Phosphorylation at Ser10 triggered p27 degradation and promoted gallbladder carcinoma cell migration and invasion by regulating stathmin1 under glucose deficiency.

Gallbladder carcinoma (GBC) is a considerable challenge because of its high metastatic potential. The tumor microenvironment is characterized by nutrient starvation, which promotes tumor metastasis. Stathmin1, an important microtubuleregulating protein, is overexpressed and promotes metastasis in GBC. It remains unclear how the harsh tumor microenvironment regulates stathmin1 expression to affect GBC metastasis. We employed glucose deficiency to mimic nutrient starvation and found that glucose deficiency upregulated stathmin1 transcription. However, glucose deficiency also promoted p27 degradation. There was a significant negative correlation between stathmin1 and p27 protein levels under glucose deficiency. Further study revealed that, under glucose deficiency, human kinase interacting with stathmin (hKIS) induced phosphorylation at Ser10 of p27 and its translocation to the cytoplasm for degradation, which upregulated the transcription factor E2F1 to promote stathmin1 transcription. hKIS knockdown significantly inhibited p27 cytoplasmic translocation and its consequent degradation. Stathmin1 knockdown significantly inhibited GBC cell migration and invasion in vitro. Our study revealed the role of the hKIS/p27/E2F1 axis in upregulating stathmin1 transcription to promote GBC cell migration and invasion under glucose deficiency conditions.

Oncoprotein 18 is necessary for malignant cell proliferation in bladder cancer cells and serves as a G3-specific non-invasive diagnostic marker candidate in urinary RNA.

BACKGROUND: Urine-based diagnostics indicated involvement of oncoprotein 18 (OP18) in bladder cancer. In cell culture models we investigated the role of OP18 for malignant cell growth. METHODS: We analyzed 113 urine samples and investigated two human BCa cell lines as a dual model: RT-4 and ECV-304, which represented differentiated (G1) and poorly differentiated (G3) BCa. We designed specific siRNA for down-regulation of OP18 in both cell lines. Phenotypes were characterized by cell viability, proliferation, and expression of apoptosis-related genes. Besides, sensitivity to cisplatin treatment was evaluated. RESULTS: Analysis of urine samples from patients with urothelial BCa revealed a significant correlation of the RNA-ratio OP18:uroplakin 1A with bladder cancer. High urinary ratios were mainly found in moderately to poorly differentiated tumors (grade G2-3) that were muscle invasive (stage T2-3), whereas samples from patients with more differentiated non-invasive BCa (G1) showed low OP18:UPK1A RNA ratios. Down-regulation of OP18 expression in ECV-304 shifted its phenotype towards G1 state. Further, OP18-directed siRNA induced apoptosis and increased chemo-sensitivity to cisplatin. CONCLUSIONS: This study provides conclusive experimental evidence for the link between OP18-derived RNA as a diagnostic marker for molecular staging of BCa in non-invasive urine-based diagnostics and the patho-mechanistic role of OP18 suggesting this gene as a therapeutic target.

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.

High STMN1 Expression is Associated with Cancer Progression and Chemo-Resistance in Lung Squamous Cell Carcinoma.

BACKGROUND: Known as a microtubule-destabilizing protein, STMN1 (gene symbol: STMN1) regulates the dynamics of microtubules, cell cycle progress, and chemo-resistance against taxane agents. It is highly expressed in various human cancers and involved in cancer progression as well as poor prognosis. METHODS: Expression of STMN1 was examined by immunohistochemistry using FFPE tissue sections from 186 patients with lung squamous cell carcinoma (LSCC). Analysis of STMN1 suppression was performed for STMN1 small interfering RNA (siRNA)-transfected LSCC cell lines to determine the change in proliferation, invasive and apoptosis abilities, and paclitaxel sensitivity. RESULTS: The cytoplasmic STMN1 expression in LSCC was higher than in normal tissues. The high expression was significantly associated with vascular invasion (P = 0.0477) and poor prognosis. In addition, the proliferating and invasive abilities were decreased, and the apoptosis ability and paclitaxel sensitivity were increased in STMN1-suppressed LSCC cells compared with control cells. CONCLUSION: The results suggest that STMN1 is a prognostic factor that also is associated with caner progression and chemo-resistance. Therefore, STMN1 could be a predictor for poor prognosis and a potential therapeutic target in LSCC.

Stathmin is overexpressed and regulated by mutant p53 in oral squamous cell carcinoma.

BACKGROUND: The aim of this study was to investigate the oncogenic function and regulatory mechanism of stathmin in oral squamous cell carcinoma (OSCC). METHODS: Two-dimensional electrophoresis and liquid chromatography-tandem mass chromatography were applied to screen differentiated proteins during carcinogenesis in OSCC. Cell Counting Kit-8 (CCK-8) assays, colony formation, migration, flow cytometry, immunofluorescence and a xenograft model were used to detect the function of stathmin. The correlation between stathmin and p53 expression was analyzed using immunohistochemistry. Mutant/wild type p53 plasmids and small interfering RNA were used to examine the regulation of stathmin. Chromatin immunoprecipitation assays and luciferase assays were performed to detect the transcriptional activation of stathmin by p53. RESULTS: Overexpression of stathmin was screened and confirmed in OSCC patients and cell lines. Silencing expression of stathmin inhibited proliferation, colony formation and migration and promoted apoptosis. Poly ADP ribose polymerase (PARP) and cyclin-dependent kinase 1 (cdc2) were activated after silencing the expression of stathmin. Suppression of tumorigenicity was also confirmed in vivo. Mutant p53 transcriptionally activated the expression of stathmin in HN6 and HN13 cancer cells, but not in HN30 cells harboring wild type p53. CONCLUSIONS: These results suggest that stathmin acts as an oncogene and is transcriptionally regulated by mutant p53, but not by wild-type p53. Stathmin could be a potential anti-tumor therapeutic target in OSCC.

Stathmin mediates neuroblastoma metastasis in a tubulin-independent manner via RhoA/ROCK signaling and enhanced transendothelial migration.

Neuroblastoma, the most common solid tumor of young children, frequently presents with aggressive metastatic disease and for these children the 5-year survival rates are dismal. Metastasis, the movement of cancer cells from one site to another, involves remodeling of the cytoskeleton including altered microtubule dynamics. The microtubule-destabilizing protein, stathmin, has recently been shown to mediate neuroblastoma metastasis although precise functions remain poorly defined. In this study we investigated stathmin's contribution to the metastatic process and potential mechanism(s) by which it exerts these effects. Stathmin suppression significantly reduced neuroblastoma cell invasion of 3D tumor spheroids into an extracellular matrix. Moreover, inhibiting stathmin expression significantly reduced transendothelial migration in two different neuroblastoma cell lines in vitro. Inhibition of ROCK, a key regulator of cell migration, in neuroblastoma cells highlighted that stathmin regulates transendothelial migration through ROCK signaling. Reduced stathmin expression in neuroblastoma cells significantly increased the activation of the RhoA small GTPase. Notably, re-expression of either wild type or a phospho-mimetic stathmin mutant (4E) made defective in tubulin binding returned cell migration and transendothelial migration back to control levels, indicating that stathmin may influence these processes in neuroblastoma cells independent of tubulin binding. Finally, stathmin suppression in neuroblastoma cells significantly reduced whole body, lung, kidney and liver metastases in an experimental metastases mouse model. In conclusion, stathmin suppression interferes with the metastatic process via RhoA/ROCK signaling in neuroblastoma cells. These findings highlight the importance of stathmin to the metastatic process and its potential as a therapeutic target for the treatment of neuroblastoma.

Preclinical Biodistribution and Safety Evaluation of a pbi-shRNA STMN1 Lipoplex after Subcutaneous Delivery.

Stathmin-1 (STMN1) is a microtubule-destabilizing protein which is overexpressed in cancer. Its overexpression is associated with poor prognosis and also serves as a predictive marker to taxane therapy. We have developed a proprietary bi-functional shRNA (bi-shRNA) platform to execute RNA interference (RNAi)-mediated gene silencing and a liposome-carrier complex to systemically deliver the pbi-shRNA plasmids. In vitro and in vivo testing demonstrated efficacy and specificity of pbi-shRNA plasmid in targeting STMN1 (Phadke, A. P., Jay, C. M., Wang, Z., Chen, S., Liu, S., Haddock, C., Kumar, P., Pappen, B. O., Rao, D. D., Templeton, N. S., et al. (2011). In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein. DNA Cell Biol. 30, 715-726.). Biodistribution and toxicology studies in bio-relevant Sprague Dawley rats with pbi-shRNA STMN1 lipoplex revealed that the plasmid DNA was delivered to a broad distribution of organs after a single subcutaneous injection. Specifically, plasmid was detected within the first week using QPCR (threshold 50 copies plasmid/1 µg genomic DNA) at the injection site, lung, spleen, blood, skin, ovary (limited), lymph nodes, and liver. It was not detected in the heart, testis or bone marrow. No plasmid was detected from any organ 30 days after injection. Treatment was well tolerated. Minimal inflammation/erythema was observed at the injection site. Circulating cytokine response was also examined by ELISA. The IL-6 levels were induced within 6 h then declined to the vehicle control level 72 h after the injection. TNFα induction was transiently observed 4 days after the DNA lipoplex treatment. In summary, the pbi-shRNA STMN1 lipoplex was well tolerated and displayed broad distribution after a single subcutaneous injection. The pre-clinical data has been filed to FDA and the pbi-shRNA STMN1 lipoplex is being investigated in a phase I clinical study.

Silencing Op18/stathmin by RNA Interference Promotes the Sensitivity of Nasopharyngeal Carcinoma Cells to Taxol and High-Grade Differentiation of Xenografted Tumours in Nude Mice.

Nasopharyngeal carcinoma (NPC) is a refractory tumour, and chemotherapy is one of the primary treatment modalities. Oncoprotein 18 (Op18)/stathmin is a conserved small cytosolic phosphoprotein and highly expressed in tumours, which plays a vital role in maintaining the malignant phenotype of tumours. Taxol is a clinically widely used chemotherapeutic agent for a broad range of taxol-resistant tumours. This study showed that Op18/stathmin silencing by RNA interference (RNAi) combined taxol cooperatively improved cellular apoptosis in CNE1 cells mainly via initiating endogenous death receptor pathway, impaired the capabilities of cellular proliferation and cellular migration and down-regulated the half maximal inhibitory concentration (IC50 ) of taxol, meanwhile decreased the expression of the upstream extracellular regulated kinase 1 (ERK1) in vitro. Evidence also showed that taxol cytotoxicity was markedly augmented for Op18/stathmin RNAi in other NPC cells. In vivo animal experiments have demonstrated that early combination of Op18/stathmin silencing and taxol evidently inhibited tumourigenicity of CNE1 cells and growth of xenografted tumours in nude mice. Remarkably, silencing Op18/stathmin by RNAi still promoted transformation of late-stage CNE1 cells in NPC-xenografted tumours from moderately to highly differentiated and inhibited the pleiotropic cytokine interleukin-10 (IL-10) autocrine by transplanted tumours. These findings suggest that silencing Op18/stathmin by RNAi promotes chemosensitization of NPC to taxol and reverses malignant phenotypes of NPC, which provides a new clue for treating drug-resistant tumours.

Anti-STMN1 therapy improves sensitivity to antimicrotubule drugs in esophageal squamous cell carcinoma.

Stathmin (STMN1) regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Several studies have shown that overexpression of STMN1 has been linked to chemoresistance of paclitaxel and vinblastine in tumor cells. This study aimed to investigate the effects of STMN1 silencing on chemosensitivities of paclitaxel or vinblastine in esophageal squamous cell carcinoma (ESCC). Immunocytochemistry and immunofluorescence assays showed that STMN1 gene was highly expressed in Eca109 and TE-1 cells. We demonstrated that lentiviral-mediated STMN1 short hairpin RNA (shRNA) specifically and efficiently downregulated STMN1 expression in Eca109 and TE-1 cells. The sensitivity of STMN1-silencing shRNA-transfected Eca109 and TE-1 cells increased 191.4- and 179.3-fold to paclitaxel, and 21.3- and 28.4-fold to vincristine, respectively. Flow cytometry and mitotic index assays showed that knockdown of STMN1 in Eca109 and TE-1 cells led to cell cycle arrest in G2/M phase. After treatment with paclitaxel or vincristine, STMN1-silencing shRNA-transfected Eca109 and TE-1 cells were more likely to enter G2 but less likely to enter mitosis than control cells. Therefore, these data suggests that silencing STMN1 gene could increase sensitivity of ESCC to paclitaxel and vincristine through G2/M phase block.

ANKHD1 silencing inhibits Stathmin 1 activity, cell proliferation and migration of leukemia cells.

ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.

miR-223 inhibits dengue virus replication by negatively regulating the microtubule-destabilizing protein STMN1 in EAhy926 cells.

The pathogenesis of dengue virus (DENV) infection is not completely understood. Endothelial cells may act as a target of the virus and be involved in disease pathogenesis. Therefore, the identification of host cell components involved in DENV replication would provide useful information for better understanding DENV infection. In this study, a significantly decreased level of miR-223 was found in DENV2-infected EAhy926 cells, a human endothelial-like cell line, whereas miR-223 overexpression inhibited DENV2 replication. Furthermore, we identified that miR-223 directly targeted the 3' untranslated region (3'UTR) of the messenger RNA (mRNA) for microtubule-destabilizing protein stathmin 1 (STMN1), thereby reducing its mRNA and protein levels. The depletion of miR-223 or overexpression of STMN1 enhanced DENV2 replication, whereas the opposite (increased miR-223 or decreased STMN1) suppressed DENV2 replication, indicating that miR-223 down-regulates STMN1 expression by targeting the 3'UTR of the STMN1 gene to inhibit DENV2 replication. Finally, we demonstrated that two transcription factors, C/EBPα and E2F1, are involved in the regulation of miR-223 levels after DENV2 infection in EAhy926 cells. Collectively, our results suggest that miR-223 may act as a novel antiviral factor, which may open an avenue to limit DENV infection.

Stathmin 1 in normal and malignant hematopoiesis.

Stathmin 1 is a microtubule destabilizer that plays an important role in cell cycle progression, segregation of chromosomes, clonogenicity, cell motility and survival. Stathmin 1 overexpression has been reported in malignant hematopoietic cells and Stathmin 1 inhibition reduces the highly proliferative potential of leukemia cell lines. However, during the differentiation of primary hematopoietic cells, Stathmin 1 expression decreases in parallel to decreases in the proliferative potential of early hematopoietic progenitors. The scope of the present review is to survey the current knowledge and highlight future perspectives for Stathmin 1 in normal and malignant hematopoiesis, with regard to the expression, function and clinical implications of this protein.

MicroRNA-9 inhibits vasculogenic mimicry of glioma cell lines by suppressing Stathmin expression.

The purpose of this study was to investigate the functions of microRNA-9, which is a tissue-specific microRNA in central nervous system, in the vasculogenic mimicry (VM) of glioma cell lines in vitro and in vivo. Glioma cell lines U87MG, U251 and SHG44 were transfected with microRNA-9 mimic, microRNA-9 inhibitor or scramble sequences. The amount of microRNA-9 and Stathmin (STMN1) mRNA was determined by quantitative real-time PCR, and the protein expression of STMN1 was determined by western blot. Cell proliferation and apoptosis were assessed. The interactions between the 3'UTR of STMN1 and miR-9 was determined by luciferase reporter assay. The VM capacity in vitro was evaluated using VM formation assay, and the rescue experiment of STMN1 was carried out in U251 cells. The in vivo experiment was applied with animal models implanted with U87MG cells.MicroRNA-9 mimic transfection reduced proliferation and increased apoptosis in glioma cell lines (p < 0.05). MicroRNA-9 mimic up-regulated STMN1 mRNA levels but reduced its protein levels (p < 0.05), and luciferase activity of STMN1 was suppressed by microRNA-9 mimic transfection (p < 0.05). Furthermore, microRNA-9 mimic transfection suppressed tumor volume growth, as well as VM both in vitro and in vivo. The cell viability and microtube density were upregulated in U251 cells after STMN1 up-regulation (p < 0.05). STMN1 is a target of microRNA-9, and microRNA-9 could modulate cell proliferation, VM and tumor volume growth through controlling STMN1 expression. MicroRNA-9 and its targets may represent a novel panel of molecules for the development of glioma treatment.

Cell cycle inhibition therapy that targets stathmin in in vitro and in vivo models of breast cancer.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that have a critical role in the regulation of mitosis. Stathmin is expressed at high levels in breast cancer and its overexpression is linked to disease progression. Although there is a large body of evidence to support a role for stathmin in breast cancer progression, the validity of stathmin as a viable therapeutic target for breast cancer has not been investigated. Here, we used a bicistronic adenoviral vector that co-expresses green fluorescent protein and a ribozyme that targets stathmin messenger RNA in preclinical breast cancer models with different estrogen receptor (ER) status. We examined the effects of anti-stathmin ribozyme on the malignant phenotype of breast cancer cells in vitro and in xenograft models in vivo both as a single agent and in combination with chemotherapeutic agents. Adenovirus-mediated gene transfer of anti-stathmin ribozyme resulted in a dose-dependent inhibition of proliferation and clonogenicity associated with a G2/M arrest and increase in apoptosis in both ER-positive and ER-negative breast cancer cell lines. This inhibition was markedly enhanced when stathmin-inhibited breast cancer cells were exposed to low concentrations of taxol, which resulted in virtually complete loss of the malignant phenotype. Interestingly, breast cancer xenografts treated with low doses of anti-stathmin therapy and taxol showed regression in a majority of tumors, while some tumors stopped growing completely. In contrast, combination of anti-stathmin ribozyme and adriamycin resulted in only a modest inhibition of growth in vitro and in breast cancer xenografts in vivo. Although inhibition of tumor growth was observed in both the combination treatment groups compared with groups treated with single agent alone, combination of anti-stathmin therapy and taxol had a more profound inhibition of tumorigenicity, as both agents target the microtubule pathway. Clinically, these findings are highly relevant because taxol is one of the most active chemotherapeutic agents in breast cancer. These studies provide the proof-of-principle that stathmin provides an attractive molecular target, which could serve as a primary focus of novel approaches to breast cancer.

Stathmin regulates mutant p53 stability and transcriptional activity in ovarian cancer.

Stathmin is a p53-target gene, frequently overexpressed in late stages of human cancer progression. Type II High Grade Epithelial Ovarian Carcinomas (HG-EOC) represents the only clear exception to this observation. Here, we show that stathmin expression is necessary for the survival of HG-EOC cells carrying a p53 mutant (p53(MUT) ) gene. At molecular level, stathmin favours the binding and the phosphorylation of p53(MUT) by DNA-PKCS , eventually modulating p53(MUT) stability and transcriptional activity. Inhibition of stathmin or DNA-PKCS impaired p53(MUT) -dependent transcription of several M phase regulators, resulting in M phase failure and EOC cell death, both in vitro and in vivo. In primary human EOC a strong correlation exists between stathmin, DNA-PKCS , p53(MUT) overexpression and its transcriptional targets, further strengthening the relevance of the new pathway here described. Overall our data support the hypothesis that the expression of stathmin and p53 could be useful for the identification of high risk patients that will benefit from a therapy specifically acting on mitotic cancer cells.

Overcoming paclitaxel resistance in lung cancer cells via dual inhibition of stathmin and Bcl-2.

Lung cancer is the leading cause of death from malignancy in people and over 85% of these patients eventually die from disseminated disease. Paclitaxel (TAX) is widely used as an antimicrotubule agent for the treatment of lung cancer. Unfortunately, the resistance to this antimicrotubule agent occurs frequently. Stathmin (STMN1) is a ubiquitous microtubule destabilizing protein linked to cancer and cell health and its expression level often correlates with cancer stage progression and prognosis for survival. Overexpression of the antiapoptotic protein Bcl-2 has been shown to prolong drug-induced growth arrest, potentially inducing resistance. In this study, we used a short hairpin RNA (shRNA) approach to evaluate the effect of STMN1 and Bcl-2 downregulation in the sensitivity to TAX in lung cancer cells. We achieved significant downregulation of STMN1 and Bcl-2 mRNA and protein expression by a combination of double shRNA treatment strategy. Our experimental data showed that inhibition of STMN1 and Bcl-2 expression with RNA interference can sensitize lung cancer cells to TAX. These findings suggest a novel approach to improve the efficacy of certain antimicrotubule agents against lung cancer by regulating the function of STMN1 and Bcl-2.

Inhibition of stathmin1 accelerates the metastatic process.

The oncoprotein stathmin 1 (STMN1) is upregulated in most, if not all, cancers of epithelial cell origin; therefore STMN1 is considered a target for cancer therapy. However, its role during metastasis has not been investigated. Here, we report for the first time that STMN1 strongly inhibits metastatic behavior in both normal epithelial and cancerous epithelial cells. Initially, loss-of-STMN1 compromises cell-cell adhesion. This is followed by epithelial-to-mesenchymal transition (EMT), increased cell migration, and metastasis via cooperative activation of p38 and through TGF-ß-independent and -dependent mechanisms. In contrast, expressing STMN1 restores cell-cell adhesion and reverses the metastatic cascade. Primary prostate epithelial cell cultures from benign to undifferentiated adenocarcinoma (UA) clinical biopsies show that EMT-like cells arise while the cancer is still organ-confined and that their emergence is tumor-stage specific. Furthermore, primary EMT-like cells exhibit metastatic behavior both in vitro and in vivo as compared with their non-EMT counterpart. These observations predict that using STMN1 as a generic therapeutic target might accelerate metastasis. Instead, there may be a tumor stage-specific window-of-opportunity in which conserving STMN1 expression is required to inhibit emergence of metastatic disease.

Stathmin1 plays oncogenic role and is a target of microRNA-223 in gastric cancer.

Stathmin1 (STMN1) is a candidate oncoprotein and prognosis marker in several kinds of cancers. This study was aimed to analyze its expression and biological functions in gastric cancer. The expression of STMN1 was evaluated by qRT-PCR, western blot and immunohistochemistry. The biological function of STMN1 was determined by MTT proliferation assays, monolayer colony formation and cell invasion assays using small interference RNA technique in gastric cancer cell lines. We also explored the regulation of STMN1 expression by microRNA-223. STMN1 was upregulated in gastric cancer cell lines and primary gastric adenocarcinomas. STMN1-positive tumors were more likely to be found in old age group and associated with p53 nuclear expression. In diffuse type gastric adenocarcinomas, STMN1 expression was correlated with age (p = 0.043), T stage (p = 0.004) and lymph node metastasis (p = 0.046). Expression of STMN1 in diffuse type gastric adenocarcinoma was associated with poor disease specific survival by univariate analysis (p = 0.01). STMN1 knockdown in AGS and MKN7 cell lines suppressed proliferation (p<0.001), reduced monolayer colony formation (p<0.001), inhibited cell invasion and migration ability (p<0.001) and induced G1 phase arrest. siSTMN1 could also suppress cell growth in vivo (p<0. 01). We finally confirmed that STMN1 is a putative downstream target of miR-223 in gastric cancer. Our findings supported an oncogenic role of STMN1 in gastric cancer. STMN1 might serve as a prognostic marker and a potential therapeutic target for gastric cancer.

The microtubule cytoskeleton is required for a G2 cell cycle delay in cancer cells lacking stathmin and p53.

In several cancer cell lines, depleting the microtubule (MT)-destabilizing protein stathmin/oncoprotein18 leads to a G2 cell cycle delay and apoptosis. These phenotypes are observed only in synergy with low levels of p53, but the pathway(s) activated by stathmin depletion to delay the cell cycle are unknown. We found that stathmin depletion caused greater MT stability in synergy with loss of p53, measured by the levels of acetylated α-tubulin and the rate of centrosomal MT nucleation. Nocodazole or vinblastine-induced MT depolymerization abrogated the stathmin-depletion induced G2 delay, measured by the percentage of cells staining positive for several markers (TPX2, CDK1 with inhibitory phosphorylation), indicating that MTs are required to lengthen G2. Live cell imaging showed that stathmin depletion increased time in G2 without an impact on the duration of mitosis, indicating that the longer interphase duration is not simply a consequence of a previous slowed mitosis. In contrast, stabilization of MTs with paclitaxel (8 nM) slowed mitosis without lengthening the duration of interphase, demonstrating that increased MT stability alone is not sufficient to delay cells in G2.