Genomic and global gene expression profiling in pediatric and young adult acute leukemia with PICALM::MLLT10 Fusion.

PICALM: MLLT10 fusion is a rare but recurrent genetic driver in acute leukemias. To better understand the genomic landscape of PICALM::MLLT10 (PM) positive acute leukemia, we performed genomic profiling and gene expression profiling in twenty PM-positive patients, including AML (n = 10), T-ALL/LLy (n = 8), Mixed-phenotype acute leukemia (MPAL), T/B (n = 1) and acute undifferentiated leukemia (AUL) (n = 1). Besides confirming the known activation of HOXA, differential gene expression analysis compared to hematopoietic stem cells demonstrated the enrichment of genes associated with cell proliferation-related pathways and relatively high expression of XPO1 in PM-AML and PM-T-ALL/LLy. Our study also suggested PHF6 disruption as a key cooperating event in PICALM::MLLT10-positive leukemias. In addition, we demonstrated differences in gene expression profiles as well as remarkably different spectra of co-occurring mutations between PM-AML and PM-T-ALL/LLy. Alterations affecting TP53 and NF1, hallmarks of PM-AML, are strongly associated with disease progression and relapse, whereas EZH2 alterations are highly enriched in PM-T-ALL/LLy. This comprehensive genomic and transcriptomic profiling provides insights into the pathogenesis and development of PICALM::MLLT10 positive acute leukemia.

Beta 2 adrenergic receptor and mu opioid receptor interact to potentiate the aggressiveness of human breast cancer cell by activating the glycogen synthase kinase 3 signaling.

BACKGROUND: Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer cells. However, the underlying cell signaling mechanism remained unclear. METHODS: To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, CRISPR or chemical inhibition or activation of beta-adrenergic receptors (B2AR) and mu-opioid receptors (MOR) gene, and PCR array technology and studied aggressive tumor phenotype and signaling cascades. RESULTS: We show here that in triple-negative breast cancer cells, the reduction in expression B2AR and MOR by genetic and pharmacological tools leads to a less aggressive phenotype of triple-negative breast cancer cells in vitro and in animal xenografts. Genomic analysis indicates the glycogen synthase kinase 3 (GSK3) pathway as a possible candidate messenger system involved in B2AR and MOR cross talk. GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. CONCLUSIONS: These data confirm a role of B2AR and MOR interaction in the control of breast cancer cell growth and identify a possible role of the GSK3 signaling system in mediation of these two receptors' cross talk. Screening for ligands targeting B2AR and MOR interaction and/or the GSK3 system may help to identify novel drugs for the prevention of triple-negative breast cancer cell growth and metastasis.

Transgenerational inheritance of fetal alcohol effects on proopiomelanocortin gene expression and methylation, cortisol response to stress, and anxiety-like behaviors in offspring for three generations in rats: Evidence for male germline transmission.

Previously it has been shown that fetal alcohol exposure increases the stress response partly due to lowering stress regulatory proopiomelanocortin (Pomc) gene expression in the hypothalamus via epigenetic mechanisms for multiple generations in mixed-breed rats. In this study we assess the induction of heritable epigenetic changes of Pomc-related variants by fetal alcohol exposure in isogenic Fischer 344 rats. Using transgenerational breeding models and fetal alcohol exposure procedures, we determined changes in hypothalamic Pomc gene expression and its methylation levels, plasma corticosterone hormone response to restraint stress, and anxiety-like behaviors using elevated plus maze tests in fetal alcohol-exposed offspring for multiple generations in isogenic Fischer rats. Fetal alcohol-exposed male and female rat offspring showed significant deficits in POMC neuronal functions with increased Pomc gene methylation and reduced expression. These changes in POMC neuronal functions were associated with increased plasma corticosterone response to restraint stress and increased anxiety-like behavior. These effects of fetal alcohol exposure persisted in the F1, F2, and F3 progeny of the male germline but not of the female germline. These data suggest that fetal alcohol exposure induces heritable changes in Pomc-related variants involving stress hyperresponsiveness and anxiety-like behaviors which perpetuate into subsequent generations through the male germline via epigenetic modifications.

Covalent Cysteine Targeting of Bruton's Tyrosine Kinase (BTK) Family by Withaferin-A Reduces Survival of Glucocorticoid-Resistant Multiple Myeloma MM1 Cells.

Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells' uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs. Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA's promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.

miR-21 Plays a Dual Role in Tumor Formation and Cytotoxic Response in Breast Tumors.

Breast cancer (BrCa) relies on specific microRNAs to drive disease progression. Oncogenic miR-21 is upregulated in many cancers, including BrCa, and is associated with poor survival and treatment resistance. We sought to determine the role of miR-21 in BrCa tumor initiation, progression and treatment response. In a triple-negative BrCa model, radiation exposure increased miR-21 in both primary tumor and metastases. In vitro, miR-21 knockdown decreased survival in all BrCa subtypes in the presence of radiation. The role of miR-21 in BrCa initiation was evaluated by implanting wild-type miR-21 BrCa cells into genetically engineered mouse models where miR-21 was intact, heterozygous or globally ablated. Tumors were unable to grow in the mammary fat pads of miR-21-/- mice, and grew in ~50% of miR-21+/- and 100% in miR-21+/+ mice. The contribution of miR-21 to progression and metastases was tested by crossing miR-21-/- mice with mice that spontaneously develop BrCa. The global ablation of miR-21 significantly decreased the tumorigenesis and metastases of BrCa, while sensitizing tumors to radio- and chemotherapeutic agents via Fas/FasL-dependent apoptosis. Therefore, targeting miR-21 alone or in combination with various radio or cytotoxic therapies may represent novel and efficacious therapeutic modalities for the future treatment of BrCa patients.

Personalized Nutrition as a Key Contributor to Improving Radiation Response in Breast Cancer.

Understanding metabolic and immune regulation inherent to patient populations is key to improving the radiation response for our patients. To date, radiation therapy regimens are prescribed based on tumor type and stage. Patient populations who are noted to have a poor response to radiation such as those of African American descent, those who have obesity or metabolic syndrome, or senior adult oncology patients, should be considered for concurrent therapies with radiation that will improve response. Here, we explore these populations of breast cancer patients, who frequently display radiation resistance and increased mortality rates, and identify the molecular underpinnings that are, in part, responsible for the radiation response and that result in an immune-suppressive tumor microenvironment. The resulting immune phenotype is discussed to understand how antitumor immunity could be improved. Correcting nutrient deficiencies observed in these populations should be considered as a means to improve the therapeutic index of radiation therapy.

Is Host Metabolism the Missing Link to Improving Cancer Outcomes?

For the past 100 years, oncologists have relentlessly pursued the destruction of tumor cells by surgical, chemotherapeutic or radiation oncological means. Consistent with this focus, treatment plans are typically based on key characteristics of the tumor itself such as disease site, histology and staging based on local, regional and systemic dissemination. Precision medicine is similarly built on the premise that detailed knowledge of molecular alterations of tumor cells themselves enables better and more effective tumor cell destruction. Recently, host factors within the tumor microenvironment including the vasculature and immune systems have been recognized as modifiers of disease progression and are being targeted for therapeutic gain. In this review, we argue that-to optimize the impact of old and new treatment options-we need to take account of an epidemic that occurs independently of-but has major impact on-the development and treatment of malignant diseases. This is the rapidly increasing number of patients with excess weight and its' attendant metabolic consequences, commonly described as metabolic syndrome. It is well established that patients with altered metabolism manifesting as obesity, metabolic syndrome and chronic inflammation have an increased incidence of cancer. Here, we focus on evidence that these patients also respond differently to cancer therapy including radiation and provide a perspective how exercise, diet or pharmacological agents may be harnessed to improve therapeutic responses in this patient population.

Transgenerational inheritance of fetal alcohol exposure adverse effects on immune gene interferon-ϒ.

BACKGROUND: Alcohol exposures in utero have been shown to alter immune system functions in the offspring which persists into adulthood. However, it is not apparent why the in utero alcohol effect on the immune system persists into adulthood of fetal alcohol-exposed offspring. The objective of this study was to determine the long-term effects of fetal alcohol exposure on the production of interferon-ϒ (IFN-ϒ), a cytokine known to regulate both innate and adaptive immunity. METHODS: Isogenic Fisher 344 rats were bred to produce pregnant dams, which were fed with a liquid diet containing 6.7% alcohol between gestation days 7 and 21 and pair-fed with an isocaloric liquid diet or fed ad libitum with rat chow; their male and female offspring were used for the study. F1-F3 generation rats were used when they were 2 to 3 months old. Fetal alcohol exposure effects on the Ifn-ɣ gene was determined by measuring the gene promoter methylation and mRNA and protein expression in the spleen. Additionally, transgenerational studies were conducted to evaluate the germline-transmitted effects of fetal alcohol exposure on the Ifn-ɣ gene. RESULTS: Fetal alcohol exposure reduced the expression of Ifn-ɣ mRNA and IFN-ϒ protein while it increased the proximal promoter methylation of the Ifn-ɣ gene in both male and female offspring during the adult period. Transgenerational studies revealed that the reduced levels of Ifn-ɣ expression and increased levels of its promoter methylation persisted only in F2 and F3 generation males derived from the male germ line. CONCLUSION: Overall, these findings provide the evidence that fetal alcohol exposures produce an epigenetic mark on the Ifn-ɣ gene that passes through multiple generations via the male germ line. These data provide the first evidence that the male germ line transmits fetal alcohol exposure's adverse effects on the immune system.

MicroRNA-21 is Required for Hematopoietic Cell Viability After Radiation Exposure.

PURPOSE: Radiation therapy is an essential intervention used in the treatment of more than half of cancer patients. With the increasing use of hypofractionated radiation regimens, concurrent use of radiation and chemotherapy, targeted agents and immunotherapy, the risk of radiation-induced toxicities is increased. However, much remains unknown about the molecular underpinnings responsible for radiation-induced toxicity. MicroRNA (miRNA) are small, non-coding RNA involved in post-transcriptional regulation of gene expression. miR-21 is an oncomiR that is dysregulated in a significant fraction of human malignancies, and its overexpression is linked to poor overall survival, chemoresistance, and radioresistance in several human cancers. However, the contribution of miR-21 in governing radiation sensitivity in normal, untransformed cells, and the impact of silencing this miRNA in normal tissues remains largely unexplored. MATERIALS AND METHODS: miR-21 levels were evaluated in tissues by qRT-PCR without and after total body irradiation (TBI). Mice lacking miR-21 were genetically engineered, subjected to TBI, and monitored for survival. Hematopoietic stem and progenitor cell (HSPC) numbers and function were assessed using flow cytometry, histology, complete blood cell counts, and bone marrow transplantation. RESULTS: miR-21 expression was increased in radiosensitive tissues, but not in radioinsensitive tissues following TBI in wild-type mice, suggesting it may have a critical function in the normal tissue response to irradiation. Compared to wild-type mice, mice lacking one or both alleles of miR-21 showed reduced numbers of HSPCs and increased sensitivity to an LD50/30 dose of TBI with evidence of bone marrow failure. Transplantation of wild-type bone marrow into irradiated miR-21-deficient mice rescued the mice from death. CONCLUSIONS: Our data identify miR-21 as a critical component of HSPC viability and essential for bone marrow recovery following irradiation. Further investigation is warranted to determine whether miR-21 can be used to stratify patients at risk for hematopoietic toxicity following irradiation.

Caloric restriction counteracts chemotherapy-induced inflammation and increases response to therapy in a triple negative breast cancer model.

Triple negative breast cancer (TNBC) is a heterogeneous disease that has no available targeted therapies. Previously, we have shown that caloric restriction (CR) can augment the effects of radiation therapy in a TNBC mouse model. To build upon this, we now present data regarding the combination of chemotherapy and CR in the same 4T1 model. Chemotherapy can induce inflammation that breeds resistance to therapy. We propose CR as a mechanism to decrease chemotherapy-induced inflammation and increase efficacy of therapy. 12-week old Balb/c mice were orthotopically injected with a syngeneic triple negative breast cancer cell line (4T1) and were treated in one of six cohorts: ad lib fed (AL), 30% reduction in calorie intake (CR), cisplatin or docetaxol alone or a combination CR+cisplatin/docetaxol. Mice in the cohorts receiving chemotherapy+CR had longer overall survival (12 ± 2 days) as compared to the AL group. These mice also demonstrated less lung metastases at the final time point of in vivo imaging. In addition, downregulation of the IGF-1R and IRS signaling pathways were noted most significantly in those mice receiving combination therapy. Lastly, serum from these mice demonstrated an increase in inflammatory cytokines TNF-α and IL-1ß in response to chemotherapy alone. This increase was dampened by the addition of CR. Taken together, these data suggest that while chemotherapy is effective in TNBC, it can cause inflammation, which can be a driver of resistance to therapy. This chemotherapy-induced inflammation can be reversed with the use of CR as a nontoxic adjunct to treatment.

Selective Glucocorticoid Receptor Properties of GSK866 Analogs with Cysteine Reactive Warheads.

Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR- and NFκB-dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders.

Caloric restriction coupled with radiation decreases metastatic burden in triple negative breast cancer.

PURPOSE: Metastatic breast cancer is devastating and triple negative breast cancers (TNBC) have a higher propensity for metastasis. Improved local control upfront in this aggressive cancer could potentially decrease its propensity toward metastasis. We sought to determine if using caloric restriction (CR) as a systemic therapy, combined with radiation therapy (IR) to the primary tumor, may impact metastatic disease. METHODS: An orthotopic mouse model using a highly metastatic, luciferase-tagged TNBC cell line (4T1), was used to generate palpable tumors. Mice were then treated with CR, IR, and a combination of the two. In vivo imaging was performed for metastatic evaluation. Molecular evaluation of the tumors was performed, generating a mechanistic hypothesis for CR, which was then tested with pertinent pathway inhibition in the model. RESULTS: CR significantly increased the time to developing metastases, decreased the overall number and volume of lung metastases, and increased survival. CR decreased proliferation, increased apoptosis and globally downregulated the IGF-1R signaling pathway. Adding an IGF-1R/INSR inhibitor to local IR in vivo accomplished a decrease in metastases similar to CR plus IR, demonstrating the importance of the IGF-1R signaling pathway, and underscoring it as a possible mechanism for CR. CONCLUSIONS: CR decreased metastatic burden and therefore may complement cytotoxic therapies being used in the clinical setting for metastatic disease. Downregulation of the IGF-1R pathway, is in part responsible for this response and modulating IGF-1R directly resulted in similar improved progression-free survival. The novel use of CR has the potential to enhance clinical outcomes for patients with metastatic breast cancer.

Epigenetic control of cardiovascular health by nutritional polyphenols involves multiple chromatin-modifying writer-reader-eraser proteins.

Nowadays, epigenetic mechanisms involving DNA methylation, histone modifications and microRNA regulation emerge as important players in cardiovascular disease (CVD). Epigenetics may provide the missing link between environment, genome and disease phenotype and be responsible for the strong interindividual variation in disease risk factors underlying CVD. Daily diet is known to have a major influence on both the development and the prevention of CVD. Interestingly, the dietary lifestyle of our (grand)parents and of us contributes to CVD risk by metabolic (re)programming of our epigenome in utero, after birth or during life. In contrast to genetic mutations, the plasticity of CVD related epigenetic changes makes them attractive candidates for nutritional prevention or pharmacological intervention. Although a growing number of epidemiologic studies have shown a link between the ingestion of nutritional polyphenols and cardiovascular health benefits, potential involvement of epigenetic mechanisms has been underexplored. In this review, we will give an overview of epigenetic alterations in atherosclerosis, with the focus on DNA and histone modifications by chromatin-modifying proteins. Finally, we illustrate that cocoa flavanols and other classes of dietary molecules may promote cardiovascular health by targeting multiple classes of chromatin writer-reader-eraser proteins related to histone acetylation-methylation and DNA methylation.

Inhibition of the NF-κB signaling pathway by a novel heterocyclic curcumin analogue.

In this study a series of curcumin analogues were evaluated for their ability to inhibit the activation of NF-κΒ, a transcription factor at the crossroads of cancer-inflammation. Our novel curcumin analogue BAT3 was identified to be the most potent NF-κB inhibitor and EMSA assays clearly showed inhibition of NF-κB/DNA-binding in the presence of BAT3, in agreement with reporter gene results. Immunofluorescence experiments demonstrated that BAT3 did not seem to prevent nuclear p65 translocation, so our novel analogue may interfere with NF-κB/DNA-binding or transactivation, independently of IKK2 regulation and NF-κB-translocation. Gene expression studies on endogenous NF-κB target genes revealed that BAT3 significantly inhibited TNF-dependent transcription of IL6, MCP1 and A20 genes, whereas an NF-κB independent target gene heme oxygenase-1 remained unaffected. In conclusion, we demonstrate that BAT3 seems to inhibit different cancer-related inflammatory targets in the NF-κB signaling pathway through a different mechanism in comparison to similar analogues, previously reported.

Ectopic microRNA-150-5p transcription sensitizes glucocorticoid therapy response in MM1S multiple myeloma cells but fails to overcome hormone therapy resistance in MM1R cells.

Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.

Redox and epigenetic regulation of the APE1 gene in the hippocampus of piglets: The effect of early life exposures.

Oxidative stress via redox reactions can regulate DNA repair pathways. The base excision repair (BER) enzyme apurinic/apyrimidinic endonuclease 1 (APE1) is a key player in the redox regulation of DNA repair. Environmental factors can alter the methylation of DNA repair genes, change their expression and thus modulate BER activity and susceptibility to oxidative DNA damage. Therefore, we hypothesized that epigenetic modifications play a role in the redox regulation of APE1 in hippocampi of newborns and investigated the effect of supplementation of pregnant sows with a diet enriched in antioxidants and other nutrients on oxidative stress, DNA methylation and DNA repair in their offspring. High levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and low levels of glutathione were detected in control piglets after birth compared with supplemented piglets, indicating the presence of oxidative stress. In control animals, this oxidative stress was associated with genomic DNA demethylation, decreased APE1 promoter methylation, increased APE1 expression and with slightly but not statistically significant increased BER-related DNA incision activity. Supplementation of piglets with antioxidants and other nutrients significantly lowered 8-oxodG levels compared to control animals, which was accompanied by overall lower APE1 promoter methylation and enhanced APE1 expression at day 7-28 after birth in supplemented piglets, although DNA incision activity was not significantly different between groups. Preliminary attempts to study the interaction between redox and epigenetic regulatory mechanisms revealed an inverse correlation between APE1 expression and methylation of CpG-sites 11 and 13 in the promoter region, which according to Genomatix "MatInspector" are located in the core binding sites of redox-sensitive transcription factors. We are the first to study methylation of the APE1 promoter and its role in mediating the functional effects of redox reactions induced by oxidative stress. Epigenetic and redox mechanisms may interact in regulating APE1-related DNA repair processes, involving redox-sensitive TFs.

Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium.

TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a⁺⁺/⁻ or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.

Dietary curcumin inhibits atherosclerosis by affecting the expression of genes involved in leukocyte adhesion and transendothelial migration.

SCOPE: The aim of the study was to examine the atheroprotective effect of dietary curcumin in a mouse model of atherosclerosis and to identify its cellular and molecular targets at the vascular level. METHODS AND RESULTS: ApoE(-/-) mice were fed with curcumin at 0.2% (wt/wt) in diet for 4 months. This supplementation reduced the extent of atherosclerotic lesion by 26% and induced changes in expression of genes implicated in cell adhesion and transendothelial migration or cytoskeleton organization, as revealed by a transcriptomic analysis in the aorta. Expression profile of these genes suggests reduction in both leukocyte adhesion and transendothelial migration. In agreement with this hypothesis, we observed a reduction (-37%) in macrophage infiltration in the plaque, as measured by immunohistochemistry, and, in vitro, a lower adhesion of monocytes to TNF-α-stimulated endothelial cells (-32%) after exposure to a nutritionally achievable concentration of curcumin. These changes in gene expression could be related to the observed increased expression of IκB protein and decrease of TNF-α-induced NF-κB/DNA binding and NF-κB-transcriptional activity upon exposure to curcumin. CONCLUSION: Our findings pointed out that the antiatherogenic effect of curcumin could be linked to its effect on gene networks and cell functions related to leukocyte adhesion and transendothelial migration via NF-κB-dependent pathways.

Identification and functional characterization of the human EXT1 promoter region.

BACKGROUND: Mutations in Exostosin-1 (EXT1) or Exostosin-2 (EXT2) cause the autosomal dominant disorder multiple osteochondromas (MO). This disease is mainly characterized by the appearance of multiple cartilage-capped protuberances arising from children's metaphyses and is known to display clinical inter- and intrafamilial variations. EXT1 and EXT2 are both tumor suppressor genes encoding proteins that function as glycosyltransferases, catalyzing the biosynthesis of heparan sulfate. At present, however, very little is known about the regulation of these genes. Two of the most intriguing questions concerning the pathogenesis of MO are how disruption of a ubiquitously expressed gene causes this cartilage-specific disease and how the clinical intrafamilial variation can be explained. Since mutations in the EXT1 gene are responsible for ~65% of the MO families with known causal mutation, our aim was to isolate and characterize the EXT1 promoter region to elucidate the transcriptional regulation of this tumor suppressor gene. METHODS: In the present study, luciferase reporter gene assays were used to experimentally confirm the in silico predicted EXT1 core promoter region. Subsequently, we evaluated the effect of single nucleotide polymorphisms (SNP's) on EXT1 promoter activity and transcription factor binding using luciferase assays, electrophoretic mobility shift assays (EMSA), and enzyme-linked immunosorbent assays (ELISA). Finally, a genotype-phenotype study was performed with the aim to identify one or more genetic modifiers influencing the clinical expression of MO. RESULTS: Transient transfection of HEK293 cells with a series of luciferase reporter constructs mapped the EXT1 core promoter at approximately -917 bp upstream of the EXT1 start codon, within a 123 bp region. This region is conserved in mammals and located within a CpG-island containing a CAAT- and a GT-box. A polymorphic G/C-SNP at -1158 bp (rs34016643) was demonstrated to be located in a USF1 transcription factor binding site, which is lost with the presence of the C-allele resulting in a ~56% increase in EXT1 promoter activity. A genotype-phenotype study was suggestive for association of the C-allele with shorter stature, but also with a smaller number of osteochondromas. CONCLUSIONS: We provide for the first time insight into the molecular regulation of EXT1. Although a larger patient population will be necessary for statistical significance, our data suggest the polymorphism rs34016643, in close proximity of the EXT1 promoter, to be a potential regulatory SNP, which could be a primary modifier that might explain part of the clinical variation observed in MO patients.

Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols.

BACKGROUND: Multidrug resistance (MDR) is a major obstacle in cancer treatment and is often the result of overexpression of the drug efflux protein, P-glycoprotein (P-gp), as a consequence of hyperactivation of NFkappaB, AP1 and Nrf2 transcription factors. In addition to effluxing chemotherapeutic drugs, P-gp also plays a specific role in blocking caspase-dependent apoptotic pathways. One feature that cytotoxic treatments of cancer have in common is activation of the transcription factor NFkappaB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFkappaB inhibitors may promote apoptosis in cancer cells and could be used to overcome resistance to chemotherapeutic agents. RESULTS: Although the natural withanolide withaferin A and polyphenol quercetin, show comparable inhibition of NFkappaB target genes (involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance) in doxorubicin-sensitive K562 and -resistant K562/Adr cells, only withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation and apoptosis is delayed only. Interestingly, although withaferin A and quercetin treatments both decrease intracellular protein levels of Bcl2, Bim and P-Bad, only withaferin A decreases protein levels of cytoskeletal tubulin, concomitantly with potent PARP cleavage, caspase 3 activation and apoptosis, at least in part via a direct thiol oxidation mechanism. CONCLUSIONS: This demonstrates that different classes of natural NFkappaB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis.