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BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma.

Nettersheim D, Jostes S, Sharma R, Schneider S, Hofmann A, Ferreira HJ, Hoffmann P, Kristiansen G, Esteller MB, Schorle H - PLoS Genet. (2015)

Bottom Line: Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated.We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling.In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.

ABSTRACT
Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

No MeSH data available.


Related in: MedlinePlus

BMP, NODAL and WNT signaling in GCC tissues and cell lines.(A) cDNA microarray expression data of BMP, NODAL and WNT signaling-associated genes in GCC tissues. SOX2 and SOX17 were used to determine zero level of expression intensity (black line). (B) Pie diagrams summarizing ID1 IHC data of GCC-TMAs. Stainings were classified as ID1 positive (+), negative (-), mixed with much more positive than negative cells (+ >-) and mixed with much more negative than positive cells (+ <-). (C) ZIC3 protein levels in indicated GCC cell lines and tissues. (D) Pie diagrams summarizing beta-CATENIN IHC data of GCC-TMAs. Beta-CATENIN staining was classified as membraneous (m), cytoplasmic (c) and nuclear (n). (E) IF /IHC staining of beta-CATENIN in in parental and xenografted TCam-2 cells (1, 2 and 6 weeks). Scale bar: 100 μm.
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pgen.1005415.g005: BMP, NODAL and WNT signaling in GCC tissues and cell lines.(A) cDNA microarray expression data of BMP, NODAL and WNT signaling-associated genes in GCC tissues. SOX2 and SOX17 were used to determine zero level of expression intensity (black line). (B) Pie diagrams summarizing ID1 IHC data of GCC-TMAs. Stainings were classified as ID1 positive (+), negative (-), mixed with much more positive than negative cells (+ >-) and mixed with much more negative than positive cells (+ <-). (C) ZIC3 protein levels in indicated GCC cell lines and tissues. (D) Pie diagrams summarizing beta-CATENIN IHC data of GCC-TMAs. Beta-CATENIN staining was classified as membraneous (m), cytoplasmic (c) and nuclear (n). (E) IF /IHC staining of beta-CATENIN in in parental and xenografted TCam-2 cells (1, 2 and 6 weeks). Scale bar: 100 μm.

Mentions: Next, we screened GCC tissues for expression of BMP and NODAL signaling keyplayers by re-analyzing cDNA microarray data and performing IHC as well as western blots (Fig 5A–5C) [15]. For IHC, only TFAP2C positive and SOX2 negative CIS and semiomas as well as SOX2 positive ECs were analyzed (S7A Fig). In CIS, seminomas and ECs, expression of BMP8B, BMPR1A /2, SMAD1 /4 and ID1 /2 was detected, while ID3 expression was restricted to ECs (Fig 5A). In line with this expression profile, ID1 was detectable in the vast majority of CIS, seminomas and ECs by IHC of GCC tissue microarrays (GCC-TMA), showing that in these GCC entities BMP signaling is active (Fig 5B and S7A Fig).


BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma.

Nettersheim D, Jostes S, Sharma R, Schneider S, Hofmann A, Ferreira HJ, Hoffmann P, Kristiansen G, Esteller MB, Schorle H - PLoS Genet. (2015)

BMP, NODAL and WNT signaling in GCC tissues and cell lines.(A) cDNA microarray expression data of BMP, NODAL and WNT signaling-associated genes in GCC tissues. SOX2 and SOX17 were used to determine zero level of expression intensity (black line). (B) Pie diagrams summarizing ID1 IHC data of GCC-TMAs. Stainings were classified as ID1 positive (+), negative (-), mixed with much more positive than negative cells (+ >-) and mixed with much more negative than positive cells (+ <-). (C) ZIC3 protein levels in indicated GCC cell lines and tissues. (D) Pie diagrams summarizing beta-CATENIN IHC data of GCC-TMAs. Beta-CATENIN staining was classified as membraneous (m), cytoplasmic (c) and nuclear (n). (E) IF /IHC staining of beta-CATENIN in in parental and xenografted TCam-2 cells (1, 2 and 6 weeks). Scale bar: 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520454&req=5

pgen.1005415.g005: BMP, NODAL and WNT signaling in GCC tissues and cell lines.(A) cDNA microarray expression data of BMP, NODAL and WNT signaling-associated genes in GCC tissues. SOX2 and SOX17 were used to determine zero level of expression intensity (black line). (B) Pie diagrams summarizing ID1 IHC data of GCC-TMAs. Stainings were classified as ID1 positive (+), negative (-), mixed with much more positive than negative cells (+ >-) and mixed with much more negative than positive cells (+ <-). (C) ZIC3 protein levels in indicated GCC cell lines and tissues. (D) Pie diagrams summarizing beta-CATENIN IHC data of GCC-TMAs. Beta-CATENIN staining was classified as membraneous (m), cytoplasmic (c) and nuclear (n). (E) IF /IHC staining of beta-CATENIN in in parental and xenografted TCam-2 cells (1, 2 and 6 weeks). Scale bar: 100 μm.
Mentions: Next, we screened GCC tissues for expression of BMP and NODAL signaling keyplayers by re-analyzing cDNA microarray data and performing IHC as well as western blots (Fig 5A–5C) [15]. For IHC, only TFAP2C positive and SOX2 negative CIS and semiomas as well as SOX2 positive ECs were analyzed (S7A Fig). In CIS, seminomas and ECs, expression of BMP8B, BMPR1A /2, SMAD1 /4 and ID1 /2 was detected, while ID3 expression was restricted to ECs (Fig 5A). In line with this expression profile, ID1 was detectable in the vast majority of CIS, seminomas and ECs by IHC of GCC tissue microarrays (GCC-TMA), showing that in these GCC entities BMP signaling is active (Fig 5B and S7A Fig).

Bottom Line: Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated.We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling.In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.

ABSTRACT
Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

No MeSH data available.


Related in: MedlinePlus