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. 2019 Aug 12;36(2):139-155.e10.
doi: 10.1016/j.ccell.2019.06.009. Epub 2019 Jul 18.

The Polycomb Repressor Complex 1 Drives Double-Negative Prostate Cancer Metastasis by Coordinating Stemness and Immune Suppression

Wenjing Su  1 , Hyun Ho Han  2 , Yan Wang  3 , Boyu Zhang  3 , Bing Zhou  4 , Yuanming Cheng  1 , Alekya Rumandla  3 , Sreeharsha Gurrapu  3 , Goutam Chakraborty  5 , Jie Su  6 , Guangli Yang  7 , Xin Liang  8 , Guocan Wang  8 , Neal Rosen  1 , Howard I Scher  9 , Ouathek Ouerfelli  7 , Filippo G Giancotti  10
Affiliations

The Polycomb Repressor Complex 1 Drives Double-Negative Prostate Cancer Metastasis by Coordinating Stemness and Immune Suppression

Wenjing Su et al. Cancer Cell. .

Abstract

The mechanisms that enable immune evasion at metastatic sites are poorly understood. We show that the Polycomb Repressor Complex 1 (PRC1) drives colonization of the bones and visceral organs in double-negative prostate cancer (DNPC). In vivo genetic screening identifies CCL2 as the top prometastatic gene induced by PRC1. CCL2 governs self-renewal and induces the recruitment of M2-like tumor-associated macrophages and regulatory T cells, thus coordinating metastasis initiation with immune suppression and neoangiogenesis. A catalytic inhibitor of PRC1 cooperates with immune checkpoint therapy to reverse these processes and suppress metastasis in genetically engineered mouse transplantation models of DNPC. These results reveal that PRC1 coordinates stemness with immune evasion and neoangiogenesis and point to the potential clinical utility of targeting PRC1 in DNPC.

Keywords: MDSCs; Tregs; angiogenesis; combination therapy; epigenetics; immune evasion; immune microenvironment; metastasis; preclinical compound; stemness.

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Figures

Figure 1.
Figure 1.. Activation of PRC1 in DNPC.
(A) cPRC1 and ncPRC1 complexes. (B and C) Genetic aberrations of PRC1 and PRC2 components in primary and metastatic prostate cancers in the Grasso dataset (n=94) (Grasso et al., 2012) (B) and the SU2C dataset (n=118) (Robinson et al., 2015) (C). (D) Similarity matrix of the expression of AR and its target genes (n=11), RNF2-regulated genes (n=20), and NE signature genes (n=10) in the SU2C dataset. (E) Scatter plot of the expression of AR and NE signatures in M-CRPC samples (Kumar et al., 2016), prostate cancer cell lines, and PtenPC-/- and PtenPC-/-Smad4PC-/- tumors (Wang et al., 2015). (F) Immunoblotting of the indicated cell lines. See also Figure S1.
Figure 2.
Figure 2.. PRC1 is required for tumor initiation and metastasis.
(A) Control or RNF2- and BMI1-silenced PC3 cells were subjected to immunoblotting. (B) Representative images (top) and quantification of luciferase counts (bottom) of male nude mice injected intracardially (i.c.) with 5×105 luciferase labeled control and RNF2-silenced PC3 cells at week 4; Error bars, mean±SEM. (C) H&E (top) and IHC (middle) staining of bone sections and X ray imaging of hindlegs (bottom) of mice in (B). Scale bar=50 μm. Dash oval indicates osteolytic lesion. (D and E) Representative images (D) and quantification of luciferase counts (E) of male nude mice at 3 weeks after injected i.c. with 1×105 PtenPC-/-Smad4PC-/- cells expressing the indicated constructs. Bars, mean±SEM. (F and G) Quantification (F) and representative images (G) of control and RNF2-silenced PC3 cells subjected to sphere assay at day 7. Scale bar=200 μm. Error bars, mean±SD of triplicate experiments, **** p<0.0001 two-tailed Student t test. (H) Control and BMI1-silenced PC3 cells were subjected to sphere assay and the results quantified at day 7. Bars, mean±SD. (I) Control and RNF2-silenced PC3 cells were transfected with empty, WT Rnf2-, or Ring domain-truncated Rnf2-expressing vector and subjected to sphere assay. Bars, mean±SD, n.s.: not significant. (J and K) Quantification (J) and representative images (K) of control and RNF2-silenced PC3 cells subjected to 3D growth for 14 days. Error bars, mean±SD. Scale bar=200 μm. (L and M) Quantification of Ki67 and CC3 positive cells (L) and representative images (M) of PC3 cells from 3D growth. Error bars, mean±SD. Scale bar=50 μm. (N) Tumor volumes of control and RNF2-silenced PC3 cells injected subcutaneously in NOG mice as indicated. Bars, mean±SD, *** p < 0.001, **** p < 0.0001. See also Figure S2.
Figure 3.
Figure 3.. PRC1 promotes the expression of CCL2 and other pro-metastatic genes.
(A) Hierarchical clustering of H3K4me3 and H3K27me3 read densities (Taberlay et al., 2014) across the promoters of RNF2-induced or repressed genes and the heatmap of the read densities of indicated histone modifications and PRC1 subunits. (B) Global analysis of gene expression in each cluster in control and RNF2-silenced PC3 cells. A standard boxplot was applied to display the z-score of the value of “fragments per kilobase of transcript per million mapped reads” (FPKM) based on a 5-number summary (“minimum”, first quartile (Q1), median, third quartile (Q3), and “maximum”). The median value is depicted as a line splitting the box in half. The middle “box” represents the middle 50% of scores for the group. The upper and lower whickers represent scores outside the middle 50%. Outlier as points extending beyond the whiskers. (C) Boxplot of the average read densities for H3K4me3, H3K27me3, RNF2 and KDM2B among the core promoters of genes in clusters 1 and 3. Standard boxplot was applied to display the distribution of the values of average reads densities based on a 5-number summary as described in (B). (D) UCSC Genome Browser view of the H3K4me3, H3K27me3, H2AUb, RNF2, BMI1, PHC2 and KDM2B peaks for the indicated representative genes in clusters 1 and 3. (E) GO enrichment and KEGG pathway analysis of genes differentially expressed upon RNF2 knockdown. Heatmap represents the top 29 genes from the ES pathway. (F) ssGSEA score correlations of RNF2-activated geneset and EMT, AR, Stemness, NEPC geneset in the SU2C dataset (n=118). (G) Representative bioluminescent images of male nude mice injected i.c. with 5×105 RNF2-silenced PC3 cells infected with the custom library sacrificed after 4 weeks. (H) Heatmap of the library gene expression fold change in bone metastases from (G). (I) Relative expression levels of CCL2 in control and RNF2- or BMI1-silenced PC3 and RM1 cells. Bars, mean±SD. (J and K) Representative images (J) and quantification of luciferase counts (K) of male nude mice at 4 weeks after injected i.c. with 5×105 PC3 cells expressing the indicated constructs. Bars, mean±SEM. (L) Survival analysis of mice from (J). (M and N) Relative occupancy of RNF2, H2AK119Ub, H3K27me3, H3K9Ac, H3K27Ac and IgG control on the CCL2 (M) or ATF3 (N) promoter from ChIP-qPCR in control or RNF2-silenced PC3 cells; bars, mean±SD. * p < 0.05, ** p < 0.01, *** p < 0.005. See also Figure S3.
Figure 4.
Figure 4.. Targeting PRC1-CCL2 signaling impairs bone metastasis.
(A) Bone tissues from mice injected with control and RNF2-silenced PC3 cells were subjected to IHC staining. CD68+ TAMs, CD31+ endothelial cells, and NKp46+ NK cells were imaged (left, bar=50 μm) and quantified (right, bars, mean±SD, * p < 0.05, ** p < 0.01). (B) Representative images (top) and quantification of luciferase counts (bottom) of male nude mice injected i.c. with 2.5×105 control and CCR4-silenced PC3 cells at week 5; bars, mean±SEM. (C) Representative images (top) and quantification of luciferase counts (bottom) at week 5 of male nude mice injected i.c. with 2.5×105 PC3 cells and treated with vehicle (captisol), RS504393 (10 mg/kg, twice/week) or BLZ945 (200 mg/kg, QD) 7 days after injection. Bars, mean±SEM. (D) Bone tissues from mice injected i.c. with control and RNF2-silenced RM1 cells were subjected to IHC staining. TAMs, endothelial cells, Foxp3+ Tregs, and B220+ B cells were imaged (left, bar=50 μm) and quantified (right, bars, mean±SD). (E and F) ssGSEA of RNF2 activity (E) or CCL2 mRNA levels (F) in ARPC, DNPC and NEPC in the SU2C dataset. Bars, mean±SD, one-way ANOVA and Tukey's multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; (G) Correlation of RNF2 activity with intratumoral abundancy of immune cell subsets. ** p < 0.01; * p < 0.05 (two-tailed). See also Figure S4.
Figure 5.
Figure 5.. Development of a Catalytic Inhibitor of PRC1.
(A) Chemical structures of PRT4165 and GW-516. (B) Acid-extracted histones from PC3 cells treated as indicated for 24 hours were subjected to immunoblotting followed by densitometry. H2AUb was normalized to control and the IC50 determined. (C) PC3 cells were subjected to sphere assay in the presence of PRT4165 or GW-516. The inhibition of sphere formation was normalized to control and the IC50 determined. (D) Histone H2A was incubated with RNF2 in the presence of E1 and E2. Reactions were carried out in the presence of PRT4165 or GW-516. (E and F) Cells were treated with GW-516 or PTC209 at the indicated concentrations for 6 hours (E), or treated with GW-516 or PTC209 for the indicated times (F). (G and H) Expression of RNF2 target genes upon RNF2 depletion or GW-516 treatment in PC3 (G) and RM1 cells (H). Bars, mean±SD. (I and J) Representative images (I) and quantification of luciferase counts (J) of mice injected i.c. with PC3 cells and treated with vehicle (DMSO) or GW-516 as indicated. Mice were dosed twice per week. Bars, mean±SEM. (K) Representative images (left, bar=50 μm) and percentages of staining intensity (right, Bars, mean±SD) of IHC staining for CCL2 or H2AUb of bone tissues from (I). See also Figure S5.
Figure 6.
Figure 6.. Pharmacological inhibition of PRC1 cooperates with immunotherapy to suppress metastasis.
(A-C) Representative images (A), cumulative luciferase counts (B) and survival analysis (C) of FVB mice injected i.c. with 1×105 PtenPC-/-Smad4PC-/- cells and treated with vehicle (control IgG), PD-1 + CTLA-4 antibodies (PD-1: 200 μg/mouse; CTLA-4: 250 μg/mouse), GW-516 (10 mg/kg) or combination starting at day 7 post-injection. Mice were dosed twice per week. Bars, mean±SEM. * p < 0.05; ** p < 0.01; **** p < 0.0001. (D-F) Quantification of luciferase counts in the bone (D), liver (E), and brain (F) of end point mice from (A); bars, mean±SEM. (G) FACS analysis of immune cell subsets in the blood and bone marrow of end point mice from (A). Bars, mean±SD. * p < 0.05, ** p < 0.01. See also Figure S6.
Figure 7.
Figure 7.. Pharmacological inhibition of PRC1 reverses immune suppression and cooperates with immunotherapy to suppress metastasis.
(A-C) Representative staining images (left, bar=50 μm) and quantifications of positive cells (right, bars, mean±SD, **** p < 0.0001) for immune-suppressive cells (A), T cells (B), and endothelial cells and proliferating/apoptotic tumor cells (C) in bone tissues from mice inoculated i.c. with PtenPC-/-Smad4PC-/- cells collected after 1 week of treatment and subjected to IHC or IF staining. See also Figure S7.
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