Stathmin is a Marker of Progression and Poor Prognosis in Esophageal Carcinoma

Stathmin, also called oncoprotein 18, is a founding member of the family of microtubule-destabilizing proteinsthat play a critical role in the regulation of mitosis. At the same time stathmin has been recognized as one ofresponsible factors in cancer cells. The aim of this study was to assess stathmin status, its correlations withclinicopathological parameters and its role as a progosnostic marker in EC patients. The protein and mRNAlevels of stathmin were examined byimmunohistochemistry (IHC) and in situ hybridization in 100EC tissuesand adjacent noncancerous tissues. mRNA and protein expression of stathmin in three EC cell lines(EC9706,ECa109, EC1 commonly used in research) were also analyzed using immunocytochemistry, western blot andin situ hybridization. The prognostic value of Stathmin expression within the tumor tissues were assessed byCox regression and Kaplan-Meier analysis. We showed that stathmin expression was significantly higher in ECtissues than in adjacent noncancerous tissues. High stathmin immunostaining score in the EC was positivelycorrelated with tumor differentiation, Tumor invasion, Lymph node metastases, and TNM stage. In addition, wedemonstrated that three EC cell lines examined, were constitutively expressing a high level of stathmin. Of those,EC-1 showed the strongest mRNA and protein expression for the stathmin analyzed. Kaplan-Meier analysisshowed that significantly longer 5-year survival rate was seen in EC patients with high Stathmin expression,compared to those with low expression of Stathmin expression. Furthermore, multivariate Cox proportionalhazard analyses revealed that Stathmin was an independent factors affecting the overall survival probability.In conclusion, our data provide a basis for the concept that stathmin might be associated with EC developmentand progression.. High levels of Stathmin expression in the tumor tissues may be a good prognostic marker forpatients with EC.     

Effect of stathmin on the sensitivity to antimicrotubule drugs in human breast cancer

Stathmin is a p53-regulated protein known to influence microtubule dynamics. Because several chemotherapeutic agents used to treat breast cancer alter the dynamic equilibrium of tubulin polymerization, stathmin may play an important role in determining the sensitivity to these drugs. Therefore, we evaluated the effect of stathmin expression on the action of taxanes and Vinca alkaloids using a panel of human breast cancer cell lines. Cell lines harboring mutant p53 expressed high levels of stathmin. Two cell lines with different levels of endogenous stathmin expression and isogenic-paired cell lines transfected to overexpress stathmin were used to determine whether or not stathmin modulated the sensitivity to drugs. Overexpression of stathmin decreased polymerization of microtubules, markedly decreased binding of paclitaxel, and increased binding of vinblastine. Stathmin overexpression decreased sensitivity to paclitaxel and, to a lesser extent, to vinblastine. In contrast, stathmin content had no significant effect on the sensitivity to chemotherapeutic drugs that do not target microtubules. Cell lines overexpressing stathmin were more likely to enter G(2) but less likely to enter mitosis as determined by fluorescence-activated cell sorting and mitotic index. This effect was magnified when stathmin-overexpressing cells were treated with vinblastine as measured by the detection of proteins phosphorylated in early mitosis. These data suggest that the action of antimicrotubule drugs can be affected by stathmin in at least two ways: (a) altered drug binding; and (b) growth arrest at the G(2) to M boundary. Mutant p53 breast cancers exhibiting high levels of stathmin may be resistant to antimicrotubule agents.     

Stathmin基因在人成骨肉瘤细胞中的表达及意义

stathmin作为细胞信号转导分子在细胞分化及恶性肿瘤发生、发展上发挥重要作用。本研究旨在探讨Stathmin基因在人成骨肉瘤细胞中的表达,并探讨阻断其表达对该肿瘤细胞的生物学行为的影响。方法：RT-PCR及原位杂交法检测2个人成骨肉瘤细胞系及45例人成骨肉瘤组织中Stathmin基因表达情况;以高表达Stathmin基因的人成骨肉瘤细胞系SOSP-9607为靶细胞、反义Stathmin(ASODN)为阻断剂,通过MTT实验观察ASODN对该细胞系的生长抑制作用,并用流式细胞仪分析其对细胞增殖周期的影响。     

Stathmin guides personalized therapy in oral squamous cell carcinoma

The survival benefit from docetaxel, cisplatin and 5‐fluorouracil (TPF) induction chemotherapy in oral squamous cell carcinoma (OSCC) patients is not satisfactory. Previously, we identified that stathmin, a microtubule‐destabilizing protein, is overexpressed in OSCC. Here, we further investigated its role as a biomarker that impacts on OSCC chemosensitivity. We analyzed the predictive value of stathmin on TPF induction chemotherapy and its impact on OSCC cell chemosensitivity. Then, we further investigated the therapeutic effects of the combination therapy of TPF chemotherapy and PI3K‐AKT‐mTOR inhibitors in vitro and in vivo. We found that OSCC patients with low stathmin expression benefited from TPF induction chemotherapy, while OSCC patients with high stathmin expression could not benefit from TPF induction chemotherapy. Stathmin overexpression promoted cellular proliferation and decreased OSCC cell sensitivity to TPF treatment. In addition, inhibition of the PI3K‐AKT‐mTOR signaling pathway decreased stathmin expression and phosphorylation. The combination therapy of TPF chemotherapy and PI3K‐AKT‐mTOR inhibitors exhibited a potent antitumor effect both in vitro and in vivo. Therefore, stathmin can be used as a predictive biomarker for TPF induction chemotherapy and a combination therapy regimen based on stathmin expression might improve the survival of OSCC patients.     

微管解聚蛋白Ｓｔａｔｈｍｉｎ与肿瘤相关性的研究进展

微管解聚蛋白Ｓｔａｔｈｍｉｎ具有微管解聚活性,在细胞周期的转换过程中起着重要作用。在多种恶性肿瘤细胞中都可以检测到Ｓｔａｔｈｍｉｎ高表达。已有研究证实,Ｓｔａｔｈｍｉｎ蛋白的高表达能够影响某些作用于微管的化疗药物的疗效,抑制其表达可以提高这些药物的疗效。Ｓｔａｔｈｍｉｎ基因为肿瘤基因治疗提供了新的分子靶点。     

Stathmin siRNA表达载体的构建及对LM8细胞生物学行为的影响

背景与目的:Stathmin在多种恶性肿瘤细胞中都有高水平表达,通过抑制其表达可以干扰恶性肿瘤细胞的分裂,影响肿瘤细胞的增殖与凋亡。本研究构建并观察stathmin基因RNA干扰表达载体转染LM8细胞系后对stathmin表达及细胞生物学行为的影响。方法:构建靶向stathmin干扰质粒pGenesil-1-Stathmin和通用对照质粒pGenesil-1-HK,以脂质体法将2个重组质粒分别转染骨肉瘤LM8细胞系。实时荧光定量PCR和Western印迹技术检测各组LM8细胞系stathmin在mRNA和蛋白水平的表达,MTT(噻唑蓝)法比色分析检测细胞体外生长抑制率,软琼脂集落形成实验观察单个细胞体外增殖活力,细胞HE染色观察细胞形态学的变化,Hoechst染色观察细胞凋亡特征并计算凋亡率,流式细胞术定量分析细胞周期变化,C3H小鼠移植瘤实验显示肿瘤细胞成瘤能力。结果:DNA测序证实成功构建pGenesil-1-Stathmin重组质粒。转染LM8细胞后,明显抑制stathmin基因表达;细胞体外生长抑制率显著增加;单个细胞体外增殖活力显著下降;细胞HE染色部分细胞呈典型的凋亡细胞形态学改变;Hoechst染色显示细胞凋亡特征,细胞凋亡率明显高于对照组;流式细胞术检测显示细胞周期阻滞于G2/M期,明显高于对照组;C3H小鼠移植瘤实验显示特异转染组致瘤率下降,肿瘤生长减缓。结论:成功构建的靶向干扰质粒pGenesil-1-Stathmin能有效抑制stathmin基因在骨肉瘤LM8细胞的表达并影响其相关生物学行为,其抑制骨肉瘤细胞的增殖与生长,为stathmin基因应用于骨肉瘤的基因治疗研究奠定了理论基础。     

微管不稳定蛋白Stathmin--肿瘤基因治疗新靶点

微管不稳定蛋白Stathmin在细胞周期的不同阶段对微运动力平衡的调节发挥重要作用。多种恶性肿瘤中Stathmin都有高水平表达，抑制其表达可以干扰恶性细胞的细胞分裂、Stathmin的过表达可干扰紫杉醇与微管的结合，但增加长春碱类药物与微管的结合能力，临床用药时应予考虑。另外．Stathmin为肿瘤基因治疗提供了一个分子新靶点，腺病毒介导抗stathmin核酶治疗联合察素药物治疗可能获得更强有力的抗增殖和抗肿瘤效应。     

Silencing stathmin gene expression by survivin promoter-driven siRNA vector to reverse malignant phenotype of tumor cells

Stathmin gene overexpression has been shown to play an important role in maintenance of malignant phenotype in tumor cells, and the blocking efficacy and tumor specificity of this target has been concerned in clinical trails. In this report, we designed survivin promoter-driven siRNA eukaryotic expression vector that expressed the small interfering RNA targeting stathmin gene to selectively knock down the stathmin gene expression in two different kinds of tumor cell lines while sparing normal cell lines. The therapeutic potential of this recombinant vector was tested in human cervical cancer Hela cells and osteosarcoma SSOP-9607 cells, and in human umbilical vein endothelial cell line ECV304 cells as control. The siRNA vector- transfected Hela cells and SSOP-9607 cells revealed marked inhibition of stathmin expression and a dramatic growth inhibition comparing with ECV304 cells, parental-vector transfected cells and untransfected cells. Cell cycle analysis of siRNA vector transfected tumor cells by Flow Cytometry showed G(2)/M phase block, while morphologic analysis by TURNEL staining method showed marked increase of apoptosis. Our study indicates that survivin gene promoter-driven stathmin siRNA expression vector may have potential use in tumor gene therapy with targeted tumor gene silencing effect.     

Stathmin蛋白:一个潜在的肿瘤标志物

人stathmin也称原癌基因蛋白18(oncogene protein 18, Op18), 是一种广泛存在于细胞质的蛋白质,其相对分子质量约为19×10~3,可与微管蛋白结合,参与微管和纺锤体的组装;与细胞的增殖、分化、再生和运动均有关,并具有信号活性调节的功能.近年来有研究发现,stathmin在多种肿瘤中高表达,并可通过调节微管的解聚,促进肿瘤细胞的运动及侵袭.Stathmin翻译后修饰状态的改变,可影响与p53蛋白的相互作用,参与肿瘤的发生发展.目前单独或者联合化疗药物使用的抗stathmin效应剂已用于某些肿瘤的治疗.虽然stathmin与肿瘤病因学的内在联系尚不十分清楚,但其作为一个潜在的肿瘤标志物或药物靶点值得进一步研究探讨.     

Stathmin! An immunohistochemical analysis of the novel marker in Oral Squamous Cell Carcinoma and Oral Leukoplakia.

Background: Stathmin is an intracellular phosphoprotein that controls the microtubule dynamics by further regulating proper attachment and alignment of chromosomes in a dividing cell. Thus, any mutation or aberrantly expressed protein that reduces the fidelity of spindle assembly will enhance chromosomal instability contributing to aneuploidy. Oral Squamous Cell Carcinoma is an extensively studied malignancy that occurs due to accumulated genetic changes due to carcinogens. The current study is done to evaluate the stathmin role and its expression in OSCC and Oral epithelial dysplasia (OED). Objective: The aim of the present study is to evaluate the role of stathmin in OSCC and Oral dysplasia and also to correlate the expression of Stathmin with respect to the different histopathological grades of OED and OSCC. Materials and Methods: 30 neutral buffered formalin fixed, paraffin embedded (FFPE) tissues of Oral Leukoplakia/OED and 30 FFPE tissues of OSCC were subjected to immunohistochemistry with stathmin antibody. Five fields of each case with 300 cells were examined and a mean percentage of positive–stained slides were determined. The percentages were recorded accordingly with their respective histological grades. The results were analysed statistically. Results: The results of the present study demonstrated higher mean values of stathmin in tissues with OSCC (2.50) compared to leukoplakia (2.11) and normal tissues (0.00) with a high level of statistical significance (0.0001). There is also an increase in the percentage levels of stathmin with increase in the histological grade of differentiation in OSCC as well as leukoplakia. Conclusion: The present study found a statistical correlation between increased grades of the disease with expression levels of stathmin. This confirms that stathmin expression can contribute to disease progression and that stathmin might have a potential role as an early diagnostic biomarker and can be a therapeutic target for OSCC.     

微管不稳定蛋白Stathmin在食管鳞癌组织中的表达及意义

目的 探讨Stathmin蛋白在食管鳞癌组织中的表达及意义.方法 收集新乡医学院2006年4月至9月期间食管鳞癌切除标本及其相应癌旁组织31例,以免疫组织化学法检测Stathmin蛋白在所取标本中的表达情况.结果 在31例食管鳞癌标本中,Stathmin蛋白表达阳性的标本为23例,阳性率为74.2%,阳性颗粒可见于细胞质;分别按年龄、性别、组织类型、分化程度、淋巴结转移、肿瘤浸润深度及TNM分期将31例食管癌标本分组,Stathmin蛋白表达差异有统计学意义的因素有分化程度、淋巴结转移、肿瘤浸润深度及TNM分期(P＜0.05).结论 食管鳞癌组织中Stathmin蛋白高表达,其表达与食管鳞癌的分化程度、淋巴结转移、肿瘤浸润深度及TNM分期显著相关,Stathmin蛋白可能为人食管癌的生物治疗提供一个新靶点.     

Prognostic value of maspin mRNA expression in ER alpha-positive postmenopausal breast carcinomas

Maspin, a member of the serpin family, has a role in cell migration, angiogenesis and apoptosis. Little is known of the clinical significance of maspin gene expression in human cancers. We developed a real-time quantitative RT-PCR assay to quantify the full range of maspin mRNA copy numbers in a series of 10 ER alpha-positive and 10 ER alpha-negative breast tumours. We observed a statistical link between low maspin mRNA levels and positive oestrogen status (P=0.0012). In consequence, to better assess the prognostic value of maspin gene expression in breast cancer, we then quantified maspin mRNA content in an additional independent well-defined cohort of 105 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. Maspin expression varied widely in tumour tissues (by nearly four orders of magnitude), being underexpressed in 33 out of 105 tumours (31.4%) and overexpressed in 24 out of 105 tumours (22.9%) relative to normal breast tissues. Immunohistochemical studies demonstrated that maspin protein was strictly expressed in myoepithelial cells of normal breast tissue and in tumour epithelial cells, exclusively in maspin-overexpressing tumours. Patients with tumours overexpressing the maspin gene had significantly shorter relapse-free survival after surgery than patients whose tumours normally expressed or underexpressed maspin (P=0.0011). The prognostic significance of maspin overexpression persisted in Cox multivariate regression analysis (P=0.0024). These findings show that the maspin mRNA level can have important prognostic significance in human breast cancer, and point to the maspin gene as a putative molecular predictor of hormone responsiveness in breast cancer.     

Stathmin expression in glioma-derived microvascular endothelial cells: a novel therapeutic target

The purpose of this study was to investigate stathmin expression and its mechanisms of action in GDMEC. Microvascular endothelial cells were isolated from human gliomas (n=68) and normal brain specimans (n=20), and purified by magnetic beads coated with anti-CD105 antibody. The expression of stathmin mRNA and protein were detected by RT-PCR and western blotting, respectively. Stathmin expression was silenced by application of specific siRNA in high grade GDMEC. The proliferation, apoptosis and invasion behavior of GDMEC were investigated. The stathmin positive rate of endothelial cells in normal brain, grade I-II glioma and grade III-IV glioma was 20, 66 and 95.5%, respectively (P<0.05). When cells were treated with siRNA to silence stathmin, cell viability was reduced, the apoptosis rate increased and the migration of vascular endothelial cells was suppressed significantly (P<0.05). Down-regulation of stathmin suppressed neoangiogenesis of glioma and provides a potential target for glioma treatment.     

Involvement of adenomatous polyposis coli (APC)/beta-catenin signalling in human breast cancer

We studied the relevance of adenomatous polyposis coli (APC)/beta-catenin signalling in the development of breast cancer by analysing the expression of beta-catenin in 54 primary breast tumours (34 ductal and 20 lobular). We showed that 13% of the tumours exhibited upregulated levels of beta-catenin in the cytosol suggesting that defects in APC/beta-catenin signalling components had lowered the rate of beta-catenin degradation. No mutations were observed in the amino-terminal region of beta-catenin, which comprises conserved serine residues important for phosphorylation-dependent degradation of the protein, but the APC protein was altered in 6% of the tumours. Tyrosine phosphorylation of beta-catenin was detected in only one tumour and could, therefore, not have been responsible for the observed increased levels of this protein. Although 9% of the tumours displayed upregulation of c-MYC protein, there was no correlation with beta-catenin overexpression, suggesting that increased beta-catenin expression is not the major cause of c-myc gene activation in breast cancer. It is imperative that elements that selectively drive the oncogenic activity of beta-catenin in breast cancer be identified.     

Specific induction of pp125 focal adhesion kinase in human breast cancer

The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT-PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51+/-0.84 (mean+/-standard deviation), which was strongly induced to 2.91 (+/-1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0+/-0.63 vs 2.27+/-0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation.     

Overexpression of stathmin1 in the diffuse type of gastric cancer and its roles in proliferation and migration of gastric cancer cells

Background:

Stathmin1 is a microtubule-regulating protein that has an important role in the assembly and disassembly of the mitotic spindle. The roles of stathmin1 in carcinogenesis of various cancers, including prostate and breast cancer, have been explored. However, its expression and roles in gastric cancer have not yet been described.

Methods:

Stathmin1 expression in paraffin-embedded tissue sections from 226 patients was analysed by immunohistochemistry. Roles of stathmin1 were studied using a specific small interfering RNA (siRNA).

Results:

The expression of stathmin1 was positively correlated with lymph node metastasis, TNM stages and vascular invasion, and negatively with recurrence-free survival, in the diffuse type of gastric cancer. The median recurrence-free survival in patients with a negative and positive expression of stathmin1 was 17.0 and 7.0 months, respectively (P=0.009). When the expression of stathmin1 was knocked down using siRNA, the proliferation, migration and invasion of poorly differentiated gastric cancer cells in vitro were significantly inhibited. Moreover, stathmin1 siRNA transfection significantly slowed the growth of xenografts in nude mice.

Conclusion:

These results suggest that stathmin1 can be a good prognostic factor for recurrence-free survival rate and is a therapeutic target in diffuse-type gastric cancer.