PTBP1 enhances exon11a skipping in Mena pre-mRNA to promote migration and invasion in lung carcinoma cells

Alternative splicing (AS) events occur in the majority of human genes. AS in a single gene can give rise to different functions among multiple isoforms. Human ortholog of mammalian enabled (Mena) is a conserved regulator of actin dynamics that plays an important role in metastasis. Mena has been shown to have multiple splice variants in human tumor cells due to AS. However, the mechanism mediated Mena AS has not been elucidated. Here we showed that polypyrimidine tract-binding protein 1 (PTBP1) could modulate Mena AS. First, PTBP1 levels were elevated in metastatic lung cancer cells as well as during epithelial-mesenchymal transition (EMT) process. Then, knockdown of PTBP1 using shRNA inhibited migration and invasion of lung carcinoma cells and decreased the Mena exon11a skipping, whereas overexpression of PTBP1 had the opposite effects. The results of RNA pull-down assays and mutation analyses demonstrated that PTBP1 functionally targeted and physically interacted with polypyrimidine sequences on both upstream intron11 (TTTTCCCCTT) and downstream intron11a (TTTTTTTTTCTTT). In addition, the results of migration and invasion assays as well as detection of filopodia revealed that the effect of PTBP1 was reversed by knockdown of Mena but not Mena11a+. Overexpressed MenaΔ11a also rescued the PTBP1-induced migration and invasion. Taken together, our study provides a novel mechanism that PTBP1 modulates Mena exon11a skipping, and indicates that PTBP1 depends on the level of Mena11a− to promote lung cancer cells migration and invasion. The regulation of Mena AS may be a potential prognostic marker and a promising target for treatment of lung carcinoma.     

多聚嘧啶区结合蛋白1通过调控基因的可变剪接促进胆管癌细胞的生长、迁移及侵袭能力

胆管癌是一种起病隐匿、侵袭性强、致死率高的原发性恶性肿瘤。多聚嘧啶区结合蛋白1(polypyrimidine tract-binding protein 1, PTBP1)已被报道,在多种类型肿瘤组织中异常高表达并参与癌症进展,但其在胆管癌中的作用仍未见报道。该研究旨在探讨PTBP1在胆管癌中的生物学功能,并初步解析其分子机制。本文利用公开的癌症基因组图谱(the cancer genome atlas, TCGA)数据,分析了胆管癌及癌旁组织中的PTBP1 mRNA表达水平。结果显示,PTBP1在胆管癌组织中的表达水平显著高于癌旁组织(P < 0.05)。随后,在胆管癌细胞系RBE和HuH28中,通过CCK-8和细胞平板克隆实验,评价了PTBP1对胆管癌细胞生长能力的影响。结果显示,过表达PTBP1可显著促进胆管癌细胞的生长(P < 0.01),而敲低PTBP1显著抑制胆管癌细胞的生长(P < 0.001)。Transwell和Invasion实验结果显示,过表达PTBP1可显著促进胆管癌细胞的迁移和侵袭(P < 0.001),而敲低PTBP1显著抑制胆管癌细胞的迁移和侵袭(P < 0.001)。转录物组测序和通路富集分析结果显示,在胆管癌细胞中,敲低PTBP1后上调表达的基因显著富集于p53信号通路;而下调表达的基因显著富集于胆固醇代谢、Rho GTPase和TGF-β等信号通路。基于上述转录物组测序数据,本文还分析发现,敲低PTBP1可导致一系列基因发生异常的mRNA可变剪接事件,例如参与TGF-β调控的TGIF1及与p53活性相关的GNAS基因等。综上所述,PTBP1可能通过调控一系列基因的可变剪接而影响多个癌症相关的信号通路,从而促进胆管癌的进展。     

HN1L promotes migration and invasion of breast cancer by up-regulating the expression of HMGB1

Recent reports showed that haematological and neurological expressed 1-like (HN1L) gene participated in tumorigenesis and tumour invasion. However, the expression and role of HN1L in breast cancer remain to be investigated. Here, bioinformatics, western blot and immunohistochemistry were used to detect the expression of HN1L in breast cancer. Wound healing, transwell assay, immunofluorescence assay and mass spectrum were used to explore the role and mechanism of HN1L on the migration and invasion of breast cancer, which was confirmed in vivo using a nude mice model. Results showed that HN1L was significantly over-expressed in breast cancer tissues, which was positively correlated with M metastasis of breast cancer patients. Silencing HN1L significantly inhibited the invasion and metastasis of breast cancer cells in vitro and lung metastasis in nude mice metastasis model of breast cancer. Mechanistically, HN1L interacted with HSPA9 and affected the expression of HMGB1, playing a key role in promoting the invasion and metastasis of breast cancer cell. These results suggested that HN1L was an appealing drug target for breast cancer.     

Akt1基因沉默降低小鼠乳腺癌细胞的肺转移能力

Akt1是一种丝氨酸/苏氨酸蛋白激酶,参与调节细胞的代谢、生长和发育。作为一种原癌基因,Akt1在许多人类肿瘤中表达显著增高,促进肿瘤转移;但也有研究表明,Akt1的活化可抑制乳腺癌细胞的侵袭和转移。为了深入探讨Akt1在肿瘤发生发展过程中的作用,采用RNA干扰技术沉默了高转移小鼠乳腺癌细胞4T1中Akt1的表达。MTT法检测发现,Akt1沉默不影响4T1细胞的增殖能力;Transwell法检测证明,Akt1沉默可降低4T1细胞的迁移能力。与以上结果相一致,Akt1沉默不影响乳腺癌形成原位瘤的能力,但显著降低其体内肺转移能力。结果表明,Akt1在小鼠乳腺癌细胞转移过程中发挥重要作用,并提示Akt1可能成为乳腺癌治疗的靶点。     

中心体蛋白70促进乳腺癌细胞的侵袭和转移

中心体蛋白70（centrosomal protein 70, CEP70）可通过介导内皮细胞的迁移影响血管新生,肿瘤的转移能力与肿瘤细胞的迁移密切相关,CEP70是否影响肿瘤细胞的侵袭转移尚不明确。结合前期淋巴结转移和未发生淋巴结转移原位乳腺癌组织的基因表达芯片的比较结果,本研究通过免疫组化染色,检测CEP70在淋巴结转移和未发生淋巴结转移的原位乳腺癌组织中的表达情况,以及real-time PCR和Western 印迹检测不同乳腺癌细胞系中CEP70的表达,结果提示,淋巴结转移患者的乳腺癌组织中CEP70强阳性的比例明显高于未发生淋巴结转移的乳腺癌组织,同时CEP70在侵袭能力强的乳腺癌细胞中表达较高。利用慢病毒转染构建CEP70稳定下调的MDA-MB-231细胞系,划痕实验以及侵袭转移的结果显示,下调CEP70的表达,可明显抑制MDA-MB-231细胞系的细胞迁移和侵袭能力。上述结果证明,CEP70的表达与乳腺癌的侵袭转移呈正相关,下调CEP70可抑制乳腺癌的侵袭转移,因此CEP70有望成为乳腺癌临床诊断及治疗的新靶点。     

Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5′ splice site selection

Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5′ splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5′ splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5′ splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5′ splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5′ splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5′ splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator.     

Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin

Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.     

Long intergenic noncoding RNA 00641 inhibits breast cancer cell proliferation,migration, and invasion by sponging miR-194-5p

Long noncoding RNAs have an essential role in the tumorigenesis of breast cancer (BC). Nonetheless, the consequences of long intergenic noncoding RNA 00641 (LINC00641) in BC remain unidentified. This study shows that LINC00641 expression level was decreased in BC tissues. LINC00641 expression level was negatively related to tumor size, lymph-node metastasis, as well as clinical stage. LINC00641 overexpression inhibited cell proliferation, migration, and invasion but stimulated apoptosis in BC cells. LINC00641 overexpression also remarkably reduced BC growth and metastasis in vivo. LINC00641 acts as a competitive endogenous RNA to sponge miR-194-5p. miR-194-5p level was higher in BC tissues and cells compared with normal-adjacent tissues and normal breast epithelial cell. miR-194-5p expression was negatively correlated with LINC00641 expression in BC tissues. miR-194-5p overexpression reversed the effects of LINC00641 on cell proliferation, cycle, apoptosis, migration, as well as invasion. In conclusion, LINC00641 inhibits BC cell proliferation, migration, as well as invasion by sponging miR-194-5p.     

转录激活因子1(ATF1)内部核糖体进入位点的鉴定及活性分析

蛋白质翻译起始通常有两种机制,一是依赖帽结构的翻译,另一种是依赖5′非翻译区的内部核糖体进入位点(IRES).在后一种方式中,在某些IRES反式作用因子,如La蛋白、多聚嘧啶串结合蛋白1等的参与下,直接招募核糖体小亚基到mRNA的翻译起始位点,启始翻译.研究发现,参与细胞生长、分化、细胞周期进程、凋亡和压力调控的相关蛋白中通常含有IRES元件.基于功能,我们提出假说：转录激活因子1(ATF1)的5′-UTR可能具有IRES活性.为验证假说,首先构建了含全长ATF1 5′-UTR的双荧光素酶报告质粒|质粒转染结合报告酶活性分析显示,ATF1 5′-UTR在Bel7402、HCT-8和HEK293细胞中表现出不同的IRES活性|而此IRES活性与5′-UTR中的隐藏启动子无关.同时还发现,ATF1 5′-UTR在NIH3T3细胞中却没有IRES活性.与此结果相一致,Western印迹检测ATF1在这几种细胞系中的表达.结果显示,Bel7402、HCT 8和HEK293中ATF1蛋白质表达水平较高,而在NIH3T3中却极低. ATF1 5′-UTR的系列5′-删除突变及报告酶分析证明,ATF1 5′-UTR的完整性对其IRES活性大小发挥重要作用|其中5′端的204 bp序列对其IRES活性贡献较大. RNA-蛋白免疫共沉淀实验揭示,ATF1 5′-UTR可与La和PTBP1蛋白结合|抑制La和PTBP1蛋白质的表达,并可减低HEK293细胞中ATF1蛋白质表达水平.这些结果提示,La和PTBP1蛋白（两种ITAFs）为ATF1 5′-UTR发挥IRES活性所必需.总之,上述结果证明,ATF1 5′-UTR具有IRES活性,其活性发挥依赖与La和PTBP1蛋白的结合.上述发现为进一步研究La和PTBP1表达及亚细胞定位对ATF1 IRES调控机制的影响奠定了基础.     

KiSS1 suppresses TNFα‐induced breast cancer cell invasion via an inhibition of RhoA‐Mediated NF‐κB activation

Tumor necrosis factor‐alpha (TNFα) induces cancer development and metastasis, which is prominently achieved by nuclear factor‐kappa B (NF‐κB) activation. TNFα‐induced NF‐κB activation enhances cellular mechanisms including proliferation, migration, and invasion. KiSS1, a key regulator of puberty, was initially discovered as a tumor metastasis suppressor. The expression of KiSS1 was lost or down‐regulated in different metastatic tumors. However, it is unclear whether KiSS1 regulates TNFα‐induced NF‐κB activation and further tumor cell migration. In this study, we demonstrate that KiSS1 suppresses the migration of breast cancer cells by inhibiting TNFα‐induced NF‐κB pathway and RhoA activation. Both KiSS1 overexpression and KP10 (kisspeptin‐10) stimulation inhibited TNFα‐induced NF‐κB activity, suppressed TNFα‐induced cell migration and cell attachment to fibronectin in breast cancer cells while KP10 has little effect on cancer cell proliferation. Furthermore, KP10 inhibited TNFα‐induced cell migration and RhoA GTPase activation. Therefore, our data demonstrate that KiSS1 inhibits TNFα‐induced NF‐κB activation via downregulation of RhoA activation and suppression of breast cancer cell migration and invasion. J. Cell. Biochem. 107: 1139–1149, 2009. © 2009 Wiley‐Liss, Inc.     

CT45A1 acts as a new proto-oncogene to trigger tumorigenesis and cancer metastasis

Cancer/testis antigen (CTA)-45 family (CT45) belongs to a new family of genes in phylogenetics and is absent in normal tissues except for testis, but is aberrantly overexpressed in various cancer types. Whether CT45 and other CTAs act as proto-oncogenes has not been determined. Using breast cancer as a model, we found that CT45A1, a representative CT45 family member, alone had a weak tumorigenic effect. However, its neoplastic potency was greatly enhanced in the presence of growth factors. Overexpression of CT45A1 in breast cancer cells markedly upregulated various oncogenic and metastatic genes, constitutively activated ERK and CREB signaling pathways, promoted epithelial–mesenchymal transition, and increased cell stemness, tumorigenesis, invasion, and metastasis, whereas silencing CT45A1 significantly reduced cancer cell migration and invasion. We propose that CT45A1 functions as a novel proto-oncogene to trigger oncogenesis and metastasis. CT45A1 and other CT45 members are therefore excellent targets for anticancer drug discovery and targeted tumor therapy, and valuable genes in the study of a molecular phylogenetic tree.     

LncRNA FIRRE functions as a tumor promoter by interaction with PTBP1 to stabilize BECN1 mRNA and facilitate autophagy

Long non-coding RNAs (lncRNAs) play critical functions in various cancers. Firre intergenic repeating RNA element (FIRRE), a lncRNA located in the nucleus, was overexpressed in colorectal cancer (CRC). However, the detailed mechanism of FIRRE in CRC remains elusive. Results of RNA sequence and qPCR illustrated overexpression of FIRRE in CRC cell lines and tissues. The aberrant expression of FIRRE was correlated with the migration, invasion, and proliferation in cell lines. In accordance, it was also associated with lymphatic metastasis and distant metastasis in patients with CRC. FIRRE was identified to physically interact with Polypyrimidine tract-binding protein (PTBP1) by RNA pull-down and RNA immunoprecipitation (RIP). Overexpression of FIRRE induced the translocation of PTBP1 from nucleus to cytoplasm, which was displayed by immunofluorescence and western blot. In turn, delocalization of FIRRE from nucleus to cytoplasm is observed after the loss of PTBP1. The RNA-protein complex in the cytoplasm directly bound to BECN1 mRNA, and the binding site was at the 3'' end of the mRNA. Cells with FIRRE and PTBP1 depletion alone or in combination were treated by Actinomycin D (ACD). Results of qPCR showed FIRRE stabilized BECN1 mRNA in a PTBP1-medieated manner. In addition, FIRRE contributed to autophagy activity. These findings indicate FIRRE acts as an oncogenic factor in CRC, which induces tumor development through stabilizing BECN1 mRNA and facilitating autophagy in a PTBP1-mediated manner.Subject terms: Cancer genomics, Oncogenes     

Lycorine inhibits breast cancer growth and metastasis via inducing apoptosis and blocking Src/FAK-involved pathway

Breast cancer is the most commonly diagnosed cancer type worldwide among women and more than 90% of patients die from tumor metastasis. Lycorine, a natural alkaloid, has been widely reported possessing potential efficacy against cancer proliferation and metastasis. In our study, the anti-tumor potency on breast cancer was evaluated in vitro and in vivo for the first time. Our results indicated that lycorine inhibited breast cancer cells growth, migration and invasion as well as induced their apoptosis.In in vivo study, lycorine not only suppressed breast tumor growth in xenograft models and inhibited breast tumor metastasis in MDA-MB-231 tail vein model. More importantly, we found lycorine had less toxicity than first-line chemotherapy drug paclitaxel at the same effective dose in vivo. Furthermore, on mechanism, lycorine inhibited tumor cell migration and invasion via blocking the Src/FAK(focal adhesion kinase)-involved pathway. In conclusion, our study implied lycorine was a potential candidate for the treatment of breast cancer by inhibition of tumor growth and metastasis.     

Angiopoietin-2, an angiogenic regulator, promotes initial growth and survival of breast cancer metastases to the lung through the integrin-linked kinase (ILK)-AKT-B cell lymphoma 2 (Bcl-2) pathway

The early onsets of breast cancer metastasis involve cell retention, survival, and resistant to apoptosis and subsequent growth at target vascular beds and tissues in distant organs. We previously reported that angiopoietin-2 (Ang2), an angiogenic regulator stimulates MCF-7 breast tumor metastasis from their orthotopic sites to distant organs through the α(5)β(1) integrin/integrin-linked kinase (ILK)/Akt pathway. Here, by using an experimental tumor metastasis model and in vitro studies, we further dissect the underlying mechanism by which Ang2 promotes the initial growth and survival of MCF-7 breast cancer metastasis in the lung of animals. We show that Ang2 increases cell survival and suppresses cell apoptosis through ILK-induced phosphorylation of Akt1, Akt2, and up-regulation of Bcl-2 in breast cancer cells. Inhibition of ILK, Akt1, and Akt2, and their effector Bcl-2 diminishes Ang2-stimulated breast cancer cell survival and Ang2-attenuated apoptosis in vitro, and initial survival and growth of breast cancer metastasis in the lung of animals. Additionally, siRNA knockdown of endogenous Ang2 in three human metastatic breast cancer cell lines also inhibits phosphorylation of Akt, expression of Bcl-2, and tumor cell survival, migration, and increases cell apoptosis. Since increased expression of Ang2 correlates with elevated potential of human breast cancer metastasis in clinic, our data underscore the importance that up-regulated Ang2 not only stimulates breast cancer growth and metastasis at late stages of the process, but is also critical at the initiating stages of metastases onset, thereby suggesting Ang2 as a promising therapeutic target for treating patients with metastatic breast cancer.     

Distinct mechanisms mediate the initial and sustained phases of cell migration in epidermal growth factor receptor-overexpressing cells

Elevated levels of epidermal growth factor receptor (EGFR) are predictive of increased invasion and metastasis in many human cancers. In the present study, we have shown that two distinct pathways regulate cell migration in EGFR-overexpressing invasive cells such as MDA 468 breast cancer cells: mitogen-activated protein kinase (MAPK or ERK 1 and 2) pathways play a major role in early stages to cell migration; and protein kinase C delta isoforms (PKC-delta) play a significant role in later stages of sustained cell migration. Inhibition of MAPK activity with MAP kinase kinase (MEK) inhibitor PD98059 blocks early stages of cell migration (up to 4 h); however, cells revert back to enhanced cell migration after 4 h. While inhibition of PKC-delta activity with rottlerin or dominant-negative PKC-delta expression blocks sustained cell migration after 4 h and up to 12 h, the combination of MAPK and PKC inhibitors completely blocked transforming growth factor alpha (TGF-alpha)-induced cell migration in EGFR-overexpressing breast cancer cells. However, inhibition of MAPK activity completely blocked cell migration in low EGFR-expressing non-invasive breast cancer cells such as MCF-7 cells. Forced overexpression of EGFR in MCF-7 cells (EGFR/MCF-7 cells) resulted in cell migration patterns seen in MDA 468 cells, that is, MAPK pathways play a major role in early stages to cell migration, and PKC-delta plays a major role in later stages of sustained cell migration. The above data demonstrate that EGFR-overexpressing invasive cells have the ability to compensate the loss of MAPK-mediated signaling through activation of PKC-delta signaling for cell migration, which plays a major role in invasion and metastasis. In addition, data suggest that inhibition of MAPK and PKC-delta signaling pathways should abrogate cell migration and invasion in EGFR-overexpressing human breast cancer cells.     

Retracted: Long noncoding RNA HCG22 suppresses proliferation and metastasis of bladder cancer cells by regulation of PTBP1

Multifarious biological functions of long noncoding RNAs (lncRNAs) have been reported in various cancers including bladder cancer (BCa). This study aims to determine the biological role of a certain lncRNA in BCa. Consistent with the data of The Cancer Genome Atlas database, it was validated that lncRNA HLA complex group 22 (HCG22) was weakly expressed in BCa samples and lowly expressed HCG22 was closely correlated with low overall survival of the BCa patient. To verify the role of HCG22 in BCa progression, functional experiments were carried out in two representative BCa cells (J82 and T24) and the negative effects of HCG22 expression on the cell proliferation, migration, and epithelial–mesenchymal transition were identified. Mechanistically, polypyrimidine tract-binding protein 1 (PTBP1), which was highly expressed in BCa tissues and cell lines, was negatively regulated by HCG22 and the PTBP1-mediated Warburg effect was also obstructed by HCG22. Furthermore, HCG22 modulated the expression of PTBP1 through destabilizing human antigen R (HuR). And functional rescue assays confirmed that HCG22 functioned in bladder cancer through downregulating PTBP1. In conclusion, the present study revealed that HCG22 inhibited BCa progression via the HuR/PTBP1 axis, opening new prospects for potent therapeutic regimens for BCa patients.     

Stanniocalicin 2 Suppresses Breast Cancer Cell Migration and Invasion via the PKC/Claudin-1-Mediated Signaling

Stanniocalcin (STC), a glycoprotein hormone, is expressed in a wide variety of tissues to regulate Ca²⁺ and PO4^- homeostasis. STC2, a member of STC family, has been reported to be associated with tumor development. In this study, we investigated whether the expression of STC2 is associated with migration and invasion of breast cancer cells. We found that breast cancer cell line 231 HM transfected with STC2 shRNA displayed high motility, fibroblast morphology, and enhanced cell migration and invasion. Introduction of STC2 in 231 cells reduced cell migration and invasion. In response to irradiation, silencing of STC2 in 231 HM cells reduced apoptosis, whereas overexpression of STC2 in 231 cells promoted apoptosis, compared with in control cells. Mechanistic study showed that STC2 negatively regulated PKC to control the expression of Claudin-1, which subsequently induced the expressions of EMT-related factors including ZEB1, ZO-1, Slug, Twist, and MMP9. Suppression of PKC activity by using a PKC inhibitor (Go 6983) restored the normal motility of STC2-silenced cells. Furthermore, in vivo animal assay showed that STC2 inhibited tumorigenesis and metastasis of breast cancer cells. Collectively, these results indicate that STC2 may inhibit EMT at least partially through the PKC/Claudin-1-mediated signaling in human breast cancer cells. Thus, STC2 may be exploited as a biomarker for metastasis and targeted therapy in human breast cancer.