Regulatory T cells (Treg) and tumor-exosomes are thought to play a role in preventing the rejection of malignant cells in patients bearing nasopharyngeal carcinoma (NPC).
Treg recruitment by exosomes ...derived from NPC cell lines (C15/C17-Exo), exosomes isolated from NPC patients' plasma (Patient-Exo), and CCL20 were tested in vitro using Boyden chamber assays and in vivo using a xenograft SCID mouse model (n = 5), both in the presence and absence of anti-CCL20 monoclonal antibodies (mAb). Impact of these NPC exosomes (NPC-Exo) on Treg phenotype and function was determined using adapted assays (FACS, Q-PCR, ELISA, and MLR). Experiments were performed in comparison with exosomes derived from plasma of healthy donors (HD-Exo). The Student's t test was used for group comparisons. All statistical tests were two-sided.
CCL20 allowed the intratumoral recruitment of human Treg. NPC-Exo also facilitated Treg recruitment (3.30 ± 0.34 fold increase, P < .001), which was statistically significantly inhibited (P < .001) by an anti-CCL20 blocking mAb. NPC-Exo also recruited conventional CD4(+)CD25(-) T cells and mediated their conversion into inhibitory CD4(+)CD25(high) cells. Moreover, NPC-Exo enhanced (P = .0048) the expansion of human Treg, inducing the generation of Tim3(Low) Treg with increased expression of CD25 and FOXP3. Finally, NPC-Exo induced an overexpression of cell markers associated with Treg phenotype, properties and recruitment capacity. For example, GZMB mean fold change was 21.45 ± 1.75 (P < .001). These results were consistent with a stronger suppression of responder cells' proliferation and the secretion of immunosuppressive cytokines (IL10, TGFB1).
Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC.
DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly ...understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.
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•Dnmt3a is phosphorylated by CK2 at two residues, reducing its activity•CK2 modulates CpG methylation of several repeats, most notably of Alu SINEs•CK2-mediated phosphorylation of Dnmt3a favors its heterochromatic localization•Phosphorylation of Dnmt3a by CK2 shapes the CpG methylome
The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Deplus et al. now demonstrate that CK2 phosphorylates Dnmt3a, downregulating its ability to methylate DNA at several repeats, most notably Alu SINEs. The authors also show that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. This study highlights phosphorylation as a mode of regulation of de novo DNA methyltransferase and sheds light on the origin of DNA methylation patterns.
Bone metastasis is the major deleterious event in prostate cancer (PCa). TMPRSS2-ERG fusion is one of the most common chromosomic rearrangements in PCa. However, its implication in bone metastasis ...development is still unclear. Since bone metastasis starts with the tropism of cancer cells to bone through specific migratory and invasive processes involving osteomimetic capabilities, it is crucial to better our understanding of the influence of TMPRSS2-ERG expression in the mechanisms underlying the bone tropism properties of PCa cells. We developed bioluminescent cell lines expressing the TMPRSS2-ERG fusion in order to assess its role in tumor growth and bone metastasis appearance in a mouse model. First, we showed that the TMPRSS2-ERG fusion increases cell migration and subcutaneous tumor size. Second, using intracardiac injection experiments in mice, we showed that the expression of TMPRSS2-ERG fusion increases the number of metastases in bone. Moreover, TMPRSS2-ERG affects the pattern of metastatic spread by increasing the incidence of tumors in hind limbs and spine, which are two of the most frequent sites of human PCa metastases. Finally, transcriptome analysis highlighted a series of genes regulated by the fusion and involved in the metastatic process. Altogether, our work indicates that TMPRSS2-ERG increases bone tropism of PCa cells and metastasis development.
Summary
Epstein–Barr Virus (EBV) is present in the neoplastic cells of around 20–30% of patients with Hodgkin Lymphoma (HL). Although, an immunosuppressive environment is currently described in HL ...patients, little is known concerning the regulatory mechanism induced by EBV proteins expression in tumour cells. This study aimed to investigate an association between regulatory Type 1 cells (Tr1) and EBV tissue positivity in HL patients. Transcriptomic analysis of both EBV‐positive and EBV‐negative tumours showed that EBV infection increased gene expression of Tr1‐related markers (ITGA2, ITGB2, LAG3) and associated‐immunosuppressive cytokines (IL10). This up‐regulation was associated with an over‐expression of several chemokine markers known to attract T‐helper type 2 (Th2) and regulatory T cells thus contributing to immune suppression. This Tr1 cells recruitment in EBV‐positive HL was confirmed by immunohistochemical analysis of frozen nodes biopsies and by flow cytometric analysis of peripheral blood mononuclear cells of EBV‐positive patients. Additionally, we showed that IL10 production was significantly enhanced in tumours and blood of EBV‐positive HL patients. Our results propose a new model in which EBV can recruit Tr1 cells to the nodes' microenvironment, suggesting that the expression of EBV proteins in tumour cells could enable the escape of EBV‐infected tumour cells from the virus‐specific CTL response.
The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is ...overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis.
Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples.
Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer.
MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression.
Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF-κB signaling in development and ...progression of CAC were derived from preclinical studies, whereas data from human are rare. The aim of this work was to study the contribution of NF-κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF-α, TLR4, and NF-κB, in colonic mucosa. We also studied NO/TNF-α modulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF-κB, TNF-α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF-α/NO production, as well as the role of NF-κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF-α induction through NF-κB pathway was suggested. AtRA downregulates NOS2 and TNF-α expression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF-κB signaling pathway targeting NOS2 and TNF-α expression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression.
The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to ...virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kappaB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5' Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kappaB and degradation of cytoplasmic NF-kappaB inhibitor, IkappaBalpha . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8(+)-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4(+) T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.
Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour ...development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss‐of‐function genetic screen in primary human fibroblasts. We report that knockdown of the M‐type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress‐induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species–DNA damage–p53‐dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.
Little is known about the physiological role of the phospholipase A2 receptor (PLA2R1). PLA2R1 has been described as regulating the replicative senescence, a telomerase-dependent proliferation ...arrest. The downstream PLA2R1 signaling and its role in cancer are currently unknown. Senescence induction in response to activated oncogenes is a failsafe program of tumor suppression that must be bypassed for tumorigenesis. We now present evidence that PLA2R1 functions in vitro as a tumor suppressor, the depletion of which is sufficient to escape oncogene-induced senescence (OIS), thereby facilitating oncogenic cell transformation. Furthermore, mice that are genetically deficient in PLA2R1 display increased sensitivity to RAS-induced tumorigenesis by facilitating OIS escape, highlighting its physiological role as a tumor suppressor. Unexpectedly, PLA2R1 activated JAK2 and its effector signaling, with PLA2R1-mediated inhibition of cell transformation largely reverted in JAK2-depleted cells. This finding was unexpected as the JAK2 pathway has been associated mainly with protumoral functions and several inhibitors are currently in clinical trials. Taken together, our findings uncover an unanticipated tumor suppressive role for PLA2R1 that is mediated by targeting downstream JAK2 effector signaling.
The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc ...silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Here we show that Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, we observe specific reactivation of the Myc‐repressed p21Cip1 gene, whereas the expression of Myc‐activated E‐boxes genes is unchanged. In addition, we find that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA‐binding factor Miz‐1. Consistent with this, we observe that Myc and Dnmt3a form a ternary complex with Miz‐1 and that this complex can corepress the p21Cip1 promoter. Finally, we show that DNA methylation is required for Myc‐mediated repression of p21Cip1. Our data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell.
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