Hanan Armanious

Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma

Aberrations of the Wnt canonical pathway (WCP) are known to contribute to the pathogenesis of various types of cancer. We hypothesize that these defects may exist in mantle cell lymphoma (MCL). Both the upstream and downstream aspects of WCP were examined in MCL cell lines and tumors. Using WCP-specific oligonucleotide arrays, we found that MCL highly and consistently expressed Wnt3 and Wnt10. beta-catenin, a transcriptional factor that is a downstream target of WCP, is localized to the nucleus and transcriptionally active in all 3 MCL cell lines examined. By immunohistochemistry, 33 (52%) of 64 MCL tumors showed nuclear localization of beta-catenin, which significantly correlated with the expression of the phosphorylated/inactive form of GSK3beta (p-GSK3beta; P = .011, Fisher). GSK3beta inactivation is directly linked to WCP stimulation, since addition of recombinant sFRP proteins (a naturally occurring decoy for the Wnt receptors) resulted in a significant decrease in p-GSK3beta. Down-regulation of DvL-2 (an upstream signaling protein in WCP) by siRNA or selective inhibition of beta-catenin using quercetin significantly decreased cell growth in MCL cell lines. To conclude, WCP is constitutively activated in a subset of MCL and it appears to promote tumorigenesis in MCL.

Constitutive activation of metalloproteinase ADAM10 in mantle cell lymphoma promotes cell growth and activates the TNFα/NFκB pathway

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.

Clinical and biological significance of GSK-3β inactivation in breast cancer—an immunohistochemical study

Glycogen synthase kinase 3β, recently found to be functionally abnormal in various types of human disease, is negatively regulated by the PI3K/Akt signaling pathway. As Akt is constitutively activated in a subset of breast cancer, we hypothesized that glycogen synthase kinase 3β is inappropriately inactivated in these cases. In this study, we aimed to assess (1) the overall frequency of glycogen synthase kinase 3β inactivation in breast cancer; (2) whether there is an association between Akt activation and glycogen synthase kinase 3β inactivation; and (3) whether there is a correlation between glycogen synthase kinase 3β inactivation and various pathologic and clinical parameters. The phosphorylated form of glycogen synthase kinase 3β (pGSK-3β) and Akt (pAkt) were used as surrogate markers for glycogen synthase kinase 3β inactivation and Akt activation, respectively. Immunohistochemistry applied to paraffin-embedded tissues was used to assess 72 consecutive invasive mammary carcinomas, of which 50 were estrogen receptor positive. Overall, pGSK-3β and pAkt were positive in 34 (47.2%) and 35 (48.6%) cases, respectively. These 2 markers were significantly correlated with each other in the overall group and in the estrogen receptor-positive subgroup (P = .01 and .003, Spearman, respectively). Importantly, pGSK-3β, but not pAkt, significantly correlated a worse clinical outcome in this cohort (P = .004, log rank). In summary, evidence of glycogen synthase kinase 3β inactivation was found in approximately half of the invasive mammary carcinomas. Our data suggest that this abnormality is likely attributed to Akt activation and that glycogen synthase kinase 3β inactivation confers a worse clinical outcome.

Disheveled proteins promote cell growth and tumorigenicity in ALK-positive anaplastic large cell lymphoma.

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.

Co-ingestion of willow bark tea and acetaminophen associated with fatal infantile fulminant liver failure.

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Abstract A1: Clinical and biological significance of GSK-3β inactivation in breast cancer: An immunohistochemical study

Glycogen synthase kinase 3β (GSK-3β), recently found to be functionally abnormal in various types of human disease, is negatively regulated by the PI3K/Akt signaling pathway. Since Akt is constitutively activated in a subset of breast cancer, we hypothesized that GSK-3β is inappropriately inactivated in these cases. In this study, we aimed to assess (1) the overall frequency of GSK-3β inactivation in breast cancer; (2) if there is an association between Akt activation and GSK-3β inactivation; and (3) whether there is correlation between GSK-3β inactivation and various pathologic and clinical parameters. The expression of the phosphorylated form of GSK-3β (pGSK-3β) and Akt (pAkt) were used as surrogate markers of GSK-3β inactivation and Akt activation, respectively. Immunohistochemistry applied to paraffin-embedded tissues was used to assess 72 consecutive invasive mammary carcinomas, of which 50 were estrogen receptor (ER)-positive. Overall, pGSK-3β and pAkt were positive in 34 (47.2%) and 35 (48.6%) cases, respectively. These two markers were significantly correlated with each other in the overall group and in the ER-positive subgroup (p=0.01 and 0.003, Spearman, respectively). Importantly, pGSK-3β, but not pAkt, significantly correlated a worse clinical outcome in this cohort (p=0.004, Log rank). In summary, evidence of GSK-3β inactivation was found in approximately half of the invasive mammary carcinomas. Our data suggest that this abnormality is likely attributed to Akt activation and that GSK-3β inactivation confers a worse clinical outcome. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A1.