Expression of the p53 target CDIP correlates with sensitivity to TNF?-induced apoptosis in cancer cells.
ABSTRACT: TNF? is a pleiotropic cytokine that signals for both survival and apoptotic cell fates. It is still unclear that the dual role of TNF? can be regulated in cancer cells. We previously described an apoptotic pathway involving p53?CDIP?TNF? that was activated in response to genotoxic stress. This pathway operated in the presence of JNK activation; therefore, we postulated that CDIP itself could sensitize cells to a TNF? apoptotic cell fate, survival, or death. We show that CDIP mediates sensitivity to TNF?-induced apoptosis and that cancer cells with endogenous CDIP expression are inherently sensitive to the growth-suppressive effects of TNF? in vitro and in vivo. Thus, CDIP expression correlates with sensitivity of cancer cells with TNF?, and CDIP seems to be a regulator of the p53-mediated death versus survival response of cells to TNF?. This CDIP-mediated sensitivity to TNF?-induced apoptosis favors pro- over antiapoptotic program in cancer cells, and CDIP may serve as a predictive biomarker for such sensitivity.
Project description:We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.
Project description:Tumor necrosis factor-? (TNF-?) plays a significant role in inflammation and cancer-related apoptosis. We identified a TNF-?-mediated epigenetic mechanism of apoptotic cell death regulation in estrogen receptor-? (ER?)-positive human breast cancer cells. To assess the apoptotic effect of TNF-?, annexin V/ propidium iodide (PI) double staining, cell viability assays, and Western blotting were performed. To elucidate this mechanism, histone deacetylase (HDAC) activity assay and immunoprecipitation (IP) were conducted; the mechanism was subsequently confirmed through chromatin IP (ChIP) assays. Finally, we assessed HDAC3-ER?-mediated apoptotic cell death after TNF-? treatment in ER?-positive human breast cancer (MCF-7) cells via the transcriptional activation of p53 target genes using luciferase assay and quantitative reverse transcription PCR. The TNF-?-induced selective apoptosis in MCF-7 cells was negatively regulated by the HDAC3-ER? complex in a caspase-7-dependent manner. HDAC3 possessed a p53-binding element, thus suppressing the transcriptional activity of its target genes. In contrast, MCF-7 cell treatment with TNF-? led to dissociation of the HDAC3-ER? complex and substitution of the occupancy on the promoter by the p53-p300 complex, thus accelerating p53 target gene expression. In this process, p53 stabilization was accompanied by its acetylation. This study showed that p53-mediated apoptosis in ER?-positive human breast cancer cells was negatively regulated by HDAC3-ER? in a caspase-7-dependent manner. Therefore, these proteins have potential application in therapeutic strategies.
Project description:Isolated limb perfusion with TNF-α and melphalan is used with remarkable efficiency to treat unresectable limb sarcomas. Here we tested the ability of TNF-α to directly induce apoptosis of sarcoma cells. In addition, we investigated the impact of p53 in the regulation of such effect.We first analysed the ability of TNF-α to induce apoptosis in freshly isolated tumour cells. For this purpose, sarcoma tumours (n = 8) treated ex vivo with TNF-α were processed for TUNEL staining. It revealed substantial endothelial cell apoptosis and levels of tumour cell apoptosis that varied from low to high. In order to investigate the role of p53 in TNF-α-induced cell death, human sarcoma cell lines (n = 9) with different TP53 and MDM2 status were studied for their sensitivity to TNF-α. TP53(Wt) cell lines were sensitive to TNF-α unless MDM2 was over-expressed. However, TP53(Mut) and TP53(Null) cell lines were resistant. TP53 suppression in TP53(Wt) cell lines abrogated TNF-α sensitivity and TP53 overexpression in TP53(Null) cell lines restored it. The use of small molecules that restore p53 activity, such as CP-31398 or Nutlin-3a, in association with TNF-α, potentiated the cell death of respectively TP53(Mut) and TP53(Wt)/MDM2(Ampl). In particular, CP-31398 was able to induce p53 as well as some of its apoptotic target genes in TP53(Mut) cells. In TP53(Wt)/MDM2(Ampl) cells, Nutlin-3a effects were associated with a decrease of TNF-α-induced NF-κB-DNA binding and correlated with a differential regulation of pro- and anti-apoptotic genes such as TP53BP2, GADD45, TGF-β1 and FAIM.More effective therapeutic approaches are critically needed for the treatment of unresectable limb sarcomas. Our results show that restoring p53 activity in sarcoma cells correlated with increased sensitivity to TNF-α, suggesting that this strategy may be an important determinant of TNF-α-based sarcomas treatment.
Project description:This model is from the article:
Heterogeneity Reduces Sensitivity of Cell Death for TNF-Stimuli
Schliemann M, Bullinger E, Borchers S, Allgower F, Findeisen R, Scheurich P. BMC Syst Biol.
2011 Dec 28;5(1):204. 22204418
Apoptosis is a form of programmed cell death essential for the maintenance of homeostasis and the removal of potentially damaged cells in multicellular organisms. By binding its cognate membrane receptor, TNF receptor type 1 (TNF-R1), the proinflammatory cytokine Tumor Necrosis Factor (TNF) activates pro-apoptotic signaling via caspase activation, but at the same time also stimulates nuclear factor kappaB (NF-kappaB)-mediated survival pathways. Differential dose-response relationships of these two major TNF signaling pathways have been described experimentally and using mathematical modeling. However, the quantitative analysis of the complex interplay between pro- and anti-apoptotic signaling pathways is an open question as it is challenging for several reasons: the overall signaling network is complex, various time scales are present, and cells respond quantitatively and qualitatively in a heterogeneous manner.
This study analyzes the complex interplay of the crosstalk of TNF-R1 induced pro- and anti-apoptotic signaling pathways based on an experimentally validated mathematical model. The mathematical model describes the temporal responses on both the single cell level as well as the level of a heterogeneous cell population, as observed in the respective quantitative experiments using TNF-R1 stimuli of different strengths and durations. Global sensitivity of the heterogeneous population was quantified by measuring the average gradient of time of death versus each population parameter. This global sensitivity analysis uncovers the concentrations of Caspase-8 and Caspase-3, and their respective inhibitors BAR and XIAP, as key elements for deciding the cell's fate. A simulated knockout of the NF-kappaB-mediated anti-apoptotic signaling reveals the importance of this pathway for delaying the time of death, reducing the death rate in the case of pulse stimulation and significantly increasing cell-to-cell variability.
Cell ensemble modeling of a heterogeneous cell population including a global sensitivity analysis presented here allowed us to illuminate the role of the different elements and parameters on apoptotic signaling. The receptors serve to transmit the external stimulus; procaspases and their inhibitors control the switching from life to death, while NF-kappaB enhances the heterogeneity of the cell population. The global sensitivity analysis of the cell population model further revealed an unexpected impact of heterogeneity, i.e. the reduction of parametric sensitivity.
SBML model generated from Matlab system description
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Project description:Angiogenin, a 14-kDa multifunctional pro-angiogenic growth factor, is upregulated in several types of cancers. Anti-angiogenin monoclonal antibodies used as antagonists inhibited the establishment, progression and metastasis of human cancer cells in athymic mice (Olson et al., 1994). Silencing angiogenin and inhibition of angiogenin's nuclear translocation blocked cell survival and induced cell death in B-lymphoma and endothelial cells latently infected with Kaposi sarcoma-associated herpesvirus (Sadagopan et al., 2009), suggesting that actively proliferating cancer cells could be inducing angiogenin for inhibiting apoptotic pathways. However, the mechanism of cell survival and apoptosis regulation by angiogenin and their functional significance in cancer is not known. We demonstrate that angiogenin interacts with p53 and colocalizes in the nucleus. Silencing endogenous angiogenin induced p53 promoter activation and p53 target gene (p53, p21 and Bax) expression, downregulated anti-apoptotic Bcl-2 gene expression and increased p53-mediated cell death. In contrast, angiogenin expression blocked pro-apoptotic Bax and p21 expression, induced Bcl-2 and blocked cell death. Angiogenin also co-immunoprecipitated with p53 regulator protein Mdm2. Angiogenin expression resulted in the inhibition of p53 phosphorylation, increased p53-Mdm2 interaction, and consequently increased ubiquitination of p53. Taken together, these studies demonstrate that angiogenin promotes the inhibition of p53 function to mediate anti-apoptosis and cell survival. Our results reveal for the first time a novel p53 interacting function of angiogenin in anti-apoptosis and survival of cancer cells and suggest that targeting angiogenin could be an effective therapy for several cancers.
Project description:Human embryonic stem cells (hESCs) are highly sensitive to DNA damage and have low survival ability relative to differentiated cells. We investigated the source of this difference by comparing damage response pathways in hESCs and differentiated cells. We found that hESCs undergo more rapid p53-dependent apoptosis after DNA damage than differentiated cells do. However, p53 localization and function are similar between hESCs and differentiated cells, suggesting that p53 alone cannot explain the difference in sensitivity. Instead, we show that mitochondrial readiness for apoptosis, known as mitochondrial priming, differs between hESCs and differentiated cells. Specifically, the balance between proapoptotic and antiapoptotic proteins is shifted closer to the apoptotic threshold in hESCs than in differentiated cells. Altering this balance in differentiated cells increases their sensitivity and results in cell death, suggesting that manipulation of mitochondrial priming could potentially alter the sensitivity of other stem cells, including cancer stem cells.
Project description:Renal cell carcinoma (RCC) is the most common type of kidney cancer. It has a poor prognosis, with approximately 20-30% of patients developing recurrent and/or metastatic diseases that is relatively high resistant to conventional therapy. Resisting cell death is a hallmark of cancer cells. Apoptosis is a form of programmed cell death mediated by the activation of caspases. Necroptosis is a form of regulated necrosis that relies on the activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like protein (MLKL), the substrate of RIPK3. Cancer cells often display apoptosis resistance via upregulation of anti-apoptotic genes and defective necroptosis due to the epigenetic silence of Ripk3. MicroRNAs (miRNAs) are non-coding small RNAs that are involved in numerous biological processes including cell proliferation, differentiation and death. In this study, we screened a set of ?120 miRNAs for apoptosis-regulating miRNAs and identified miR-381-3p as a suppressor of TNF-induced apoptosis in various cancer cells. Ectopic expression of miR-381-3p inhibits the activation of caspase-8 and caspase-3. The expression level of miR-381-3p inversely correlates with the sensitivity of cancer cells to TNF-induced apoptosis. Moreover, we found that overexpression of miR-381-3p blocks TNF-induced necroptosis by inhibiting the activation of RIPK3 and MLKL. Of note, Kaplan-Meier Plotter analysis demonstrates that papillary RCC patients with high miR-381-3p expression have a lower overall survival than those with low expression level of miR-381-3p. Importantly, miR-381-3p overexpression promotes colony formation in human renal cancer cells. Thus, miR-381-3p acts as an oncogenic miRNA that counteracts both apoptotic and necroptotic signaling pathways. Our findings highlight miR-381-3p as a biomarker for predicting sensitivity to apoptosis and necroptosis, and as a possible therapeutic target for RCC.
Project description:BACKGROUND: The incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours. RESULTS: Here we show that LiCl induces apoptosis of tumour cells both in vitro and in vivo. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-? and FasL expression. Inhibition of TNF-? induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth. CONCLUSIONS: Induction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-? and FasL.Key words: LiCl, TNF-?, FasL, apoptosis, GSK-3, FasL.
Project description:Glioblastoma multiforme (GBM) are resistant to TNF?-induced apoptosis and blockade of TNF?-induced NF-?B activation sensitizes glioma cells to apoptosis. As Casein kinase-2 (CK2) induces aberrant NF-?B activation and as we observed elevated CK2 levels in GBM tumors, we investigated the potential of CK2 inhibitors (CK2-Is) - DRB and Apigenin in sensitizing glioma cells to TNF?-induced apoptosis. CK2-Is and CK2 small interfering RNA (siRNA) reduced glioma cell viability, inhibited TNF?-mediated NF-?B activation, and sensitized cell to TNF?-induced apoptosis. Importantly, CK2-Is activated p53 function in wild-type but not in p53 mutant cells. Activation of p53 function involved its increased transcriptional activation, DNA-binding ability, increased expression of p53 target genes associated with cell cycle progression and apoptosis. Moreover, CK2-Is decreased telomerase activity and increased senescence in a p53-dependent manner. Apoptotic gene profiling indicated that CK2-Is differentially affect p53 and TNF? targets in p53 wild-type and mutant glioma cells. CK2-I decreased MDM2-p53 association and p53 ubiquitination to enhance p53 levels. Interestingly, CK2-Is downregulated SIRT1 activity and over-expression of SIRT1 decreased p53 transcriptional activity and rescued cells from CK2-I-induced apoptosis. This ability of CK2-Is to sensitize glioma to TNF?-induced death via multiple mechanisms involving abrogation of NF-?B activation, reactivation of wild-type p53 function and SIRT1 inhibition warrants investigation.
Project description:Cisplatin-based chemotherapy is currently the standard treatment for locally advanced esophageal cancer. Cisplatin has been shown to induce both apoptosis and necrosis in cancer cells, but the mechanism by which programmed necrosis is induced remains unknown. In this study, we provide evidence that cisplatin induces necrotic cell death in apoptosis-resistant esophageal cancer cells. This cell death is dependent on RIPK3 and on necrosome formation via autocrine production of TNF?. More importantly, we demonstrate that RIPK3 is necessary for cisplatin-induced killing of esophageal cancer cells because inhibition of RIPK1 activity by necrostatin or knockdown of RIPK3 significantly attenuates necrosis and leads to cisplatin resistance. Moreover, microarray analysis confirmed an anti-apoptotic molecular expression pattern in esophageal cancer cells in response to cisplatin. Taken together, our data indicate that RIPK3 and autocrine production of TNF? contribute to cisplatin sensitivity by initiating necrosis when the apoptotic pathway is suppressed or absent in esophageal cancer cells. These data provide new insight into the molecular mechanisms underlying cisplatin-induced necrosis and suggest that RIPK3 is a potential marker for predicting cisplatin sensitivity in apoptosis-resistant and advanced esophageal cancer.