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Hegazy S.A.,University of Alberta | Hegazy S.A.,Taibah University | Alshareef A.,University of Alberta | Alshareef A.,Taibah University | And 10 more authors.
Cellular Signalling | Year: 2013

Our previous oligonucleotide array studies revealed that ALK-positive anaplastic large cell lymphoma (ALK+ALCL) express high levels of the disheveled proteins (Dvls), a family of proteins that is integral to the Wnt signaling pathways. In this study, we assessed whether the Dvls are important in the pathogenesis of ALK+ALCL. By Western blotting, Dvl-2 and Dvl-3 were found to be highly expressed in ALK+ALCL cell lines and patient samples. The higher molecular weight forms, consistent with phosphorylated/active Dvl proteins, were observed in these lysates. siRNA knock-down of Dvls did not affect the Wnt canonical pathway, as assessed by the β-catenin protein levels and nuclear localization. In contrast, the same treatment led to changes in the transcriptional activity of NFAT and the phosphorylation status of Src, both of which are known to be regulated by the Wnt non-canonical signaling pathways in other cell types.Coupled with these biochemical changes, there was a significant decrease in cell growth and soft agar colony formation. NPM-ALK, the oncogenic tyrosine kinase characteristic of ALK+ALCL, was found to bind to the Dvls and enhance their tyrosine phosphorylation. In conclusion, our data suggest that the Dvls contribute to the pathogenesis of ALK+ALCL via signaling in the Wnt non-canonical pathways. To our knowledge, this is the first report demonstrating a physical and functional interaction between the Dvls and an oncogenic tyrosine kinase. © 2012 Elsevier Inc. Source

Armanious H.,University of Alberta | Gelebart P.,University of Alberta | Anand M.,University of Alberta | Belch A.,University of Alberta | And 2 more authors.
Blood | Year: 2011

One of the main functions of A Disintegrin and Metalloproteinase 10 (ADAM10) is to regulate the bioavailability of adhesion molecules and ligands to various cellular-signaling receptors. Constitutive activation of ADAM10 has been implicated in the pathogenesis of several types of solid tumors. In this study, we found that mantle cell lymphoma (MCL) cell lines and all 12 patient samples examined expressed the active/mature form of ADAM10. In contrast, PBMCs from healthy donors (n = 5) were negative. Using immunohistochemistry, ADAM10 was readily detectable in 20 of 23 (87%) MCL tumors, but absent in 5 reactive tonsils. Knockdown of ADAM10 using short interfering RNA (siRNA) in MCL cells significantly induced growth inhibition and cell-cycle arrest, and these changes were correlated with down-regulation of cyclin D1, up-regulation of p21 waf1, and significant reductions in the TNFα production/transcriptional activity of NFκBp65. The addition of recombinant ADAM10 to MCL cells led to the opposite biologic effects. Lastly, down-regulation of ADAM10 using siRNA enhanced the growth-suppressing effects mediated by the proteasome inhibitors MG132 and bortezomib. We conclude that constitutive activation of ADAM10 contributes to the growth of MCL and therefore inhibition of ADAM10 may be a useful strategy to enhance the response of MCL to other therapeutic agents. © 2011 by The American Society of Hematology. Source

Gelebart P.,University of Alberta | Hegazy S.A.,University of Alberta | Wang P.,University of Alberta | Bone K.M.,University of Alberta | And 8 more authors.
Blood Cancer Journal | Year: 2012

Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK +-positive anaplastic large cell lymphoma (ALK +ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK +ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK +ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2active cells, but not Sox2inactive cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK +ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells. © 2012 Macmillan Publishers Limited All rights reserved. Source

Jung K.,University of Alberta | Sabri S.,University of Alberta | Sabri S.,McGill University | Hanson J.,Cross Cancer Institute | And 7 more authors.
Cancer Biology and Therapy | Year: 2015

Radiation therapy (RT) the front-line treatment after surgery for early breast cancer patients is associated with acute skin toxicities in at least 40% of treated patients. Monocyte-derived macrophages are polarized into functionally distinct (M1 or M2) activated phenotypes at injury sites by specific systemic cytokines known to play a key role in the transition between damage and repair in irradiated tissues. The role of M1 and M2 macrophages in RT-induced acute skin toxicities remains to be defined. We investigated the potential value of M1 and M2 macrophages as predictive factors of RT-induced skin toxicities in early breast cancer patients treated with adjuvant RT after lumpectomy. Blood samples collected from patients enrolled in a prospective clinical study (n = 49) were analyzed at baseline and after the first delivered 2Gy RT dose. We designed an ex vivo culture system to differentiate patient blood monocytes into macrophages and treated them with M1 or M2-inducing cytokines before quantitative analysis of their “M1/M2” activation markers, iNOS, Arg1, and TGFß1. Statistical analysis was performed to correlate experimental data to clinical assessment of acute skin toxicity using Common Toxicity Criteria (CTC) grade for objective evaluation of skin reactions. Increased ARG1 mRNA significantly correlated with higher grades of erythema, moist desquamation, and CTC grade. Multivariate analysis revealed that increased ARG1 expression in macrophages after a single RT dose was an independent prognostic factor of erythema (p = 0.032), moist desquamation (p = 0.027), and CTC grade (p = 0.056). Interestingly, multivariate analysis of ARG1 mRNA expression in macrophages stimulated with IL-4 also revealed independent prognostic value for predicting acute RT-induced toxicity factors, erythema (p = 0.069), moist desquamation (p = 0.037), and CTC grade (p = 0.046). To conclude, our findings underline for the first time the biological significance of increased ARG1 mRNA levels as an early independent predictive biomarker of RT-induced acute skin toxicities. © 2015 Taylor & Francis Group, LLC. Source

Young L.C.,University of Alberta | Bone K.M.,University of Alberta | Wang P.,University of Alberta | Wu F.,University of Alberta | And 8 more authors.
American Journal of Pathology | Year: 2011

The fusion tyrosine kinase NPM-ALK is central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK+ALCL). We recently identified that MSH2, a key DNA mismatch repair (MMR) protein integral to the suppression of tumorigenesis, is an NPM-ALKinteracting protein. In this study, we found in vitro evidence that enforced expression of NPM-ALK in HEK293 cells suppressed MMR function. Correlating with these findings, six of nine ALK +ALCL tumors displayed evidence of microsatellite instability, as opposed to none of the eight normal DNA control samples (P = 0.007, Student's t-test). Using co-immunoprecipitation, we found that increasing levels of NPM-ALK expression in HEK293 cells resulted in decreased levels of MSH6 bound to MSH2, whereas MSH2·NPM-ALK binding was increased. The NPM-ALK·MSH2 interaction was dependent on the activation/ autophosphorylation of NPM-ALK, and the Y191 residue of NPM-ALK was a crucial site for this interaction and NPM-ALKmediated MMR suppression. MSH2 was found to be tyrosine phosphorylated in the presence of NPM-ALK. Finally, NPM-ALK impeded the expected DNA damage-induced translocation of MSH2 out of the cytoplasm. To conclude, our data support a model in which the suppression of MMR by NPM-ALK is attributed to its ability to interfere with normal MSH2 biochemistry and function. © 2011 American Society for Investigative Pathology. Source

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