DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells

C Pistore, E Giannoni, T Colangelo, F Rizzo, E Magnani, L Muccillo, G Giurato, M Mancini, S Rizzo, M Riccardi, N Sahnane, V Del Vescovo, K Kishore, M Mandruzzato, F Macchi, M Pelizzola, M A Denti, D Furlan, A Weisz, V Colantuoni & 2 others P Chiarugi, I M Bonapace

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

Original languageEnglish
Pages (from-to)5551-5566
Number of pages16
JournalOncogene
Volume36
Issue number40
DOIs
Publication statusPublished - Oct 5 2017

Fingerprint

Epithelial-Mesenchymal Transition
DNA Methylation
Prostatic Neoplasms
Methylation
CpG Islands
Genes
Maintenance
Multigene Family
Cancer-Associated Fibroblasts
Genome
Neoplastic Stem Cells
Nucleic Acid Regulatory Sequences
Castration
Conditioned Culture Medium
Cluster Analysis
Epithelium

Keywords

  • Cancer-Associated Fibroblasts
  • Cell Line, Tumor
  • Cell Transformation, Neoplastic
  • Culture Media, Conditioned
  • Cytosine
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA Methylation
  • DNA, Neoplasm
  • Epithelial Cells
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Male
  • Mesoderm
  • Prostatic Neoplasms
  • Stem Cells
  • Transcriptional Activation
  • Journal Article

Cite this

Pistore, C., Giannoni, E., Colangelo, T., Rizzo, F., Magnani, E., Muccillo, L., ... Bonapace, I. M. (2017). DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells. Oncogene, 36(40), 5551-5566. https://doi.org/10.1038/onc.2017.159

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells. / Pistore, C; Giannoni, E; Colangelo, T; Rizzo, F; Magnani, E; Muccillo, L; Giurato, G; Mancini, M; Rizzo, S; Riccardi, M; Sahnane, N; Del Vescovo, V; Kishore, K; Mandruzzato, M; Macchi, F; Pelizzola, M; Denti, M A; Furlan, D; Weisz, A; Colantuoni, V; Chiarugi, P; Bonapace, I M.

In: Oncogene, Vol. 36, No. 40, 05.10.2017, p. 5551-5566.

Research output: Contribution to journalArticle

Pistore, C, Giannoni, E, Colangelo, T, Rizzo, F, Magnani, E, Muccillo, L, Giurato, G, Mancini, M, Rizzo, S, Riccardi, M, Sahnane, N, Del Vescovo, V, Kishore, K, Mandruzzato, M, Macchi, F, Pelizzola, M, Denti, MA, Furlan, D, Weisz, A, Colantuoni, V, Chiarugi, P & Bonapace, IM 2017, 'DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells', Oncogene, vol. 36, no. 40, pp. 5551-5566. https://doi.org/10.1038/onc.2017.159
Pistore, C ; Giannoni, E ; Colangelo, T ; Rizzo, F ; Magnani, E ; Muccillo, L ; Giurato, G ; Mancini, M ; Rizzo, S ; Riccardi, M ; Sahnane, N ; Del Vescovo, V ; Kishore, K ; Mandruzzato, M ; Macchi, F ; Pelizzola, M ; Denti, M A ; Furlan, D ; Weisz, A ; Colantuoni, V ; Chiarugi, P ; Bonapace, I M. / DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells. In: Oncogene. 2017 ; Vol. 36, No. 40. pp. 5551-5566.
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AU - Pistore, C

AU - Giannoni, E

AU - Colangelo, T

AU - Rizzo, F

AU - Magnani, E

AU - Muccillo, L

AU - Giurato, G

AU - Mancini, M

AU - Rizzo, S

AU - Riccardi, M

AU - Sahnane, N

AU - Del Vescovo, V

AU - Kishore, K

AU - Mandruzzato, M

AU - Macchi, F

AU - Pelizzola, M

AU - Denti, M A

AU - Furlan, D

AU - Weisz, A

AU - Colantuoni, V

AU - Chiarugi, P

AU - Bonapace, I M

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N2 - Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

AB - Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

KW - Cancer-Associated Fibroblasts

KW - Cell Line, Tumor

KW - Cell Transformation, Neoplastic

KW - Culture Media, Conditioned

KW - Cytosine

KW - DNA (Cytosine-5-)-Methyltransferases

KW - DNA Methylation

KW - DNA, Neoplasm

KW - Epithelial Cells

KW - Gene Expression Regulation, Neoplastic

KW - Humans

KW - Male

KW - Mesoderm

KW - Prostatic Neoplasms

KW - Stem Cells

KW - Transcriptional Activation

KW - Journal Article

U2 - 10.1038/onc.2017.159

DO - 10.1038/onc.2017.159

M3 - Article

VL - 36

SP - 5551

EP - 5566

JO - Oncogene

JF - Oncogene

SN - 0950-9232

IS - 40

ER -