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Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4+CD25+FoxP3+ regulatory T cells activation.

ABSTRACT: Rationale: Loss of histone macroH2A1 induces appearance of cancer stem cells (CSCs)-like cells in hepatocellular carcinoma (HCC). How CSCs interact with the tumor microenvironment and the adaptive immune system is unclear. Methods: We screened aggressive human HCC for macroH2A1 and CD44 CSC marker expression. We also knocked down (KD) macroH2A1 in HCC cells, and performed integrated transcriptomic and secretomic analyses. Results: Human HCC showed low macroH2A1 and high CD44 expression compared to control tissues. MacroH2A1 KD CSC-like cells transferred paracrinally their chemoresistant properties to parental HCC cells. MacroH2A1 KD conditioned media transcriptionally reprogrammed parental HCC cells activated regulatory CD4+/CD25+/FoxP3+ T cells (Tregs). Conclusions: Loss of macroH2A1 in HCC cells drives cancer stem-cell propagation and evasion from immune surveillance.

SUBMITTER: Lo Re O

PROVIDER: S-EPMC6929991 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4+CD25+FoxP3+ regulatory T cells activation.

Lo Re Oriana O Mazza Tommaso T Giallongo Sebastiano S Sanna Paola P Rappa Francesca F Vinh Luong Tu T Li Volti Giovanni G Drovakova Adela A Roskams Tania T Van Haele Matthias M Tsochatzis Emmanuel E Vinciguerra Manlio M

Theranostics 20200101 2

Rationale: Loss of histone macroH2A1 induces appearance of cancer stem cells (CSCs)-like cells in hepatocellular carcinoma (HCC). How CSCs interact with the tumor microenvironment and the adaptive immune system is unclear. Methods: We screened aggressive human HCC for macroH2A1 and CD44 CSC marker expression. We also knocked down (KD) macroH2A1 in HCC cells, and performed integrated transcriptomic and secretomic analyses. Results: Human HCC showed low macroH2A1 and high CD44 ...[more]

PMID: 31903159

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Project description:We recently described the phenotype of HepG2 and Huh-7, hepatocellular carcinoma cells, knocked down for histone variant macroH2A1. Both cell lines acquire a cancer stem cell phenotype (Lo Re O et al., Hepatology 2017, PMID: 28913935; Lo Re O et al., Epigenetics 2018, PMID: 30165787). We found that short hairpin RNA-mediated macroH2A1 knockdown induced acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolytic pathways. As macroH2A1 is a potent transcriptional modifier we asked how KD of this histone variant might affect patterns of gene expression, and whether we could identify potential mechanistic links to the observed in vitro and in vivo HCC phenotypes. Here we used RNA-Seq to gain deep mechanistic insight into the distinct functions of macroH2A1 KD and conditioned medium (CM)-exposed Huh-7 cells. Heatmap analysis of the differentially expressed genes between the three groups revealed a similar transcriptomic profile between KD and CM, compared to the control condition. Genes were considered differentially expressed between groups if their expression values significantly differed by >2 fold. Statistical differences in gene expression were assessed by the ANOVA test. Correction for multiple test was achieved by the Benjamini-Hochberg procedure. The significance threshold was set to 0.05. Using a cut-off of 2 fold change, assessment of differentially expressed genes for Huh-7 macroH2A1 KD or Huh-7 CM KD versus CTL showed no transcriptional overlap between the different Huh-7 cell lines. 783 and 987 genes were instead significantly differentially-expressed in macroH2A1 KD or CM KD versus control cells, respectively. Interestingly, the significantly enriched transcripts over-represented in KD and CM-exposed cells compared to control cells were implicated in a number of functions and diseases that were also shared between CM versus control comparisons. Specifically, Ingenuity pathway analysis (IPA) identified categories of cancer, gastrointestinal diseases, lipid metabolism, cell-to-cell signaling, nucleic acid metabolism, cell death & survival and others, in common between KD versus CTL and CM versus CTL. These data support a paracrine modulation of gene expression by macroH2A1 KD in HCC cells.

Project description:BackgroundMesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells (iTregs) in specific inflammatory contexts. Stable Foxp3 expression plays a major role in the phenotypic and functional stability of iTregs. However, how MSCs induce stable Foxp3 expression remains unknown.MethodsWe first investigated the role of cell-cell contact and cytokine secretion by bone marrow-derived MSCs (BM-MSCs) on the induction, stability, and suppressive functions of Tregs under various experimental conditions that lead to Foxp3 generation by flow cytometry and ELISA respectively. Second, we studied the effect of MSCs on TRAF6, GRAIL, USP7, STUB1, and UBC13 mRNA expression in CD4+ T cells in correlation with the suppressive function of iTregs by real-time PCR; also, we investigated Foxp3 Treg-specific demethylated region (TSDR) methylation in correlation with Foxp3 stability by the high-resolution melting technique. Third, we studied the effect of ex-vivo-expanded BM-MSCs on the induction of transplant tolerance in a model of fully allogeneic skin transplantation. We further analyzed the cytokine secretion patterns in grafted mice as well as the mRNA expression of ubiquitination genes in CD4+ T cells collected from the spleens of protected mice.ResultsWe found that in-vitro MSC-induced Tregs express high mRNA levels of ubiquitination genes such as TRAF6, GRAIL, and USP7 and low levels of STUB1. Moreover, they have enhanced TSDR demethylation. Infusion of MSCs in a murine model of allogeneic skin transplantation prolonged allograft survival. When CD4+ T cells were harvested from the spleens of grafted mice, we observed that mRNA expression of the Foxp3 gene was elevated. Furthermore, Foxp3 mRNA expression was associated with increased TRAF6, GRAIL, UBC13, and USP7 and decreased STUB1 mRNA levels compared with the levels observed in vitro.ConclusionsOur data suggest a possible ubiquitination mechanism by which MSCs convert Tconvs to suppressive and stable iTregs.

Project description:Systemic inflammation associated with major surgery rapidly accelerates atherosclerotic plaque progression in mice. Regulatory T cells (Tregs) have emerged as important modulators of atherogenesis. In coronary artery disease patients, low frequency of Tregs constitutes an independent risk factor for cardiovascular complications after non-cardiac surgery. In this exploratory analysis, we investigate whether preoperative Treg levels affect surgery-induced atherosclerotic lesion destabilization in a murine model of perioperative stress. After 9 weeks of high-cholesterol diet, atherosclerotic apolipoprotein E-deficient mice with modulated Treg levels were subjected to a 30-minute surgical procedure consisting of general isoflurane anesthesia, laparotomy and moderate blood loss. Controls underwent general anesthesia only. Brachiocephalic arteries were harvested 3 days after the intervention for histomorphological analyses of atherosclerotic plaques. Tregs were depleted by a single dose of anti-CD25 monoclonal antibody (mAb) administered 6 days prior to the intervention. Expansion of Tregs was induced by daily injections of IL-2/anti-IL-2 complex (IL-2C) on three consecutive days starting 3 days before surgery. Isotype-matched antibodies and PBS served as controls. Antibody-mediated modulation was Treg-specific. IL-2C treatment resulted in an eight-fold elevation of peripheral CD4+CD25+Foxp3+ Tregs compared to mice administered with anti-CD25 mAb. In mice treated with PBS and anti-CD25 mAb, surgical stress response caused a significant increase of atherosclerotic plaque necrosis (PBS: p < 0.001; anti-CD25 mAb: p = 0.037). Preoperative Treg expansion abrogated perioperative necrotic core formation (p = 0.556) and significantly enhanced postoperative atherosclerotic plaque stability compared to PBS-treated mice (p = 0.036). Postoperative plaque volume (p = 0.960), stenosis (p = 0.693), lesional collagen (p = 0.258), as well as the relative macrophage (p = 0.625) and smooth muscle cell content (p = 0.178) remained largely unaffected by preoperative Treg levels. In atherosclerotic mice, therapeutic expansion of Tregs prior to major surgery mitigates rapid effects on perioperative stress-driven atherosclerotic plaque destabilization. Future studies will show, whether short-term interventions modulating perioperative inflammation qualify for prevention of cardiovascular events associated with major non-cardiac surgery.

Dataset Information

Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4+CD25+FoxP3+ regulatory T cells activation.

Publications

Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> regulatory T cells activation.

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