[Show abstract][Hide abstract] ABSTRACT: Senescent cells secrete a combination of factors collectively known as the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence and activates an immune surveillance response, but it can also show pro-tumorigenic properties and contribute to age-related pathologies. In a drug screen to find new SASP regulators, we uncovered the mTOR inhibitor rapamycin as a potent SASP suppressor. Here we report a mechanism by which mTOR controls the SASP by differentially regulating the translation of the MK2 (also known as MAPKAPK2) kinase through 4EBP1. In turn, MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1 during senescence, inhibiting its ability to degrade the transcripts of numerous SASP components. Consequently, mTOR inhibition or constitutive activation of ZFP36L1 impairs the non-cell-autonomous effects of senescent cells in both tumour-suppressive and tumour-promoting contexts. Altogether, our results place regulation of the SASP as a key mechanism by which mTOR could influence cancer, age-related diseases and immune responses.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) are professional antigen-presenting cells playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of BM hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
European Journal of Immunology 07/2015; 45(10). DOI:10.1002/eji.201545530 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction Oncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism, but its impact on tumorigenesis is largely dependent on the nature of SASP, senescence-associated secretory phenotype. Major components of the SASP include TGFβ1 and pro-inflammatory cytokines, such as IL1A, IL6, and IL8 that have pleiotropic context-dependent effects.
Method We utilised the well validated ER:RasG12VIMR90 HDF in vitromodel which undergo Ras-induced senescence (RIS) with 4OHT. In vivohepatocyte RIS was achieved with hydrodynamic tail-vein injection of NRasG12V-containing transposons.
Results Previously, we have shown that Notch1, a highly conserved receptor is up-regulated in RIS. In contrast to the up-regulation of Notch1, downstream signalling is dynamically regulated; Notch-target genes were transiently up-regulated at an early phase of RIS, but down-regulated at full senescence. The dynamic expression pattern of N1ICD and TGF-β1 expression were nearly identical, and inversely correlated with the cytokines, IL1A and IL8. Inhibition of Notch1 signalling, through expression of a dominant-negative form of the Notch1 binding partner MAML1, led to a reduction in TGF-β1, but increased IL1A and IL8 expression during RIS, suggesting Notch1 signalling plays a critical role in secretome switching.
It has been shown that the SASP in RIS is regulated by two major transcription factors, NFkB and CEBPβ. Strikingly, over-expression of N1ICD strongly down-regulated CEBPβ, but not NFkB, in fully established RIS cells. Further, expression of ectopic CEBPβ in N1ICD-expressing cells partially restored levels of IL6/8. N1ICD was also able to suppress pro-inflammatory cytokine expression in TNF-α stimulated cells, through repression of CEBPβ. These data indicate that Notch1 represses pro-inflammatory cytokines by down-regulating CEBPβ. Finally, Notch1 up-regulation in OIS was confirmed in Nras-driven hepatocyte senescence; Notch1 inhibition through dnMAML1 promoted immune-mediated clearance of senescent hepatocytes.
Conclusion We propose that the transition to OIS is correlated with a switch from Notch1-driven TGFβ-rich secretome to a CEBPβ-driven IL1/8 rich secretome, and that dynamic Notch1 signalling modulates senescence and its long-term fate strictly through a non-cell-autonomous fashion.
Disclosure of interest None Declared.
[Show abstract][Hide abstract] ABSTRACT: Genomic analysis of human hepatocellular carcinoma (HCC) is potentially confounded by the differentiation state of the hepatic cell-of-origin. Here we integrated genomic analysis of mouse HCC (with defined cell-of-origin) along with normal development. We found a major shift in expression of Wnt and RXR-α pathway genes (up and down, respectively) coincident with the transition from hepatoblasts to hepatocytes. A combined Wnt and RXR-α gene signature categorized HCCs into two subtypes (high Wnt, low RXR-α and low Wnt, high RXR-α), which matched cell-of-origin in mouse models and the differentiation state of human HCC. Suppression of RXR-α levels in hepatocytes increased Wnt signaling and enhanced tumorigenicity, whereas ligand activation of RXR-α achieved the opposite. These results corroborate that there are two main HCC subtypes that correspond to the degree of hepatocyte differentation and that RXR-α, in part via Wnt signaling, plays a key functional role in the hepatocyte-like subtype and potentially could serve as a selective therapeutic target.
PLoS ONE 03/2015; 10(3):e0118480. DOI:10.1371/journal.pone.0118480 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.
Nature Medicine 09/2014; 20(10). DOI:10.1038/nm.3679 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner.
The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: The p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.
[Show abstract][Hide abstract] ABSTRACT: In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.
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Mouse Double Minute homolog 4 (MDM4) gene up-regulation often occurs in human hepatocellular carcinoma (HCC), but the molecular mechanisms responsible for its induction remain poorly understood. Here we investigated the role of the phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (PI3K/AKT/mTOR) axis in the regulation of MDM4 levels in HCC. The activity of MDM4 and the PI3K/AKT/mTOR pathway was modulated in human HCC cell lines by way of silencing and overexpression experiments. Expression of main pathway components was analyzed in an AKT mouse model and human HCCs. MDM4 inhibition resulted in growth restraint of HCC cell lines both in vitro and in vivo. Inhibition of the PI3K-AKT and/or mTOR pathways lowered MDM4 protein levels in HCC cells and reactivated p53-dependent transcription. Deubiquitination by ubiquitin-specific protease 2a and AKT-mediated phosphorylation protected MDM4 from proteasomal degradation and increased its protein stability. The eukaryotic elongation factor 1A2 (EEF1A2) was identified as an upstream inducer of PI3K supporting MDM4 stabilization. Also, we detected MDM4 protein up-regulation in an AKT mouse model and a strong correlation between the expression of EEF1A2, activated/phosphorylated AKT, and MDM4 in human HCC (each rho > 0.8, P < 0.001). Noticeably, a strong activation of this cascade was associated with shorter patient survival.
The EEF1A2/PI3K/AKT/mTOR axis promotes the protumorigenic stabilization of the MDM4 protooncogene in human HCC by way of a posttranscriptional mechanism. The activation level of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis significantly influences the survival probability of HCC patients in vivo and may thus represent a promising molecular target.
[Show abstract][Hide abstract] ABSTRACT: Immunotherapy of solid tumors is often hampered by the low frequency of tumor-specific T cells elicited by current vaccination strategies. Here we describe a prime-boost vaccination protocol based on the administration of antigen conjugated to PLGA microspheres followed by booster vaccination with Listeria monocytogenes vectors, which rapidly generates potent immune responses within two weeks. Compared with conventional vaccination with antigen-pulsed dendritic cells, the use of PLGA microspheres resulted in immune responses of significantly higher magnitude, which could be further enhanced via co-injection of Toll-like receptor 3 agonists. In an immunocompetent model of subcutaneous hepatocellular carcinoma, PLGA/Listeria vaccination resulted in complete remission of established tumors and prolonged survival. To further test the efficacy of the novel vaccination for the treatment of solid tumors, we developed an orthotopic liver cancer model based on the injection of transposon-flanked plasmids expressing oncogenes and model antigens. In this transgenic mouse model of liver cancer, PLGA/Listeria vaccination resulted in eradication of liver tumors, long-term survival of animals and establishment of stable cancer-specific memory CD8(+) T-cell populations. Therefore, combined PLGA/Listeria vaccination holds promise as a novel immunotherapeutic option for the treatment of solid cancers and as a means to boost the therapeutic efficacy of established cancer vaccines. This article is protected by copyright. All rights reserved.
European Journal of Immunology 04/2014; 44(4). DOI:10.1002/eji.201343794 · 4.03 Impact Factor