Stathmin 1 inhibition amplifies ruxolitinib-induced apoptosis in JAK2V617F cells

The JAK/STAT pathway is constitutively activated in myeloproliferative neoplasms and can be inhibited by ruxolitinib, a selective JAK1/2 inhibitor. The JAK2^V617F mutation leads to constitutive STAT3 phosphorylation and potentially leads to inhibition of Stathmin 1 activity via STAT3. In support of this hypothesis, we found that, in HEL JAK2^V617F cells, ruxolitinib treatment decreased STAT3 and Stathmin 1 association, induced Stathmin 1 activation and microtubule instability. Silencing of Stathmin 1 significantly reduced cell proliferation and clonal growth, and increased apoptosis induced by ruxolitinib. Stathmin 1 silencing also prevented ruxolitinib-induced microtubule instability. To phenocopy the effect of Stathmin 1 inhibition, cells were treated with paclitaxel, a microtubule-stabilizing drug, in association or not with ruxolitinib; combined treatment significantly increased apoptosis, when compared to monotherapy. Notably, Stathmin 1 mRNA levels were highly expressed in CD34⁺ cells from primary myelofibrosis patients. We then proposed that an undesired effect of ruxolitinib treatment may constitute Stathmin 1 activation and microtubule instability in JAK2^V617F cells. Induction of microtubule stability, through Stathmin 1 silencing or paclitaxel treatment, combined with ruxolitinib could be an effective strategy for promoting apoptosis in JAK2^V617F cells.     

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.     

A combination of paclitaxel and siRNA-mediated silencing of Stathmin inhibits growth and promotes apoptosis of nasopharyngeal carcinoma cells

Background

Stathmin, a microtubule associated protein (MAP), is an important molecular target for cancer therapy. Paclitaxel is one of the primary antitumor drugs targeting microtubules (MTs). We hypothesized that decreasing the expression level of Stathmin might improve the effectiveness of paclitaxel in the treatment of nasopharyngeal carcinoma (NPC).

Methods

NPC cell lines, CNE1-LMP1 and HNE2, and a CNE1-LMP1 tumor xenograft mouse model were used to test both in vitro and in vivo our siRNA-based Stathmin silencing strategy. The effects of Stathmin silencing on cell proliferation, apoptosis, and viability were investigated using MTT, AO/EB staining, TUNEL, caspase protein detection, and FCM assays. Cell migration and invasion were assayed using a Transwell assay. The combined effects of Stathmin silencing and paclitaxel were investigated using MTT, FCM, Western blot and indirect immunofluorescence assays. The effect of paclitaxel on Stathmin expression in NPC cells and, in addition, A375, MGC and HeLa cells was determined by RT-PCR and Western blotting.

Results

We found that siRNA-mediated silencing of Stathmin suppresses proliferation, induces apoptosis through the mitochondrial pathway, and causes G2/M-phase cell cycle arrest in the NPC cell lines CNE1-LMP1 and HNE2. Also, the migration and invasion of the respective NPC cells were found to be inhibited. In addition, we show that a combination of Stathmin silencing and paclitaxel is more effective than either agent alone in inhibiting proliferation and inducing apoptosis, cell cycle arrest, and MT polymerization. Furthermore, we found that Stathmin expression in the tumor cells is down-regulated by paclitaxel treatment.

Conclusion

siRNA-mediated silencing of Stathmin suppresses the proliferation, invasion and metastasis, and induces the apoptosis of NPC cells. Paclitaxel reduces the expression of Stathmin, and combining Stathmin silencing with paclitaxel treatment enhances MT polymerization. This combined strategy may provide a new approach for clinical NPC treatment.     

CircLASP1 silence strengthens the therapeutic effects of MK-2206 on nasopharyngeal cancer through upregulating miR-625

Therapeutic effects of MK-2206 are largely limited due to the complexity of the pathogenesis of nasopharyngeal cancer (NPC). Here, we aimed to investigate whether and how circLASP1 is involved in the therapeutic effects of MK-2206 on NPC. We showed circLASP1 was increased while miR-625 was decreased in NPC tissues and cell lines. CircLASP1 silence strengthened the therapeutic effects of MK-2206 via suppressing NPC cell proliferation and inducing autophagy and apoptosis in vitro. In mechanism analyses, we found that circLASP1 indirectly released AKT by directly binding to miR-625 in NPC cells, and miR-625 acted as a tumor suppressor in NPC and activated cell autophagy through inhibiting the AKT/mTOR pathway. Most importantly, knockdown of circLASP1 was revealed to enhance the therapeutic effects of MK-2206 on NPC in vivo. Our results suggest that the circLASP1/miR-625 axis is involved the therapeutic effects of MK-2206 on NPC by regulating autophagy, proliferation, and apoptosis through the AKT/mTOR pathway. miR-625 is involved in NPC tumorigenesis.     

Aurora-A interacts with Cyclin B1 and enhances its stability

The mitotic regulator Aurora-A is an oncogenic protein that is over-expressed in many types of human tumors. However, the underlying mechanism through which Aurora-A promotes tumorigenesis remains unclear. Here, we show that overexpression of Aurora-A causes an elevation of Cyclin B1 expression. Cyclin B1 degradation is delayed in Aurora-A over-expressing cells, which depends on Aurora-A kinase activity. In contrast, Aurora-A RNAi enhances Cyclin B1 degradation. Furthermore, we found that Aurora-A interacts with Cyclin B1, and that Aurora-A overexpression reduces the interaction of Cyclin B1 with APC subunits. In human esophageal squamous cell carcinomas (ESCC), overexpression of Aurora-A was correlated with deregulated expression of Cyclin B1. Taken together, these findings suggest that overexpression of Aurora-A may stabilize Cyclin B1 through inhibiting its degradation. These results provide new insight into the mechanism of how deregulated Aurora-A contributes to genomic instability and carcinogenesis.     

STUB1 suppresseses tumorigenesis and chemoresistance through antagonizing YAP1 signaling

Yes‐associated protein (YAP) is a component of the canonical Hippo signaling pathway that is known to play essential roles in modulating organ size, development, and tumorigenesis. Activation or upregulation of YAP1, which contributes to cancer cell survival and chemoresistance, has been verified in different types of human cancers. However, the molecular mechanism of YAP1 upregulation in cancer is still unclear. Here we report that the E3 ubiquitin ligase STUB1 ubiquitinates and destabilizes YAP1, thereby inhibiting cancer cell survival. Low levels of STUB1 expression were correlated with increased protein levels of YAP1 in human gastric cancer cell lines and patient samples. Moreover, we revealed that STUB1 ubiquitinates YAP1 at the K280 site by K48‐linked polyubiquitination, which in turn increases YAP1 turnover and promotes cellular chemosensitivity. Overall, our study establishes YAP1 ubiquitination and degradation mediated by the E3 ligase STUB1 as an important regulatory mechanism in gastric cancer, and provides a rationale for potential therapeutic interventions.     

Overexpression of the stathmin gene in a subset of human breast cancer.

Stathmin is a highly conserved cytosolic phosphoprotein that destabilizes microtubules. Stathmin, which has been proposed as a relay protein integrating diverse cell signalling pathways, acts in vitro as a tubulin-sequestering protein, and its activity is dramatically reduced by phosphorylation. Interestingly, stathmin expression and phosphorylation are regulated during the control of cell growth and differentiation, and there is much evidence suggesting that in vivo stathmin plays a role in the control of microtubule dynamics during mitosis. Stathmin may thus be considered as one of the key regulators of cell division. We examined 50 human primary breast tumours for stathmin mRNA and protein expression and screened for abnormalities in the chromosome region harbouring the stathmin gene. Overexpression of stathmin was found in 15 tumours (30%). At the present stage, no clear correlation emerged between stathmin expression and several prognosis markers. Interestingly, perfect matching was observed between stathmin mRNA overexpression, protein overexpression and strong staining for stathmin on paraffin-embedded tumour sections when specimens were available. Furthermore, a tentative link between loss of heterozygosity (LOH) in the 1p32-1pter region and stathmin overexpression was observed. Our results suggest that stathmin might play a role in breast carcinogenesis and that stathmin-overexpressing tumours may represent a new subtype of breast cancer.     

Stathmin基因表达与肿瘤的研究进展

Stathmin在多种恶性肿瘤细胞中都有高水平表达，Stathmin蛋白的主要作用是通过促进微管的解聚或阻止微管的聚合从而影响有丝分裂纺锤体的形成，通过抑制其表达可以干扰恶性肿瘤细胞的有丝分裂，影响肿瘤细胞的增殖与凋亡。同时，抑制stathmin表达能够协同增效某些化疗药物的抗癌疗效。Stathmin基因正成为肿瘤基因治疗的一个新靶点。     

Tissue inhibitor of metalloproteinase‐1 protects MCF‐7 breast cancer cells from paclitaxel‐induced apoptosis by decreasing the stability of cyclin B1

Paclitaxel (PTX) is a very effective drug in treating tumors. It disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis, leading to cell death by apoptosis. However, the effectiveness of PTX in cancer chemotherapy is hampered by drug resistance in some patients. Tissue inhibitor of metalloproteinase‐1 (TIMP‐1) is well known to be capable of inhibiting apoptosis. Elevated tumor tissue TIMP‐1 levels have been significantly associated with a poor response to chemotherapy. We hypothesized that TIMP‐1 could reduce the sensitivity of breast cancer cells to PTX by inhibiting apoptosis. To test this hypothesis, we first examined the effects of TIMP‐1 on the apoptosis induced by PTX and investigated the effects of TIMP‐1 on the expression and stability of cyclin B1 that critically regulates the metaphase to anaphase transition during mitosis in MCF‐7 breast cancer cells. Our data demonstrate that TIMP‐1 could significantly decrease the sensitivity of MCF‐7 cells to PTX‐induced apoptosis, attenuate mitotic blockage in G₂/M, and enhance the degradation of cyclin B1. To further investigate whether the inhibitory effect of TIMP‐1 on PTX‐induced apoptosis is mediated by lowering levels of cyclin B1, a cyclin B1‐expression plasmid was transfected into clone overexpressing TIMP‐1. The levels of PTX‐induced apoptosis were then analyzed. The data showed that the TIMP‐1‐based decrease in PTX‐induced apoptosis was reversed by cyclin B1. Our data indicate that TIMP‐1 can protect breast cancer cells from PTX‐induced apoptosis by decreasing the stability of cyclin B1.     

Stathmin在上皮性卵巢癌组织中的表达及意义

背景与目的:Stathmin是一种重要的微管调节蛋白,参与多种肿瘤的发生、发展.本研究旨在验证并探讨Stathmin在上皮性卵巢癌中的表达及意义.方法:利用免疫组化和逆转录聚合酶链反应(RT-PCR)方法检测22例卵巢正常上皮组织,16例良性肿瘤和50例上皮性卵巢癌组织中Stathmin的表达.结果:上皮性卵巢癌中Stathmin mRNA的表达明显高于卵巢良性肿瘤(P<0.05)和正常卵巢上皮(PO.05).进一步利用x2检验统计分析得出,Stathmin蛋白与卵巢癌的临床分期、病理分级、淋巴转移相关(P<0.05),而与年龄、病理类型及腹水量无相关性(P>0.05).结论:Stathmin在上皮性卵巢癌中高表达,可能与卵巢癌的发生、发展相关.     

PIWIL 2 在人类膀胱尿路上皮癌中的表达及其功能研究

 目的 研究PIWIL2在人类膀胱尿路上皮癌（BTCC）中的表达及siRNA对人类膀胱癌细胞PIWIL2表达的影响。方法 采用反转录聚合酶链反应（RT-PCR）检测BTCC 46例、腺性膀胱炎21例、癌旁组织17例、正常膀胱组织14例中PIWIL2 mRNA的表达;设计合成针对PIWIL2的3个特异性siRNA,转入人类膀胱癌BIU-87细胞,分别采用四甲基偶氮唑蓝、DNA原位末端标记法、RT-PCR和Western blot检测siRNA对BIU-87细胞生长抑制率（IR）、凋亡指数（AI）和PIWIL2 mRNA及其蛋白表达的影响。结果 BTCC 组织中PIWIL2 mRNA的表达率为76.08 %（35／40）,均显著高于腺性膀胱炎组织[42.86 %（9／21）]、癌旁组织[41.17 %（7／17）]和正常膀胱组织[7.14 %（1／14）]（P＝0.008,P＝0.010,P＝0.000）。BTCC组织中PIWIL2的高表达与肿瘤淋巴结转移、病理分级均密切相关。siRNA1～3组细胞的IR[（37.52±8.84）%、（64.36±9.64）%、（62.94±8.43）%]和AI[（26.18±5.42）%、（38.75±6.19）%、（40.02±5.64）%]均分别显著高于对照组[（1.97±0.02）%、（3.35±0.47）%]（均P＝0.000）,PIWIL2 mRNA及其蛋白表达水平均显著低于对照组;其中siRNA 2、3组细胞的IR、AI和对PIWIL2表达的抑制作用均显著高于siRNA 1组。结论 PIWIL2在BTCC中的高表达,提示其与膀胱癌的发生、发展密切相关;体外转录合成的siRNA可抑制BIU-87细胞PIWIL2的表达,诱导肿瘤细胞凋亡,从而抑制肿瘤细胞生长,提示PIWIL2可作为膀胱肿瘤基因治疗的重要靶点。     

WIF-1启动子甲基化与肿瘤

WIF-1（Wnt Inhibitory Factor-1）是Wnt信号通路的一种拮抗物,能在胞外与Wnt蛋白结合而抑制Wnt信号转导。近年来的研究表明,多种肿瘤中均存在WIF-1基因启动子的异常甲基化。WIF-1启动子甲基化使WIF-1蛋白表达降低,从而降低了抑制Wnt信号转导的作用,使细胞发生增殖、分化,促进肿瘤的发生发展。检测组织中WIF-1甲基化程度,可作为肿瘤早期诊断和评估预后的生物学标志物;运用去甲基化剂如5-氮杂胞苷,可逆转WIF-1的甲基化状态,从而成为肿瘤治疗的一个新靶点。     

NUSAP1 influences the DNA damage response by controlling BRCA1 protein levels

NUSAP1 has been reported to function in mitotic spindle assembly, chromosome segregation, and regulation of cytokinesis. In this study, we find that NUSAP1 has hitherto unknown functions in the key BRCA1-regulated pathways of double strand DNA break repair and centrosome duplication. Both these pathways are important for maintenance of genomic stability, and any defects in these pathways can cause tumorigenesis. Depletion of NUSAP1 from cells led to the suppression of double strand DNA break repair via the homologous recombination and single-strand annealing pathways. The presence of NUSAP1 was also found to be important for the control of centrosome numbers. We have found evidence that NUSAP1 plays a role in these processes through regulation of BRCA1 protein levels, and BRCA1 overexpression from a plasmid mitigates the defective phenotypes seen upon NUSAP1 depletion. We found that after NUSAP1 depletion there is a decrease in BRCA1 recruitment to ionizing radiation-induced foci. Results from this study reveal a novel association between BRCA1 and NUSAP1 and suggests a mechanism whereby NUSAP1 is involved in carcinogenesis.     

The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer

The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.     

Overexpression of PAFAH1B1 is associated with tumor metastasis and poor survival in non-small cell lung cancer

Our previous array-comparative genomic hybridization study showed that PAFAH1B1 gene locus was amplified in lung cancer patients, suggesting that PAFAH1B1 is a potential oncogene in lung cancer. Here, we investigate the oncogenic mechanisms of PAFAH1B1 in lung cancer. PAFAH1B1 was characterized in cell and animal models of lung cancer by in vitro migration and invasion assays and in vivo metastasis studies. The mRNA and protein expression levels of PAFAH1B1 were further determined and the prognostic effects of PAFAH1B1 overexpression in lung cancer patients were analyzed. Overexpression of PAFAH1B1 enhanced migration and invasion in lung cancer cells, whereas knockdown of PAFAH1B1 decreased cell migration and invasion, and disrupted cell microtubule organization and pericellular poly-fibronectin assemblies. In vivo tumor metastasis assay confirmed that PAFAH1B1 knockdown in lung cancer cells markedly reduced their metastasis capabilities in animals. The frequencies of overexpressed PAFAH1B1 mRNA and protein were 62.4% (63/101) and 57.4% (58/101) in lung cancer patients, respectively. The clinical correlation results showed that overexpression of PAFAH1B1 was significantly associated with late stage (mRNA: P=0.008, protein: P=0.008) and poor survival in lung adenocarcinoma (P=0.020) and male patients (P=0.049). Our results provide the first evidence that PAFAH1B1 overexpression contributes to lung tumorigenesis and poor prognosis. These effects are partly mediated through disruption of microtubule network and pericellular poly-fibronectin assembly to promote migration and invasiveness of lung cancer cells.     

PD-L1表达调控机制及其对抗肿瘤免疫反应的影响

肿瘤的发生是一个多因素、多步骤的过程.肿瘤细胞通过抑制机体的抗肿瘤免疫应答,实现免疫逃逸,促进肿瘤生长.肿瘤免疫逃逸的分子机制是肿瘤免疫研究的核心问题之一.在T细胞介导的免疫反应中,程序性死亡受体-1(programmed death receptor-1,PD-1)是关键的抑制性免疫检查点.肿瘤通过表达程序性死亡配体-1(programmed death ligand-1,PD-L1),可以增强PD-1抑制信号,促进肿瘤免疫逃逸.研究肿瘤细胞如何调节PD-L1表达,有助于阐明肿瘤发生免疫逃逸的分子机制.近年来的研究发现,肿瘤细胞在基因转录、转录后调节以及蛋白翻译后修饰等多个环节对PD-L1表达进行调节,并且通过调控PD-L1的表达影响抗肿瘤免疫反应.这些研究为肿瘤免疫治疗提供了新的靶点.     

PRKAA1 Promotes Proliferation and Inhibits Apoptosis of Gastric Cancer Cells Through Activating JNK1 and Akt Pathways

PRKAA1 (protein kinase AMP-activated catalytic subunit 1) is a catalytic subunit of AMP-activated protein kinase (AMPK), which plays a key role in regulating cellular energy metabolism through phosphorylation, and genetic variations in the PRKAA1 have been found to be associated with gastric cancer risk. However, the effect and underlying molecular mechanism of PRKAA1 on gastric cancer tumorigenesis, especially the proliferation and apoptosis, are not fully understood. Our data showed that PRKAA1 is highly expressed in BGC- 823 and MKN45 cells and is expressed low in SGC-7901 and MGC-803 cells in comparison with the other gastric cancer cells. PRKAA1 downregulation by shRNA or treatment of AMPK inhibitor compound C significantly inhibited proliferation as well as promoted cell cycle arrest and apoptosis of BGC-823 and MKN45 cells. Moreover, the expression of PCNA and Bcl-2 and the activity of JNK1 and Akt signaling were also reduced in BGC-823 and MKN45 cells after PRKAA1 downregulation. In vivo experiments demonstrated that tumor growth in nude mice was significantly inhibited after PRKAA1 silencing. Importantly, inactivation of JNK1 or Akt signaling pathway significantly inhibited PRKAA1 overexpression-induced increased cell proliferation and decreased cell apoptosis in MGC-803 cells. In conclusion, our findings suggest that PRKAA1 increases proliferation and restrains apoptosis of gastric cancer cells through activating JNK1 and Akt pathways.     

Stathmin1 regulates p27 expression,proliferation and drug resistance,resulting in poor clinical prognosis in cholangiocarcinoma

Patients with extrahepatic cholangiocarcinoma (EHCC) have a poor prognosis; postoperative survival depends on cancer progression and therapeutic resistance. The mechanism of EHCC progression needs to be clarified to identify ways to improve disease prognosis. Stathmin1 (STMN1) is a major cytosolic phosphoprotein that regulates microtubule dynamics and is associated with malignant phenotypes and chemoresistance in various cancers. Recently, STMN1 was reported to interact with p27, an inhibitor of cyclin‐dependent kinase complexes. Eighty EHCC cases were studied using immunohistochemistry and clinical pathology to determine the correlation between STMN1 and p27 expression; RNA interference to analyze the function of STMN1 in an EHCC cell line was also used. Cytoplasmic STMN1 expression correlated with venous invasion (P = 0.0021) and nuclear p27 underexpression (P = 0.0011). Patients in the high‐STMN1‐expression group were associated with shorter recurrence‐free survival and overall survival than those in the low‐expression group. An in vitro protein‐binding assay revealed that cytoplasmic STMN1 bound to p27 in the cytoplasm, but not in the nucleus of EHCC cells. Moreover, p27 accumulated in EHCC cells after STMN1 suppression. STMN1 knockdown inhibited proliferation and increased the sensitivity of EHCC cells to paclitaxel. STMN1 contributes to a poor prognosis and cancer progression in EHCC patients. Understanding the regulation of p27 by STMN1 could provide new insights for overcoming therapeutic resistance in EHCC.     

HKDC1 upregulation promotes glycolysis and disease progression,and confers chemoresistance onto gastric cancer

There is increasing evidence that hexokinase is involved in cell proliferation and migration. However, the function of the hexokinase domain containing protein-1 (HKDC1) in gastric cancer (GC) remains unclear. Immunohistochemistry analysis and big data mining were used to evaluate the correlation between HKDC1 expression and clinical features in GC. In addition, the biological function and molecular mechanism of HKDC1 in GC were studied by in vitro and in vivo assays. Our study indicated that HKDC1 expression was upregulated in GC tissues compared with adjacent nontumor tissues. High expression of HKDC1 was associated with worse prognosis. Functional experiments demonstrated that HKDC1 upregulation promoted glycolysis, cell proliferation, and tumorigenesis. In addition, HKDC1 could enhance GC invasion and metastasis by inducing epithelial–mesenchymal transition (EMT). Abrogation of HKDC1 could effectively attenuate its oncogenic and metastatic function. Moreover, HKDC1 promoted GC proliferation and migration in vivo. HKDC1 overexpression conferred chemoresistance to cisplatin, oxaliplatin, and 5-fluorouracil (5-Fu) onto GC cells. Furthermore, nuclear factor kappa-B (NF-κB) inhibitor PS-341 could attenuate tumorigenesis, metastasis, and drug resistance ability induced by HKDC1 overexpression in GC cells. Our results highlight a critical role of HKDC1 in promoting glycolysis, tumorigenesis, and EMT of GC cells via activating the NF-κB pathway. In addition, HKDC1-mediated drug resistance was associated with DNA damage repair, which further activated NF-κB signaling. HKDC1 upregulation may be used as a potential indicator for choosing an effective chemotherapy regimen for GC patients undergoing chemotherapy.