Increased lanosterol turnover: a metabolic burden for daunorubicin-resistant leukemia cells.

The cholesterol metabolism is essential for cancer cell proliferation. We found the expression of genes involved in the cholesterol biosynthesis pathway up-regulated in the daunorubicin-resistant leukemia cell line CEM/R2, which is a daughter cell line to the leukemia cell line CCRF-CEM (CEM). Cellular (2)H2O labelling, mass spectrometry, and isotopomer analysis revealed an increase in lanosterol synthesis which was not accompanied by an increase in cholesterol flux or pool size in CEM/R2 cells. Exogenous addition of lanosterol had a negative effect on CEM/R2 and a positive effect on sensitive CEM cell viability. Treatment of CEM and CEM/R2 cells with cholesterol biosynthesis inhibitors acting on the enzymes squalene epoxidase and lanosterol synthase, both also involved in the 24,25-epoxycholesterol shunt pathway, revealed a connection of this pathway to lanosterol turnover. Our data highlight that an increased lanosterol flux poses a metabolic weakness of resistant cells that potentially could be therapeutically exploited.

Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism.

Cancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them.We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation.We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid ß-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death.Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy.

Rewired metabolism in drug-resistant leukemia cells: a metabolic switch hallmarked by reduced dependence on exogenous glutamine.

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid ß-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.

Neutralization of human papillomavirus by specific nanobodies against major capsid protein L1.

The human papillomavirus (HPV) is the main cause of cervical cancer in developing countries. Rapid diagnosis and initiation of treatment of the HPV infection are critical. Various methods have been employed to reduce the immunogenicity of antibodies targeting HPV serotypes. Nanobodies are the smallest fragments of naturally occurring single-domain antibodies with their antigenbinding site compromised into a single domain. Nanobodies have remarkable properties such as high stability, solubility, and high homology to the human VH3 domain. In this study, a phagemid library was employed to enrich for nanobodies against the L1 protein of the human papilloma virus. Binding reactivity of the selected clones was evaluated using phage enzyme-linked immunosorbent assay (phage-ELISA). Finally, two nanobodies (sm5 and sm8) with the best reactivity against the Gardasil vaccine and the purified HPV-16 L1 protein were expressed and purified using a Ni(+)-NTA column. The accuracy of expression and purification of the nanobodies was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting assays. In vitro studies demonstrated that neutralization was achieved by the selected nanobodies. The ease of generation and unique features of these molecules make nanobodies promising molecules for the new generation of HPV diagnosis and therapy.

Regulation of acute graft-versus-host disease by microRNA-155.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD. Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155-deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic anti-miR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.

MiR-21 is up-regulated in psoriasis and suppresses T cell apoptosis.

MicroRNAs are short non-coding RNAs that regulate gene expression. Previously, in a genome-wide screen, we found deregulation of microRNA expression in psoriasis skin. MicroRNA-21 (miR-21) is one of the microRNAs significantly up-regulated in psoriasis skin lesions. To identify the cell type responsible for the increased miR-21 level, we compared expression of miR-21 in epidermal cells and dermal T cells between psoriasis and healthy skin and found elevated levels of miR-21 in psoriasis in both cell types. In cultured T cells, expression of miR-21 increased markedly upon activation. To explore the function of miR-21 in primary human T helper cells, we inhibited miR-21 using a tiny seed-targeting LNA-anti-miR. Specific inhibition of miR-21 increased the apoptosis rate of activated T cells. Our results suggest that miR-21 suppresses apoptosis in activated T cells, and thus, overexpression of miR-21 may contribute to T cell-derived psoriatic skin inflammation.

Silencing of microRNA families by seed-targeting tiny LNAs.

The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.

Evidence for impaired CARD15 signalling in Crohn's disease without disease linked variants.

BACKGROUND: Sensing of muramyl dipeptide (MDP) is impaired in Crohn's disease (CD) patients with disease-linked variants of the CARD15 (caspase activation and recruitment domain 15) gene. Animal studies suggest that normal CARD15 signalling prevents inflammatory bowel disease, and may be important for disease development in CD. However, only a small fraction of CD patients carry the disease linked CARD15 variants. The aim of this study was thus to investigate if changes could be found in CARD15 signalling in patients without disease associated CARD15 variants. METHODOLOGY/PRINCIPAL FINDINGS: By mapping the response to MDP in peripheral monocytes obtained from CD patients in remission not receiving immunosuppresives, an impaired response to MDP was found in patients without disease linked CARD15 variants compared to control monocytes. This impairment was accompanied by a decreased activation of IkappaB kinase alpha/beta (IKKalpha/beta), the initial step in the nuclear factor kappaB (NFkappaB) pathway, whereas activation of mitogen-activated protein (MAP)-kinases was unaffected. MDP additionally stimulates the inflammasome which is of importance for processing of cytokines. The inflammasome was constitutively activated in CD, but unresponsive to MDP both in CD and control monocytes. CONCLUSIONS/SIGNIFICANCE: These results suggest that inhibited MDP-dependent pathways in CD patients not carrying the disease-associated CARD15 variants might be of importance for the pathogenesis of CD. The results reveal a dysfunctional immune response in CD patients, not able to sense relevant stimuli on the one hand, and on the other hand possessing constitutively active cytokine processing.

CD47 regulates collagen I-induced cyclooxygenase-2 expression and intestinal epithelial cell migration.

Increased epithelial cell expression of the cyclooxygenase-2 (COX-2) enzyme is a characteristic event of both inflammatory bowel disease and colon cancer. We here report the novel findings that collagen I-induced de novo synthesis of COX-2 in intestinal epithelial cells is inhibited by pertussis toxin (PTX) and by an inhibitory peptide selective for the heterotrimeric G alpha(i3)-protein. These findings could be explained by a regulatory involvement of the G-protein-dependent integrin-associated protein CD47. In support of this notion, we observed a collagen I-induced association between CD47 and alpha2 integrins. This association was reduced by a blocking anti-CD47 antibody but not by PTX or a control anti-beta2 antibody. Furthermore, a blocking antibody against CD47, dominant negative CD47 or specific siRNA knock down of CD47, significantly reduced collagen I-induced COX-2 expression. COX-2 has previously been shown to regulate intestinal epithelial cell adhesion and migration. Morphological analysis of intestinal cells adhering to collagen I revealed a co-localisation of CD47 and alpha2 integrins to non-apoptotic membrane blebs enriched in Rho A and F-actin. The blocking CD47 antibody, PTX and a selective COX-2 inhibitor, dramatically inhibited the formation of these blebs. In accordance, migration of these cells on a collagen I-coated surface or through a collagen I gel were significantly reduced by the CD47 blocking antibody, siRNA knock down of CD47 and the COX-2 inhibitor NS-398. In conclusion, we present novel data that identifies the G-protein-dependent CD47 protein as a key regulator of collagen I-induced COX-2 expression and a promoter of intestinal epithelial cell migration.

[The role of cysteinyl leukotrienes in chronic inflammation and neoplasia of the intestine]. / Cysteinylleukotrieners betydning for kronisk inflammation og neoplasi i tarmen.

It has recently been demonstrated that cysteinyl leukotrienes are involved in a variety of proinflammatory and neoplastic functions. This article gives an up-to-date overview of the present knowledge of these bioactive lipid molecules. Inflammatory bowel disease (IBD) and colorectal cancer (CRC) are applied as models of their cellular actions. It is described how signalling cascades initiated by cysteinyl leukotrienes represent alternative ways in which IBD might progress and lead to the development and propagation of CRC, and as such represent potential targets for future therapeutic regimens to manage chronic inflammatory disorders and cancer.

Alpha2beta1 integrin signalling enhances cyclooxygenase-2 expression in intestinal epithelial cells.

Inflammatory bowel diseases (IBD) are linked to an increased risk of developing colon cancer, by inflammatory mediators and alterations to the extracellular matrix (ECM). The events induced by inflammatory mediators lead to dysregulated activation and induction of inflammatory genes such as cyclooxygenase-2 (COX-2). COX-2 is involved in the conversion of arachidonic acid to biologically active prostanoids and is highly upregulated in colon cancer. Since inflammation-induced changes to the extracellular matrix could affect integrin activities, we here investigated the effect of integrin signalling on the level of COX-2 expression in the non-transformed intestinal epithelial cell lines, Int 407 and IEC-6. Adhesion of these cells to a collagen I- or IV-coated surface, increased surface expression of alpha2beta1 integrin. Activation of integrins with collagen caused an increased cox-2 promoter activity, with a subsequent increase in COX-2 expression. The signalling cascade leading to this increased expression and promoter activity of cox-2, involves PKCalpha, the small GTPase Ras and NFkappaB but not Erk1/2 or Src activity. The integrin-induced increase in cellular COX-2 activity is responsible for an elevated generation of reactive oxygen species (ROS) and increased cell migration. This signalling pathway suggests a mechanism whereby inflammation-induced modulations of the ECM, can promote cancer transformation in the intestinal epithelial cells.

The pro-inflammatory mediator leukotriene D4 induces phosphatidylinositol 3-kinase and Rac-dependent migration of intestinal epithelial cells.

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.