CD44 variant,but not standard CD44 isoforms,mediate disassembly of endothelial VE-cadherin junction on metastatic melanoma cells

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8-v10 induced junction disassembly and vascular endothelial (VE)-cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)-mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8-v10 promoted endothelial junction disruption. Overexpression of CD44v8-v10 but not of standard CD44 (CD44s) promoted gap formation in the non-metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE-cadherin mutant Y658E supported transmigration of CD44-silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.     

Polypyrimidine tract-binding protein 1 regulates the alternative splicing of dopamine receptor D(2)

Dopamine receptor D(2) (DRD2) has two splicing isoforms, a long form (D2L) and short form (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is unknown. In this study, we examined which splicing factors regulate the expression of D2L and D2S by over-expressing several RNA-binding proteins in HEK293 cells. In a cellular splicing assay, the over-expression of polypyrimidine tract-binding protein 1 (PTBP1) reduced the expression of D2S, whereas the knockdown of PTBP1 increased the expression of D2S. We also identified the regions of DRD2 that are responsive to PTBP1 using heterologous minigenes and deletion mutants. Our results indicate that PTBP1 regulates the alternative splicing of DRD2. Considering that DRD2 inhibits cAMP-dependent protein kinase A, which modulates the intracellular localization of PTBP1, PTBP1 may contribute to the autoregulation of DRD2 by regulating the expression of its isoforms.     

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。     

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 μM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 μg/ml and 50 μg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.     

Met receptors induce Sam68-dependent cell migration by activation of alternate extracellular signal-regulated kinase family members

The hepatocyte growth factor (HGF)/Met receptor signaling pathway is deregulated in diverse human malignancies and plays a central role in oncogenesis, tumor progression, and invasive cancer growth. Similarly, altered expression and splicing (i.e. inclusion of variant exon 5, "v5") of the cell adhesion marker, CD44, is associated with advanced cancer phenotypes. We sought to further understand how HGF regulates CD44v5 expression. Immortalized nontumorigenic keratinocyte (HaCaT) cells abundantly express both Met receptors and CD44v5 transmembrane glycoproteins. HGF stimulated CD44v5 protein expression and HaCaT cell migration; these events required activation of the ERK1/2 MAPK module and Sam68, a protein involved in RNA processing, splicing, and v5 inclusion. Similar to HaCaT cells, highly migratory MDA-MB-231 breast cancer cells also required Sam68 expression for HGF-induced migration. However, MDA-MB-231 cell migration occurred independently of ERK1/2 and CD44v5 expression and instead required ERK5 signaling to Sam68. Phospho-mutant, but not WT-Sam68, blocked HGF-induced cell migration in both cell types; MDA-MB-435 cells behaved similarly. These results suggest that Sam68 acts as a convergence point for ERK signaling to cell migration; blockade of phospho-Sam68 may provide a new avenue for therapeutic inhibition of metastatic cancers.     

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion.     

Characterization of the expression of variant and standard CD44 in prostate cancer cells: Identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface

CD44 is a glycosylated adhesion molecule and osteopontin is one of its ligand. CD44 undergoes alternative splicing to produce variant isoforms. Our recent studies have shown an increase in the surface expression of CD44 isoforms (sCD44 and v4–v10 variant CD44) in prostate cancer cells over‐expressing osteopontin (PC3/OPN). Formation of CD44/MMP9 complex on the cell surface is indispensable for MMP9 activity. In this study, we have characterized the expression of variant CD44 using RT‐PCR, surface labeling with NHS–biotin, and immunoblotting. Expression of variant CD44 encompassing v4–v10 and sCD44 at mRNA and protein levels are of the same levels in PC3 and PC3/OPN cells. However, an increase in the surface expression of v6, v10, and sCD44 in PC3/OPN cells suggest that OPN may be a ligand for these isoforms. We then proceeded to determine the role of sCD44 in MMP9 activation. Based on our previous studies in osteoclasts, we hypothesized that phosphorylation of CD44 has a role on its surface expression and subsequent activation of MMP9. We have prepared TAT‐fused CD44 peptides comprising unphosphorylated and constitutively phosphorylated serine residues at positions Ser323 and Ser325. Transduction of phosphopeptides at Ser323 and Ser323/325 into PC3 cells reduced the surface levels of CD44, MMP9 activity, and cell migration; but had no effect on the membrane localization of MMP9. However, MMP9 knock‐down PC3 cells showed reduced CD44 at cellular and surface levels. Thus we conclude that surface expression of CD44 and activation of MMP9 on the cell surface are interdependent. J. Cell. Biochem. 108: 272–284, 2009. © 2009 Wiley‐Liss, Inc.     

CD44 acts through RhoA to regulate YAP signaling

The Hippo pathway plays an important role in both physical and pathogenesis processes. As crucial downstream effectors of Hippo pathway, YAP is inhibited by Lats1/2 through phosphorylation. However, upstream signals that regulate the Hippo pathway have been still poorly understood. Here, we found that knockdown of CD44 reduced YAP expression and nuclear localization, but nearly had no effect on its upstream effectors, Mst1 and Lats1. Downregulated CD44 expression also significantly decreased the expression of YAP downstream effectors CTGF, Cyr61 and EDN1 at mRNA level. Our next study showed that knockdown of CD44 inhibited RhoA expression, which was consistent with RhoA knockdown mediated YAP downregulation. Furthermore, we demonstrated that over expression of the constitutively active RhoA (RhoA-V14) could block the YAP expression decrease mediated by CD44 knockdown. Moreover, downregulation of CD44 significantly promoted cell apoptosis and inhibited cell proliferation, cell cycle progression and migration, which were consistent with the effects of RNAi-mediated YAP knockdown in both A549 and HepG2 cells. Overall, data are presented showing that CD44 could act through RhoA signaling to regulate YAP expression and this study also provide new insights into the regulatory mechanisms of the Hippo–YAP pathway.     

Epac1 increases migration of endothelial cells and melanoma cells via FGF2‐mediated paracrine signaling

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N‐sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N‐sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2‐mediated cell–cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM‐induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N‐sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS–FGF2‐mediated cell–cell communication.     

CD44v6对急性白血病细胞HL-60和THP-1增殖和迁移的影响

目的:探讨CD44变异亚型对急性白血病细胞增殖和迁移的影响。方法:选择对数生长期的急性白血病细胞株HL-60、THP-1和慢性白血病细胞株K562,采用荧光定量PCR法检测CD44v6mRNA的表达。通过电转的方法转染CD44v6siRNA到HL-60和THP-1细胞抑制细胞的CD44v6表达,通过western方法检测CD44v6蛋白的抑制情况。将实验分成HL-60+N-siRNA、HL-60+CD44V6-siRNA、THP-1+N-si RNA、THP-1+CD44V6-siRNA共4组,培养24、48、72 h后分别取细胞悬液用台盼蓝染色后计数活细胞数检测细胞的增殖情况;使用Transwell小室培养法观察HL-60和THP-1细胞的迁移率。结果:通过荧光定量PCR方法检测THP-1和HL-60细胞均高表达CD44v6(分别为0.0037±0.0007和0.00292±0.0002),明显高于K562的表达(P0.01);转染后的HL-60和THP-1细胞株中CD44v6蛋白表达水平明显下调,细胞计数结果显示转染CD44v6-siRNA的HL-60和THP-1细胞在24、48和72 h增殖均明显下降。迁移实验结果显示THP-1+N-si RNA和HL-60+N-si RNA细胞的迁移率为17%和23%,与相应对照组相比THP-1+CD44v6-siRNA和HL60+CD44v6-siRNA组细胞24 h迁移率明显下降(分别降至11%和14%)。结论:CD44v6可以通过干预白血病细胞的增殖和迁移能力,参与调解白血病细胞的增殖和髓外进展。     

Cell surface-expressed Thomsen-Friedenreich antigen in colon cancer is predominantly carried on high molecular weight splice variants of CD44.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.     

CD44v6: a target for antibody-based cancer therapy

The human CD44 gene encodes type 1 transmembrane glycoproteins involved in cell-cell and cell-matrix interactions. The structural heterogeneity of the gene products is caused primarily by alternative splicing of at least 10 out of 20 exons. Certain CD44 variant isoforms, in particular those containing CD44 variant domain 6 (CD44v6), have been implicated in tumourigenesis, tumour cell invasion and metastasis. Here we will give an overview of immunohistochemically determined CD44v6 expression in human malignancies (primary epithelial and nonepithelial tumours as well as metastases) and normal tissues, and review several examples of the clinical use of CD44v6-specific antibodies. In nonmalignant tissues, CD44v6 expression is essentially restricted to a subset of epithelia. Intense and homogeneous expression of CD44v6 was reported for the majority of squamous cell carcinomas and a proportion of adenocarcinomas of differing origin, but was rarely seen in nonepithelial tumours. This expression pattern has made CD44v6 an attractive target for antibody-guided therapy of various types of epithelium-derived cancers.Abbreviations CD44 type 1 transmembrane glycoprotein, cell surface receptor for hyaluronate - CD44s (CD44H) standard form of CD44 - CD44v6 splice variant exon 6 of CD44 - CTC common toxicity criteria - 2F10, VFF4, VFF7, VFF18 (BIWA 1), U36, V6B3, HB-256, Var 3.1 monoclonal antibodies targeting the CD44v6 antigen - SCC squamous cell carcinoma     

Alternative splicing regulation by interaction of phosphatase PP2Cgamma with nucleic acid-binding protein YB-1

Kinases and phosphatases participate in precursor messenger RNA (pre-mRNA) splicing regulation, but their precise roles and the identities of their cofactors and substrates remain poorly understood. The human Ser/Thr phosphatase PP2Cgamma promotes spliceosome assembly. We show that PP2Cgamma's distinctive acidic domain is essential for its activity in splicing and interacts with YB-1, a spliceosome-associated factor. Moreover, PP2Cgamma is a phosphoprotein in vivo, and its acidic domain is phosphorylated under splicing conditions in vitro. PP2Cgamma phosphorylation enhances its interaction with YB-1 and is reversed by the phosphatase in cis. PP2Cgamma knockdown leaves constitutive splicing unaffected but inhibits cell proliferation and affects alternative splicing of CD44, a YB-1 target. This effect on splicing regulation is mediated by PP2Cgamma's acidic domain, which is essential to promote inclusion of CD44 exons v4 and v5 in vivo. We propose that PP2Cgamma modulates alternative splicing of specific pre-mRNAs coregulated by YB-1.