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1 Division of Hematology/Oncology, 3 Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, and 4 Department of Pathology, UCLA School of Medicine, Los Angeles, California, and 2 Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, Shanghai, China
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß3, which have been shown to interact with Cyr61 and ILK. The activity of ILK was increased dramatically in these cells. Overexpression of Cyr61 also resulted in the phosphorylation of glycogen synthase kinase-3ß and accumulation and nuclear translocation of ß-catenin, leading to activation of the ß-catenin-TCF/Lef-1 signaling pathway. Furthermore, forced expression of Cyr61 in the glioma cells activated phosphatidylinositol 3'-kinase pathway, resulting in prominent phosphorylation of Akt and the antiapoptotic protein Bad. Cyr61 appears to stimulate several signaling pathways in the development of gliomas. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cyr61 is a member of a growing family of growth factors termed the CCN (Cysteine-rich 61/Connective tissue growth factor/Nephroblastoma overexpressed) gene family that is characterized by a high degree of amino acid sequence homology ranging from 50–90%. This family is composed of Cyr61 (cysteine-rich protein), connective tissue growth factor (9 , 10) , nov (nephroblastoma overexpressed gene; Refs. 11 , 12 ), and Wnt-1-induced secreted protein 1 (WISP-1; Ref. 13 ). All of the members of the CCN gene family possess a secretory signal peptide, indicating that they are secreted proteins. Cyr61 is a cysteine-rich, heparin-binding protein that associates with the cell surface and the extracellular matrix and can interact with various cellular integrins (14, 15, 16) . Expression of Cry61 is induced by a variety of growth factors, hormones, and drug components, including serum, epidermal growth factor, basic fibroblast growth factor, transforming growth factor ß, 17ß-estradiol, muscarinic acetylcholine receptors, and vitamin D3 (17, 18, 19, 20, 21, 22) . Purified Cyr61 protein has been reported to mediate cell adhesion, stimulate chemotaxis, augment growth factor-induced DNA synthesis, foster cell survival, and enhance angiogenesis in vivo (16 , 18 , 19 , 23) .
Several lines of evidence support a role for CCN molecules in tumorigenesis. Elevated expression of avian nov mRNA was found consistently in all of the myeloblastosis-associated virus 1- and myeloblastosis-associated virus 2-induced avian nephroblastomas (11) . The human homologue of avian nov is overexpressed mainly in tumors of predominantly stromal origin, such as Wilms tumors (43) . Consistent with its profibrotic properties, connective tissue growth factor is overexpressed in pancreatic cancers (25) , melanomas (26) , and mammary tumors (27 , 28) . WISP-1 is expressed strongly in the fibrovascular stroma of breast tumors developing in Wnt-1 transgenic mice and primary human colon cancers (13) . Moreover, forced overexpression of WISP-1 in normal rat kidney fibroblasts (NRK-49F) was sufficient to induce their transformation (29) . We and several other groups have shown recently that Cyr61 was overexpressed in breast cancers and might be involved in estrogen-mediated tumor development (22 , 28 , 30 , 31) .
In this study, we found that Cyr61 was highly expressed in primary gliomas and in cell lines derived from high-grade gliomas. Stable expression of Cyr61 under the regulation of a constitutive promoter in U343 cells accelerated cell proliferation in culture, enhanced anchorage-independent cell growth in soft agar, and significantly increased tumorigenicity and vascularization in nude mice. Furthermore, characterization of the oncogenic activity of Cyr61 demonstrated that it might contribute to tumorigenesis through activation of integrin-linked kinase (ILK)-mediated ß-catenin-TCF/Lef and Akt signaling pathways.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA Preparation and Northern Analysis.
Total RNA was isolated from cell lines and patient tissue by using TRIzol reagent (Life Technologies) according to the standard protocol. Cyr61 cDNA probe was labeled with 32P-dCTP using random primers (Life Technologies). Total cellular RNA was separated on 1.2% formaldehyde-agarose gels and was immobilized on a hybond-N+ membrane by standard capillary transfer and UV cross-linking. The membrane was hybridized with the Cyr61 probe by standard protocol and was rehybridized with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase cDNA or ß-actin cDNA to confirm equal loading of the samples.
Cell Transfection and Soft Agar Assays.
The Cyr61 expression constructs were transfected into U343 cells using Lipofectamine (Invitrogen, Carlsbad, CA) as described previously, and transfectants were selected for G418 resistance (500 ìg/ml; Ref. 22
). The selected clones were confirmed to have prominent expression of Cyr61 by Northern and Western blot analysis. For clonogenic assay, cells were plated into 24-well flat-bottomed plates using a two-layer soft agar system with 1 x 103 cells/well in a volume of 400 µl/well as described previously (22)
. After 14 days of incubation, the colonies were counted and measured. All of the experiments were done at least three times using triplicate plates per experimental point.
Cell Migration Assays.
Cell migration assays were performed as described previously (22)
. Cells were allowed to migrate to the underside of the top chamber for 4–8 h. The migratory cells attached to the bottom surface of the membrane were stained with 0.1% crystal violet in 0.1 M borate (pH 9.0) and 2% ethanol for 20 min at room temperature. The stained cells were extracted using extraction buffer (Chemicon). The number of migratory cells per membrane was determined by absorbance at 550 nm.
Flow Cytometric Analysis.
Fluorescence-activated cell sorting (FACS) analysis was performed using LM609 antibody (1:500; Chemicom). After washing the primary antibody with PBS, the cells were incubated with FITC-conjugated antimouse IgG (5 µg/ml) at 4°C for 30 min. The cells again were washed with PBS. FACS sorting was performed using a FACScan (Becton Dickinson, Mountain View, CA), and analysis was performed using CellQuest 2.0 (Becton Dickinson).
Tumorigenicity Assay.
Stably transfected U343/Cyr61 and U343/V cells (1.0 x 105 cells/flank) were injected s.c. into 8-week-old female nude mice. Each animal was injected at two sites in the flanks. The resulting tumors were measured once a week, and tumor volume (mm3) was calculated using the standard formula: length x width x height x 0.5236. Tumors were harvested 8 weeks after injection and individually weighed before fixation. Data were presented as tumor volume (mean ± SD) and tumor weight (mean ± SD). Statistical analysis was performed by computer program software (GraphPad, San Diego, CA) using the Student’s t test.
Cell Proliferation and Cycle Analysis.
For the cell proliferation assay, U343/Cyr61 and U343/V cells were plated into 96-well plates at 2.0 x 103 cells/well and cultured for various durations; cell numbers were measured by MTT assay according to the protocol provided by Roche Molecular Biochemicals (Basel, Switzerland). For cell cycle analysis, cells were plated in 100-mm dishes and trypsinized when they reached 60% confluence. After washing twice with PBS, cells were fixed in 70% ice-cold ethanol overnight. After staining with propidium iodide, samples were analyzed using a FACScan.
Purification of the Cyr61 Protein from Sf9 Cells.
The pcDNA61 was cloned into the transfer plasmid pVL1392 (BD Biosciences, Franklin Lakes, NJ), and baculovirus-encoding Cyr61 was generated using the BaculoGold system (BD Biosciences). Sf9 insect cells were maintained in TNM-FH Insect Medium (BD Biosciences). His6-tagged Cyr61 protein was produced in Sf9 cells by infecting these cells with Cyr61-baculovirus according to Baculovirus Expression Vector System (BD Biosciences). The Cyr61 was purified from the condition media using a two-step purification method. First, the Cyr61 was markedly enriched after application of the condition media to HiTrap Heparin HP columns (Amersham Bioscience, Piscataway, NJ), and the eluted fraction then was purified using His-Bind Resin (Novagen, Madison, WI). Finally, the eluted fraction from the His-Bind Resin column was applied to the PD-10 column (Amersham Bioscience) to exchange the eluting buffer, which includes imidazole for isotonic PBS.
Real-Time PCR Assay.
Primers and probes for Cyr61 and ß-actin genes were designed using PRIMER3 software (http://www.genome.wi.nit.edu/cgi-bin/primer/primer3_www.cgi). Primers were purchased from Life Technologies, and probes were from Perkin-Elmer Applied Biosystems (Boston, MA). Amplification reactions contained 5 µl of cDNA, 12.5 µl of the Universal Taqman 2x PCR mastermix (Applied Biosystems, Foster City, CA), and 2.5 µl of each of the specific primers and the probe. Primer and TaqMan probe concentrations in the final volume of 25 µl were 500 nM and 100 nM, respectively. All of the reactions were performed in triplicate in an iCycler iQ system (Bio-Rad, Hercules, CA), and the thermal cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min.
ILK and Akt Kinase Assay.
The ILK kinase assays were performed using a rabbit immunoaffinity-purified ILK antibody (Upstate Biotechnology, Lake Placid, NY) and myelin basic protein as a substrate. U343/Cyr61 and U343/V cell lysates were centrifuged at 10,000 x g for 5 min, and protein concentrations were determined using a modified Bradford assay protocol (Bio-Rad). The supernatants (100 µg) were precipitated for 12 h at 4°C with protein A-agarose beads precoated with saturating amounts of the antibody. Immunoprecipitated proteins on beads were washed twice with 1 ml of lysis buffer and twice with kinase buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 5 mM MnCl2, and 1 mM DTT]. The beads then were resuspended in 40 µl of kinase buffer containing the protein substrate (2 µg of myelin basic protein), 10 mM ATP, and 5 mCi of [
-32P]ATP (6,000 Ci/mmol; 1 Ci = 37 GBq; Amersham Pharmacia Biotech). The samples were incubated for 30 min at 30°C with occasional mixing, boiled in polyacrylamide gel sample buffer containing SDS, and separated by electrophoresis. Phosphorylated proteins were quantified after exposure to autoradiographic film. The Akt kinase assay was analyzed using IP/kinase assay following the manufacturer’s protocol (Cell Signaling Technology, Beverly, MA). Briefly, U343/Cyr61 and U343/V cell extracts (200 ìl) were incubated 2 h with immobilized Akt 1G1 monoclonal antibody. After extensive washing, the kinase reaction was performed in the presence of 200 µM of cold ATP and glycogen synthase kinase-3ß (GSK-3ß) substrate. Phosphorylation of GSK-3ß was measured by Western blot analysis using phospho-GSK-3ß antibody.
Immunohistochemical and Immunofluorescence Staining.
Immunohistochemical staining for Cyr61 was performed with polyclonal antiserum from Santa Cruz Biotechnology (Santa Cruz, CA). Heat-induced epitope retrieval was performed with a pressure cooker and Tris buffer (pH 9.0) for 2 min. Localization was performed with Dako Envision (Dako, Carpinteria, CA) conjugated to horseradish peroxidase, followed by the diaminobenzidine reaction. Negative controls consisted of substitution of the primary antiserum with normal rabbit serum at the same dilution. For immunofluorescence staining of ß-catenin, cells were cultured on a four-well chamber Lab-Tek slide (Nunc, Naperville, IL). After 8 h, cells were fixed in 3% paraformaldehyde in PBS at room temperature for 8 min, permeabilized with 0.3% NP40 in PBS for another 8 min, washed in PBS, and incubated with rabbit polyclonal ß-catenin antibody (1 ìg/ml; Santa Cruz Biotechnology) at 4°C overnight. The immunoreactivity was revealed using Rhodamine Red-X-conjugated rabbit IgG (Molecular Probes, Eugene, OR). The cells were examined under a Nikon fluorescence microscope (Image Systems, Columbia, MD).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% of the brain tumors (Fig. 1B)
. The highest levels were found in the GBM, which are the most malignant gliomas. Twenty-seven of 40 (68%) GBMs overexpressed Cyr61 compared with only 4 of 19 (21%) astrocytomas and 1 of 7 (14%) oligodendrogliomas. Four of 16 (25%) meningiomas and 2 of 16 (12.5%) other subtypes of brain tumors also had prominent level of Cyr61.
To determine whether overexpression of Cyr61 mRNA was associated with an increased protein level of Cyr61, proteins were extracted for Western blot analysis from normal brain tissue and several human primary glioma samples with different expression levels of Cyr61 mRNA. The results showed that expression of Cyr61 at the protein level paralleled expression level of Cyr61 mRNA measured by real-time PCR in normal brain and human gliomas (Fig. 1C)
.
Immunohistochemical staining was evaluated in three GBM tumors and three normal brain samples. The normal brain tissue was negative for Cyr61 except for sparse cytoplasmic staining in a few glial cells and neurons. In contrast, strong staining for Cyr61 occurred in the neoplastic astrocytoma cells (Fig. 1D)
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Cyr61 Stimulates Cell Growth in Culture, Colony Formation in Soft Agar, and Cell Migration in Glioma Cells.
We first examined the effects of Cyr61 protein on the growth of U343 cells by adding Cyr61 protein into the cultures. The Cyr61 protein stimulated the growth of U343 cells (Fig. 1A)
. Our earlier study showed that forced expression of Cyr61 promoted cell proliferation and anchorage-independent growth in the normal breast cell line MCF-12A and the breast cancer cell line MCF-7 (22)
. To study whether similar activities occurred in glioma cells, U343 cells, which have low motility and invasiveness, were stably transfected with pcDNA61 containing either full-length Cyr61 or empty vector pcDNA3.1 as a control (32
, 33)
. As expected, Cyr61 was highly expressed in pcDNA61 vector-transfected cells (U343/Cyr61–1 and U343/Cyr61–2) but not in the pcDNA3.1-transfected cells (U343/V) as examined by Northern (data not shown) and Western blot analysis (Fig. 2B)
. Western blot analysis was used to compare expression of Cyr61 in Cyr61-transfected cells (U343/Cyr61) with that observed in the human brain tumor samples. As shown in Fig. 1C
, the amount of Cyr61 produced in the U343/Cyr61 was comparable with the level of Cyr61 in those human gliomas that highly expressed Cyr61 mRNA. The ability of Cyr61 to enhance growth of cultured cells was investigated by MTT assay. When cells were plated at a density of 1.5 x 105 in RPMI supplemented with 10% fetal bovine serum, the rate of growth was 2.5-fold greater for the U343/Cyr61 compared with the U343/V control cells. When these cells were grown in RPMI with 0.5% fetal bovine serum, the difference in cell growth rate was even greater (mean, 3.8- ± 0.8-fold; P < 0.05; Fig. 2D
). Cell cycle analysis showed that U343/Cyr61 cells had a much lower percentage of cells in the G1 phase (56%) and a higher percentage of cells in the S phase (36%) compared with U343/V cells (82% and 16%, respectively; Fig. 2E
).
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). The colonies formed by U343/Cyr61 also were substantially larger than those formed by U343/V (data not shown).
To determine whether increased expression of Cyr61 enhanced directed cell movement, migration assays of U343/V and U343/Cyr61 were performed in vitronectin-coated Boyden chambers. As shown in Fig. 3
, the Cyr61 stably transfected U343/Cyr61 cells had a significantly increased migration compared with the empty vector-transfected U343/V cells (P < 0.05). Cyr61 antibody significantly inhibited migration of the U343/Cyr61 cells (P < 0.05), suggesting that Cyr61 promoted migration of U343 cells, and this stimulation depended at least in part on Cyr61 being extracellular (Fig. 3)
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and ß subunits. These receptors play a role in cell migration, proliferation, and gene transcription and can affect growth and invasion of cancer cells (34, 35, 36)
. Previous studies have shown that CYR61 interacts with various integrins, including ß3, 6ß3, ß5, and IIß3, which can enhance disparate activities, such as cell migration, angiogenesis, and tumorigenesis (14, 15, 16
, 37)
. To determine whether overexpression of Cyr61 alters the level of integrin expression in U343 cells, we performed an RNase protection assay to quantify simultaneously 10 mRNA species of distinct integrins from U343/V and U343/Cyr61 cells. Seven of the 10 integrins were expressed more abundantly in the U343/Cyr61 cells compared with U343/V control cells, including integrin and ß1, which were elevated dramatically in the Cyr61 stably transfected cells (Fig. 4A)
. In contrast, glyceraldehyde-3-phosphate dehydrogenase (control) was expressed equivalently in both types. We also used immunofluorescent staining and flow cytometry to examine expression of the integrin ß3, a known receptor of Cyr61 (16)
. It was expressed at a higher level on the cellular surface of U343/Cyr61 cells compared with U343/V cells (Fig. 4B)
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, protein levels of ILK in U343/Cyr61 cells were only slightly higher than those in either the U343 or U343/V cells. In contrast, the ILK activity in both U343/Cyr61 sublines was increased dramatically compared with those in either U343 or U343/V control cells (Fig. 4D)
. These results together showed that overexpression of Cyr61 in U343/Cyr61 cells increased expression of distinct integrins, leading to the activation of the ILK.
Cyr61 Stimulates Nuclear Translocation and Transcriptional Activity of ß-Catenin.
Previous studies have shown that ILK can activate the ß-catenin-TCF/Lef signaling pathway perhaps by phosphorylating GSK-3ß, which inhibits its activity, allowing accumulation and translocation of ß-catenin into the nucleus (40
, 41)
. Furthermore, ILK can up-regulate cyclin D1, also through phosphorylation and inactivation of GSK-3ß (42)
. To assess whether overexpression of Cyr61, which leads to stimulation of ILK activity, altered the ß-catenin signaling pathway, we performed Western blot analysis to evaluate the expression of ß-catenin and GSK-3ß in the Cyr61 stably transfected cells (U343/Cyr61–1 and U343/Cyr61–2). Protein levels of ß-catenin (Fig. 5A)
were elevated in the U343/Cyr61 cells compared with the U343/V cells. Whereas the level of expression of total GSK-3ß was similar in the two cell types, phosphorylated GSK-3ß was markedly increased in the U343/Cyr61 cells (Fig. 5B)
. To determine whether the increase of ß-catenin can alter its cellular localization, we examined the cells by immunofluorescence. Overexpression of Cyr61 altered dramatically the subcellular localization of ß-catenin. ß-Catenin remained in the cytoplasm and cell membrane with minimal nuclear localization in the empty vector-transfected U343/V cells; in contrast, the Cyr61 stably transfected U343/Cyr61 cells showed prominent nuclear localization of ß-catenin (Fig. 5C)
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, the complex of ß-catenin/TCF-4 was detected easily in both sublines of U343/Cyr61 cells but rarely was found in U343/V cells.
Previous studies have shown that the NH2 terminus of TCF is required for binding to ß-catenin and that TCF mutant proteins lacking N-terminal sequences retain DNA binding activity but function in a dominant-negative fashion (46)
. Hence, we sought to test the effects of such a dominant-negative TCF-4 protein on the Cyr61-overexpressing U343/Cyr61 cells. These cells were transfected with dominant-negative TCF-4 (pcDNA3/
N-TCF) and pMACS Kk (codes for cell surface expressed, truncated H-2Kk) or with pMACS Kk alone. Cells expressing the truncated H-2Kk were collected using beads coated with an antibody to H-2Kk. Expression of the dominant-negative TCF-4 in U343/Cyr61 cells (U343/Cyr61-N-TCF) caused their severe growth arrest compared with control cells transfected with only pMACS Kk(U343/Cyr61-Kk) as measured by MTT (Fig. 5E)
and colony formation assay (Fig. 5F)
. In the soft agar assay, cells expressing the dominant-negative TCF-4 formed 2.5-fold fewer colonies that also were markedly smaller than the colonies formed by U343/Cyr61 cells (Fig. 5F
; data for colony size not shown).
We also determined whether the elevated level of endogenous cyclin D1 expression in U343/Cyr61 cells could be inhibited by transient expression of a dominant-negative TCF-4. The dominant-negative TCF-4 reduced dramatically the prominent expression of cyclin D1 induced by overexpression of Cyr61 in U343/Cyr61 cells (Fig. 5G)
. Taken together, these results suggest that Cyr61 stimulates ß-catenin-TCF/Lef signaling to enhance expression of proteins associated with cell proliferation. Reduction of levels of cyclin D1 caused by disrupting signaling from ß-catenin to TCF probably contributed to the slowing of cell growth because it is one of the rate-limiting factors for progression through the G1 phase of the cell cycle (47)
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Cyr61 Activates Akt through PI3k.
Previous studies demonstrated that ILK phosphorylated directly Akt on serine 473 in vitro via the binding of PtdIns (3, 4, 5)
with a PH-like domain of ILK; in contrast, a kinase-deficient form of ILK inhibited severely Akt serine 473 phosphorylation in vivo (40)
. Therefore, we wanted to determine whether overexpression of Cyr61 could regulate phosphorylation and activation of Akt in U343 cells. Because phosphorylation of Akt on serine 473 is required for its activation, we monitored this site using an antibody that reacted specifically with phosphorylated serine 473. Akt was phosphorylated constitutively on serine 473 in Cyr61 stably transfected U343/Cyr61 cells (Fig. 6A)
. Therefore, because we had shown that ILK activity was stimulated constitutively in U343/Cyr61 cells, Cyr61 might regulate the serine 473 of Akt through activation of ILK. Because others have shown that engagement of integrins stimulated PI3k activity leading to activation of Akt (48
, 49)
and that GSK-3ß activity can be regulated by Akt in a PI3k-dependent manner (50)
, we measured kinase activity of Akt using GSK-3ß as a substrate. Akt 1G1 monoclonal antibody was used selectively to immunoprecipitate Akt, and the resulting immunoprecipitate then was incubated with the GSK-3ß fusion protein in the presence of ATP. Overexpression of Cyr61 increased kinase activity of Akt and resulted in phosphorylation of GSK-3 (Fig. 6B)
. To test whether these events were linked to PI3k activation, we treated U343/Cyr61 and U343/V cells with two distinct PI3k inhibitors, wortmannin and LY294002. Both PI3k inhibitors blocked Cyr61-induced increase in the Akt activity (Fig. 6B)
, suggesting that activation of Akt by Cyr61 is mediated by a PI3k-dependent mechanism.
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, suggesting that Cyr61 also may be involved in the Akt-Bad antiapoptotic signaling pathway.
Cyr61 Promotes Tumorigenicity and Vascularization in Nude Mice.
Our previous studies showed that stable expression of Cyr61 under the regulation of a constitutive promoter in the breast cancer cell MCF-7 significantly increased its tumorigenicity in nude mice. Moreover, overexpression of Cyr61 in normal breast cells (MCF-12A) induced their transformation and formed tumors in nude mice (22)
. On the basis of our in vitro studies indicating that overexpression of Cyr61 promoted anchorage-independent clonogenic proliferation in soft agar and cell migration, we examined whether forced expression of Cyr61 in U343 cells (U343/Cyr61) could enhance their ability to form tumors and stimulate neovascularization in nude mice compared with their controls. These cells were injected s.c. into 8-week-old nude mice, and tumor growth was measured once a week. The U343/Cyr61 cells (sublines 1 and 2), expressing Cyr61 at a high level, developed tumors with a significantly shorter latency (2 weeks after injection; P < 0.05) and with a markedly larger size during the 8 weeks of observation compared with the tumors formed by the control U343/V cells (Fig. 7, A, B, and C)
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DISCUSSION |
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In this investigation, we have found that Cyr61 assumed a similar role in gliomas as in breast cancers. It was expressed prominently in the highly tumorigenic astrocytoma cell lines U87, U373, and T98G and expressed very weakly in the less tumorigenic cell U343. Furthermore, overexpression of Cyr61 occurred in 68% of the highly malignant GBM compared with 22% of the astrocytomas and 14% of the oligodendrogliomas, suggesting that Cyr61 might be involved in tumor progression of astrocytomas and oligodendrogliomas into GBM.
The role of Cyr61 in the growth of gliomas was evaluated in several experimental models. Its forced expression in U343 cells (U343/Cyr61) markedly stimulated their proliferation in liquid culture and in an anchorage-independent manner in soft agar and significantly enhanced their tumorigenicity and vascularization in vivo. These cells developed larger, more vascularized tumors in nude mice. These cells also migrated more readily. All of these properties are reminiscent of our earlier observations in Cyr61 stably transfected MCF-7 breast cancer cells (22) .
An important finding of this study is the characterization of Cyr61 as a tumorigenic enhancer of gliomas through activation of Akt and stabilization of ß-catenin and its nuclear translocation, resulting in stimulating ß-catenin-TCF/Lef signaling pathway. Overexpression of Cyr61 in the U343 cells resulted in enhanced levels of distinct integrins, including ß3 and ß1, which are known to be the receptors of Cyr61 (14
, 16)
. We concurrently found that the Cyr61-expressing glioma cells activated the PI3k pathway through ILK. To our knowledge, this is the first report showing that Cyr61 can up-regulate various integrins and activate ILK. Overexpression of Cyr61 in U343 cells also inhibited the activity of GSK-3ß by its phosphorylation and induced nuclear translocation of ß-catenin. Both of these effects were most likely through the activation of ILK. A previous study showed that activated ILK could inhibit GSK-3ß by directly phosphorylating GSK-3ß, resulting in translocation of ß-catenin into the nucleus in mammary epithelial cells (41)
. ß-Catenin plays a signaling role as a key mediator in the Wnt signaling pathway. The ultimate mediator of this pathway is the nuclear complex of ß-catenin acting as a coactivator with TCF/Lef transcription factors to stimulate transcription of a variety of target genes (54)
. These genes often are associated with stimulating cell proliferation, including cyclin D1, c-myc, c-Jun, Fra-1, urokinase-type plasminogen activator receptor, and E-cadherin (55, 56, 57)
. Our studies showed that forced expression of Cyr61 in glioma cells up-regulated transcription of cyclin D1 but not the c-myc (data not shown). Whether Cyr61 results in the up-regulation of other TCF/Lef-1 target genes remains to be elucidated.
WISP-1, another CNN family member closely related to Cyr61, was found recently to be a Wnt-1- and ß-catenin-responsive oncogene (29) . Transfected and overexpressed WISP-1 in a normal rat kidney fibroblast cell line (NRK-49F) induced their morphologic transformation, accelerated their cell growth, enhanced their saturation density in vitro, and permitted the cells to form tumors in nude mice. Considering that Cyr61 has four identical structural domains and is related closely to WISP-1, both of these genes may be involved in similar signaling pathways in the development and progression of tumors.
Another interesting finding of this study is that Cyr61 can activate Akt and inhibit the apoptotic effector Bad by its phosphorylation, suggesting that Cyr61 may be involved in more than one signaling pathway. Akt is a key regulator of many intracellular processes implicated in progression of brain tumors (5 , 58) . Studies have found that Akt-dependent phosphorylation of Bad resulted in its cytosolic sequestration by the ô form of the 14-3-3 proteins and prevented its binding to the survival factor Bcl-XL at intracellular membrane sites (60) . Because Bad exerts its death-promoting effects by heterodimerizing with and inhibiting the death antagonist Bcl-XL, phosphorylation of Bad by Akt can preclude its binding to the membrane-anchored Bcl-XL, leading to increased cell survival (61) . Thus, phosphorylation of Bad by Akt is a possible mechanism by which Cyr61 delivers a survival signal, leading to the inhibition of apoptosis. Interestingly, WISP-1 also was shown recently to activate the Akt signaling pathway and up-regulate the antiapoptotic Bcl-XL protein (62) ..
We also have demonstrated that Cyr61-dependent activation of Akt can phosphorylate GSK-3ß, leading to its inactivation (Fig. 6B)
. Thus, the nuclear accumulation of ß-catenin caused by overexpression of Cyr61 may result from inactivation of GSK-3ß by Akt. Several studies have found that Akt could decrease GSK-3ß activity, but the change was not sufficient to cause translocation of ß-catenin into the nucleus in the absence of Wnt signaling (63
, 64)
. In contrast, several other studies have found that the Akt-mediated phosphorylation and inhibition of GSK-3ß led to accumulation and nuclear translocation of ß-catenin (65, 66, 67, 68, 69)
. ILK has been reported to phosphorylate directly the serine 473 of Akt, resulting in phosphorylation and inhibition of GSK-3ß, stimulating nuclear translocation of ß-catenin and activation of TCF/Lef transcription factor (40
, 42
, 59
, 70) . Furthermore, Fukumoto et al. (66)
have shown that activated Akt bound to the Axin-GSK-3ß complex in the presence of Dishevelled resulted in phosphorylation of GSK-3ß and increased free ß-catenin, which could be blocked by a dominant-negative Akt.
We showed that overexpression of Cyr61 enhanced tumorigenicity of gliomas cells. Overexpression of Cyr61 results in up-regulation of distinct integrins and activation of ILK mediated by PI3k. Activated ILK inhibits GSK-3ß activity by either phosphorylating it directly or first phosphorylating and activating AKT, which then phosphorylates and inactivates GSK-3ß. This causes the accumulation of ß-catenin in the cytoplasm, resulting in its translocation into nucleus, where it binds to the transcription factors Tcf/Lef, increasing transcriptional activation of cyclin D1 and other target genes. Meanwhile, activated ILK can directly phosphorylate and activate Akt, resulting in inhibition of apoptosis by phosphorylating and suppressing the proapoptotic protein Bad.
In summary, our data indicate that overexpression of Cyr61 may be involved in development of gliomas through activation of the ILK-mediated ß-catenin-TCF/Lef and the Akt signaling pathways. This comprehensive elucidation of Cyr61 signaling in brain tumors is an important step to explore the mechanism and function of this protein in the development of gliomas.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Dong Xie, Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048. Phone: 310-423-7740; Fax: 310-423-0225; E-mail: xied{at}ucla.edu
Received 3/16/03. Revised 1/14/04. Accepted 1/15/04.
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 C. T. Walsh, J. Radeff-Huang, R. Matteo, A. Hsiao, S. Subramaniam, D. Stupack, and J. H. Brown Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61 FASEB J, November 1, 2008; 22(11): 4011 - 4021. [Abstract] [Full Text] [PDF]
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 P. C. McDonald, A. B. Fielding, and S. Dedhar Integrin-linked kinase - essential roles in physiology and cancer biology J. Cell Sci., October 1, 2008; 121(19): 3121 - 3132. [Abstract] [Full Text] [PDF]
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 C. T. Walsh, D. Stupack, and J. H. Brown G Protein-Coupled Receptors Go Extracellular: RhoA Integrates the Integrins Mol. Interv., August 1, 2008; 8(4): 165 - 173. [Abstract] [Full Text] [PDF]
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 M.-T. Lin, I-H. Kuo, C.-C. Chang, C.-Y. Chu, H.-Y. Chen, B.-R. Lin, M. Sureshbabu, H.-J. Shih, and M.-L. Kuo Involvement of Hypoxia-inducing Factor-1{alpha}-dependent Plasminogen Activator Inhibitor-1 Up-regulation in Cyr61/CCN1-induced Gastric Cancer Cell Invasion J. Biol. Chem., June 6, 2008; 283(23): 15807 - 15815. [Abstract] [Full Text] [PDF]
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 A. Kumar, A. Zloza, R. T. Moon, J. Watts, A. R. Tenorio, and L. Al-Harthi Active {beta}-Catenin Signaling Is an Inhibitory Pathway for Human Immunodeficiency Virus Replication in Peripheral Blood Mononuclear Cells J. Virol., March 15, 2008; 82(6): 2813 - 2820. [Abstract] [Full Text] [PDF]
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Y.-Z. Deng, P.-P. Chen, Y. Wang, D. Yin, H. P. Koeffler, B. Li, X.-J. Tong, and D. Xie Connective Tissue Growth Factor Is Overexpressed in Esophageal Squamous Cell Carcinoma and Promotes Tumorigenicity through -Catenin-T-cell Factor/Lef Signaling J. Biol. Chem., December 14, 2007; 282(50): 36571 - 36581. [Abstract] [Full Text] [PDF]
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M.-T. Lin, C.-C. Chang, B.-R. Lin, H.-Y. Yang, C.-Y. Chu, M.-H. Wu, and M.-L. Kuo Elevated Expression of Cyr61 Enhances Peritoneal Dissemination of Gastric Cancer Cells through Integrin {alpha}2beta1 J. Biol. Chem., November 23, 2007; 282(47): 34594 - 34604. [Abstract] [Full Text] [PDF]
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B.-R. Lin, C.-C. Chang, L.-R. Chen, M.-H. Wu, M.-Y. Wang, I-H. Kuo, C.-Y. Chu, K.-J. Chang, P.-H. Lee, W.-J. Chen, et al. Cysteine-Rich 61 (CCN1) Enhances Chemotactic Migration, Transendothelial Cell Migration, and Intravasation by Concomitantly Up-Regulating Chemokine Receptor 1 and 2 Mol. Cancer Res., November 1, 2007; 5(11): 1111 - 1123. [Abstract] [Full Text] [PDF]
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 Y. Wang, D.-P. Liu, P.-P. Chen, H. P. Koeffler, X.-J. Tong, and D. Xie Involvement of IFN Regulatory Factor (IRF)-1 and IRF-2 in the Formation and Progression of Human Esophageal Cancers Cancer Res., March 15, 2007; 67(6): 2535 - 2543. [Abstract] [Full Text] [PDF]
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 Y. Hong, K. S. Ho, K. W. Eu, and P. Y. Cheah A Susceptibility Gene Set for Early Onset Colorectal Cancer That Integrates Diverse Signaling Pathways: Implication for Tumorigenesis Clin. Cancer Res., February 15, 2007; 13(4): 1107 - 1114. [Abstract] [Full Text] [PDF]
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J. Jiang, X. Chen, J. Shen, Y. Wei, T. Wu, Y. Yang, H. Wang, H. Zong, J. Yang, S. Zhang, et al. beta1,4-Galactosyltransferase V Functions as a Positive Growth Regulator in Glioma J. Biol. Chem., April 7, 2006; 281(14): 9482 - 9489. [Abstract] [Full Text] [PDF]
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N. Nguyen, A. Kuliopulos, R. A. Graham, and L. Covic Tumor-Derived Cyr61(CCN1) Promotes Stromal Matrix Metalloproteinase-1 Production and Protease-Activated Receptor 1-Dependent Migration of Breast Cancer Cells. Cancer Res., March 1, 2006; 66(5): 2658 - 2665. [Abstract] [Full Text] [PDF]
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 D. Mahadevan, C. Spier, K. Della Croce, S. Miller, B. George, C. Riley, S. Warner, T. M. Grogan, and T. P. Miller Transcript profiling in peripheral T-cell lymphoma, not otherwise specified, and diffuse large B-cell lymphoma identifies distinct tumor profile signatures Mol. Cancer Ther., December 1, 2005; 4(12): 1867 - 1879. [Abstract] [Full Text] [PDF]
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U. R. Pendurthi, T. T. Tran, M. Post, and L. V. M. Rao Proteolysis of CCN1 by Plasmin: Functional Implications Cancer Res., November 1, 2005; 65(21): 9705 - 9711. [Abstract] [Full Text] [PDF]
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T. H. Barker, G. Baneyx, M. Cardo-Vila, G. A. Workman, M. Weaver, P. M. Menon, S. Dedhar, S. A. Rempel, W. Arap, R. Pasqualini, et al. SPARC Regulates Extracellular Matrix Organization through Its Modulation of Integrin-linked Kinase Activity J. Biol. Chem., October 28, 2005; 280(43): 36483 - 36493. [Abstract] [Full Text] [PDF]
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S. Gery, D. Xie, D. Yin, H. Gabra, C. Miller, H. Wang, D. Scott, W. S. Yi, M. L. Popoviciu, J. W. Said, et al. Ovarian Carcinomas: CCN Genes Are Aberrantly Expressed and CCN1 Promotes Proliferation of these Cells Clin. Cancer Res., October 15, 2005; 11(20): 7243 - 7254. [Abstract] [Full Text] [PDF]
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M. Bredel, C. Bredel, D. Juric, G. R. Harsh, H. Vogel, L. D. Recht, and B. I. Sikic Functional Network Analysis Reveals Extended Gliomagenesis Pathway Maps and Three Novel MYC-Interacting Genes in Human Gliomas Cancer Res., October 1, 2005; 65(19): 8679 - 8689. [Abstract] [Full Text] [PDF]
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 Y. Jin, H. P. Kim, E. Ifedigbo, L. F. Lau, and A. M. K. Choi Cyr61 Protects against Hyperoxia-Induced Cell Death via Akt Pathway in Pulmonary Epithelial Cells Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 297 - 302. [Abstract] [Full Text] [PDF]
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M.-T. Lin, C.-Y. Zuon, C.-C. Chang, S.-T. Chen, C.-P. Chen, B.-R. Lin, M.-Y. Wang, Y.-M. Jeng, K.-J. Chang, P.-H. Lee, et al. Cyr61 Induces Gastric Cancer Cell Motility/Invasion via Activation of the Integrin/Nuclear Factor-{kappa}B/Cyclooxygenase-2 Signaling Pathway Clin. Cancer Res., August 15, 2005; 11(16): 5809 - 5820. [Abstract] [Full Text] [PDF]
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 D. Zhou, D. J. Herrick, J. Rosenbloom, and B. Chaqour Cyr61 mediates the expression of VEGF, {alpha}v-integrin, and {alpha}-actin genes through cytoskeletally based mechanotransduction mechanisms in bladder smooth muscle cells J Appl Physiol, June 1, 2005; 98(6): 2344 - 2354. [Abstract] [Full Text] [PDF]
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J. M. Nigro, A. Misra, L. Zhang, I. Smirnov, H. Colman, C. Griffin, N. Ozburn, M. Chen, E. Pan, D. Koul, et al. Integrated Array-Comparative Genomic Hybridization and Expression Array Profiles Identify Clinically Relevant Molecular Subtypes of Glioblastoma Cancer Res., March 1, 2005; 65(5): 1678 - 1686. [Abstract] [Full Text] [PDF]
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C. Y.F. Yau, J. J. Wheeler, K. L. Sutton, and D. W. Hedley Inhibition of Integrin-Linked Kinase by a Selective Small Molecule Inhibitor, QLT0254, Inhibits the PI3K/PKB/mTOR, Stat3, and FKHR Pathways and Tumor Growth, and Enhances Gemcitabine-Induced Apoptosis in Human Orthotopic Primary Pancreatic Cancer Xenografts Cancer Res., February 15, 2005; 65(4): 1497 - 1504. [Abstract] [Full Text] [PDF]
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 M. Hagedorn, S. Javerzat, D. Gilges, A. Meyre, B. de Lafarge, A. Eichmann, and A. Bikfalvi Accessing key steps of human tumor progression in vivo by using an avian embryo model PNAS, February 1, 2005; 102(5): 1643 - 1648. [Abstract] [Full Text] [PDF]
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W. Chien, T. Kumagai, C. W. Miller, J. C. Desmond, J. M. Frank, J. W. Said, and H. P. Koeffler Cyr61 Suppresses Growth of Human Endometrial Cancer Cells J. Biol. Chem., December 17, 2004; 279(51): 53087 - 53096. [Abstract] [Full Text] [PDF]
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N. Cordes Overexpression of Hyperactive Integrin-Linked Kinase Leads to Increased Cellular Radiosensitivity Cancer Res., August 15, 2004; 64(16): 5683 - 5692. [Abstract] [Full Text] [PDF]
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