Dual role of Ski in pancreatic cancer cells: tumor-promoting versus metastasissuppressive

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1 Carcinogenesis vol.30 no.9 pp , 2009 doi: /carcin/bgp154 Advance Access publication June 22, 2009 Dual role of Ski in pancreatic cancer cells: tumor-promoting versus metastasissuppressive function Peng Wang 1,2, Zhen Chen 1,2, Zhi-Qiang Meng 1,2, Jie Fan 3, Jian-Min Luo 4, Wang Liang 5, Jun-Hua Lin 1,2, Zhen-Hua Zhou 1,2, Hao Chen 1,2, Kun Wang 1,2, Ye-Hua Shen 1,2, Zu-De Xu 3 and Lu-Ming Liu 1,2, 1 Department of Hepatobiliary and Pancreatic Oncology, Cancer Hospital, Fudan University, 270 Dong An Road, Shanghai , People s Republic of China, 2 Department of Oncology, Shanghai Medical College, Fudan University, 270 Dong An Road, Shanghai , People s Republic of China, 3 Department of Pathology, Huashan Hospital, Fudan University, 12 Wulumuqi Road Central, Shanghai , People s Republic of China, 4 Central Laboratory, Cancer Hospital/Cancer institute, Fudan University, 270 Dong An Road, Shanghai , People s Republic of China and 5 Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, 130 Dong An Road, Shanghai , China To whom correspondence should be addressed. Tel: þ ; Fax: þ ; llm1010@yahoo.com.cn Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-b (TGFb)-induced growth arrest. As TGF-b has a dual effect on tumor growth with both tumor-suppressing and -promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 cell line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-b-induced transcriptional activity, which is associated with increased TGFb-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-b-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P ). Our results suggest that Ski may act as a tumor proliferation-promoting factor or as a metastatic suppressor in human pancreatic cancer. Introduction Abbreviations: MMP, matrix metalloproteinase; PCR, polymerase chain reaction; PDAC, Pancreatic ductal adenocarcinoma; TGF-b, transforming growth factor-b; VEGF, vascular endothelial growth factor. Ski was originally described as the oncogene present in the avian Sloan Kettering viruses (1); however, its oncogenic activity was rationalized upon discovering that Ski directly interacts with Smad2, Smad3 and Smad4 (2 5). Ski blocks the positive transcriptional activity of the Smads and confers resistance to the transforming growth factor-b (TGF-b) cytostatic response (2 5). Recent studies indicated that the expression of Ski is evident in several human malignancies, and the overexpression of Ski correlates with the more advanced stages of tumors in melanoma, esophageal and colorectal cancers (6 10). All these results suggest that Ski might be an oncogene that contributes to the resistance of tumor cells to TGF-b-induced growth arrest. Pancreatic ductal adenocarcinoma (PDAC) is highly invasive and metastatic and one of the leading causes of cancer deaths in the world, with an overall cure rate of merely 4% (11). Moreover, it is the type of human cancer in which the TGF-b-/Smad-signaling pathway is most frequently affected (12 14). Clinical studies have shown that pancreatic tumors overexpress all three TGF-b isoforms, and this correlates with decreased patient survival (15). As is the case for most cancer cell lines, exogenous TGF-b failed to suppress the growth of pancreatic cancer cells (16,17). Treatment of pancreatic cancer cells with TGF-b1 stimulated migration and invasion in vitro, whereas blocking TGF-b signaling using a soluble type II exoreceptor or a TbRI kinase inhibitor, SD-093 or SD-208, inhibited cellular migration and invasion (17 19). In addition to these experimental studies, several clinical observations support the idea that the metastatic phenotype of pancreatic cancer is driven by activation of TGF-b signaling, particularly in the context of Smad4 loss. For example, Teraoka et al. (20) reported an association between TGF-b1 overexpression and the presence of liver metastases. On the basis of these findings, we proposed that the increased expression of Ski may lead to reduced signaling induced by TGF-b, typically resulting in the diminished sensitivity of pancreatic cancer cells to growth inhibition by TGF-b. The downregulation of Ski expression may restore TGF-b signaling and growth inhibition induced by TGF-b. At the same time, another important question we need to answer is whether the increased TGF-b-induced transcriptional activities through Ski knockdown enhances invasion or metastases since studies have shown that the same TGF-b-signaling pathway dependent on Smad2 and/or Smad3 mediates both the tumor suppressor activities of TGF-b and its pro-oncogenic activities on invasion and metastasis (21,22). Therefore, in this study, we investigated the effect of Ski not only on the proliferative but also on the invasive and metastatic potential of the pancreatic cancer cell line SW1990, which contains wild-type Smad4 expression, and BxPC3, which is Smad4 deficient. In addition, we studied the association of Ski expression with the clinicopathological characteristics and prognosis in patients with pancreatic adenocarcinoma. Materials and methods Cell lines and mice Human pancreatic cancer cell lines SW1990, BxPC3 and PANC-1 were obtained from the American Type Culture Collection and grown in complete growth medium as recommended by the manufacturer. PC3, which was isolated from a Chinese woman with histologically confirmed PDAC, was obtained from the laboratory of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and PUMC, Beijing, China (23). The PC3 cells were grown in RPMI1640/10% fetal calf serum. All of the cultured cells were maintained in a humidified 5% CO 2 atmosphere at 37 C. Female BALB/c-nu/nu nude mice, 4 6 weeks old, were obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China), and housed in laminar flow cabinets under specific pathogen-free conditions with food and water ad libitum. All experiments on mice were conducted in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. The study protocol was also approved by the Shanghai Medical Experimental Animal Care Committee. Reagents Human recombinant TGF-b1 was obtained from PeproTech Asia (Peprotech, Rocky Hill, NJ). The following antibodies were used, anti-phosphorylated Smad2 and anti-phosphorylated Smad3 (Ser465/467 and Ser423/425, respectively; both from Cell Signaling Technology, Beverly, MA), anti-gapdh (ProteinTech Group, Chicago, IL). Other primary antibodies, including ant- Ski (G8), anti-tbrii (C16), anti-tbri (V22), anti-smad2 (S20), anti-smad3 (FL425) and anti-smad4 (B8), were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Lentivirus-mediated Ski RNA interference Sequences for targeting the Ski gene (GenBank accession no. NM_003036) using the BLOCK-iT RNA interference (RNAi) Designer (Invitrogen, Carlsbad, CA) were selected. Small interfering RNA targeting the following sequences were synthesized: Ski sense 5#-GATGAAAGAGGCCAACGAG-3#, Ski antisense 5#- CTACTTTCTCCGGTTGCTC-3# and control sense 5#-TTCTCCGAAC- GTGTCACGT-3#, control antisense 5#-AAGAGGCTTGCACAGTGCA-3#. Ó The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 1497

2 P.Wang et al. Fig. 1. Expression of Ski in human pancreatic cancer cell lines. (A) Western blot showing the expression of Ski in human pancreatic cancer cell lines. (B) Reverse transcription PCR analysis showing the expression of TGF-b pathway components in human pancreatic cancer cell lines. (C) Western blot analysis showing successful Ski knockdown in SW1990 and BxPC3 cell lines. Anti-GAPDH antibodies served as loading control. Following that the DNA oligomers were annealed and inserted into the BamHI (GGATCC) and EcoRI (GAATTC) sites of the psih1-h1-copgfp short hairpin RNA Vector. The recombinant vectors were identified by automated sequencing analysis. Next, according to the instructions of the SBI Lentivector Expression System (catalog number K ; Invitrogen), a lentiviral expression construct and ppack packaging plasmid mix were cotransfected into 293T cells. Then viral particles were collected and the titer was determined. The recombinant lentivector was used for infecting the target cells. SW1990 and BxPC3 cells were exposed to lentivirus-containing supernatant for 16 h in the presence of 6 mg/ml of Polybrene (Sigma, St Louis, MO). Pooled stable transfectants were established using puromycin selection. Cell proliferation assays A total of cells were plated in duplicate wells of 96-well plates and allowed to adhere overnight. Cells were then treated with human recombinant TGF-b1 at concentrations ranging from 0 to 300 pm. After 48 h, indices of cell proliferation were determined with Cell Counting Kit-8 (Dojindo, Molecular Technologies, Gaithersburg, MD). Wound closure assays Wound closure assays were conducted as described previously (17). Cells were plated in wells of 6-well plates. Confluent cell monolayers were wounded by manually drawing a furrow across the monolayer with a 1 20 ll pipette tip The cell culture medium was then replaced with fresh medium, 200 pm TGFb1 or vehicle was added as required, and wound closure was monitored by phase contrast microscopy at various times. The wound area at each time point after wounding was quantified using Adobe Photoshop version 7.0 (Adobe Systems) and ImageJ version 1.29 (National Institutes of Health) software. The experiments were performed in duplicate. Invasion assay Cell invasion was tested using the Transwell chamber invasion assay (Matrigel-coated membrane, BD Biosciences, Bedford, MA). Cells ( ) were seeded in serum-free medium into the upper chamber and allowed to invade toward 10% fetal calf serum as a chemoattractant in the lower chamber. TGFb1 (200 pm) or vehicle alone was added to both upper and lower chambers. After 24 or 48 h, respectively, Cells that had invaded through the Matrigel matrix and adhered to the underside of the membrane were counted as described previously (17). Assessment of tumorigenicity in vivo Cells ( cells in 200 ll) were injected subcutaneously into the right axilla of each BALB/c-nu/nu nude mouse. The length and width of tumors (in millimeters) were measured weekly with calipers. Tumor volume was calculated by the formula (a b 2 ) 0.5, where a and b were the long and short dimensions, respectively. Mice were killed when tumors reached 1.5 cm in diameter. The tumors were then removed and weighed. Each group had at least six mice. In vivo metastasis assay In vivo metastasis assays were conducted as described previously (24). Female nude mice received intravenous injection of cells in 0.2 ml of normal saline via the tail vein. Seven weeks after injection, mice were examined grossly at necropsy for the presence of metastases in the lungs. We evaluated tumor metastasis by counting the number of metastatic colonies in one histological section of the midportion of each sample of the lung from each mouse, by measuring lung weight and by determining the ratio of the metastatic area to the total area in histological sections from the midportion of each lung (25). The ratio of metastatic area to total area in the histological section was calculated by using Adobe Photoshop version 7.0 (Adobe Systems) and ImageJ version 1.29 (National Institutes of Health) software. The result was expressed as a percentage. Reverse transcription polymerase chain reaction analysis Total RNA was isolated from cultured cells or tissues using the TRIzolÒ Reagent (Invitrogen, San Diego, CA) according to the manufacturer s instructions. Semiquantitative real-time reverse transcription polymerase chain reaction (PCR) using SYBR green I to compare the relative expression of specific gene messenger RNA was done as described previously (26). The primer sequences and PCR conditions are described in supplementary Table I (available at Carcinogenesis Online). Western blot analysis Western blot analysis was performed according to the previous method, with some modifications (16). Briefly, proteins were extracted from the cultured cells and then quantitated using the bicinchoninic acid assay kit (Pierce, Rockford, IL), with bovine serum albumin as a standard. Equal amounts of protein from different cells were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then incubated with antihuman monoclonal antibodies. Target proteins were detected by the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and exposure to X-ray films (Eastman Kodak, Rochester, NY). Images were analyzed by ImageJ version 1.29 (National Institutes of Health) software. Immunohistochemical analysis of Ski and Smad4 Specimens of resected PDAC tissue obtained during surgery were fixed in 10% formalin and embedded in paraffin wax. Unstained 3 mm sections were then cut from paraffin blocks for immunohistochemical analysis. The sections were stained with anti-ski and Smad4 polyclonal antibodies as described previously and the evaluation of Ski and Smad4 expression was also performed accordingly (7,27). All procedures were done by two independent assessors and one pathologist, all of whom had no previous knowledge of the clinical outcome for this series of cases. Statistical analysis Analysis of variance and Student s t-test were used to determine the statistical significance of differences between experimental groups. The Kaplan Meier method was used to calculate the overall survival rate and the prognostic significance was evaluated by the log-rank test. The correlation of Ski

3 Dual role of Ski in pancreatic cancer cells immunoreactivity with the patients clinicopathological variables were analyzed by Fisher s exact test. Differences were considered significant at P, Results Generation of Ski knockdown in pancreatic cancer cells We analyzed Ski expression in a panel of pancreatic cancer cell lines. We found that pancreatic cancer cell lines differentially expressed Ski protein, which was 1.1-, 1.5- and 0.6-fold in BxPC3, PANC-1 and PC3, respectively, compared with that in SW1990 (Figure 1A). The expressions of Ski-related TGF-b-signaling pathway components, including TbRII, TbRI, Smad2, Smad3 and Smad4, were detected in the four human pancreatic cancer cell lines, except for Smad4 in BxPC3 and TbRII in PC3 (Figure 1B). SW1990, which contains wild-type Smad4 expression, and BxPC3, which is Smad4 deficient, were chosen for further Ski RNAi studies. Western blot analysis revealed that lentiviral small interfering RNA expression reduced endogenous Ski protein expression by 90% compared with the small interfering RNA control cells (Figure 1C). Downregulation of Ski inhibits the growth of pancreatic cancer cells both in vitro and in vivo First, we determined the effects of Ski downregulation on TGF-b-induced growth inhibition. The growth of parental and control RNAi SW1990 cells was resistant to the inhibitory effects of TGF-b1. In contrast, SW1990/Ski RNAi cells were inhibited up to 40% when exposed to 200 pm TGF-b1 (Figure 2A). The growth of parental and control RNAi BxPC3 cells was slightly suppressed by TGF-b1 in a dose-dependent manner, and the growth of RNAi expressing BxPC3 cells was inhibited up to 50% when the cells were exposed to 200 pm TGF-b1 (Figure 2A). Next, we investigated whether the downregulation of Ski could affect the growth of pancreatic cancer cells in nude mice. As shown in Figure 2C, all mice injected with pancreatic cancer cells developed subcutaneous tumors. The tumors formed by Ski RNAi SW1990 and BxPC3 cells were, however, much smaller than those formed by parental and control RNAi cells (Figure 2B and C). Downregulation of Ski enhances pancreatic cancer cell migration and invasion We evaluated the effect of Ski RNAi on TGF-b-mediated pancreatic cancer cell migration using a wound closure assay. In this assay, Ski RNAi cells displayed faster rate of wound closure in both cell lines, compared with control RNAi cells (Figure 3A). Representative images are shown in supplementary Figure 1 (available at Carcinogenesis Online). Because cell migration is a process that promotes tumor invasion, we tested the effect of Ski knockdown on cell invasion using Matrigel-coated Transwell chambers. Ski RNAi compared with control RNAi strongly increased SW1990 and BxPC3 cell invasion (Figure 3B). Representative images are shown in supplementary Figure 2 (available at Carcinogenesis Online). These results show that Ski is an inhibitor of pancreatic cancer cell migration and invasion. Fig. 2. Downregulation of Ski inhibits the growth of pancreatic cancer cells both in vitro and in vivo.(a) Cellular proliferation assay. Triplicates containing of parental, control RNAi and Ski RNAi cells were treated with increasing concentrations of TGF-b1 and cultured for 24 h. The growth of cells was determined by Cell Counting Kit 8 assay and expressed as absorbance value (OD) value. Points indicate mean; bars indicate standard error; P, 0.05 versus control. (B) Tumor growth curves. A total of viable cells were injected subcutaneously into the right axilla of each mouse. These curves show tumor growth until day 31 (for SW1990 group) or week 8 (for BxPC3 group). Points indicate mean; bars indicate standard error; P, 0.05 versus control. (C) Analysis of tumor weights and incidence. Tumor was removed and weighed at study termination. Columns indicate mean; bars indicate standard error; P, 0.05 versus control. 1499

4 P.Wang et al. Effect of Ski on lung metastasis of pancreatic cancer cells To examine the role of Ski in metastasis, we injected SW1990 and BxPC3 cells with different statuses of Ski expression into the tail veins of nude mice. Mice bearing tumors derived from Ski RNAi cells displayed significantly more metastasis than control mice, as determined by an increase in the number of metastatic colonies, organ weight and the ratio of metastatic area to total area (Figure 3C) in the lungs of nude mice. Representative images are shown in Figure 3D. Downregulation of Ski increases TGF-b-induced transcriptional activity To test whether the downregulation of Ski compromised TGF-b signaling, we measured its effects on the TGF-b stimulation of PAI- 1 and CTGF, the target genes of TGF-b that are widely used and highly TGF-b responsive (16,19), by using real-time PCR analysis. The inductions of PAI-1 and CTGF were very weak and transient in both cell lines. By comparison, cells with Ski downregulation showed strong and sustained inductions of PAI-1 and CTGF messenger RNA after TGF-b1 stimulation (Figure 4A). To explore the mechanism that leads to increased TGF-b-induced transcriptional activities after the downregulation of Ski, we examined the expression of TGF-b pathway components. The results showed that the downregulation of Ski did not significantly alter the expression levels of TbRI, TbRII, Smad2, Smad3 and Smad4 in either cell line (Figure 4B). The very transient inductions of PAI-1 and CTGF observed in both cell lines prompted us to investigate whether the TGF-b-signaling pathway was attenuated by the expression of Ski in these cells relative to Ski RNAi cells. To do this, we investigated the temporal pattern of phosphorylated Smads upon stimulation of TGF-b1. In parental and control RNAi cells activated with TGF-b1, we observed an increase in phosphorylated Smad2/3 upon TGF-b1 stimulation, but the levels of phosphorylated Smad2/3 disappeared abruptly after 1 and 2 h, respectively. The behavior of Ski RNAi cells was very different. A rapid increase of phosphorylated Smad2/3 upon TGF-b1 stimulation was observed, which persisted, and then diminished 24 h after induction (Figure 4B). Downregulation of Ski results in an altered expression profile of TGFb-inducible genes required for cell cycle arrest, epithelial mesenchymal transition, invasion and metastasis To elucidate the molecular basis underlying the Ski RNAi-mediated tumor suppression and metastasis promotion, we further examined the expression change of a panel of genes, including genes involved in TGF-b-induced growth arrest, cyclin D1, cyclin E and the cyclin dependent kinase inhibitor, p21waf1/cip1 (hereafter, p21), wellcharacterized epithelial mesenchymal transition (EMT) related markers and several extensively recognized invasive- and metastatic-associated genes, matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. Semiquantitative real-time reverse transcription PCR analyses showed that Ski RNAi was constitutive with the upregulation of p21, MMP2, MMP9 and VEGF and with the downregulation of cyclin D1 in both PC cell lines (Figure 5a). Similar effects of Ski RNAi on p21, MMP2, MMP9, VEGF and cyclin D1 expression noted in the in vitro studies were also observed in in vivo studies (Figure 5B). Furthermore, in both in vitro and in vivo studies, we observedareducedexpressionofe-cadherin, which is an epithelial marker, and a concomitant increase of N-cadherin, fibronectin and vimentin, which are mesenchymal markers (Figure 5C and D). Association of Ski expression with clinicopathological characteristics as well as prognosis in patients with PDAC A group of 71 cases with histologically confirmed PDAC treated surgically were investigated with immunohistochemical analysis. Fig. 3. Downregulation of Ski promotes migration and invasion in vitro and lung metastasis in vivo.(a) Wound closure assay. Confluent cell monolayers in 6-well plates were wounded by manually drawing a furrow across the monolayer with a micropipette tip as described in Materials and Methods. A total of 200 pm TGFb1 or vehicle only was added. Wound closure at various time intervals following wounding was quantitated using Adobe Photoshop 7.0 and ImageJ software and expressed graphically. Vehicle-treated parental, control RNAi and Ski RNAi cells, open circles, open squares and open rectangles, respectively; TGF-b1-treated parental, control RNAi and Ski RNAi cells, closed circles, closed triangles and closed rectangles, respectively. Points indicate mean; bars indicate standard error. (B) Invasion assay. Cells (10 5 /well) were plated onto Matrigel precoated cell culture inserts in the presence of TGF-b1 (200 pm) or vehicle only. After 24 or 48 h, the number of cells that had migrated through the filters was counted as described in Materials and Methods. Columns indicate mean for three experiments; bars indicate standard error. (C) Lung metastasis assay. Cells were injected into the tail veins of 4-week-old female nude mice as described in Materials and Methods. After 7 weeks, their lungs were removed. The metastatic lesions were quantitatively evaluated by the number of metastatic colonies, organ weight and ratio of metastatic area to total area in lungs of mice (n 6 mice/group); (D) Representative macroscopic and microscopic images of hematoxylin eosin-stained sections of lung from mice injected with pancreatic cancer cells are shown. Original magnification of microscopic findings, 40 (upper) and 400 (lower). Asterisk indicates statistically significant difference compared with control. 1500

5 Dual role of Ski in pancreatic cancer cells Fig. 3. Continued. Clinical examples of positive or negative immunostaining for Ski and Smad4 are presented in Figure 6A. The patients characteristics and their correlation with Ski expression is shown in supplementary Table II (available at Carcinogenesis Online). We found that Ski expression was significantly correlated with depth of invasion (P ) and overall survival time (P ) but not with pathological stage, histopathological grading or lymph node metastasis (supplementary Table II is available at Carcinogenesis Online and Figure 6B). We also determined Smad4 expression in tumors by immunostaining analysis. Based on this immunohistochemical analysis, we classified patients into either a Smad4-negative group or a Smad4-positive group. We found that in both groups, the survival of patients whose cancer cells expressed Ski was significantly worse than those whose cancer cells did not express Ski (Figure 6c and d). Discussion TGF-b has a dual effect on tumor growth, and the same TGF-bsignaling pathway dependent on Smad2 and/or Smad3 mediates both the tumor suppressor activities of TGF-b and its pro-oncogenic activities on invasion and metastasis (21,22). Previously, one study published by Heider et al. (9) showed that Ski expression contributes 1501

6 P.Wang et al. Fig. 4. Effect of Ski downregulation on the expression of TGF-b signaling. (A) Downregulation of Ski perturbed the induction of specific TGF-b-responsive target genes PAI-1 and CTGF. Cells were treated with 200 pm TGF-b1 for the indicated times. Total RNA was extracted and the expression of PAI-1 and CTGF were detected by real-time reverse transcription PCR. Columns indicate mean for three experiments; bars indicate standard error. (B) Downregulation of Ski increased the induction of phosphorylated Smad2 in PC cells. Cells were either uninduced or induced with TGF-b1 (200 pm) for 1 h. Whole-cell extracts were analyzed by western blotting using antibodies against TbRII, TbRI, Smad2, Smad3, Smad4, phosphorylated smad2 and GAPDH served as a loading control. 1502

7 Dual role of Ski in pancreatic cancer cells Fig. 5. Ski downregulation results in an altered expression profile of TGF-b-inducible genes. (A) Cells were either uninduced or induced with TGF-b1 (200 pm) for 24 h. Total RNA was extracted and the expression of MMPs, VEGF, basic fibroblast growth factor (bfgf), p21, cyclin D1 and cyclin E were detected by realtime reverse transcription PCR. Columns indicate mean for three experiments; bars indicate standard error. (B) Total RNA was extracted from tumor tissue from xenografts and the expression of MMPs, VEGF, bfgf, p21, cyclin D1 and cyclin E messenger RNA (mrna) were detected by real-time reverse transcription PCR. Columns indicate mean for three experiments; bars indicate standard error. (C) Cells were either uninduced or induced with TGF-b1 (200 pm) for 24 h. Total RNA was extracted and the expression of EMT-related markers were detected by real-time reverse transcription PCR. Columns indicate mean for three experiments; bars indicate standard error. (D) Total RNA was extracted from tumor tissue from xenografts and the expression of EMT related markers were detected by real-time reverse transcription PCR. Columns indicate mean for three experiments; bars indicate standard error. 1503

8 P.Wang et al. Fig. 6. Ski expression in PDAC and its association with patient survival time. (A) Immunohistochemical staining for Ski and Smad4 in PDAC tissues. Original magnification, 400. (B) Kaplan Meier survival curve of patients with different Ski expression in PDAC. The survival of patients whose cancer cells express Ski (n 5 49) was significantly (P ) worse than that of patients whose cancer cells did not express Ski (n 5 22). (Cand D) Kaplan Meier survival curve of patients with different Ski expression in the Smad4-negative group (C) and the Smad4-positive group (D) of PDAC. to tumor growth through the inhibition of TGF-b signaling in the pancreatic cancer cell line PANC-1, which contains wild-type Smad4 expression. The effect of TGF-b signaling mediated by Ski on the invasion and metastasis of pancreatic cancer cells remains to be elucidated since study have shown that downregulation of Ski expression enhances tumor metastasis in MDA-MB-231 and A549 cell lines (28). Furthermore, Smad4 undergoes a loss of heterozygosity in.90% of pancreatic cancer (13,29). Whether the expression of Ski contributes to the growth of pancreatic cancer cells through a Smad4-independentsignaling pathway remains unknown. In our study, we have shown that the downregulation of Ski significantly inhibited the growth of the pancreatic cancer cell line SW1990, which is consistent with Heider s study (9). In contrast with Heider s study, we introduced the pancreatic cancer cell line BxPC3, which is Smad4 deficient, to evaluate whether Ski is still functional. We also observed a similar result in BxPC3 cells. Mutation or deletion of the Smad4 gene has been detected in 50% of all PDAC (29), and many pancreatic cancer cell lines have impaired TGF-b/Smad signaling due to a functionally inactivated Smad4 (30). However, recent studies have shown that the TGF-b signal may pass via the TbRI- Smad2/3 pathway, and p21/waf1 may be upregulated by TGF-b in a Smad4-independent manner through TbRI-Smad2/3 signaling and not via the mitogen-activated protein kinase cascade (31). Ski efficiently blocks TGF-b superfamily signaling inside the cells, and it primarily inhibits Smad-mediated signaling but not other types of signaling (32). Given our results, we may conclude that Ski may inhibit the growth of pancreatic cancer cells independent of Smad4. Perhaps, our most important observation is that the downregulation of Ski also promoted invasion and metastasis in both cell lines. Previous studies have shown that blocking TGF-b signaling using a soluble type II exoreceptor or TbRI kinase inhibitor inhibits cellular migration and invasion (17 19), suggesting that the inhibition of TGF-b signaling is essential for the prevention of invasion and 1504 metastasis. Therefore, we suspected that the increased TGFb-induced transcriptional activity through Ski downregulation would promote pancreatic cancer cell invasion and metastasis. In agreement with our speculation, we found that Ski prevented pancreatic cancer cell migration and invasion in vitro and metastasis in a mouse model. It is well known that both Smad-dependent and Smad-independentsignaling pathways are involved in mediating the effects of TGF-b on the pancreatic cancer phenotype. Therefore, which signaling pathway mediates cancer cell migration and invasion remains unclear. In our study, we observed that downregulation of Ski had little effect on in vitro growth or the invasive capacity of pancreatic cancer cells without TGF-b induction. And the downregulation of Ski did not lead to increased induction of PAI-1 and CTGF messenger RNA without TGF-b1 stimulation. Since Ski may efficiently block TGF-b superfamily signaling inside the cells, and it primarily inhibits Smadmediated signaling but not other types of signaling (32), we may conclude that Ski-mediated pancreatic cancer cell invasion and metastasis occurs mainly through a Smad-dependent-signaling pathway. Recent studies also support our conclusion. For example, Decker s study showed that Smad signaling is required for the TGFb-induced bone metastasis of breast cancer cells. Additionally, RNA interference-mediated depletion of Smad4 in breast cancer cell line inhibited bone metastasis in a mouse xenograft model, and this could be restored by the ectopic expression of Smad4 (33,34). The overexpression of Smad3 had prometastatic effects on breast cancer cell lines (21), whereas overexpression of Smad7 (25) or ectopic expression of a Smad-binding defective mutant of TbRI (35) suppressed the metastatic capacity. TGF-b signaling is frequently attenuated in pancreatic cancer because of alterations in components of the pathway (36). Even in PANC-1, which has functional Smad4, it has been reported to have attenuated Smad signaling compared with epithelial cells that are responsive to the antiproliferative effects of TGF-b (16). As

9 Dual role of Ski in pancreatic cancer cells a transcriptional co-repressor through binding to Smad proteins, Ski may negatively regulate the target gene transcription induced by TGF-b. The attenuated signaling in pancreatic cancer cells may be caused by high expression of Ski. In our study, we observed that the inductions of PAI-1 and CTGF, target genes of TGF-b signaling, were strong and sustained for a long time when Ski was downregulated. Consistent with this, we also observed that cells with Ski downregulation showed strong and sustained expression of phosphorylated Smad2/3 after TGF-b1 stimulation. All of these results confirmed that Ski is a potent inhibitor of TGF-b/Smad signaling. TGF-b arrests the cell cycle of epithelial cells at the early G 1 phase via Smad-mediated transcriptional regulation of critical regulators of the cell cycle (37). In our study, the same G 1 phase arrest effect happened when Ski was downregulated (supplementary Figure 3 is available at Carcinogenesis Online). Furthermore, to gain insight into the molecular basis underlying the influence of Ski on the cell cycle distribution, the expression change of TGF-b signaling downstream effectors, p21 and the cyclins, both contributing to the control of cell cycle progression, were detected. We found that cells with Ski downregulation showed an upregulated and sustained p21 expression and downregulated cyclin D1 expression. Previous studies have shown that pancreatic cancer cells that growth arrest upon TGF-b stimulation usually showed strong inductions of p21. And sustained production of p21 in response to TGF-b led to enhanced accumulation of cells in the G 1 phase (16). We conclude that high Ski expression may account for the loss of TGF-b-induced growth arrest in pancreatic cancer cells. The basis of the altered outcome of signaling through the TGF-b/ Smad pathway in PC cells that have acquired the ability to invade and metastasize must be considered. Previous studies have demonstrated exogenous TGF-b-enhanced migration and invasiveness of pancreatic cancer cells through the activation of TGF-b-regulated genes specifically involved in migration and invasion, for example, MMP-2 and the urokinase plasminogen activator system (38 40). In our study, we found that the downregulation of Ski positively regulated the TGF-b-induced MMP2, MMP9 and VEGF expression, which are involved in cell invasion and metastasis. EMT is an important process in the invasion and metastasis of pancreatic cancer cells (41,42). EMT in response to TGFb1 is characterized phenotypically by downregulation of epithelial markers and upregulation of mesenchymal markers (43). In our study, we found that downregulation of Ski led to a reduced expression of E- cadherin, which is an epithelial marker, and a concomitant increase of N-cadherin, fibronectin and vimentin, which are mesenchymal markers. This indicates that upon activation of the TGF-b pathway induced by Ski knockdown, cells exhibit more overt EMT, which may lead to more aggressive and metastatic tumors. We also examined Ski protein expression in 71 cases of human PDAC samples. We observed a close association between high Ski expression and poor prognosis in pancreatic adenocarcinoma, independent of Smad4 expression. There was a positive correlation between Ski expression and depth of invasion. However, we did not find any correlation between Ski expression and lymph node metastasis. These data are consistent with previous clinical studies that defined Ski as an oncogene. Based on this clinical observation, Ski seems to act as a promoter of tumor progression in patients with PDAC. At present, it is difficult to reconcile these seemingly contradictory observations between experimental and clinical studies. These contradictory findings may suggest that the tumor-enhancing properties of Ski predominate over the tumor suppression of invasion and metastasis within a patient s tumor mass. Since this dual role of Ski in tumorigenesis mimics the role of TGF-b in human cancer, whether Ski functions differently depending on the stage of carcinogenesis and the responsiveness of the tumor cell remains to be further studied. In conclusion, our results indicate that Ski downregulation enhances the antiproliferative activity induced by TGF-b yet promotes pancreatic cell migration and invasiveness in vitro and metastasis in vivo in the pancreatic cancer cell lines SW1990 and BxPC3. However, continued researches are needed before understanding the mechanisms by which Ski functions as a tumor promoter or as a metastasis suppressor. Supplemantary material Supplementary material Tables I and II and Figures 1 3 can be found at Funding Climbing Up Project of Shanghai Municipal Commission for Science and Technology, Shanghai, China (GJ-KW0601); Shanghai Nature Science Fund, Shanghai, China (09ZR ). Acknowledgements Conflict of Interest Statement: None declared. References 1. Li,Y. et al. (1986) Unique sequence, ski, in Sloan-Kettering avian retroviruses with properties of a new cell-derived oncogene. J. Virol., 57, Akiyoshi,S. et al. (1999) c-ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads. J. Biol. Chem., 274, Luo,K. et al. (1999) The Ski oncoprotein interacts with the Smad proteins to repress TGFbeta signaling. Genes. Dev., 13, Sun,Y. et al. (1999) Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling. Mol. Cell, 4, Xu,W. et al. 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