and Pc (l) mutant discs.

Figure 3. Unpaired is a direct target of PcG-mediated silencing in imaginal discs

(a) Real-time PCR analysis measuring transcription of JAK/STAT pathway components in Psc-Su(z)2 (orange), ph (yellow), Pc (light green) and Sce (dark green) mutant eye discs. Ligand-encoding upd transcripts are upregulated while other pathway components are relatively unchanged. (b) ChIP quantifying H3K27me3 enrichment (SD, n=3) and Pc-binding at upd, upd2 and AbdB relative to the unmethylated control hsp68 in WT imaginal discs. (c) Eye color induced by a mini-white transgene inserted in the upd regulatory region. (d) Darker eye color indicates reduction of silencing of the mini-white gene in Pc heterozygous males.

Figure 4. JAK/STAT signaling drives PRC1 mutant overgrowth

(a,b) Wing imaginal discs expressing GFP (green) under the control of MS1096GAL4 stained for DNA (red). Characteristic landmarks of wing discs are indicated by colored dots: cyan – dorsal-ventral boundary; magenta – second fold; yellow – peripodial stalk. Compared to discs expressing GFP alone (a), discs coexpressing Unpaired (b) show expansion of the disc epithelium along with increased epithelial folding. (c–l) Phenotype of PRC1 mutant eye imaginal discs with reduced JAK/STAT activity, stained for Actin. Compared to Psc-Su(z)2 mutant eye discs (c), Psc-Su(z)2 mutant discs heterozygous for Stat92E (d) show reduced size. Control imaginal discs expressing no transgene (e), DomeΔCyt (f) or SOCS36E (g) under the control of eyFLP FLPout-GAL4. Expression in Sce (h) or Pc (k) mutant discs of DomeΔCyt (i, l) or SOCS36E (j) reduces overgrowth of mutant eyes. (m–n) Quantitation of disc size: individual discs (circles/squares), average (grey bar) and SD are shown with P values (Student's T-test). (m) Psc-Su(z)2 disc size (0.87 +/− 0.13 ×10⁵ μm², n=12) is reduced when heterozygous for Stat92E (0.78 +/− 0.12 ×10⁵ μm², n=12). (n) Sce disc size (1.59 +/− 0.32 ×10⁵ μm², n=20) is reduced when discs express SOCS36E (1.05 +/− 0.33 ×10⁵ μm², n=22). (o) Rate of eclosion in animals carrying Psc-Su(z)2 mutant eye discs alone, heterozygous for Stat92E, upd, or the os[1A] deficiency deleting upd, upd2, and upd3. > 15-fold increase in eclosion rate is seen.