Cyr61, a member of CCN family, is a tumor suppressor in non-small cell lung cancer.

Cysteine-rich protein 61 (Cyr61) is a member of a family of growth factor-inducible immediate-early genes. It regulates cell adhesion, migration, proliferation, and differentiation and is involved in tumor growth. In our experiments, the role of Cyr61 in non-small cell lung cancer (NSCLC) was examined. Expression of Cyr61 mRNA was decreased markedly in four of five human lung tumor samples compared with their normal matched lung samples. NSCLC cell lines NCI-H520 and H460, which have no endogenous Cyr61, formed 60-90% fewer colonies after being transfected with a Cyr61 cDNA expression vector than cells transfected with the same amount of empty vector. After stable transfection of a Cyr61 cDNA expression vector, proliferation of both H520-Cyr61 and H460-Cyr61 sublines decreased remarkably compared with the cells stably transfected with empty vector. The addition of antibody against Cyr61 partially rescued the growth suppression of both H520-Cyr61 and H460-Cyr61 cells. Cell cycle analysis revealed that both H520-Cyr61 and H460-Cyr61 cells developed G(1) arrest, prominently up-regulated expression of p53 and p21(WAF1), and had decreased activity of cyclin-dependent kinase 2. The increase of pocket protein pRB2/p130 was also detected in these cells. Notably, both of the Cyr61-stably transfected lung cancer cell lines developed smaller tumors than those formed by the wild-type cells in nude mice. Taken together, we conclude that Cyr61 may play a role as a tumor suppressor in NSCLC.

mous lung cancer) and H460 (large cell lung cancer) were plated in 100-mm dishes and transfected with Cyr61 cDNA cloned into a pcDNA3.1 expression vector by using GenePorter transfection reagent (GTS, Inc.) according to the protocol provided by GTS. After 48 h, the cells were replated in the medium containing 400 g/ml G418. Two weeks later, well separated colonies were isolated and plated into 24-well plates. The expression of Cyr61 was detected by either Northern (H520 cells) or Western (H460 cells) blot. Neomycin-resistant cells (Neo cells) were obtained by transfecting them with empty pcDNA3.1 vector.
Colony Formation Assay-The H520 or H460 cells were split evenly into two 100-mm dishes. After growing to ϳ60% confluence, cells were transfected with 8 g of either pcDNA3.1-Cyr61 or pcDNA3.1 empty vector. After 48 h, cells were resuspended in the medium containing 400 g/ml G418 and replated into 12-well plates. After treatment with G418 for 2 weeks, the cells were stained with 0.1% crystal violet to assess colony formation. Colonies containing more than 40 cells were counted.
Cell Proliferation Assay-The cells, which had been stably transfected with either Cyr61 (H520-Cyr61 and H460-Cyr61) or pcDNA3.1 empty vector, which contains the neomycin-resistant gene (H520-Neo and H460-Neo), were plated into 96-well plates at 3.0 ϫ 10 3 cells/well. After culturing for various durations, cell numbers were measured by MTT assay according to the protocol provided by Roche Molecular Biochemicals. To examine the effect of Cyr61 antibody (14) on the growth of the cells, the antibody was added at a ratio of 1:50 (v/v) into the wells after the cells were plated as described above. Two days later, the growth of the cells was assessed by MTT assay, and the cell number was calculated according to the standard curve.
Cell Cycle Analysis-Cells were plated in 100-mm dishes and trypsinized when they reached 60% confluence. After washing twice with phosphate-buffered saline, cells were fixed in 70% ice-cold ethanol overnight. After staining with propidium iodide, samples were analyzed by flow cytometry.
Immunoprecipitation and in Vitro Kinase Assay-Cell lysates for immunoprecipitation were made as described above. Endogenous CDK2 was precipitated from an equal amount of total protein using anti-CDK2 antibody (Santa Cruz, sc-163) and protein A/G-Sepharose (Santa Cruz, sc-2003) at 4°C for 1 h. The Sepharose beads were washed three times with protease containing RIPA buffer. Kinase activity was assayed by using histone H1 (Roche Molecular Biochemicals) as the substrate in a 10-l mixture containing 20 mM Tris-HCl, pH 7.5, 75 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, 20 M ATP, 2 g of histone H1, and 5 Ci of [␥-32 P]ATP. The mixture was incubated at 30°C for 15 min and then stopped by adding 10 l of 2ϫ Laemmli loading buffer. Labeled substrate was separated from free [␥-32 P]ATP by 4 -15% SDSpolyacrylamide gel electrophoresis. The gel was dried and exposed to x-ray film (20).
Tumorigenesis Assay-H520-Cyr61 and H520-Neo cells (2 ϫ 10 6 ) were injected subcutaneously in the left and right flank, respectively, of 5-week-old nude mice. Identical experiments were repeated using H460-Cyr61 and H460-Neo cells. Five weeks after injection, the mice were sacrificed, and the tumors were weighed.

Expression of Cyr61 in Normal Lung and NSCLC Samples and Cell
Lines-Expression of Cyr61 in various normal tissues and cell lines was examined by Northern blot (Fig. 1). Cyr61 was highly expressed in normal lung and heart; much lower levels were present in placenta, liver, skeletal muscle, and kidney; and negligible levels were in brain and pancreas (Fig.  1A).
We also examined Cyr61 levels in five matched samples of normal lung and NSCLC from the same individuals. Compared with normal lung tissues, expression of Cyr61 was decreased markedly in four of the five NSCLC samples (Fig. 1B). In lung cancer cell lines, expression of Cyr61 varied dramatically among the various lines (Fig. 1C). It was totally absent in three small cell lung cancer lines (H446, H187, and H526) and in the NSCLC cell lines H520 (squamous) and H460 (large cell). Each of these lines has been reported to have a high potential to form tumors in nude mice (data from ATCC). In contrast, the NSCLC cell lines H157 (squamous), H125 (adenosquamous), and H1299 (large cell), which had low potential to form tumors (data not shown), had high expression of Cyr61 mRNA.
After stably transfecting H520 and H460 NSCLC cell lines with Cyr61, we isolated two sublines for each cell line. Prominent expression of Cyr61 in these sublines was confirmed by Northern or Western blots (Fig. 2B). Both of the Cyr61-trans-fected sublines of H520 and H460 had a markedly lower proliferative rate than the Neo-containing control cells in liquid culture as measured by MTT assay (Fig. 2C).
Antibody against Cyr61 was added to cultures containing H520 and H460 cells. Compared with the cells cultured in normal medium, the addition of the Cyr61 antibody had nearly no effect on the growth of the control Neo cells, but the proliferation of both H520-Cyr61 and H460-Cyr61 cells increased by 73.6% Ϯ 1.8% (mean Ϯ S.D., p Ͻ 0.0001) and 48.1% Ϯ 2.3% (p Ͻ 0.0001), respectively (Fig. 2D).
H520-Cyr61 and H460-Cyr61 Cells Exhibited G 1 Arrest, Upregulation of p21 WAF1 , and Decrease of CDK2 Kinase Activity-Because our proliferation assays indicated that the growth of the Cyr61-transfected cells slowed, cell cycle analysis was performed to clarify which phases of the cell cycle were blocked (Fig. 3A). Both H520-Cyr61 and H460-Cyr61 cells had a much higher percentage of cells in the G 1 phase (82 and 70%, respectively) compared with the Neo cells (54 and 42%, respectively). The results showed that cells transfected with Cyr61 formed fewer colonies than those transfected with the same amount of empty vector. The mean (Ϯ S.D.) colony numbers are shown in the right panels. Panel B, expression of Cyr61 in stably transfected sublines. pcDNA3.1-Cyr61 was stably transfected into both H520 and H460 cells. After treating with G418 for 2 weeks, single colonies were isolated, and Cyr61 expression was checked by either Northern (H520 cells) or Western (H460 cells) blot. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Panel C, effect of Cyr61 on the proliferation of H520 and H460 cells. Lung cancer cells stably transfected with either empty vector (Neo cell) (squares) or Cyr61 (triangles and circles) were plated at 3 ϫ 10 3 in 96-well plates. After culturing for different durations, growth rates were measured by MTT assays. Data represent the mean Ϯ S.D. of four culture wells. Each experiment was repeated at least three times, and similar results were obtained each time. Panel D, Cyr61 antibody partially rescued the growth suppression of Cyr61-stably transfected cells. The antibody was added to the culture medium at a ratio of 1:50 (v/v). Two days later, cell growth was measured by MTT assays, and the cell number was calculated according to the standard curve of the correlation between the absorbance and cell number (curves not shown). Data represent the mean Ϯ S.D. of four culture wells. Each experiment was repeated three times, and similar results were obtained on each occasion.

Cyr61 Is a Tumor Suppressor in NSCLC
As a consequence, the percentage of Cyr61-expressing cells in S phase decreased proportionally. The number of cells in G 2 /M did not change markedly (Fig. 3A). Moreover, these Cyr61stably transfected lung cancer cells became larger and flatter, with a greater amount of cytoplasm than the Neo cells (data not shown).
Cell cycle arrest is often caused by up-regulation of CDK inhibitors. We examined several proteins related to the cell cycle (Fig. 3, B and C). p21 WAF1 , a major CDK inhibitor, was up-regulated in both H520-Cyr61 and H460-Cyr61 cells. Levels of the tumor suppressor proteins pRB and pRB2/p130 were also examined by Western blot (Fig. 3B). Expression of pRB was not different between the Cyr61-transfected NSCLC cell lines and their Neo controls. However, pRB2/p130, another member of pRB family, was prominently up-regulated in H520-Cyr61 cells and modestly increased in the H460-Cyr61 cells.
Levels of the tumor suppressor protein p53 were also examined. Because p53 is frequently mutated in NSCLC cell lines, single strand conformation polymorphism analysis was performed, and we confirmed that both H520 and H460 cells had the wild-type p53 gene (data not shown). A marked increase of expression of p53 was observed in Cyr61-stably transfected H520 cells, and a modest increase of p53 was found in H460-Cyr61 (Fig. 3C). The H1299 NSCLC cell had no p53 protein as reported previously (21).
Cyr61 Inhibited Tumor Growth in Nude Mice-To study whether this protein can inhibit tumor growth, the paired Cells were collected at 60% confluence, fixed with 70% cold ethanol, and stained with propidium iodide. The cell cycle was analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle is indicated. Panel B, Western blot for p21 WAF1 , p130, and pRB. Cells were harvested, cell lysates were run on a 4 -15% polyacrylamide gel, transferred onto polyvinylidene difluoride membrane, and cell cycle regulatory proteins were detected using specific antibodies. Expression levels for each protein relative to the Neo cells in each group are recorded beneath each band. GAPDH, glyceraldehyde-3phosphate dehydrogenase. Panel C, Western blot for p53 and CDK2 kinase assay. The kinase assay was performed by immunoprecipitating CDK2 from the same amount of cell lysates of each of the cell subtypes, incubating with histone H1 and [␥-32 P]ATP, separating the product by 4 -15% polyacrylamide gel, and exposing the gel to x-ray film. Expression levels for each protein and kinase activity relative to the Neo cells in each cell type are recorded beneath each band. -, signal undetectable.
sublines, H520-Neo and H520-Cyr61, as well as H460-Neo and H460-Cyr61, were grown in nude mice as described under "Experimental Procedures." Each nude mouse was injected with the experimental cells on one flank and the control cells on the other flank. After 5 weeks, the tumors were removed and weighed. On each mouse, the Cyr61-transfected cells developed a smaller tumor than the Neo control cells (Fig. 4, A and B). Statistical analysis showed that the mean weight of the H520-Cyr61 tumors was 67 Ϯ 17% (p ϭ 0.0015) less than those formed by the H520-Neo cells (Fig. 4D). Likewise, the mean weight of the tumors formed by H460-Cyr61 was 52 Ϯ 11% (p ϭ 0.0003) less than those formed by H460-Neo cells (Fig. 4C). DISCUSSION Cyr61 was the first member cloned in the CCN family. Its expression is induced within several minutes by many growth factors (2) and by stimulation through the muscarinic acetylcholine receptors (22), estrogen receptors (14), as well as by factor VIIa and thrombin (23). The Cyr61 protein associates with the extracellular matrix and several integrins (8 -11) and promotes cellular processes. Several studies show that Cyr61 can enhance tumor growth of a gastric adenocarcinoma cell line RF-1 and breast cancer cell lines (14,15,18). Furthermore, levels of Cyr61 are highest in breast cancers that are at an advanced stage at the time of diagnosis (14). However, Cyr61 levels are not increased or are decreased in hepatomas, leiomyomas, and prostate cancers (13,19,24).
The role of Cyr61 in NSCLC was examined in our study. Expression of this CCN protein was higher in normal human lung tissue than in their matched NSCLC samples. Cyr61transfected cells formed much fewer colonies than cells transfected with empty vector, which indicated that this protein inhibited clonal growth. Proliferation was also retarded in the H520-Cyr61 and H460-Cyr61 cells stably transfected with Cyr61 expression vector. The addition of Cyr61 antibody to the culture medium was able partially to rescue the growth repression. This would suggest that growth inhibition mediated by Cyr61 is at least in part a result of Cyr61 being secreted and then acting as a ligand for an integrin receptor that is associated with growth retardation. Tumorigenesis assays in mice revealed that Cyr61-transfected cells formed smaller tumors in nude mice compared with the control Neo cells. Taken together, the evidence suggests that Cyr61 is a tumor suppressor protein for lung tissue.
Both H520-Cyr61 and H460-Cyr61 cells had a higher percentage of cells in the G 1 phase and a lower percent in the S phase of the cell cycle compared with matched control cells. To clarify the molecular mechanism of the G 1 arrest, we examined the expression of cell cycle-related proteins. The up-regulation of both p53 and p21 WAF1 was detected in the Cyr61-transfected cells, especially H520-Cyr61, which also had the most prominent inhibition of cell growth. Increased expression of p21 is intimately related to G 1 cell cycle arrest by inactivating the CDK (25)(26)(27)(28)(29). CDK2 is one of the major target proteins of p21. The complex of cyclin E-CDK2 plays a pivotal role in the transition from G 1 to S phase. High levels of p21 associate with cyclin E-CDK2 complex and inhibit the kinase activity, resulting in a G 0 /G 1 phase block of the cell. In our study, in accordance with the up-regulation of p21, a remarkably decreased kinase activity of CDK2 was detected in both H520-Cyr61 and H460-Cyr61 cells.
Interestingly, the pocket protein pRB2/p130 was also upregulated in Cyr61-transfected cells, especially in H520-Cyr61 cells. As a member of the pRB family, p130 can inhibit cell proliferation and tumor formation (30 -37), including in lung tissue (38 -42). Unlike pRB and p107, p130 is the dominant pocket protein in quiescent (G 0 ) and differentiated cells (43,44). The loss of activated cyclin-CDK complex results in the accumulation of p130. The p130 is able to impose a G 1 arrest by phosphorylation-regulated binding of E2F and phosphorylation-independent sequestration of cyclin E-CDK2 (45). Together, pRB2/p130 and p21 WAF1 probably mediated the G 1 block in Cyr61-stably transfected NSCLC cells.
The mechanism of action of Cyr61 is still poorly understood. Several studies showed that Cyr61 was a ligand of integrins (8 -11). By binding to integrin, Cyr61 stimulated several cellular events including the enhanced cellular response to growth factors and serum (2,3). However, the secondary signal pathways triggered after Cyr61 binds to integrins are not clear. Cyr61-transfected cells formed smaller tumors than control cells in nude mice. Empty vector (Neo) and Cyr61-stably transfected lung cancer cells (2 ϫ 10 6 ) were injected subcutaneously into the right and the left flanks, respectively, of BNX nude mice. Five weeks later, tumors were removed and weighed. In all eight mice injected with either H460-Neo and H460-Cyr61 cells (panel A) or H520-Neo and H520-Cyr61 cells (panel B), the Cyr61-transfected cells always developed smaller tumors than the Neo cells. Statistical analysis showed that the mean weight of tumors formed by H460-Cyr61 decreased 52 Ϯ 11% (p ϭ 0.0003) compared with those formed by H460-Neo cells (panel C). The mean weight of tumors formed by H520-Cyr61 decreased 67 Ϯ 17% (p ϭ 0.0015) compared with those formed by H520-Neo cells (panel D).
select integrins in the target cells or various pathways that could be triggered by the same integrin in different types of cells. These are questions still requiring investigation.
In our study, expression of Cyr61 resulted in a G 0 /G 1 block in the cell cycle and up-regulation of both p53 and p21, but the linkage between Cyr61 and either p53 or p21 is unclear. Prior studies found that the integrin ␣ 6 ␤ 4 was able to activate p53 and p21 and caused cell cycle arrest (46,47). Further studies are required to determine whether Cyr61 can also bind to integrin ␣ 6 ␤ 4 and stimulate p53 or p21.
Interestingly, elm1 (also known as WISP1, another member of the CCN family), similar to Cyr61, can have opposing functions depending on the cell type that expresses it. For example, WISP1 can suppress in vivo tumor growth in a melanoma cell line (48) but accelerate proliferation of renal fibroblasts (49). These examples illustrate the complexity of function of the CCN family in cell growth and differentiation.
Taken together, we have found that Cyr61 behaved as a tumor suppressor gene in NSCLC. Expression of Cyr61 in the NSCLC cell lines H520 and H460 resulted in their growth arrest and up-regulation of p53, p21 WAF1 , and pRB2/p130. The Cyr61-transfected cells also formed markedly smaller tumors in nude mice compared with the control cells. We have provided the first direct evidence suggesting that Cyr61 can provide a negative regulation of cell growth.