Carboxyl-terminal polypeptide fragment of MUC16 combing stathmin1 promotes gallbladder cancer cell migration and invasion.

CA-125, coded by MUC16 gene, is responsible to many kinds of tumor metastasis. However, the related mechanism remains unclear. We have established a novel manner to reveal gallbladder cancer metastasis related to serum CA-125 levels through the C-terminal polypeptide of MUC16 gene production. MUC16 C-terminal polypeptide significantly promoted gallbladder cancer cell migration and invasion in vitro. Mass spectrum indicated that interaction of MUC16 C-terminal fragment with the C-terminal domain of stathmin1 inhibited the phosphorylation of stathmin1 to promote the combination with tubulin. Stathmin1 knockdown inhibited the migration and invasion of gallbladder cancer cells in vitro and lung metastasis in vivo induced by MUC16 C-terminal polypeptide. The high expression level of MUC16c consistent with stathmin1 was also confirmed in gallbladder cancer tissues. Our study revealed the underlying mechanism of gallbladder cancer metastasis related to CA-125 levels, which was beneficial for CA-125 in gallbladder cancer diagnose and therapy.

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells.

Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA-mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

Stathmin recruits tubulin to Listeria monocytogenes-induced actin comets and promotes bacterial dissemination.

The tubulin cytoskeleton is one of the main components of the cytoarchitecture and is involved in several cellular functions. Here, we examine the interplay between Listeria monocytogenes (Lm) and the tubulin cytoskeleton upon cellular infection. We show that non-polymeric tubulin is present throughout Lm actin comet tails and, to a less extent, in actin clouds. Moreover, we demonstrate that stathmin, a regulator of microtubule dynamics, is also found in these Lm-associated actin structures and is required for tubulin recruitment. Depletion of host stathmin results in longer comets containing less F-actin, which may be correlated with higher levels of inactive cofilin in the comet, thus suggesting a defect on local F-actin dynamics. In addition, intracellular bacterial speed is significantly reduced in stathmin-depleted cells, revealing the importance of stathmin/tubulin in intracellular Lm motility. In agreement, the area of infection foci and the total bacterial loads are also significantly reduced in stathmin-depleted cells. Collectively, our results demonstrate that stathmin promotes efficient cellular infection, possibly through tubulin recruitment and control of actin dynamics at Lm-polymerized actin structures.

Effect of the p53-tristetraprolin-stathmin-1 pathway on trophoblasts at maternal-fetal interface.

PROBLEM: To reveal the effect of p53-tristetraprolin-stathmin-1 signaling on trophoblasts and recurrent spontaneous abortion (RSA). METHOD OF STUDY: Stathmin-1 (STMN1), p53, and tristetraprolin (TTP) expression in paraffin-embedded villus tissue was determined using immunohistochemistry. HTR-8/SVneo cells were treated with doxorubicin to activate p53; STMN1 and TTP levels were detected by quantitative reverse transcription-PCR and western blotting. Western blotting and immunofluorescence were used to investigate STMN1 expression after TTP overexpression or knockdown in HTR-8 cells. RESULTS: STMN1 was downregulated and p53 was upregulated in the villus tissue from patients with RSA. Doxorubicin decreased STMN1 expression but enhanced TTP expression in HTR-8 cells. In vitro, TTP overexpression inhibited STMN1 production; TTP knockdown promoted it. TTP downregulated STMN1 expression in trophoblasts by directly binding its 3' untranslated region. CONCLUSIONS: TTP modulates trophoblast function and interacts with STMN1 and p53, and is related to pregnancy outcomes.

Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells.

Stathmin is a prominent destabilizer of microtubules (MTs). Extensive in vitro studies have strongly suggested that stathmin could act by sequestering tubulin and/or by binding to MT tips. In cells, the molecular mechanisms of stathmin binding to tubulin and/or MTs and its implications for the MT dynamics remain unexplored. By using immunofluorescence resonance energy transfer and fluorescence recovery after photobleaching, we analyzed the ability of stathmin and its phosphorylated forms (on Ser16, -25, -38, and -63) to interact with tubulin and MTs in A549 cells. Consistent with in vitro studies, we detected stathmin-tubulin interactions at the MT plus ends and in the cytosol. Of interest, we also observed a novel pool of stathmin bound along the MT. Expression of truncated stathmin and use of MT-stabilizing taxol further showed that the C-terminal domain of stathmin is the main contributor to this binding and that the phosphorylation state of stathmin plays a role in its binding along the MT wall. Our findings demonstrate that stathmin binds directly along the MT wall. This pool of stathmin would be readily available to participate in protofilament dissociation when the moving plus end of a depolymerizing MT reaches stathmin molecules.-Nouar, R., Breuzard, G., Bastonero, S., Gorokhova, S., Barbier, P., Devred, F., Kovacic, H., Peyrot, V. Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells.

Molecular basis of major psychiatric diseases such as schizophrenia and depression.

Recently several potential susceptibility genes for major psychiatric disorders (schizophrenia and major depression) such as disrupted-in-schizophrenia 1(DISC1), dysbindin and pituitary adenylate cyclase-activating polypeptide (PACAP) have been reported. DISC1 is involved in neural development directly via adhesion molecules or via its binding partners of DISC1 such as elongation protein ζ-1 (FEZ1), DISC1-binding zinc-finger protein (DBZ) and kendrin. PACAP also regulates neural development via stathmin 1 or via regulation of the DISC1-DBZ binding. Dysbindin is also involved in neural development by regulating centrosomal microtubule network formation. All such molecules examined to date are involved in neural development. Thus, these findings provide new molecular insights into the mechanisms of neural development and neuropsychiatric disorders. On the other hand, in addition to neurons, both DISC and DBZ have been detected in oligodendrocytes and implicated in regulating oligodendrocyte differentiation. DISC1 inhibits the differentiation of oligodendrocyte precursor cells into oligodendrocytes, while DBZ has a positive regulatory role in oligodendrocyte differentiation. Evidence suggesting that disturbance of oligodendrocyte development causes major depression is also described.

siRNA targeting stathmin inhibits invasion and enhances chemotherapy sensitivity of stem cells derived from glioma cell lines.

Glioma is one of the most highly angiogenic tumors, and glioma stem cells (GSCs) are responsible for resistance to chemotherapy and radiotherapy, as well as recurrence after operation. Stathmin is substantial for mitosis and plays an important role in proliferation and migration of glioma-derived endothelial cells. However, the relationship between stathmin and GSCs is incompletely understood. Here we isolated GSCs from glioma cell lines U87MG and U251, and then used siRNA targeting stathmin for silencing. We showed that silencing of stathmin suppressed the proliferation, increased the apoptosis rate, and arrested the cell cycle at G2/M phase in GSCs. Silencing of stathmin in GSCs also resulted in inhibited the migration/invasion as well as the capability of vasculogenic mimicry. The susceptibility of GSCs to temozolomide was also enhanced by stathmin silencing. Our findings suggest stathmin as a potential target in GSCs for glioma treatment.

Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing.

Changes in the stability of microtubules regulate many biological processes, but their role in memory remains unclear. Here we show that learning causes biphasic changes in the microtubule-associated network in the hippocampus. In the early phase, stathmin is dephosphorylated, enhancing its microtubule-destabilizing activity by promoting stathmin-tubulin binding, whereas in the late phase these processes are reversed leading to an increase in microtubule/KIF5-mediated localization of the GluA2 subunit of AMPA receptors at synaptic sites. A microtubule stabilizer paclitaxel decreases or increases memory when applied at the early or late phases, respectively. Stathmin mutations disrupt changes in microtubule stability, GluA2 localization, synaptic plasticity and memory. Aged wild-type mice show impairments in stathmin levels, changes in microtubule stability and GluA2 localization. Blocking GluA2 endocytosis rescues memory deficits in stathmin mutant and aged wild-type mice. These findings demonstrate a role for microtubules in memory in young adult and aged individuals.

Stathmin 1 is involved in the highly proliferative phenotype of high-risk myelodysplastic syndromes and acute leukemia cells.

Stathmin 1 is an important cytoplasmic microtubule-destabilizing protein that plays critical roles in proliferation and accurate chromosome segregation through regulation of microtubule dynamics. High levels of Stathmin 1 expression have been reported in leukemia and solid tumors. However, Stathmin 1 has not been studied in myelodysplastic syndrome cells. We, herein, report that significantly higher Stathmin 1 levels were observed in proliferating hematopoietic cells, in high-risk MDS and acute leukemia cells. In addition, Stathmin 1 silencing in U937 and Namalwa leukemia cells reduced cell proliferation and clonogenicity. Our data suggest that Stathmin 1 expression may be related to the highly proliferative phenotype of hematopoietic cells and add new insights into the participation of Stathmin 1 in hematological malignancies.

Signal transducer and activator of transcription 3 is a proviral host factor for hepatitis C virus.

UNLABELLED: Host factors play an important role in all facets of the hepatitis C virus (HCV) life cycle and one such host factor is signal transducer and activator of transcription 3 (STAT3). The HCV core protein has been shown to directly interact with and activate STAT3, while oxidative stress generated during HCV replication in a replicon-based model also induced STAT3 activation. However, despite these findings the precise role of STAT3 in the HCV life cycle remains unknown. We have established that STAT3 is actively phosphorylated in the presence of replicating HCV. Furthermore, expression of a constitutively active form of STAT3 leads to marked increases in HCV replication, whereas, conversely, chemical inhibition and small interfering RNA (siRNA) knockdown of STAT3 leads to significant decreases in HCV RNA levels. This strongly implicates STAT3 as a proviral host factor. As STAT3 is a transcription factor, up-regulation of a distinct set of STAT3-dependent genes may create an environment that is favorable for HCV replication. However, STAT3 has recently been demonstrated to positively regulate microtubule (MT) dynamics, by way of a direct sequestration of the MT depolymerizing protein Stathmin 1 (STMN1), and we provide evidence that STAT3 may exert its effect on the HCV life cycle by way of positive regulation of MT dynamics. CONCLUSION: We have demonstrated that STAT3 plays a role in the life cycle of HCV and have clarified the role of STAT3 as a proviral host factor.

Novel role of stathmin in microtubule-dependent control of endothelial permeability.

Microtubule (MT) dynamics in vascular endothelium are modulated by vasoactive mediators and are critically involved in the control of endothelial cell (EC) permeability via Rho GTPase-dependent crosstalk with the actin cytoskeleton. However, the role of regulators in MT stability in these mechanisms remains unclear. This study investigated the involvement of the MT-associated protein stathmin in the mediation of agonist-induced permeability in EC cultures and vascular leak in vivo. Thrombin treatment of human pulmonary ECs induced rapid dephosphorylation and activation of stathmin. Inhibition of stathmin activity by small interfering RNA-based knockdown or cAMP-mediated phosphorylation abrogated thrombin-induced F-actin remodeling and Rho-dependent EC hyperpermeability, while expression of a phosphorylation-deficient stathmin mutant exacerbated thrombin-induced EC barrier disruption. Stathmin suppression preserved the MT network against thrombin-induced MT disassembly and release of Rho-specific guanine nucleotide exchange factor, GEF-H1. The protective effects of stathmin knockdown were observed in vivo in the mouse 2-hit model of ventilator-induced lung injury and were linked to MT stabilization and down-regulation of Rho signaling in the lung. These results demonstrate the mechanism of stathmin-dependent control of MT dynamics, Rho signaling, and permeability and suggest novel potential pharmacological interventions in the prevention of increased vascular leak via modulation of stathmin activity.

Stathmin regulates microtubule dynamics and microtubule organizing center polarization in activated T cells.

Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation.

Circadian variation of cell proliferation in HTR-8/SVneo cell line.

Circadian clock controls several physiological processes such as cell proliferation. Extravillous trophoblast proliferation is a tightly regulated function playing a fundamental role in maternal vessel remodeling. We recently demonstrated that clock genes Per2 and Dec1 as well as the clock-controlled genes Dbp and Vegf are rhythmically expressed in human extravillous trophoblast-derived HTR-8/SVneo cells. Analyzing the time course of HTR-8/SVneo cell proliferation, a circadian variation in cell number was found. Moreover, we showed a rhythmic expression of mRNAs for Wee1 and stathmin, two genes involved in cell cycle progression. We suggest that circadian clockwork may orchestrate the functionality of the several factors involved in the control of human trophoblast functions that are fundamental for a successfully pregnancy outcome.

A novel insight to the functional role of Stathmin 1 in IgE-mediated activation of RBL-2H3 cells.

IgE-mediated cell signaling, induced by cross-linking of high affinity receptor for IgE (FcεRI) in the presence of antigen (Ag), is a well known mechanism described for mast cell activation in allergy and hypersensitivity reactions, which induces a spectrum of cellular responses such as secretion and up-regulation of cell surface FcεRI. Although for several years IgE binding to FcεRI was considered to be a passive sensitization process, the outcomes of several recent studies have revealed a variety of different cellular responses to IgE binding compared to IgE plus Antigen binding. The present study applied a functional proteomics-based approach to investigate mast cell signaling events and provided new insights to FcεRI-mediated cell signaling in RBL-2H3.1 cells, and may point to the activation of alternative signaling pathways in response to IgE or IgE plus Ag. Comparative analysis by 2-D PAGE of RBL cells activated with IgE plus Ag for three and four hours compared to non-activated cells was followed by mass spectrometric protein identification and provided evidence for the induction of Stathmin 1 (STMN1) gene expression in response to IgE plus Ag activation.Complementary SDS-PAGE analysis showed a distinct up-regulation of STMN1 induction in response to challenge with IgE plus Ag compared to sensitization with IgE only. Phosphoproteomics analysis gave evidence for significant increase at phosphorylation of STMN1 on ser16 after 1min, though a slight rise at 5 min, and on ser38 after 1 and 5min sensitization with IgE and a similar result was observed for 1min IgE plus Ag-activation. IgE plus Ag-activation was also found to induce the phosphorylation of ser38 to a greater extent than sensitization with IgE. In contrast, IgE alone was more effective than IgE plus Ag at inducing phosphorylation of ser16. Collectively this study provides further insights into the role of stathmin 1 in FcRI-mediated activation of cells of mast cell lineage and might shed light on the diverse response of these cells to IgE or IgE plus Ag.

Stathmin overexpression identifies high-risk patients and lymph node metastasis in endometrial cancer.

PURPOSE: Overexpression of the oncogen Stathmin has been linked to aggressive endometrial carcinoma and a potential for PI3Kinase inhibitors in this disease. We wanted to validate the prognostic value of Stathmin expression in a large prospective multicenter setting. As lymph node sampling is part of current surgical staging, we also aimed to test if Stathmin expression in endometrial curettage specimens could predict lymph node metastasis. EXPERIMENTAL DESIGN: A total of 1,076 endometrial cancer patients have been recruited from 10 centers to investigate the biological tumor marker Stathmin in relation to clinicopathologic variables, including lymph node status and survival. Stathmin immunohistochemical staining was carried out in 477 hysterectomy and 818 curettage specimens. RESULTS: Seventy-one percent of the patients (n = 763) were subjected to lymph node sampling, of which 12% had metastatic nodes (n = 94). Overexpression of Stathmin was detected in 37% (302 of 818) of the curettage and in 18% (84 of 477) of the hysterectomy specimens investigated. Stathmin overexpression in curettage and hysterectomy specimens were highly correlated and significantly associated with nonendometrioid histology, high grade, and aneuploidy. Stathmin analysis in preoperative curettage samples significantly correlated with, and was an independent predictor of, lymph node metastases. High Stathmin expression was associated with poor disease-specific survival (P ≤ 0.002) both in curettage and hysterectomy specimens. CONCLUSIONS: Stathmin immunohistochemical staining identifies endometrial carcinomas with lymph node metastases and poor survival. The value, as a predictive marker for response to PI3Kinase inhibition and as a tool to stratify patients for lymph node sampling in endometrial carcinomas, remains to be determined.

Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis.

Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

Involvement of stathmin 1 in the neurotrophic effects of PACAP in PC12 cells.

Rat pheochromocytoma PC12 cells have been widely used to investigate the neurotrophic activities of pituitary adenylate cyclase-activating polypeptide (PACAP). In particular, PACAP has been shown to promote differentiation and to inhibit apoptosis of PC12 cells. In order to identify the mechanisms mediating these effects, we sought for proteins that are phosphorylated upon PACAP treatment. High-performance liquid chromatography and 2D gel electrophoresis analysis, coupled with mass spectrometry, revealed that stathmin 1 is strongly phosphorylated within only 5 min of exposure to PACAP. Western blot experiments confirmed that PACAP induced a robust phosphorylation of stathmin 1 in a time-dependent manner. On the other hand, PACAP decreased stathmin 1 gene expression. Investigations of the signaling mechanisms known to be activated by PACAP revealed that phosphorylation of stathmin 1 was mainly mediated through the protein kinase A and mitogen-activated protein kinase pathways. Blockage of stathmin 1 expression with small interfering RNA did not affect PC12 cell differentiation induced by PACAP but reduced the ability of the peptide to inhibit caspase 3 activity and significantly decreased its neuroprotective action. Taken together, these data demonstrate that stathmin 1 is involved in the neurotrophic effect of PACAP in PC12 cells.

Stathmin/oncoprotein 18, a microtubule regulatory protein, is required for survival of both normal and cancer cell lines lacking the tumor suppressor, p53.

Stathmin, a microtubule regulatory protein, is overexpressed in many cancers and required for survival of several cancer lines. In a study of breast cancer cell lines(1) proposed that stathmin is required for survival of cells lacking p53, but this hypothesis was not tested directly. Here we tested their hypothesis by examining cell survival in cells depleted of stathmin, p53 or both proteins. Comparing HCT116 colon cancer cell lines differing in TP53 genotype, stathmin depletion resulted in significant death only in cells lacking p53. As a second experimental system, we compared the effects of stathmin depletion from HeLa cells, which normally lack detectable levels of p53 due to expression of the HPV E6 protein. Stathmin depletion caused a large percentage of HeLa cells to die. Restoring p53, by depletion of HPV E6, rescued HeLa cells from stathmin-depletion induced death. Cleaved PARP was detected in HCT116(p53-/-) cells depleted of stathmin and cell death in stathmin-depleted HeLa cells was blocked by the caspase inhibitor Z-VAD-FMK, consistent with apoptotic death. The stathmin-dependent survival of cells lacking p53 was not confined to cancerous cells because both proteins were required for survival of normal human fibroblasts. In HCT116 and HeLa cells, depletion of both stathmin and p53 leads to a cell cycle delay through G(2). Our results demonstrate that stathmin is required for cell survival in cells lacking p53, suggesting that stathmin depletion could be used therapeutically to induce apoptosis in tumors without functional p53.

Predominant regulators of tubulin monomer-polymer partitioning and their implication for cell polarization.

The microtubule-system organizes the cytoplasm during interphase and segregates condensed chromosomes during mitosis. Four unrelated conserved proteins, XMAP215/Dis1/TOGp, MCAK, MAP4 and Op18/stathmin, have all been implicated as predominant regulators of tubulin monomer-polymer partitioning in animal cells. However, while studies employing the Xenopus egg extract model system indicate that the partitioning is largely governed by the counteractive activities of XMAP215 and MCAK, studies of human cell lines indicate that MAP4 and Op18 are the predominant regulators of the interphase microtubule-array. Here, we review functional interplay of these proteins during interphase and mitosis in various cell model systems. We also review the evidence that MAP4 and Op18 have interphase-specific, counteractive and phosphorylation-inactivated activities that govern tubulin subunit partitioning in many mammalian cell types. Finally, we discuss evidence indicating that partitioning regulation by MAP4 and Op18 may be of significance to establish cell polarity.