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Innate immunity and the NF-κB pathway control prostate stem cell plasticity, reprogramming and tumor initiation

Nature Cancer volume 6, pages 1537–1558 (2025)Cite this article

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Abstract

Prostate epithelium develops from multipotent stem cells, which are replaced in adult life by different lineage-restricted basal and luminal unipotent stem cells. Deletion of Pten re-induces multipotency in basal cells (BCs); however, the molecular mechanisms regulating BC plasticity and tumor initiation are poorly understood. Here we showed that Pten deletion in BCs led to distinct cell fate reprogramming and tumor initiation in a regionalized manner. Single-cell RNA sequencing, ATAC-seq and in situ characterization revealed that following Pten deletion in anterior and dorsolateral prostates, BCs were highly plastic and reprogrammed into a hillock-like state, progressing into a proximal-like luminal state before giving rise to invasive tumors. This BC reprogramming was associated with the activation of innate immunity. Pharmacological targeting of interleukin-1, JAK–STAT and NF-κB as well as genetic deletion of Nfkb inhibit Pten-induced cell plasticity and reprogramming in a cellular autonomous manner, opening new opportunities for prevention and treatment of prostate cancer.

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Fig. 1: Pten deletion induced BC plasticity and tumor initiation in a cell of origin and region-specific manners.
Fig. 2: Pten deletion in LCs does not promote multipotency and is associated with slower tumor progression in AP/DLP compared to BC-derived tumors.
Fig. 3: Pten Deletion together with oncogenic Pik3ca expression or p53 deletion accelerate BC plasticity.
Fig. 4: Region-dependent BC reprograming following Pten deletion.
Fig. 5: BC plasticity following Pten deletion is associated with the activation of interferon and TNF pathways in epithelial cells.
Fig. 6: Regional reprogramming and activation of innate immunity persist in mouse prostate tumors and human prostate cancers.
Fig. 7: Pharmacological inhibition of Nfkb, JAK/STAT and Il1 inhibits SC plasticity induced by Pten deletion.
Fig. 8: Cellular autonomous activation of Nfkb mediates BC plasticity and regional reprogramming following Pten deletion.

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Data availability

All raw sequencing datasets that support the findings of this study have been deposited in the GEO under accession number GSE270187 (scRNA-seq), GSE270189 (bulk RNA-seq, rapamycin treated), GSE270190 (bulk RNA-seq, cell fate upon PTEN deletion in BC), GSE286018 (bulk RNA-seq of P65/Pten knock BCs), GSE286019 (bulk RNA-seq of basal-derived and luminal-derived luminal tumors), GSE270191 (ATAC-seq of pooled BC/LC of WT and PTEN-deleted cells) and GSE288787 (ATAC-seq of BCs from different lobes upon PTEN deletion). Previously published human prostate cancer datasets re-analyzed in this study were available from GitHub repository of the author (https://github.com/franklinhuanglab/scRNA-seq-Analysis-of-Prostate-Cancer-Samples)38, the Prostate Cell Atlas (https://www.prostatecellatlas.org)39 and GEO GSE181294 (ref. 40) and GSE185344 (ref. 41). The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The codes used for data processing, downstream analysis and plotting for this paper are available on GitHub at https://github.com/yurasong/PTEN_codes.

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Acknowledgements

We thank the ULB animal facility and ULB genomic core facility (F. Libert and A. Lefort) for help with sequencing, DIAPath–CMMI, supported by the Fonds Yvonne Boël and by the European Regional Development Fund and the Walloon region (S. Rorive and J. Allard), for helping with histology, and J.M. Vanderwinden and LiMif for helping with microscopy. We thank M. Pasparakis, University of Cologne for providing the P65fl/fl mice. C.J. is supported by a long-term EMBO Postdoctoral Fellowships (ALTF 982-2021) and Foundation Against Cancer (2023-041). Y.S. is supported by Télévie. C.B. is supported by the WEL Research Institute, FNRS, Télévie, Fond Erasme, Fondation Contre le Cancer, ULB Foundation, European Research Council, Fonds Yvonne Boël and the Foundation Baillet Latour. A.S. is supported by KULeuven (SymBioSys – C14/18/092) and the Fondation Contre le Cancer (2015-143). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Chen Jiang, Yura Song.

  2. These authors jointly supervised this work: Alejandro Sifrim, Cédric Blanpain.

Authors and Affiliations

  1. Laboratory of Stem Cells and Cancer, Université Libre de Bruxelles (ULB), Brussels, Belgium

    Chen Jiang, Yura Song, Elisavet Tika, Zahra Zahedi, Christine Dubois & Cédric Blanpain

  2. Centre Universitaire Inter Regional d’Expertise en Anatomie Pathologique Hospitalière, CurePath (CHIREC - CHU TIVOLI -Université Libre de Bruxelles), Charleroi, Belgium

    Sandrine Rorive & Isabelle Salmon

  3. DIAPath, Center for microscopy and molecular Imaging, Université Libre de Bruxelles, Gosselies, Belgium

    Justine Allard

  4. Department of Human Genetics, University of Leuven, KU Leuven, Leuven, Belgium

    Alejandro Sifrim

  5. WEL Research Institute, Université Libre de Bruxelles (ULB), Brussels, Belgium

    Cédric Blanpain

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Contributions

C.J. and C.B. designed the experiments and performed data analysis. C.J. performed most of the experiments and data analysis. Y.S. and A.S. performed the bioinformatic analysis. S.R. performed prostate tumor scoring. I.S. provided human prostate cancer samples. J.A. performed the H&E staining on mouse prostate tumors and IHC staining on human prostate cancers. E.T. performed the experiments on bulk RNA-seq of Pten deletion at 5 weeks. Z.Z. provided technical help. C.J. and C.D. performed sorting experiments. C.B. wrote the manuscript. All authors read and approved of the final paper.

Corresponding author

Correspondence to Cédric Blanpain.

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Extended data

Extended Data Fig. 1 Pten deletion induced BC plasticity and tumor initiation in a cell of origin and region-specific manners.

(a) FACS gating strategy to analyze and isolate BCs and LCs from K5CreER/Ptenfl/fl/RosaYFP mice 6 weeks after TAM injection. Same strategy for all lineage tracing mice in this study. (b) Quantification of the % of Lin- YFP+ LCs in total YFP+ cells in VP and DLP at different time points after TAM injection in K5CreER/Ptenfl/fl/RosaYFP mice. n = 3 mice for 1w, 4w, 7w, 8w and 20w, n = 4 mice for 6w and 12w. (c) Representative FACS plot of CD49f and EpCAM expression in Lin- YFP+ prostate epithelial cells from K5CreER/RosaYFP mice 6 week after TAM administration. (d) Quantification of the % of Lin− YFP+ BCs and LCs in total BCs and LCs in VP, DLP and AP in in K5CreER/RosaYFP mice after 6-8 weeks TAM administration. n = 6 mice. (e) Quantification of the % of YFP + K14+ and YFP + K8+ in total K14+ and K8+ in VP, DLP and AP in K5CreER/RosaYFP mice after 4 weeks TAM administration. n = 2 mice. (f) Immunostainings of EdU (Red), GFP (Green), K14 (Grey) and K8 (Grey) in the prostate of K5CreER/Ptenfl/fl/RosaYFP at 4w, 6w and 12-week after TAM administration 24 h after EdU injection. Scale bar, 10 μm. n = 3 mice. (g) Quantification of percentage of EdU doublet following basal cell division giving rise BC-BC, BC-LC and LC-LC doublets. n = 152 EdU doublets (4w), n = 65 EdU doublets (6w) and n = 142 EdU (12w) doublets pooled from 3 mice. (h) Genetic strategy to lineage trace LC following Pten deletion (i) Quantification of Lin- YFP+ BCs and LCs in total BCs and LCs in VP, DLP and AP at different time points after TAM administration in K8CreER/Ptenfl/fl/RosaYFP mice. n = 3 mice. Graphs are mean ± s.e.m.

Source data

Extended Data Fig. 2 mTOR inhibition blocks multipotency and tumor initiation.

Representative immunostaining of the prostate from (a) K5CreER/Ptenfl/fl/RosaYFP mice 6 weeks after TAM administration (K5-PTEN 6w) and (d) K5CreER/Ptenfl/fl/Pik3caH1047R/RosaYFP mice 3 weeks after TAM administration (K5-PTEN-PIK 3w) treated with EtOH or Rapamycin (6 mg/kg, three injections per week) using anti-GFP (green), anti-K8 (red) and anti-K14 (grey) antibodies. Arrows indicate hybrid cells (YFP + K14 + K8 + ). Scale bar, 20 μm (upper row), 10 μm (lower row). n = 3 mice. Representative FACS plot of CD49f and EpCAM expression in Lin- YFP+ prostate epithelial cells of (b) K5-PTEN 6w mice and (e) K5-PTEN-PIK 3w mice treated with EtOH or Rapamycin. Quantification of % of LIN- YFP+ LCs in total YFP+ cells in the whole prostate of (c) K5-PTEN 6w mice and (f) K5-PTEN-PIK 3w mice treated with EtOH or Rapamycin. n = 6 for K5-PTEN 6w mice, n = 4 for K5-PTEN-PIK 3w mice. (g) H&E-stained histological sections and (h) quantification of different types of tumorigenic lesions along prostate tumor progression in DLP of K5-PTEN 6w mice treated with EtOH (n = 7 mice) or Rapamycin (n = 8 mice). Scale bar, 250 μm (upper row), 50 μm (lower row). Graphs are mean ± s.e.m. p values are derived from two-sided unpaired t-test.

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Extended Data Fig. 3 Marker genes for each cell type in control and in K5-PTEN 6w prostate epithelial cells.

Cell populations found by scRNA-seq in CTL prostate epithelial cells: (a) UMAP dimensionality reduction plots of CTL prostate epithelial cells with colors representing unsupervised clustering. (b-g) UMAP plots colored by normalized gene expression for (b) BC marker genes, (c) Proximal marker genes, (d) Ventral marker genes, (e) Lateral marker genes, (f) Antero-Dorsal marker genes and (g) Proliferative marker genes. (h) UMAP dimensionality reduction plots for K5-PTEN 6w using Seurat: UMAP dimensionality reduction plots with colors representing unsupervised clustering. (i–o) UMAP plots colored by normalized gene expression for (i) BC marker genes, (j) Proximal marker genes, (k) HY BC Prox marker genes, (l) HY Nkx3.1 marker genes, (m) Ventral marker genes, (n) Antero-Dorsal marker genes and (o) Proliferative marker genes.

Extended Data Fig. 4 Cellular trajectory and in situ characterization of Hillock, Hybrid and proximal-like LCs.

(a) Cell populations of K5-PTEN 6w, which are used for trajectory inference: UMAP dimensionality reduction plots with different colors representing unsupervised clustering. (b) UMAP plot for the K5-PTEN 6w dataset showing the trajectory from BC to proximal LCs. (c-d) UMAP plot colored by the adjusted proportion of (c) BC-specific marker genes and (d) LC-specific marker genes. (e-h) Representative immunostaining of AP/DLP of CTL, K5CreER/Ptenfl/fl/RosaYFP mice 6 weeks after TAM administration, K5CreER/Ptenfl/fl/Pik3caH1047R/RosaYFP mice 3 weeks after TAM administration and K5CreER/Ptenfl/fl/P53fl/fl/RosaYFP mice 4 weeks after TAM administration using (e) anti-K13, (f) anti-Aqp3, (g) anti-K4, (h) anti-Trop2 and anti-GFP (green), anti-K14 (grey) and anti-K8 (grey) antibodies. Arrows indicate Hillock cells, Hybrid cells and Proximal LCs as indicated markers. n = 3 mice. Dash lines marked the expanded LCs. L: prostate lumen. Scale bar, 20 μm.

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Extended Data Fig. 5 scRNA‑seq characterize hybrid basal–luminal cell states and lineage trajectory in K5-PTEN 6w prostate epithelial cells.

(a) Cell populations of K5-PTEN 6w, which are used for trajectory inference: UMAP dimensionality reduction plots with different colors representing unsupervised clustering. (b) UMAP plot for the K5-PTEN 6w dataset showing the trajectory from BC to ventral LCs. (c-d) UMAP plot colored by the adjusted proportion of (c) BC-specific marker genes and (d) LC-specific marker genes. (e) Representative images of immunostaining of the VP of K5CreER/Ptenfl/fl/RosaYFP mice 6 months after TAM administration using anti-GFP (green), anti-Nkx3.1 (red). Scale bar, 50 μm. n = 3 mice. (f) Representative images of immunostaining of the VP, DLP and AP of K8CreER/Ptenfl/fl/RosaYFP mice 6 months after TAM administration using anti-GFP (green), anti-K13 (red), anti-K4 (red) and anti-K14 (grey) antibodies. Scale bar, 50 μm. n = 3 mice. (g) Genes significantly upregulated on HY BC Prox compared to BC p63 low and BC p63 high in K5-PTEN 6w scRNA dataset. (h) Genes significantly upregulated in HY BC Prox compared to proximal luminal cells. (i) Genes significantly upregulated on HY Nkx3.1 compared to the BC Nkx3.1. (j) Genes significantly upregulated in HY Nkx3.1 compared to the ventral prostate. (k-m) UMAP plots colored by (Left) SCENIC AUCs for regulon activation and (Right) normalized gene expression of the TF for (k) Elf3, (l) Grhl3 and (m) Creb5. For g to j, red dots are significantly differentially expressed genes (FDR-adjusted P value < 0.01) and blue-labeled genes are uniquely expressed on hybrid population. P values were calculated using the Wilcoxon rank-sum test and adjusted using the Benjamini–Hochberg FDR method.

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Extended Data Fig. 6 Temporal analysis of the activation of innate immunity and ATAC-seq and bulk RNA-seq on different lobes after Pten deletion in BC.

(a) Relative mRNA expression levels of Il1a and Cxcl2 were determined by quantitative RT–PCR in FACS-isolated BCs of AP/DLP from CD1 mice and YFP+ BCs from of K5CreER/Ptenfl/fl/RosaYFP mice at indicated time after TAM injection. mean ± s.e.m. n = 3 mice. p values are derived from one-way ANOVA with Dunnett test. ATAC-seq peaks of (b) Krt13 and (c) Aqp3 genes from FACS-isolated BCs of VP, DLP and AP of K5CreER/Ptenfl/fl/RosaYFP mice 6 weeks after TAM administration. Scale for visualization: 0-52 (Krt13) and 0-30 (Aqp3). Peaks which are upregulated at least 2-fold on AP and DLP compared to VP are highlighted with orange box. (d) TF motif enrichment analysis of peaks upregulated in BCs of AP/DLP compared to BCs of VP in K5-PTEN 6w. P values were calculated using a binomial test. (e) Relative Erg expression from bulk RNA-seq in FACS-isolated CTL BC, CTL LC from CTL mice and YFP+ BCs (BC_PTEN) and YFP+ LCs arising from Pten deleted BCs (LC_PTEN) from K5CreER/Ptenfl/fl/RosaYFP mice 5 weeks after TAM injection. n = 2. (f) Relative Erg expression from bulk RNA-seq of FACS-isolated LCs from AP/DLP versus VP in K5CreER/Ptenfl/fl/RosaYFP mice 6 months following TAM administration. (n = 3 for AP/DLP, n = 2 for VP).

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Extended Data Fig. 7 Activation of innate immunity is a mTOR dependent.

(a) Venn diagram illustrating the number of 2-fold upregulated genes in FACS-isolated LIN- YFP+ BCs of K5CreER/Ptenfl/fl/Pik3caH1047R/RosaYFP mice 3 weeks after TAM injection (vehicle treated, BC_PTEN-PIK) compared to FACS-isolated LIN- BCs of CD1 mice (vehicle treated, BC_CTL) and the number of 2-fold downregulated genes in FACS-isolated LIN- YFP+ BCs of K5CreER/Ptenfl/fl/Pik3caH1047R/RosaYFP mice 3 weeks after TAM injection treated with Rapamycin (BC_PTEN-PIK Rap) compared to BC_PTEN-PIK. (b) GO analysis of genes upregulated more than 2-fold in FACS-isolated BC_PTEN-PIK compared to FACS-isolated BC_CTL. (c) GO analysis of genes downregulated more than 2-fold in FACS-isolated BC_PTEN-PIK Rap compared to FACS-isolated BC_PTEN-PIK. (d) Expression of genes on bulk RNA-seq in FACS-isolated BC_CTL, BC_PTEN-PIK and BC_PTEN-PIK Rap. n = 2 samples. (e) Representative immunostaining of myeloid cells within AP from K5CreER/Ptenfl/fl/Pik3caH1047R/RosaYFP mice treated with EtOH or Rapamycin 3 weeks after TAM injection using anti-GFP (green), anti-Ly6G (red) antibodies. Scale bar, 20 μm. (f) Representative immunostaining of myeloid cells within the DLP from K5CreER/Ptenfl/fl/RosaYFP mice and K5CreER/Ptenfl/fl/P53fl/fl/RosaYFP mice 4 weeks after TAM injection using anti-GFP (green), anti-Ly6G (red) antibodies. Scale bar, 50 μm. For b and c, P values were derived from two-tailed modified Fisher’s Exact Test and adjusted using the Benjamini–Hochberg method.

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Extended Data Fig. 8 Regional reprogramming and activation of innate immunity in BC-derived mouse prostate tumors.

(a) UMAP dimensionality reduction plots with different colors representing unsupervised clustering of scRNA-seq of FACS-isolated LIN- YFP+ epithelial cells from K5CreER/Ptenfl/fl/RosaYFP mice 10 months after TAM administration: (b-h) UMAP plots colored by normalized gene expression for (b) Proximal marker genes, (c) Proliferative marker genes, (d) HY BC Prox marker genes, (e) Distal LC marker genes, (f) Chemokine marker genes, (g) Interferons marker genes, (h) MHC Class II antigens marker genes. (i–n) Representative immunostaining and quantification of (i, j) K13, (k, l) Aqp3 and (m, n) K4 in VP, DLP and AP of K5CreER/Ptenfl/fl/RosaYFP mice 6 months following TAM administration. Scale bar, 50 μm. n = 4 mice for K13 and Aqp3, n = 3 mice for K4. (o) Relative mRNA expression from bulk RNA-seq of the indicated genes in FACS-isolated YFP+ LCs from AP/DLP and VP in K5CreER/Ptenfl/fl/RosaYFP mice 6 months following TAM administration. (n = 3 for AP/DLP, n = 2 for VP). Representative immunostainings of AdeK5Cre infected AP of (p) Ptenfl/fl/P53/RosaYFP mice and (q) Ptenfl/fl/Pik3caH1047R/RosaYFP mice 2 months after virus injection using the indicated antibodies. Scale bar, 50 μm. (r) Quantification of the % of K13 + , Aqp3+ and K4+ in total YFP+ and Ly6G+ cells in AdeK5Cre injected AP of Ptenfl/fl/Pik3caH1047R/RosaYFP mice. n = 3 infections. Graphs are mean ± s.e.m. p values are derived from one-way ANOVA with Tukey’s test.

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Extended Data Fig. 9 Regional reprogramming in human prostate tumors.

(a) Cell populations on ERG-driven human prostate cancer cells, human prostate cancer atlas, treatment-naïve prostate adenocarcinoma and invasive cribriform carcinoma and intraductal carcinoma data. (b) UMAP dimensionality reduction plots for KRT13 and WFDC2 with color scaling representing the level of gene expression. (c-f) Heatmap of reprogramming marker genes defined on K5-PTEN 6 W scRNA-seq data on (c) ERG-driven human prostate cancer cells, (d) human prostate cancer atlas, (e) treatment-naïve prostate adenocarcinoma and (f) invasive cribriform carcinoma and intraductal carcinoma data. (g-i) Slingshot pseudotime trajectory analysis illustrating the lineage trajectory on (g) ERG-driven human prostate cancer cells, (h) human prostate cancer atlas and (i) invasive cribriform carcinoma and intraductal carcinoma data. (j) Summary of IHC staining results for PIGR and AQP3 in prostate tumor samples from 136 patients, categorized by GG groups of prostate cancer. Green boxes indicate co-staining of PIGR and AQP3.

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Extended Data Fig. 10 Model of the early step of prostate tumor initiation.

Cell plasticity, lineage infidelity and tumor progression following Pten deletion occurs in a region-specific manner during the early stage of prostate cancer initiation and are mediated by the activation of innate immunity in prostate basal stem cells.

Supplementary information

Reporting Summary

Software policy checklist

Software policy checklist includes the information of public Github repository (https://github.com/yurasong/PTEN_codes) where the workflow and codes used for this publication are available. This checklist includes summary of system requirements, installation/demo steps and license details.

Supplementary Table

List of antibodies used in this study.

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Jiang, C., Song, Y., Rorive, S. et al. Innate immunity and the NF-κB pathway control prostate stem cell plasticity, reprogramming and tumor initiation. Nat Cancer 6, 1537–1558 (2025). https://doi.org/10.1038/s43018-025-00994-3

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