Early diagnostic value of Bcl-3 localization in colorectal cancer

B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. Elevated expression, sustained nuclear accumulation, and uncontrolled activation of Bcl-3 causes increased cellular proliferation or survival, dependent on the tissue and type of stimuli

Integrin α10-Antibodies Reduce Glioblastoma Tumor Growth and Cell Migration

Glioblastoma (GB) is the most common and the most aggressive form of brain tumor in adults, which currently lacks efficient treatment strategies. In this study, we investigated the therapeutic effect of function-blocking antibodies targeting integrin α10β1 on patient-derived-GB cell lines in vitro and in vivo. The in vitro studies demonstrated significant inhibiting effects of the integrin α10 antibodies on the adhesion, migration, proliferation, and sphere formation of GB cells. In a xenograft mouse model, the effect of the antibodies on tumor growth was investigated in luciferase-labeled and subcutaneously implanted GB cells. As demonstrated by in vivo imaging analysis and caliper measurements, the integrin α10-antibodies significantly suppressed GB tumor growth compared to control antibodies. Immunohistochemical analysis of the GB tumors showed lower expression of the proliferation marker Ki67 and an increased expression of cleaved caspase-3 after treatment with integrin α10 antibodies, further supporting a therapeutic effect. Our results suggest that function-blocking antibody targeting integrin α10β1 is a promising therapeutic strategy for the treatment of glioblastoma

Protein Kinase Cδ Supports Survival of MDA-MB-231 Breast Cancer Cells by Suppressing the ERK1/2 Pathway*

Mechanisms that mediate apoptosis resistance are attractive therapeutic targets for cancer. Protein kinase Cδ (PKCδ) is considered a pro-apoptotic factor in many cell types. In breast cancer, however, it has shown both pro-survival and pro-apoptotic effects. Here, we report for the first time that down-regulation of PKCδ per se leads to apoptosis of MDA-MB-231 cells. Inhibition of MEK1/2 by either PD98059 or U0126 suppressed the induction of apoptosis of PKCδ-depleted MDA-MB-231 cells but did not support survival of MCF-7 or MDA-MB-468 cells. Basal ERK1/2 phosphorylation was substantially higher in MDA-MB-231 cells than in the other cell lines. PKCδ depletion led to even higher ERK1/2 phosphorylation levels and also to lower expression levels of the ERK1/2 phosphatase MKP3. Depletion of MKP3 led to apoptosis and higher levels of ERK1/2 phosphorylation, suggesting that this may be a mechanism mediating the effect of PKCδ down-regulation. However, PKCδ silencing also induced increased MEK1/2 phosphorylation, indicating that PKCδ regulates ERK1/2 phosphorylation both upstream and downstream. Moreover, PKCδ silencing led to increased levels of the E3 ubiquitin ligase Nedd4, which is a potential regulator of MKP3, because down-regulation led to increased MKP3 levels. Our results highlight PKCδ as a potential target for therapy of breast cancers with high activity of the ERK1/2 pathway

Molecular characterization of protein kinase C delta (PKCδ)-Smac interactions

Background: Protein kinase C o (PKCo) is known to be an important regulator of apoptosis, having mainly pro-but also anti-Apoptotic effects depending on context. In a previous study, we found that PKCo interacts with the pro-Apoptotic protein Smac. Smac facilitates apoptosis by suppressing inhibitor of apoptosis proteins (IAPs). We previously established that the PKCo-Smac complex dissociates during induction of apoptosis indicating a functional importance. Because the knowledge on the molecular determinants of the interaction is limited, we aimed at characterizing the interactions between PKCo and Smac. Results: We found that PKCo binds directly to Smac through its regulatory domain. The interaction is enhanced by the PKC activator TPA and seems to be independent of PKCo catalytic activity since the PKC kinase inhibitor GF109203X did not inhibit the interaction. In addition, we found that C1 and C2 domains from several PKC isoforms have Smac-binding capacity. Conclusions: Our data demonstrate that the Smac-PKCo interaction is direct and that it is facilitated by an open conformation of PKCo. The binding is mediated via the PKCo regulatory domain and both the C1 and C2 domains have Smac-binding capacity. With this study we thereby provide molecular information on an interaction between two apoptosis-regulating proteins

NLK-mediated phosphorylation of HDAC1 negatively regulates Wnt signaling

The Wnt signaling pathway is essential in regulating various cellular processes. Different mechanisms of inhibition for Wnt signaling have been proposed. Besides β-catenin degradation through the proteasome, nemo-like kinase (NLK) is another molecule that is known to negatively regulate Wnt signaling. However, the mechanism by which NLK mediates the inhibition of Wnt signaling was not known. In the present study, we used primary embryonic fibroblast cells isolated from NLK-deficient mice and showed that these cells proliferate faster and have a shorter cell cycle than wild-type cells. In NLK-knockout cells, we observed sustained interaction between Lef1 and β-catenin, leading to elevated luciferase reporter of β-catenin/Lef1-mediated transcriptional activation. The mechanism for the reduced β-catenin/Lef1 promoter activation was explained by phosphorylation of HDAC1 at serine 421 via NLK. The phosphorylation of HDAC1 was achieved only in the presence of wild-type NLK because a catalytically inactive mutant of NLK was unable to phosphorylate HDAC1 and reduced the luciferase reporter of β-catenin/Lef1-mediated transcriptional activation. This result suggests that NLK and HDAC1 together negatively regulate Wnt signaling, which is vital in preventing aberrant proliferation of nontransformed primary fibroblast cells

Association of nuclear-localized nemo-like kinase with heat-shock protein 27 inhibits apoptosis in human breast cancer cells

Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis

Cytosolic localization of NLK induces cell death.

<p>(A) MCF7 and MCF10A cells were transfected for 24 hours with vectors encoding FLAG-tag fusions of full-length NLK (WT), or catalytically inactive NLK mutants (K155M and T286V). NLK localization (Green) was visualized by immunofluorescence staining and confocal microscopy. The nuclei of the cells were counter-stained with DAPI (Blue). (B) MCF7 cells were transfected with vectors encoding FLAG-tag fusions of full-length NLK (WT) or catalytically inactive NLK mutants (K155M and T286V), and counted 24, 48, or 72 h post transfection, using a Countess Automated Cell Counter. Data (mean ± s.e.m., p<0.05, n = 4) are expressed as number of viable cells at each time point. (C) MCF7 cells were mock-treated or transfected for 24 hours with WT-, K155M-, or T286V-NLK. The cells were fixed and stained with propidium iodide in order to count the cells displaying an apoptotic nuclear morphology. Data (mean ± s.e.m., p<0.05, n = 3) show the percentage of apoptotic cells in comparison with cells of mock-treated controls. (D) MCF7 cells were mock-treated or transfected for 24 hours with WT-, K155M-, or T286V-NLK. After 48, or 72 hours cells were subjected to DNA fragmentation assay. Data (mean ± s.e.m., p<0.05, n = 3) represent the percent of sub G0 population. Right panels shows the levels of Flag tagged WT-, K155M-, or T286V-NLK overexpressed in MCF10A and MCF7 cells.</p

Heat-shock protein 27 binds to NLK in the nucleus.

<p>(A) MCF7 cells, transfected with FLAG-tagged WT-, K155M-, or T286V-NLK plasmids, were harvested for immunoprecipitation 24 hours post-transfection, using FLAG antibody. The protein lysates were separated by SDS-PAGE, and the protein bands were cut and processed for the MALDI-TOF analysis. Arrows indicate a specific HSP27 band in NLK-K155M as well as NLK-T286V transfected cells. (B) Immunoprecipitation of FLAG in total cell lysates from MCF7 cells transfected with wild type (WT) and catalytic inactive mutants (K155M and T286V) of NLK, followed by a Western blot, using FLAG or HSP27 antibodies. (C) Endogenous immunoprecipitation of NLK in total cell lysates from MCF10A, MCF7, and MDA231 cells, and a Western blot, using NLK or HSP27 antibodies. As control we used anti-rabbit negative control IgG for immunoprecipitation in total cell lysates from MCF10A cells. (D) DNA sequencing and alignment of NLK gene using MCF10A, MCF7, MDA231, BT549 and CAMA1 cell lines. (E) The cellular distribution of NLK and HSP27 in MCF10A and MCF7 cells, using immunofluorescence staining and confocal microscopy. (F) Endogenous immunoprecipitation of NLK in nuclear fractions of MCF10A, MCF7, and MDA231 cells, and a Western blot, using NLK or HSP27 antibodies. The lysate from nuclear fraction were followed by a Western blot analysis using lamin B or tubulin antibodies. (G) Recombinant GST-NLK and His-HSP27 were incubated in a binding buffer and thereafter GST was pulled down with GSH-coupled sepharose. Pull-downs were analyzed with Western blot using GST or HSP-27 antibodies. (H) The levels of HSP27 in total cell lysates of MCF10A, MCF7, and MDA231 cells. (I) Isolation of nuclei by a subcellular fractionation assay, followed by a Western blot analysis, using HSP27, tubulin, or lamin B antibodies. (J) The levels of HSP27 and phosphorylated HSP27 in total cell lysates of MCF7 cells transfected with wild type (WT) and catalytic inactive mutants (K155M and T286V) of NLK, followed by a Western blot.</p