SOX2 is essential for in vivo reprogramming of seminoma-like TCam-2 cells to an embryonal carcinoma-like fate

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Inhibition of BMP signaling is crucial for initiation of reprogramming.

<p>(A) IF /IHC staining of pSMAD 1 /5 in parental and xenografted TCam-2 at indicated time points. Scale bar: 100 μm. (B, C) qRT-PCR analysis of indicated genes after NOGGIN treatment of TCam-2 cells for 1–8 days. (D) Western blotting of indicated proteins 8 days after NOGGIN treatment of TCam-2 cells.</p

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

<div><p>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 (<i>GDF3</i>, <i>NODAL</i>, <i>DNMT3B</i>, <i>DPPA3</i>, <i>GAL</i>, <i>AK3L1</i>) 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.</p></div

Doxycycline inhibits experimental cerebral malaria by reducing inflammatory immune reactions and tissue-degrading mediators

<div><p>Malaria ranks among the most important infectious diseases worldwide and affects mostly people living in tropical countries. Mechanisms involved in disease progression are still not fully understood and specific treatments that might interfere with cerebral malaria (CM) are limited. Here we show that administration of doxycycline (DOX) prevented experimental CM (ECM) in <i>Plasmodium berghei</i> ANKA (PbA)-infected C57BL/6 wildtype (WT) mice in an IL-10-independent manner. DOX-treated mice showed an intact blood-brain barrier (BBB) and attenuated brain inflammation. Importantly, if WT mice were infected with a 20-fold increased parasite load, they could be still protected from ECM if they received DOX from day 4–6 post infection, despite similar parasitemia compared to control-infected mice that did not receive DOX and developed ECM. Infiltration of T cells and cytotoxic responses were reduced in brains of DOX-treated mice. Analysis of brain tissue by RNA-array revealed reduced expression of chemokines and tumour necrosis factor (TNF) in brains of DOX-treated mice. Furthermore, DOX-administration resulted in brains of the mice in reduced expression of matrix metalloproteinase 2 (MMP2) and granzyme B, which are both factors associated with ECM pathology. Systemic interferon gamma production was reduced and activated peripheral T cells accumulated in the spleen in DOX-treated mice. Our results suggest that DOX targeted inflammatory processes in the central nervous system (CNS) and prevented ECM by impaired brain access of effector T cells in addition to its anti-parasitic effect, thereby expanding the understanding of molecular events that underlie DOX-mediated therapeutic interventions.</p></div

Model of the dynamics and molecular mechanisms during the SET.

<p>(A) Model summarizing the dynamics and events driving acquisition of pluripotency and epigenetic reprogramming of TCam-2 cells to an EC-like cell fate. (B) Models of the fast and slow acting NODAL feedback loop. Arrows indicate 'activation', T-shaped arrows indicate 'inhibition'.</p

Global dynamics of Gex and 5mC during reprogramming of TCam-2 cells.

<p>(A—B) Unsupervised hierarchical clustering illustrates genome-wide changes in Gex (A) and 5mC (B) over time in indicated samples. Dendrograms indicate that during SET TCam-2 cells become more similar to the EC control cells.</p

DOX administration reduced ECM development and stabilized the blood-brain barrier after PbA infection.

<p>(A, B) C57BL/6 mice received 5*10⁴ <i>PbA</i>-infected erythrocytes (PbA-iRBC) and indicated mice were injected i.v. with 80 mg/kg DOX dpi 4–6. All animals were then monitored for (A) survival and (B) ECM score (ECM phase dpi 6–9 marked in grey). (C, D) C57BL/6 mice received 5*10⁴ iRBC and indicated groups of mice were treated orally with either 80 mg/kg/day or 250 mg/kg/day from dpi 4–6. Mice treated i.v. with 80 mg/kg/day DOX (dpi 4–7) served as reference treatment group. All mice were monitored for (C) survival and (D) ECM score (ECM phase dpi 6–9 marked in grey). Data sets are representative for 2–3 individual experiments with n = 10 mice/group. Survival data were analyzed with log-rank (Mantel-Cox) test. ECM scores are displayed as median. (E, F) Mice were infected with 5*10⁴ PbA-iRBC ± 80 mg DOX/kg/day. The integrity of the BBB was analyzed with an Evans Blue assay on dpi 6. All mice were injected i.v. with 2% Evans Blue dye and one hour later, extravasation of the dye into the brain was determined. (E) Photo documentation of the discoloration and (F) quantification of dye extravasation into the brain by measuring the absorbance of brain tissue at 620nm. Data show representative results of 1 from 2 independent experiments. *p<0.05, t-test on data after analysis of normal distribution.</p

BMP, NODAL and WNT signaling in GCC tissues and cell lines.

<p>(A) cDNA microarray expression data of BMP, NODAL and WNT signaling-associated genes in GCC tissues. <i>SOX2</i> and <i>SOX17</i> 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.</p

DOX regulates inflammatory responses and tissue degrading enzyme activity in brain cells of PbA infected animals.

<p>C57BL/6 mice were left naïve or infected with PbA as described above and indicated groups received DOX (80 mg/kg/day) from dpi 4–6. Six days post PbA infection, brain tissue of naïve mice and PbA infected mice ± DOX was examined for inflammatory responses and MMP activity. (A) RNA was prepared from brain tissue and analyzed via PCR array. Volcano plot analysis of the comparison PbA versus PbA DOX: Shown is the logarithmic fold-change (x-axis) against the negative logarithmic p-value (y-axis). The dashed lines indicate where p ≤ 0.05 and FC ≥2. (B) Additionally, IFN-γ was analyzed by RT-qPCR in the brain tissue of all groups (C) Quantification of MMP-2-specific zymography. Brain tissue extracted on dpi 6 was subjected to zymography and quantified by scanning densitometry of the gelatinolytic bands of MMP-2. Representative experiments are shown with 6–7 mice/ group. Relative scanning units of MMP-2 are shown as fold change against expression of naïve animals displayed as median (D) Characterization of brain leukocytes on dpi 6 from naïve, PbA and PbA+DOX-treated animals. Flow cytometry gating scheme to identify CD8⁺T cells is shown for representative animals from naïve and infected ± 80 mg/kg/day DOX mice. (E) Absolute numbers of brain infiltrating CD45^hiCD8a⁺ cells as determined by FACS. (F) Brain tissue supernatant from dpi 6 was analyzed with an ELISA for granzyme B. (B, C, E, F) *p<0.05, **p<0.01, ***p<0.001, ANOVA with Tukey’s post test on data after analysis of normal distribution.</p

Elevated initial infective parasite dose could not reverse DOX-mediated ECM protection.

<p>C57BL/6 mice were infected with either 5*10⁴ or 1*10⁶ PbA-iRBCs and received from dpi 4–6 80 mg DOX/kg/day i.v. or not. (A) Blood parasite burden was assessed from dpi 5 on. (B) Survival and (C) ECM score (ECM phase dpi 6–9 marked in grey) were analyzed over the course of infection with low or high dose of parasites and DOX treatment. Representative data from 2–3 individual experiments with n = 10 mice/group are shown. Parasitemia is displayed as the mean/SD and ECM score as median. Survival data was analysed with log-rank (Mantel-Cox) test.</p