The scaffold protein KSR1, a novel therapeutic target for the treatment of Merlin-deficient tumors

Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell-matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1's function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4DCAF1. Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.

IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component

The bronchial epithelium is a source of both α and β chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and α2-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC50 ∼0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC

BRACHYURY confers cancer stem cell characteristics on colorectal cancer cells

Cancer stem cells (CSCs) are initiating cells in colorectal cancer (CRC). Colorectal tumours undergo epithelial to mesenchymal transition (EMT)-like processes at the invasive front, enabling invasion and metastasis, and recent studies have linked this process to the acquisition of stem cell-like properties. It is of fundamental importance to understand the molecular events leading to the establishment of cancer initiating cells and how these mechanisms relate to cellular transitions during tumourigenesis. We use an in vitro system to recapitulate changes in CRC cells at the invasive front (mesenchymal-like cells) and central mass (epithelial-like cells) of tumours. We show that the mesoderm inducer BRACHYURY is expressed in a subpopulation of CRC cells that resemble invasive front mesenchymal-like cells, where it acts to impose characteristics of CSCs in a fully reversible manner, suggesting reversible formation and modulation of such cells. BRACHYURY, itself regulated by the oncogene β-catenin, influences NANOG and other 'stemness' markers including a panel of markers defining CRC-CSC whose presence has been linked to poor patient prognosis. Similar regulation of NANOG through BRACHYURY was observed in other cells lines, suggesting this might be a pathway common to cancer cells undergoing mesenchymal transition. We suggest that BRACHYURY may regulate NANOG in mesenchymal-like CRC cells to impose a 'plastic-state', allowing competence of cells to respond to signals prompting invasion or metastasis. Copyright © 2011 UICC.

NKG2D ligands mediate immunosurveillance of senescent cells

Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis. Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis.