Mitochondrial uncoupling reveals a novel therapeutic opportunity for p53-defective cancers.

There are considerable challenges in directly targeting the mutant p53 protein, given the large heterogeneity of p53 mutations in the clinic. An alternative approach is to exploit the altered fitness of cells imposed by loss-of-wild-type p53. Here we identify niclosamide through a HTS screen for compounds selectively killing p53-deficient cells. Niclosamide impairs the growth of p53-deficient cells and of p53 mutant patient-derived ovarian xenografts. Metabolome profiling reveals that niclosamide induces mitochondrial uncoupling, which renders mutant p53 cells susceptible to mitochondrial-dependent apoptosis through preferential accumulation of arachidonic acid (AA), and represents a first-in-class inhibitor of p53 mutant tumors. Wild-type p53 evades the cytotoxicity by promoting the transcriptional induction of two key lipid oxygenation genes, ALOX5 and ALOX12B, which catalyzes the dioxygenation and breakdown of AA. Therefore, we propose a new paradigm for targeting cancers defective in the p53 pathway, by exploiting their vulnerability to niclosamide-induced mitochondrial uncoupling.

Aberrant activation of the GIMAP enhancer by oncogenic transcription factors in T-cell acute lymphoblastic leukemia.

The transcription factor TAL1/SCL is one of the most prevalent oncogenes in T-cell acute lymphoblastic leukemia (T-ALL), a malignant disorder resulting from leukemic transformation of thymus T-cell precursors. TAL1 is normally expressed in hematopoietic stem cells (HSCs) but is silenced in immature thymocytes. We hypothesize that TAL1 contributes to leukemogenesis by activating genes that are normally repressed in immature thymocytes. Herein, we identified a novel TAL1-regulated super-enhancer controlling the GIMAP locus, which resides within an insulated chromosomal locus in T-ALL cells. The GIMAP genes are expressed in HSCs and mature T cells but are downregulated during the immature stage of thymocyte differentiation. The GIMAP enhancer is activated by TAL1, RUNX1 and GATA3 in human T-ALL cells but is repressed by E-proteins. Overexpression of human GIMAP genes in immature thymocytes alone does not induce tumorigenesis but accelerates leukemia development in zebrafish. Our results demonstrate that aberrant activation of the GIMAP enhancer contributes to T-cell leukemogenesis.

Mouse models for core binding factor leukemia.

RUNX1 and CBFB are among the most frequently mutated genes in human leukemias. Genetic alterations such as chromosomal translocations, copy number variations and point mutations have been widely reported to result in the malfunction of RUNX transcription factors. Leukemias arising from such alterations in RUNX family genes are collectively termed core binding factor (CBF) leukemias. Although adult CBF leukemias generally are considered a favorable risk group as compared with other forms of acute myeloid leukemia, the 5-year survival rate remains low. An improved understanding of the molecular mechanism for CBF leukemia is imperative to uncover novel treatment options. Over the years, retroviral transduction-transplantation assays and transgenic, knockin and knockout mouse models alone or in combination with mutagenesis have been used to study the roles of RUNX alterations in leukemogenesis. Although successful in inducing leukemia, the existing assays and models possess many inherent limitations. A CBF leukemia model which induces leukemia with complete penetrance and short latency would be ideal as a platform for drug discovery. Here, we summarize the currently available mouse models which have been utilized to study CBF leukemias, discuss the advantages and limitations of individual experimental systems, and propose suggestions for improvements of mouse models.

Rap1-mediated nuclear factor-kappaB (NF-κB) activity regulates the paracrine capacity of mesenchymal stem cells in heart repair following infarction.

Paracrine effect is the major mechanism that underlies mesenchymal stem cells (MSC)-based therapy. This study aimed to examine how Rap1, telomeric repeat-binding factor 2-interacting protein 1 (Terf2IP), which is a novel modulator involved in the nuclear factor-kappaB (NF-κB) pathway, regulates the paracrine effects of MSC-mediated heart repair following infarction. NF-κB activity of stromal cells was increased by Rap1 as measured by pNF-κB-luciferase reporter activity, and this was abolished by IkB-dominant-negative protein. Knockdown of Rap1 with shRap1 resulted in diminished translocation of p65-NF-κB from the cytoplasm to nuclei in response to tumor necrosis factor-α (TNF-α) stimulation. Compared with BM-MSCs, Rap1(-/-)-BM-MSCs displayed a significantly reduced ratio of phosphorylated NF-κB to NF-κB-p65 and of Bax to Bcl-2, and increased resistance to hypoxia-induced apoptosis by the terminal deoxynucleotidal transferase-mediated dUTP nick end labeling (TUNEL) assay. In contrast, re-expression of Rap1 in Rap1(-/-)-BM-MSCs resulted in loss of resistance to apoptosis in the presence of hypoxia. Moreover, absence of Rap1 in BM-MSCs led to downregulation of NF-κB activity accompanied by reduced pro-inflammatory paracrine cytokines TNF-α, IL (interleukin)-6 and monocyte chemotactic protein-1 in Rap1(-/-)-BM-MSCs compared with BM-MSCs. The apoptosis of neonatal cardiomyocytes (NCMCs) induced by hypoxia was significantly reduced when cocultured with Rap1(-/-)-BM-MSC hypoxic-conditioned medium (CdM). The increased cardioprotective effects of Rap1(-/-)-BM-MSCs were reduced when Rap1(-/-)-BM-MSCs were reconstituted with Rap1 re-expression. Furthermore, in vivo study showed that transplantation of Rap1(-/-)-BM-MSCs significantly improved heart function, decreased infarct size, prevented cardiomyocyte apoptosis and inhibited inflammation compared with controls and BM-MSCs (P<0.01). This study reveals that Rap1 has a critical role in the regulation of MSC paracrine actions. Compared with BM-MSCs, Rap1(-/-)-BM-MSCs decreased NF-κB sensitivity to stress-induced pro-inflammatory cytokine production and reduced apoptosis. Selective inhibition of Rap1 in BM-MSCs may be a novel strategy to enhance MSC-based therapeutic efficacy in myocardial infarction.

Negative regulation of signal transducer and activator of transcription-3 signalling cascade by lupeol inhibits growth and induces apoptosis in hepatocellular carcinoma cells.

BACKGROUND: Constitutive activation of signal transducer and activator of transcription signalling 3 (STAT3) has been linked with survival, proliferation and angiogenesis in a wide variety of malignancies including hepatocellular carcinoma (HCC). METHODS: We evaluated the effect of lupeol on STAT3 signalling cascade and its regulated functional responses in HCC cells. RESULTS: Lupeol suppressed constitutive activation of STAT3 phosphorylation at tyrosine 705 residue effectively in a dose- and time-dependent manner. The phosphorylation of Janus-activated kinases (JAKs) 1 and 2 and Src was also suppressed by lupeol. Pervanadate treatment reversed the downregulation of phospho-STAT3 induced by lupeol, thereby indicating the involvement of a phosphatase. Indeed, we observed that treatment with lupeol increased the protein and mRNA levels of SHP-2, and silencing of SHP-2 abolished the inhibitory effects of lupeol on STAT3 activation. Treatment with lupeol also downregulated the expression of diverse STAT3-regulated genes and decreased the binding of STAT3 to VEGF promoter. Moreover, the proliferation of various HCC cells was significantly suppressed by lupeol, being associated with substantial induction of apoptosis. Depletion of SHP-2 reversed the observed antiproliferative and pro-apoptotic effects of lupeol. CONCLUSIONS: Lupeol exhibited its potential anticancer effects in HCC through the downregulation of STAT3-induced pro-survival signalling cascade.

RUNX1 meets MLL: epigenetic regulation of hematopoiesis by two leukemia genes.

A broad range of human leukemias carries RUNX1 and MLL genetic alterations. Despite such widespread involvements, the relationship between RUNX1 and MLL has never been appreciated. Recently, we showed that RUNX1 physically and functionally interacts with MLL, thereby regulating the epigenetic status of critical cis-regulatory elements for hematopoietic genes. This newly unveiled interaction between the two most prevalent leukemia genes has solved a long-standing conundrum: leukemia-associated RUNX1 N-terminal point mutants that exhibit no obvious functional abnormalities in classical assays for the assessment of transcriptional activities. These mutants turned out to be defective in MLL interaction and subsequent epigenetic modifications that can be examined by the histone-modification status of cis-regulatory elements in the target genes. RUNX1/MLL binding confirms the importance of RUNX1 function as an epigenetic regulator. Recent studies employing next-generation sequencing on human hematological malignancies identified a plethora of mutations in epigenetic regulator genes. These new findings would enhance our understanding on the mechanistic basis for leukemia development and may provide a novel direction for therapeutic applications. This review summarizes the current knowledge about the epigenetic regulation of normal and malignant hematopoiesis by RUNX1 and MLL.

Nuclear factor κB (NF-κB) suppresses food intake and energy expenditure in mice by directly activating the Pomc promoter.

AIMS/HYPOTHESIS: While chronic low-grade inflammation is associated with obesity, acute inflammation reduces food intake and leads to negative energy balance. Although both types of inflammation activate nuclear factor κB (NF-κB) signalling, it remains unclear how NF-κB activation results in opposite physiological responses in the two types of inflammation. The goal of this study was to address this question, and to understand the link between inflammation and leptin signalling. METHODS: We studied the ability of NF-κB to modulate Pomc transcription, and how it impinges on signal transducer and activator of transcription 3 (STAT3)-mediated leptin signalling by using a combination of animal models, biochemical assays and molecular biology. RESULTS: We report that suppression of food intake and physical movement with acute inflammation is not dependent on STAT3 activation in pro-opiomelanocortin (POMC) neurons. Under these conditions, activated NF-κB independently leads to increased Pomc transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) experiments reveal that NF-κB v-rel reticuloendotheliosis viral oncogene homologue A (avian) (RELA [also known as p65]) binds to the Pomc promoter region between -138 and -88 bp, which also harbours the trans-acting transcription factor 1 (SP1) binding site. We found significant changes in the methylation pattern at this region and reduced Pomc activation under chronic inflammation induced by a high-fat diet. Furthermore, RELA is unable to bind and activate transcription when the Pomc promoter is methylated. Finally, RELA binds to STAT3 and inhibits STAT3-mediated promoter activity, suggesting that RELA, possibly together with forkhead box-containing protein 1 (FOXO1), may prevent STAT3-mediated leptin activation of the Pomc promoter. CONCLUSIONS/INTERPRETATION: Our study provides a mechanism for the involvement of RELA in the divergent regulation of energy homeostasis in acute and chronic inflammation.

R-Roscovitine simultaneously targets both the p53 and NF-kappaB pathways and causes potentiation of apoptosis: implications in cancer therapy.

Seliciclib (CYC202, R-Roscovitine) is a 2, 6, 9-substituted purine analog that is currently in phase II clinical trials as an anticancer agent. We show in this study that R-Roscovitine can downregulate nuclear factor-kappa B (NF-kappaB) activation in response to tumor necrosis factor (TNF)alpha and interleukin 1. Activation of p53-dependent transcription is not compromised when R-Roscovitine is combined with TNFalpha. We characterize the molecular mechanism governing NF-kappaB repression and show that R-Roscovitine inhibits the IkappaB kinase (IKK) kinase activity, which leads to defective IkappaBalpha phosphorylation, degradation and hence nuclear function of NF-kappaB. We further show that the downregulation of the NF-kappaB pathway is also at the level of p65 modification and that the phosphorylation of p65 at Ser 536 is repressed by R-Roscovitine. Consistent with repression of canonical IKK signaling pathway, the induction of NF-kappaB target genes monocyte chemoattractant protein, intercellular adhesion molecule-1, cyclooxygenase-2 and IL-8 is also inhibited by R-Roscovitine. We further show that treatment of cells with TNFalpha and R-Roscovitine causes potentiation of cell death. Based on these results, we suggest the potential use of R-Roscovitine as a bitargeted anticancer drug that functions by simultaneously causing p53 activation and NF-kappaB suppression. This study also provides mechanistic insight into the molecular mechanism of action of R-Roscovitine, thereby possibly explaining its anti-inflammatory properties.

Nutlin-3 inhibits the NFkappaB pathway in a p53-dependent manner: implications in lung cancer therapy.

Nutlins were identified as the first potent and specific small molecule Mdm2 antagonists that inhibit the p53-Mdm2 interaction. We show in this study that Nutlin-3 can downregulate TNFalpha induced activation of the NFkappaB reporter in lung cancer cells. Activation of p53 dependent transcription is not compromised when Nutlin-3 is combined with TNFalpha. Instead, this combination treatment decreases cell viability in a p53 dependent manner. We show that Nutlin-3 strikingly inhibits the protein expression of NFkappaB target genes ICAM-1 and MCP-1 while other targets like Bcl-xL and FLIP are not affected, thereby suggesting that the inhibition is promoter specific. This inhibition of ICAM-1 and MCP-1 by Nutlin-3 is again dependent on the p53 status in cells. Furthermore, we show that Nutlin-3 strongly inhibits protein expression of ICAM-1 and MCP-1 induced by IL1, another NFkappaB activating stimuli. Nutlin-3 does not inhibit Akt phosphorylation, IkappaB alpha phosphorylation, IkappaB alpha degradation, p65 modification or p65 DNA binding in the cell lines tested. This study suggests the potential of Nutlin-3 as a bitargeted anti-cancer drug by simultaneously causing p53 activation and NFkappaB suppression. It also suggests that Nutlin-3 could be evaluated for treatment of lung cancer as a single agent or in combination therapy by targeting its effect on ICAM-1 and MCP-1 which are known to be critical for cancer cell invasion, thereby downregulating tumor formation and metastasis. This study also suggests biomarkers of response for evaluation of Nutlin-3 in the clinic.

Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species.

Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.

Pleiotropic effects of human papillomavirus type 16 E6 oncogene expression in human epithelial cell lines.

Two human papillomavirus (HPV)-negative epithelial cell lines, HaCaT and C33A, were transfected with HPV-16 E6 and analysed for functional consequences which are relevant to invasive tumour progression. After transfection with E6, both cell lines invaded collagen matrices, in contrast to vector-transfected control cells. The E6-expressing cells showed a marked increase in expression of the beta1 integrin subunit, with no or relatively minor alterations in the levels of a range of integrin subunits. In addition, the epithelial cell lines expressing E6 displayed resistance to apoptosis generated by serum starvation. This resistance is comparable to that generated by ras and is not generated by HPV-11 E6 or HPV-16 E7. Both C33A and HaCaT cells have mutations in the p53 loci and hence these functional consequences of E6 are probably independent of wild-type p53 function.

Cytokeratin patterns of expression in human epithelial cell lines correlate with transcriptional activity of the human papillomavirus type 16 upstream regulatory region.

A comparison of the CAT reporter activity of a plasmid which contains the 232 bp epithelial specific enhancer alone with that of plasmids which contain additional sequences from the human papillomavirus type 16 (HPV-16) upstream regulatory region (URR) revealed two markedly different patterns, in an analysis of six human epithelial cell lines. In HeLa, C33A and SiHa, the CAT reporter activities of all the constructs were comparable. In contrast, in CaSki, HK2bE6-E7 and HaCaT we detected very low levels of CAT reporter activity using the constructs with the additional HPV-16 URR sequences. The ability of HPV-16 E2 to transactivate a construct with 2 E2 binding sites also differed markedly and showed the same pattern. Cytokeratin staining revealed a correlation between cytokeratin 10 and 14 expression and the transcriptional differences observed. We also found alterations in the activity of one of the constructs on altering the growth conditions of the HaCaT cell line.