Discrete functions of GSK3α and GSK3β isoforms in prostate tumor growth and micrometastasis

Isoform specific function of glycogen synthase kinase-3 (GSK3) in cancer is not well defined. We report that silencing of GSK3α, but not GSK3β expression inhibited proliferation, survival and colony formation by the PC3, DU145 and LNCaP prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Silencing of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts. ShRNA-mediated knockdown of GSK3α and GSK3β equally inhibited the ability of prostate cancer cells to migrate and invade the endothelial-barrier in vitro, and PC3 cell micrometastasis to lungs in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro-apoptotic markers cleaved caspase-3 and cleaved caspase-9 in LNCaP, PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell-cell contacts, increased expression and membrane localization of β-catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP-9. This indicated the specific role of GSK3β in EMT, acquisition of motility and invasive potential. Overall, our data demonstrated the isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.     

Syntaxins 3 and 4 mediate vesicular trafficking of α5β1 and α3β1 integrins and cancer cell migration

Integrins, a family of heterodimeric receptors for cell adhesion to the extracellular matrix (ECM), play key roles in cell migration, cancer progression and metastasis. As transmembrane proteins, integrins are transported in vesicles and delivered to the cell surface by vesicular trafficking. The final step for integrin delivery, i.e., fusion of integrin-containing vesicles with the plasma membrane, is poorly understood at the molecular level. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxins 1, 2, 3 and 4 are present at the plasma membrane to drive vesicle fusion. In this study, we examined the roles of syntaxins?1, 2, 3 and 4 in vesicular trafficking of α5β1 and α3β1 integrins. We showed that syntaxins?2, 3 and 4 were expressed in HeLa cervical adenocarcinoma cells and PANC-1 pancreatic adenocarcinoma cells. In migrating HeLa and PANC-1 cells, syntaxins?2, 3 and 4 co-localized with the lipid raft constituent GM1 ganglioside at the leading edge. siRNA knockdown (KD) of syntaxins?3 and 4, but not of syntaxin?2, in HeLa cells reduced cell surface expression of α5β1 and α3β1 integrins and accumulated the integrins in cytoplasmic vesicles, indicating that syntaxins?3 and 4 mediate vesicular trafficking of α5β1 and α3β1 integrins to the cell surface. In addition, KD of syntaxins?3 and 4 inhibited cell adhesion to fibronectin, suppressed chemotactic cell migration and triggered apoptosis. Collectively, these data suggest that syntaxins?3- and 4-dependent integrin trafficking is important in cancer cell migration and survival, and may be a valuable target for cancer therapy.     

Twin characterisation using 2D and 3D EBSD

IntroductionTwin characterisation plays an important role inunderstanding the deformation and annealing behaviourof many metals,particularly those with hcp crystalstructures[1~8].Often,the aim is to determine the vol-ume fraction of twins within a material,perhaps as afunction of strain.However,many materials twin onseveral differentsystems and formsecondary or tertiary,as well as primary,twins.Furthermore,the variants ofa particular twin type may be activated to differing ex-tents.Analysis o…     

Role of 14-3-3σ in poor prognosis and in radiation and drug resistance of human pancreatic cancers

Background  

Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3σ mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis.     

Tamoxifen downregulation of miR-451 increases 14-3-3ζ and promotes breast cancer cell survival and endocrine resistance

Many estrogen receptor (ER)-positive breast cancers respond well initially to endocrine therapies, but often develop resistance during treatment with selective ER modulators (SERMs) such as tamoxifen. We have reported that the 14-3-3 family member and conserved protein, 14-3-3ζ, is upregulated by tamoxifen and that high expression correlated with an early time to disease recurrence. However, the mechanism by which tamoxifen upregulates 14-3-3ζ and may promote the development of endocrine resistance is not known. Our findings herein reveal that the tamoxifen upregulation of 14-3-3ζ results from its ability to rapidly downregulate microRNA (miR)-451 that specifically targets 14-3-3ζ. The levels of 14-3-3ζ and miR-451 were inversely correlated, with 14-3-3ζ being elevated and miR-451 being at a greatly reduced level in tamoxifen-resistant breast cancer cells. Of note, downregulation of miR-451 was selectively elicited by tamoxifen but not by other SERMs, such as raloxifene or ICI182,780 (Fulvestrant). Increasing the level of miR-451 by overexpression, which decreased 14-3-3ζ, suppressed cell proliferation and colony formation, markedly reduced activation of HER2, EGFR and MAPK signaling, increased apoptosis, and, importantly, restored the growth-inhibitory effectiveness of SERMs in endocrine-resistant cells. Opposite effects were elicited by miR-451 knockdown. Thus, we identify tamoxifen downregulation of miR-451, and consequent elevation of the key survival factor 14-3-3ζ, as a mechanistic basis of tamoxifen-associated development of endocrine resistance. These findings suggest that therapeutic approaches to increase expression of this tumor suppressor-like miR should be considered to downregulate 14-3-3ζ and enhance the effectiveness of endocrine therapies. Furthermore, the selective ability of the SERM tamoxifen but not raloxifene to regulate miR-451 and 14-3-3ζ may assist in understanding differences in their activities, as seen in the STAR (Study of Tamoxifen and Raloxifene) breast cancer prevention trial and in other clinical trials.     

Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression by Blocking BCR-Dependent and -Independent Activation of NF-κB and AKT

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14-3-3σ and p63 play opposing roles in epidermal tumorigenesis

14-3-3σ plays a regulatory role in epidermal epithelial differentiation and loss of 14-3-3σ leads to increased proliferation and impaired differentiation. A tumor suppressor function for 14-3-3σ has been proposed based on the fact that some epithelial-derived tumors lose 14-3-3σ expression. p63, a p53 family member, is a master regulator of epidermal epithelial proliferation and differentiation and is necessary for the epidermal development. The function of p63 in tumorigenesis is still controversial and poorly defined as multiple isoforms have been found to play either collaborative or opposing roles. By using 'repeated epilation' heterozygous (Er/+) mice containing a dominant-negative 14-3-3σ mutation, the functional relationship of p63 with 14-3-3σ in epidermal proliferation, differentiation and tumorigenesis was investigated. It was found that p63, particularly the ΔNp63α isoform, was strongly expressed in 14-3-3σ-deficient keratinocytes and knockdown of p63 remarkably inhibited proliferation in these cells. To study the functional roles of 14-3-3σ and p63 in epidermal tumorigenesis, we adopted a 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) two-stage tumorigenesis procedure to induce formation of skin papillomas and squamous cell carcinomas in Er/+ mice and identified strong p63 expression in resultant tumors. The loss of one allele of p63 caused by the generation of Er/+/p63(+/-) double compound mice decreased the sensitivity to DMBA-/TPA-induced tumorigenesis as compared with Er/+ mice. This study shows that p63 and 14-3-3σ play opposing roles in the development of skin tumors and that the accumulation of p63 is essential for Ras/14-3-3σ mutation-induced papilloma formation and squamous cell carcinoma carcinogenesis.     

Expression of Granulysin and FOXP3 in Cutaneous T Cell Lymphoma and Sézary Syndrome

Background: Multiple complex pathways are operable in the evolution of cutaneous T cell lymphomas(CTCLs). These pathways involve interaction between neoplastic T cells and cells of the immune system (especiallydendritic cells and the non-malignant T cells). Granulysin is a proinflammatory antimicrobial peptide whichhas an immune alarmin function, activating dendritic cells, as well as an active role in tumor immunology andprognosis. FOXP3+ regulatory T cells Tregs are an important player in the immune system. Much controversy isfound in the literature about the role of Tregs in CTCL. Aim: The present study aimed to investigate the expressionof granulysin and FOXP3 in mycosis fungoides (MF), its precursor lesion large plaque parapsoriasis and itsleukemic form Sézary syndrome (SS). Materials and Methods: Immunohistochemical expression of granulysinand FOXP3 were assessed in lesional skin biopsies taken from 58 patients (4 large plaque parapsoriasis, 48 MFand 6 SS). Results: Granulysin positivity was cytoplasmic and higher in MF than in parapsoriasis en plaqueand higher in the more advanced stages of MF (p<0.001). All groups showed significant differences betweeneach other except between MF tumor stage and SS. FOXP3 positivity was nuclear and higher in early stageMF (plaque and patch stages) than in tumor stages and SS (p<0.001). However the FOXP3 count was lowerin parapsoriasis en plaque than in other stages of MF. All the groups showed significant differences betweeneach other except between parapsoriasis and SS and between patch and plaque stages of MF. Conclusions: Thepresent study supports a role for granulysin in MF progression and proposes a novel hypothesis about the effectof FOXP3 +veTregs in the suppression of the activity of the neoplastic cells in MF.     

Quantification of thrombospondin-1 secretion and expression of αvβ3 and α3β1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface v3 and 31 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of v3 and 31 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of v3 and 31 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/1 × 10⁶ cells/24 h (mean ± SD=626 ± 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 g. Seven of eight non-glioma cell lines secreted less than 100 0ng of TSP-1. All glioma cell lines expressed 31 integrin and syndecan-1, and seven of 10 glioma cell lines expressed v3 integrin. Treatment of the glioma cell lines with TGF-2 did not change the expression of v3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with v3 and 31 integrins, and syndecan-1.     

ERβ and PEA3 co-activate IL-8 expression and promote the invasion of breast cancer cells

Metastasis represents the major remaining cause of mortality in human breast cancer. Interleukin-8 (IL-8), a proinflammatory chemokine, plays an important role during tumor angiogenesis and metastasis. In this study, we found that IL-8 and ERβ showed positive association. Overexpression of ERβ or PEA3 could up-regulate IL-8 promoter activity, mRNA and secretion; silencing of ERβ or PEA3 decreased IL-8 mRNA and secretion. ERβ and PEA3 increased IL-8 expression through binding to the IL-8 promoter and increased cell invasion. HER2 could increase ERβ and PEA3 expression and their binding to the IL-8 promoter. We conclude that ERβ and PEA3 play important roles in tumor invasion by regulating IL-8 expression, and HER2 maybe the upstream of ERβ and PEA3 - IL-8 pathway.     

COP1 and GSK3β Cooperate to Promote c-Jun Degradation and Inhibit Breast Cancer Cell Tumorigenesis

High abundance of c-Jun is detected in invasive breast cancer cells and aggressive breast tumor malignancies. Here, we demonstrate that a major cause of high c-Jun abundance in invasive breast cancer cells is prolonged c-Jun protein stability owing to poor poly-ubiquitination of c-Jun. Among the known c-Jun-targeting E3 ligases, we identified constitutive photomorphogenesis protein 1 (COP1) as an E3 ligase responsible for c-Jun degradation in less invasive breast cancer cells because depletion of COP1 reduced c-Jun poly-ubiquitination leading to the stabilization of c-Jun protein. In a panel of breast cancer cell lines, we observed an inverse association between the levels of COP1 and c-Jun. However, overexpressing COP1 alone was unable to decrease c-Jun level in invasive breast cancer cells, indicating that efficient c-Jun protein degradation necessitates an additional event. Indeed, we found that glycogen synthase kinase 3 (GSK3) inhibitors elevated c-Jun abundance in less invasive breast cancer cells and that GSK3β nonphosphorylable c-Jun-T239A mutant displayed greater protein stability and poorer poly-ubiquitination compared to the wild-type c-Jun. The ability of simultaneously enforced expression of COP1 and constitutively active GSK3β to decrease c-Jun abundance in invasive breast cancer cells allowed us to conclude that c-Jun is negatively regulated through the coordinated action of COP1 and GSK3β. Importantly, co-expressing COP1 and active GSK3β blocked in vitro cell growth/migration and in vivo metastasis of invasive breast cancer cells. Gene expression profiling of breast tumor specimens further revealed that higher COP1 expression correlated with better recurrence-free survival. Our study supports the notion that COP1 is a suppressor of breast cancer progression.     

Toxicity and metabolism of 3′-deoxyadenosine N1-oxide in mice and Ehrlich ascites tumor cells

Summary The toxic effect of 3-deoxyadenosine N¹-oxide (3-dANO) on mice, on their different organs, and on Ehrlich ascites tumor cells was studied. In both healthy and tumour-bearing animals, the lethal dose for 10% of the mice receiving i. p. injections (LD₁₀) of 3-dANO was estimated to be about 300 mg/kg×4 days in one mouse strain (Theiller). In another mouse strain (NMRI), we obtained a markedly higher LD₁₀ value (675 mg/kg×5 days). At nonlethal doses (250 mg/kg×4 days), we observed reversible neurological symptoms on days 4–12 after treatment, but no macroscopical or microscopical changes was detected in the brain, heart, thymus, lung, lymph node, spleen, liver, kidney, bone marrow, or gastrointestinal tract. At doses of 450 mg/kg×4 days, severe neurological symptoms were observed, and atony of the gastrointestinal canal and damage to the kidney and liver were registered. Even at doses that were lethal to the mice, no histopathological change was observed in the bone marrow or in the gastrointestinal canal. Pharmacokinetics studies showed that after the i.p. injection of 3-dANO, the maximal plasma concentration was reached after 10 min, after which it declined showing a half-life of about 40 min. A transient accumulation of 3-deoxyadenosine triphosphate (3-dATP) was observed within 24 h in the liver and kidney, with the maximal concentration being reached after about 2–3 h. 3-dANO was excreted partly as the unchanged substance and partly as the metabolite 3-deoxyinosine within 24 h. Flow-cytometric DNA analysis of Ehrlich tumor cells treated either in vitro or in vivo with 3-dANO revealed no therapy-induced change in the cell-cycle perturbations, which indicates that cells were randomly killed during all phases of the cycle.Abbreviations 3-dANO 3-deoxyadenosine N¹-oxide - 3-dA 3-deoxyadenosine - 3-dI 3-deoxyinosine - 3-dATP 3-deoxyadenosine triphosphate This work was supported by grants from the Director Ib Hendriksens Foundation and the P. Carl Petersens Foundation