TC21/R-Ras2 upregulation in esophageal tumorigenesis: potential diagnostic implications

OBJECTIVES: Early detection of esophageal cancer is hampered by paucity of molecular markers for diagnosis of this aggressive gastrointestinal malignancy in early stages. We recently identified TC21/R-Ras2, a small GTP-binding protein (SMG) in esophageal squamous cell carcinomas (ESCCs) by differential display. This study was designed to test the hypothesis that differential expression of TC21 in normal, dysplastic and malignant esophageal tissues may be of clinical relevance in esophageal tumorigenesis. METHODS: Immunohistochemical analysis of TC21 was carried out in 83 ESCCs, 37 dysplasias and 29 matched histologically normal esophageal tissues and correlated with clinicopathological parameters. The cellular localization of TC21 was determined by confocal microscopy. RESULTS: Expression of TC21 protein was observed in 60/83 (73%) ESCCs predominantly localized in tumor nuclei. Intriguingly, intense TC21 immunoreactivity was observed in all endoscopic biopsies with histological evidence of dysplasia (16 cases) as well as in dysplastic areas distant to ESCCs (21 cases), while matched distant histologically normal epithelia did not show detectable TC21 expression. Immunoblotting and semi-quantitative RT-PCR confirmed TC21 expression in dysplastias and ESCCs. Confocal microscopy showed nuclear as well as cytoplasmic TC21 expression in ESCCs and TE13 cells. CONCLUSIONS: To our knowledge, this is the first report demonstrating differential expression of TC21 in normal, dysplastic and ESCC tissues, suggesting that TC21 expression is associated with early stages of esophageal tumorigenesis. Nuclear localization of TC21 makes it the third of over 100 small SMGs identified to be localized in the nucleus.     

TC21/R-Ras 2在肝癌中的表达、亚细胞定位及意义

目的:探讨TC21基因在肝癌细胞、肝癌及癌旁组织中的表达、亚细胞定位及意义。方法:用RT-PCR检测TC 21在肝癌细胞系及人肝癌及癌旁组织中mRNA水平的表达,用免疫组化及Western印迹检测TC21蛋白在肝癌相关组织中的表达差异,用组织芯片技术结合免疫组化检测TC21蛋白的亚细胞定位。结果:RT-PCR检测结果表明TC21 mRNA在SMMC-7721、BEL-7402、Huh-7、Hep3B、HepG2、MHCC-97L、MHCC-LM3 7个肝癌细胞系及L-02和Chang liver 2株永生化人肝细胞系、7对肝癌及对应癌旁肝组织中均有较高表达;Western印迹结果表明上述细胞系中TC21蛋白表达与mRNA表达一致,TC21在4对肝癌及癌旁组织中均呈较高水平的表达;免疫组化检测结果表明TC21在人原发性肝癌、癌旁组织、硬化肝组织与正常肝组织中的表达阳性率分别为84.2%、77.2%、41.7%、0%,肝癌与肝硬化、正常肝相比显著高表达(P<0.05,P<0.01),与癌旁组织比无统计学意义(P>0.05);统计学分析结果表明TC21蛋白表达与肿瘤大小呈正相关(P<0.05),与是否肝硬化呈负相关(P<0.05);肝癌组织中TC21主要定位于肝癌细胞核,部分定位于胞质,膜定位不明显,TC21蛋白在肝癌细胞中的核定位显著高于癌旁肝细胞(P<0.001)。结论:本研究首次揭示TC21蛋白在肝癌组织中的高表达及核定位预示其在肝细胞恶性转化及肝癌发生发展中发挥重要作用。     

Signal transduction elements of TC21, an oncogenic member of the R-Ras subfamily of GTP-binding proteins

TC21 is a Ras-like GTPase with high oncogenic potential that is found mutated in some human tumors and overexpressed in breast cancer cell lines. We have conducted cellular and biochemical studies in order to understand the role of this protein in signal transduction and to unveil the signaling elements that participate in the TC21 pathway. Using gene transfer experiments, we demonstrate here that the TC21 oncogene can induce both cellular transformation in mouse fibroblasts and neuronal-like differentiation in rat PC12 cells. Interestingly, the proto-oncogenic version of TC21 shows also a lower, but significant, activity in both biological processes. We also demonstrate that the similarity of the cellular responses induced by TC21 and Ras derive from the utilization of overlapping pathways. Thus, the exchange of guanosine nucleotides in wild type TC21 is catalyzed by Ras exchange factors. Moreover, TC21 binds physically to c-Raf-1 in a GTP-dependent manner. Finally, overexpression of TC21G23V in NIH3T3 cells results in the activation of c-Raf-1 and the MAPK and the JNK branches of serine/threonine cascades. From these results, we conclude that TC21 promotes Ras-like responses in diverse cell types due to the use of overlapping, if not identical, signaling elements of the Ras oncogenic pathway.     

施万细胞在恶性肿瘤进展中的作用

施万细胞（Schwann cells,SCs）是外周神经系统中最重要的神经胶质细胞类型,在神经损伤修复过程中起重要作用。近年来研究显示,SCs作为连接神经和肿瘤细胞的“桥梁分子”,通过与肿瘤细胞、免疫细胞、间质细胞等相互作用,改善局部肿瘤微环境,进而促使肿瘤嗜神经生长和神经支配、促进肿瘤的生长、迁移和侵袭,产生免疫抑制并出现肿瘤疼痛等一系列恶性生物学行为。本文就SCs在非神经肿瘤中的作用及其机制进行综述,以期为将来抗肿瘤治疗联合靶向SCs的治疗策略提供理论基础。     

Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis

Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD⁺ -dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.     

miR-21通过靶向SOX2增强胃癌细胞的迁移能力

目的:探讨miR-21在胃癌细胞系中对SOX2的调控作用及其对胃癌细胞迁移的影响。方法:在胃癌细胞系AZ-521、AGS中通过转染miR-21 mimic或者miR-21 antagomir构建miR-21过表达和低表达模型,RT-qPCR检测转染效果,并通过蛋白免疫印迹法检测SOX2在转染细胞中表达的变化。构建miR-21双荧光素酶报告基因,在工具细胞HEK293T中检验miR-21是否通过直接结合SOX2的3′-UTR区域来对其实现调控。应用Transwell迁移实验检测miR-21及SOX2对胃癌细胞迁移能力的影响。结果:miR-21直接结合于SOX2的3′-UTR区域来抑制SOX2在胃癌细胞系中的表达。迁移实验发现上调miR-21或者下调SOX2都能促进胃癌细胞的迁移。结论:miR-21可促进胃癌细胞的迁移能力,其机制可能通过抑制SOX2的表达而实现。     

R-Ras promotes tumor growth of cervical epithelial cells

BACKGROUND: R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype. METHODS: Dominant negative (R-Ras43N) and constitutively active (R-Ras87L) mutants of R-Ras were stably transfected into human cervical epithelium C33A cells. Transfected cells were analyzed for adhesion, cell spreading, migration, and growth in culture and in nude mice. The activity of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K) also was determined by Western blot analysis and by in vitro kinase assays. RESULTS: R-Ras87L-transfected cells, but not R-Ras43 N-transfected cells, had a higher growth rate in nude mice and in culture compared with control cells. None of the transfected C33A cells showed an increase in cell adhesion to fibronectin or collagen I, nor did they show an increment of beta1 integrin affinity. However, cells that expressed R-Ras87L, but not cells that expressed R-Ras 43N, presented a marked increase in cell spreading and migration through collagen-coated membranes. Increases in cell proliferation, spreading, and migration induced by R-Ras87L were inhibited by the PI 3-K inhibitor LY294002. In addition, PI 3-K activity, but not ERK activity, was increased only in cells that expressed R-Ras87L. CONCLUSIONS: These data suggest that the oncogene R-Ras promotes tumor growth of cervical epithelial cells and increases their migration potential over collagen through a pathway that involves PI 3-K.     

Role of synaptojanin 2 in glioma cell migration and invasion

The small GTPase Rac1 is thought to play an important role in cell migration and invasion. We have previously identified synaptojanin 2, a phosphoinositide phosphatase, as an effector of Rac1. Here, we show that small interfering RNA-mediated depletion of either Rac1 or synaptojanin 2 inhibits invasion of SNB19 and U87MG glioblastoma cells through Matrigel and rat brain slices. Depletion of Rac1 or synaptojanin 2 also inhibits migration of SNB19 and U87MG cells on glioma-derived extracellular matrix. In addition, we found that depletion of Rac1 or synaptojanin 2 inhibits the formation of lamellipodia and invadopodia, specialized membrane structures that are thought to be involved in extracellular matrix degradation. These results suggest that synaptojanin 2 contributes to the role of Rac1 in cell invasion and migration by regulating the formation of invadopodia and lamellipodia. This study also identifies synaptojanin 2 as a novel potential target for therapeutic intervention in malignant tumors.     

Involvement of R-Ras and Ral GTPases in estrogen-independent proliferation of breast cancer cells

A key step in the progression of breast cancer is the conversion of cells from an estrogen-dependent to an estrogen-independent state. Yet the molecular mechanisms underlying this transition in the control of cell proliferation of breast cancer cells remain poorly understood. A potential role for Ras-related GTPases in this process was suggested by the finding that BCAR3/AND-34, a protein isolated on the basis of its ability to convert MCF-7 and ZR-75 breast cancer cell lines to estrogen independence and tamoxifen resistance, is a guanine nucleotide exchange factor with the potential to activate the Ras-related Ral, R-Ras and Rap GTPases. In this study we investigated the potential contribution of these GTPases to the generation of estrogen-independence in MCF-7 cells. We found that elevated R-Ras but not Ral or Rap activity was sufficient to induce estrogen-independent proliferation of MCF-7 cells. The effect of R-Ras was dependent upon its ability to constitutively activate the AKT kinase. Interestingly, although AKT was also constitutively activated when estrogen-independent proliferation was induced by over-expression of EGF receptors, this mechanism of hormone independence did not require AKT activation. In contrast, EGF receptors did require Ral activation to induce estrogen-independent proliferation, while Ral activation was not required for estrogen-induced proliferation of MCF-7 cells. These findings suggest that Ral activity takes on a significant role in controlling cell proliferation of breast cancer cells when progression to estrogen-independence is associated with over-expression of EGF receptor family members. Moreover, because R-Ras promotes hormone-independent growth in a manner distinct from EGF receptors, it may participate in the conversion of breast cancer cells to estrogen independence when over-expression of EGF receptor family members is not involved.     

Overexpression of activated neu/erbB2 initiates immortalization and malignant transformation of immature Schwann cells in vitro

The neu/erbB2 protooncogene is overexpressed in numerous human cancers and is mutationally activated in N-ethyl-N-nitrosourea (ENU)-induced rodent tumors of the Schwann cell lineage. We investigated whether expression of activated neu in Schwann cells is sufficient to initiate their immortalization and transformation. Clones of embryonic dorsal root ganglia cells infected with a retrovirus bearing activated neu (NID cells) were selected based on their expression of Schwann cell-specific markers. Compared to embryonic Schwann cells infected with a virus encoding empty vector, we found that NID cells have altered shapes and disorganized cytoskeletons, grow in the absence of growth factors required for normal Schwann cell survival and proliferation, and can be repeatedly passaged. Furthermore, NID cells are invasive in an in vitro matrix invasion assay and form metastatic tumors when injected into syngeneic animals. The neu-induced growth and invasive phenotypes could be reversed by drugs that inhibit Ras and Src activity. Interestingly, later stage Schwann cells infected with activated neu failed to become immortalized. These findings indicate that constitutive activation of erbB2 is sufficient to initiate the immortalization and transformation of immature Schwann cells, and support the notion that Schwann cells have particular developmental stages during which they are susceptible to immortalizing and transforming events.     

Migration-prone glioma cells show curcumin resistance associated with enhanced expression of miR-21 and invasion/anti-apoptosis-related proteins

In study, the expression patterns and functional differences between an original glioma cell population (U251 and U87) and sublines (U251-P10, U87-P10) that were selected to be migration-prone were investigated. The expressions levels of VEGF and intracellular adhesion molecule-1 (ICAM-1) were increased in the migration-prone sublines as well as in samples from patients with high-grade glioma when compared to those with low-grade glioma. In addition, cells of the migration-prone sublines showed increased expression of the oncogenic microRNA. miR-21, which was also associated with more advanced clinical pathological stages in the patient tissue specimens. Treatment of U251 cells with an miR-21 mimic dramatically enhanced the migratory activity and expression of anti-apoptotic proteins. Furthermore, treatment with curcumin decreased the miR-21 level and anti-apoptotic protein expression, and increased the expression of pro-apoptosis proteins and microtubule-associated protein light chain 3-II (LC3-II) in U251 cells. The migration-prone sublines showed decreased induction of cell death markers in response to curcumin treatment. Finally, U251-P10 cells showed resistance against curcumin treatment. These results suggest that miR-21 is associated with regulation of the migratory ability and survival in human glioma cells. These findings suggest novel mechanisms of malignancy and new potential combinatorial strategies for the management of malignant glioma.     

The COOH-terminal end of R-Ras alters the motility and morphology of breast epithelial cells through Rho/Rho-kinase

R-Ras has a high degree of sequence homology with Ras and other members of the Ras subfamily, including Rap, TC21, and M-Ras. Although R-Ras has been suggested to regulate cell adhesion, migration, and invasion, the biological mechanism has not been well assessed. In this report, we show that constitutively active R-Ras (38V) induces a more rounded cell shape and redistribution of focal adhesion, and enhances the phosphorylation of focal adhesion kinase and paxillin. Active R-Ras (38V) induces cell adhesion to type I collagen, but inhibits cell motility. In active R-Ras (38V) cells, the activity of RhoA is increased and accompanied with translocation to plasma membrane, but not that of Rac1 or Cdc42. In parallel, dominant-negative RhoA (N19RhoA) and Y27632, a specific inhibitor of Rho-associated kinase, dramatically reverse the rounded cell morphology to a spread cell shape and enhance motility. Furthermore, coincident with the formation of cortical actin filaments in active R-Ras (38V) cells, myosin light chain and Ser-19-phosphorylated myosin light chain mainly accumulate at the peripheral region, which is inhibited by the treatment of Y27632. Using H-Ras/R-Ras and R-Ras/H-Ras hybrid constructs, we show that the COOH-terminal region of R-Ras contains the specific signal for inducing changes in motility and morphology. Our results suggest that R-Ras in breast epithelial cells disrupts cell polarity and motility through the Rho/Rho-associated kinase pathway triggered by a signal from the COOH-terminal end of R-Ras.     

Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin

Background  

Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.     

Neuregulin and laminin stimulate phosphorylation of the NF2 tumor suppressor in Schwann cells by distinct protein kinase A and p21-activated kinase-dependent pathways

Mutations in the neurofibromatosis type 2 (NF2) gene cause formation of schwannomas and other tumors in the nervous system. The NF2 protein, Schwannomin/Merlin, is a cytoskeleton-associated tumor suppressor regulated by phosphorylation at serine 518 (S518). Unphosphorylated Schwannomin restricts cell proliferation in part by inhibiting Rac- and p21-activated kinase (Pak). In a negative-feedback loop, Pak phosphorylates Schwannomin inactivating its ability to inhibit Pak. Little is known about receptor mechanisms that promote Pak activity and Schwannomin phosphorylation. Here we demonstrate in primary Schwann cells (SCs) that Schwannomin is rapidly phosphorylated on S518 by Pak following laminin-1 binding to beta1 integrin, and by protein kinase A following neuregulin-1beta (NRG1beta) binding to ErbB2/ErbB3 receptors. These receptors, together with phosphorylated Schwannomin, P-Pak, Cdc42 and paxillin are enriched at the distal tips of SC processes, and can be isolated as a complex using beta1 integrin antibody. Dual stimulation with laminin-1 and NRG1beta does not synergistically increase Schwannomin phosphorylation because ErbB2 kinase partially antagonizes integrin-dependent activation of Pak. These results identify two parallel, but interactive pathways that inactivate the tumor suppressor activity of Schwannomin to allow proliferation of subconfluent SCs. Moreover, they identify ErbB2, ErbB3 and beta1 integrins as potential therapeutic targets for NF2.     

Role of STAT3/5 and Bcl-2/xL in 2-methoxyestradiol-induced endoreduplication of nasopharyngeal carcinoma cells

2‐methoxyestradiol (2ME2), an endogenous metabolite of 17‐β‐estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well‐differentiated nasopharyngeal carcinoma (NPC) HK‐1 and a poorly differentiated C666‐1 cell line. In the present study, we studied the survival factors involved in 2ME2‐induced endoreduplicating NPC cells. In the HK‐1 cells, knockdown of BcL‐xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p‐STAT5‐Y694), but not p‐STAT3 (Y705) and p‐STAT3 (S727), in the nucleus of HK‐1 cells. Pre‐treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl‐xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p‐STAT3 in the poorly differentiated C666‐1 cells. Pharmacological inhibition of STAT3 or Bcl‐2/xL resulted in a decrease in endoreduplication of C666‐1 cells. Taken together, the expression of p‐STAT5 and p‐STAT3 was upregulated in 2ME2‐induced endoreduplicating HK‐1 and C666‐1 cells, respectively. Combination of 2ME2 with Bcl‐2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.     

Role of aquaporin-4 in the regulation of migration and invasion of human glioma cells

Glioblastoma is the most aggressive form of primary brain tumor with a tendency to invade surrounding healthy brain tissues, rendering tumors of this type largely incurable. Aquaporin-4 (AQP4) is a key molecule involved in maintaining water and ion homeostasis in the central nervous system and has been recently reported to play a role in cell migration in addition to its well-known function in brain edema. Increased AQP4 expression has been demonstrated in glioblastoma multiforme (GBM), suggesting that it is also involved in malignant brain tumors. Here, we identify a novel role for aquaporin-4 in glioblastoma cell migration and invasion. In the present study, we used small-interference RNA technology and a pharmacological inhibitor to knock down the expression of AQP4, which resulted in speci?c and massive impairment of glioblastoma cell migration and invasion in vitro and in vivo. In addition, we demonstrated the possible mechanisms by which AQP4 functions in the process of glioblastoma cell invasion. The downregulation of matrix metalloprotease-2 (MMP-2) expression in LN229 cells with AQP4 reduction coincided with decreased cell invasive ability. Furthermore, our study showed that AQP4 may also be involved in the regulation of glioblastoma cell adhesion. The expression of β-catenin and connexin 43 were increased in AQP4-downregulated LN229 cells consistent with their enhanced cell-cell adhesion ability. In summary, our results indicate that AQP4 is involved in the control of glioblastoma cell migration and invasion and may be a potential therapeutic target for glioblastoma cell in?ltration.     

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration

Introduction

Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam).

Methods

We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions.

Results

Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility.

Conclusions

Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.     

Degradation of HER2/neu by ANT2 shRNA suppresses migration and invasiveness of breast cancer cells

Background  

In breast cancer, the HER2/neu oncoprotein, which belongs to the epidermal growth factor receptor family, may trigger activation of the phosphoinositide-3 kinase (PI3K)/Akt pathway, which controls cell proliferation, survival, migration, and invasion. In this study, we examined the question of whether or not adenine nucleotide translocase 2 (ANT2) short hairpin RNA (shRNA)-mediated down-regulation of HER2/neu and inhibitory effects on the PI3K/Akt signaling pathway suppressed migration and invasiveness of breast cancer cells.