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 ß-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.

Identification of a novel crosstalk between casein kinase 2α and NPM-ALK in ALK-positive anaplastic large cell lymphoma.

It was previously reported that ß-catenin contributes to the tumorigenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), and the oncogenic effects of ß-catenin in these tumors are promoted by NPM-ALK, an abnormal fusion protein characteristic of ALK(+)ALCL. In this study, we hypothesized that NPM-ALK promotes the oncogenic activity of ß-catenin via its functional interactions with the Wnt canonical pathway (WCP). To test this hypothesis, we examined if NPM-ALK modulates the gene expression of various members in the WCP. Using a Wnt pathway-specific oligonucleotide array and Western blots, we found that the expression of casein kinase 2α (CK2α) was substantially downregulated in ALK(+)ALCL cells in response to siRNA knockdown of NPM-ALK. CK2α is biologically important in ALK(+)ALCL, as its inhibition using 4,5,6,7-tetrabromobenzotriazole or siRNA resulted in a significant decrease in cell growth and a substantial decrease in the ß-catenin protein level. Furthermore, CK2α co-immunoprecipitated with NPM-ALK and regulated its level of serine phosphorylation, a feature previously shown to correlate with the oncogenic potential of this fusion protein. To conclude, this study has revealed a novel crosstalk between NPM-ALK and CK2α, and our data supports the model that these two molecules work synergistically to promote the tumorigenicity of these lymphomas.

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.

STAT3 upregulates the protein expression and transcriptional activity of ß-catenin in breast cancer.

The expression of ß-catenin detectable by immunohistochemistry has been reported to be prognostically important in breast cancer. In this study, we investigated the mechanism by which ß-catenin is regulated in breast cancer cells. Our analysis of the gene promoter of ß-catenin revealed multiple putative STAT3 binding sites. In support of the concept that STAT3 is a transcriptional regulator for ß-catenin, results from our chromatin immunoprecipitation studies showed that STAT3 binds to two of the three potential STAT3-binding sites in the gene promoter of ß-catenin (-856 and -938). Using our generated MCF-7 cell clones that carry an inducible STAT3C construct, we found that the expression levels of STAT3C significantly correlated with the transcriptional activity of ß-catenin. Similar observations were made when we subjected two breast cancer cell lines (MCF-7 and BT-474) to STAT3 knock-down or transient gene transfection of STAT3C. Using immunohistochemistry, we found that pSTAT3 and ß-catenin significantly correlated with each other (p=0.003, Fisher's exact test) in a cohort of primary breast tumors (n=129). To conclude, our results support the concept that STAT3 upregulates the protein expression and transcriptional activity of ß-catenin in breast cancer, and these two proteins may cooperate with each other in exerting their oncogenic effects in these tumors.

Clinical and biological significance of GSK-3ß inactivation in breast cancer-an immunohistochemical study.

Glycogen synthase kinase 3ß, 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 3ß is inappropriately inactivated in these cases. In this study, we aimed to assess (1) the overall frequency of glycogen synthase kinase 3ß inactivation in breast cancer; (2) whether there is an association between Akt activation and glycogen synthase kinase 3ß inactivation; and (3) whether there is a correlation between glycogen synthase kinase 3ß inactivation and various pathologic and clinical parameters. The phosphorylated form of glycogen synthase kinase 3ß (pGSK-3ß) and Akt (pAkt) were used as surrogate markers for glycogen synthase kinase 3ß 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-3ß 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-3ß, but not pAkt, significantly correlated a worse clinical outcome in this cohort (P = .004, log rank). In summary, evidence of glycogen synthase kinase 3ß 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 3ß inactivation confers a worse clinical outcome.

Biological and clinical significance of GSK-3beta in mantle cell lymphoma--an immunohistochemical study.

GSK-3beta, a biologically important signalling protein, is regulated by the Wnt canonical and the PI3K/Akt pathways. We recently reported that mantle cell lymphoma (MCL) frequently shows evidence of GSK-3beta inactivation, since GSK-3beta is phoshorylated at its functionally critical serine 9 residue in all MCL cell lines and the majority of MCL tumors examined. To further assess the clinical and biological significance of GSK-3beta inactivation in MCL, we employed immunohistochemistry to assess the expression of the phosphorylated/inactive form of GSK-3beta (pGSK-3beta) in 83 paraffin-embedded tumors, and correlated its expression with various biological and clinical parameters. Dichotomizing pGSK-3beta into 2 groups produced twenty-seven (32.5%) tumors assessed as negative and fifty-six (67.5%) as positive. Positive pGSK-3beta expression correlated significantly with positive nuclear expression of beta-catenin and high expression of cyclin D1 (p = 0.0025, 0.0032 Fisher's exact, respectively), both of which have been previously shown to be regulated by GSK-3beta regarding their expression levels and/or sub cellular localization in-vitro. However, no significant correlation was found between pGSK-3beta and Ki67. Of the clinical parameters, continuous pGSK-3beta status had a significant correlation with absolute lymphocyte count in blood (p = 0.0011, Spearman) and negative pGSK-3beta expression was significantly correlated with a longer overall survival (p= 0.045, HR = 1.89), but not with age at diagnosis, clinical stage or the international prognostic index. To conclude, our results support the concept that GSK-3beta inactivation, found in approximately two-thirds of MCL tumors, is biologically and clinically important in MCL.

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.