点击显示 收起
【关键词】 Inhibition
Transforming growth factor (TGF) is a pleiotropic factor that regulates cell proliferation, angiogenesis, metastasis, and immune suppression. Dysregulation of the TGF pathway in tumor cells often leads to resistance to the antiproliferative effects of TGF while supporting other cellular processes that promote tumor invasiveness and growth. In the present study, SD-208, a 2,4-disubstituted pteridine, ATP-competitive inhibitor of the TGF receptor I kinase (TGFRI), was used to inhibit cellular activities and tumor progression of PANC-1, a human pancreatic tumor line. SD-208 blocked TGF-dependent Smad2 phosphorylation and expression of TGF-inducible proteins in cell culture. cDNA microarray analysis and functional gene clustering identified groups of TGF-regulated genes involved in metastasis, angiogenesis, cell proliferation, survival, and apoptosis. These gene responses were inhibited by SD-208. Using a Boyden chamber motility assay, we demonstrated that SD-208 inhibited TGF-stimulated invasion in vitro. An orthotopic xenograft mouse model revealed that SD-208 reduced primary tumor growth and decreased the incidence of metastasis in vivo. Our findings suggest mechanisms through which TGF signaling may promote tumor progression in pancreatic adenocarcinoma. Moreover, they suggest that inhibition of TGFRI with a small-molecule inhibitor may be effective as a therapeutic approach to treat human pancreatic cancer.
Pancreatic cancer is the fifth leading cause of cancer-related deaths, resulting in approximately 31,000 deaths annually in the United States alone (Jemal et al., 2006). It is a highly metastatic cancer with an average survival of 3 to 8 months after diagnosis. Because of the aggressiveness of the cancer, difficulties in diagnosis, and lack of effective treatment, only 5% of patients who receive a diagnosis of pancreatic cancer survive longer than 5 years (Jemal et al., 2006). The current treatment, gemcitabine, confers only a modest survival advantage when used as a stand-alone treatment or in combination with other therapies (Eckel et al., 2006).
A number of genetic and epigenetic alterations have been identified in pancreatic cancer. The most common are mutations that affect the activity or expression of K-ras, p15, p16, p53, and DPC4/Smad4. Activating point mutations in the K-ras oncogene are believed to occur early in progression to neoplasia (Hruban et al., 2000) and are found in 85 to 95% of pancreatic cancers (Friess et al., 1999). Mutations in cell cycle inhibitor genes p15 and p16 are found at a frequency of approximately 60 and 80%, respectively (Naumann et al., 1996; Villanueva et al., 1998). Aberrations in p53 and DPC4/ Smad4 are believed to occur late in tumor progression (Hruban et al., 2000) and are found in approximately half of pancreatic cancers (Schutte et al., 1996; Friess et al., 1999).
Deletion of DPC4/Smad4, a key mediator of TGF signaling, has been associated with abnormal growth arrest by TGF (Yasutome et al., 2005). In addition to altered Smad4 expression, signaling from the Smad pathway may be disrupted by mutations that affect the expression of TGF receptors, Smad6, Smad7, and downstream genes (Friess et al., 1999). TGF signals through the Smad pathway and through Smad-independent pathways. Smad signaling is initiated upon binding of TGF to a type II receptor (TGFRII) followed by recruitment and transphosphorylation of TGFRI (Heldin et al., 1997). Activated TGFRI then phosphorylates regulatory Smads, Smad2 and Smad3. Once phosphorylated, Smad2 and Smad3 form a complex with Smad4 and translocate to the nucleus, where they activate the transcription of TGF-responsive genes. TGF signaling through the Smad pathway is tightly controlled by negative feedback loops involving Smad6 and Smad7. TGF can also signal through mitogen-activated protein kinase (ERK, JNK, p38) cascades and the phosphatidylinositol-3 kinase pathway (Derynck and Zhang, 2003; Elliott and Blobe, 2005). Cross-talk between these pathways and the TGF pathway coordinates proliferation, survival signals, and other signals.
In normal epithelial cells, TGF acts as a tumor suppressor, mediating growth arrest through down-regulation of c-Myc, and through transcriptional activation of cell cycle inhibitors p15 and p21Cip1/WAF1 (Grau et al., 1997; Donovan and Slingerland, 2000; Adhikary and Eilers, 2005). In addition to regulating survival and proliferation, TGF signaling promotes angiogenesis, fibrosis, metastasis, and immune suppression (Elliott and Blobe, 2005). Alterations that affect the expression or activity of components of the TGF path-way can render cells insensitive to TGF-mediated growth arrest while enabling other responses that support tumor progression (Dumont et al., 2003; Nicolás and Hill, 2003). During neoplastic conversion, autocrine expression of TGF is believed to promote tumorigenesis. The cellular response is also influenced by other dysregulated pathways such as the Ras-MEK-ERK-signaling cascade (Ellenrieder et al., 2001) and by stromal cell interactions, growth factors, and cytokines in the tumor cell microenvironment.
The pivotal role of TGF in promoting cellular processes that are important for tumor progression suggests that the pathway may be a good target for therapy. In this study, we investigated whether SD-208, a small-molecule inhibitor of TGFRI, can inhibit tumor progression in pancreatic cancer. We used PANC-1, a human pancreatic ductal carcinoma that harbors genetic alterations (K-ras, p15, p16, and p53) commonly found in pancreatic cancer (Villanueva et al., 1998; Moore et al., 2001). PANC-1 has also been reported to have altered TGFRI and Smad7 expression (Nicolás and Hill, 2003). It is an attractive model for the human disease because PANC-1 tumors are metastatic when grown orthotopically in nu/nu (nude) mice. Furthermore, PANC-1 secretes TGF, which is believed to promote tumor progression and desmoplasia in human pancreatic cancer. Our studies reveal that SD-208 abrogates TGF-mediated gene responses that may facilitate tumor growth and metastasis. We also demonstrate for the first time that a small-molecule inhibitor of TGFRI attenuates growth and metastasis of established tumors in an orthotopic xenograft model of pancreatic adenocarcinoma.
Reagents. Recombinant human TGF was purchased from R&D Systems (Minneapolis, MN). TGF1 and VEGF ELISA kits were from Biosource International (Camarillo, CA). The TGF2 ELISA was from R&D Systems. The PAI-1 ELISA was from American Diagnostica, Inc. (Stamford, CT). The rabbit polyclonal antibody for phospho-Smad2 (Ser465/467) was from Cell Signaling Technology (Danvers, MA). The mouse monoclonal antibody against vimentin was from Affinity Bioreagents (Golden, CO). HRP-conjugated donkey anti-rabbit secondary antibody was from Amersham (Pittsburgh, PA), and HRP-conjugated goat anti-mouse secondary antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).
CTGF ELISA. CTGF-specific polyclonal antibodies, which were generated using peptides derived from the C terminus of the protein, were absorbed on a high-binding ELISA plate. After blocking, CTGF standards and cell-culture supernatants were added and incubated overnight at 4°C. Bound CTGF was detected via its heparin-binding site by incubation with biotinylated-heparin (Sigma-Aldrich, St. Louis, MO) and streptavidin-HRP (Chemicon International, Temecula, CA). Quantitation of bound CTGF was extrapolated from a standard curve generated with recombinant human CTGF.
Cell Culture and Inhibitor Treatment. Human pancreatic cancer cell lines PANC-1 (CRL-1469) and BxPC-3 (CRL-1687) were acquired from American Type Culture Collection (Manassas, VA). PANC-1 was cultured in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum. BxPC-3 was cultured in RPMI 1640 (Mediatech) supplemented with 10% fetal bovine serum. TGFRI kinase inhibitor SD-208 (Scios, Inc., Fremont, CA) was dissolved in DMSO (1000x stock). SD-208 has an IC50 value of 49 nM when measured by direct enzymatic assay of TGFRI kinase activity in vitro. It is 100-fold less specific for TGFRII and is more than 17-fold less specific for related kinases (Kapoun et al., 2006).
Construction of PANC-1 Luciferase Cells. For constitutive expression of luciferase and the Zeocin-resistance gene, PANC-1 were cotransfected with pGL-3 (Promega, Madison, WI) and pSV40-Zeo (Invitrogen, Carlsbad, CA) using FuGENE transfection reagent (Roche Applied Science, Alameda, CA). After selection with Zeocin (Invitrogen), a clone with stable luciferase expression and normal growth characteristics was selected for studies.
Phospho-Smad2 Analysis. Cells were seeded in six-well plates at 2 x 105 cells/well and cultured in serum-containing medium. The next day, they were treated with SD-208 (31.25-1000 nM) for 15 min before the addition of TGF1 (2 ng/ml). After 65 min, cell lysates were prepared and analyzed by Western blot as described previously (Kapoun et al., 2006).
Measurement of Secreted Proteins. PANC-1 cells were seeded in six-well plates at 3 x 105 cells/well and cultured in serum-containing medium. The next day, medium was changed to serum-free DMEM containing 1x Insulin-Transferrin-Selenium (Gibco) and 0.2% bovine serum albumin. The medium also contained combinations of the following treatments: 0.1% DMSO vehicle control, 400 nM SD-208, and 5 ng/ml TGF1. After 24 h, cell supernatants were collected and assayed for TGF, VEGF, CTGF, and PAI-1 by ELISA. Cells were harvested in MPER buffer (Pierce, Rockford, IL), and protein was quantitated by bicinchoninic acid assay (Pierce). Concentrations of secreted proteins were determined by ELISA and normalized to the total cell protein.
Gene Expression Analysis. PANC-1 cells were seeded in DMEM containing 10% serum and grown to 70% confluence. The next day, cells were treated in complete medium with vehicle (0.1% DMSO), 400 nM SD-208, 2 ng/ml TGF1, or a combination of TGF1 and SD-208 for 24 h. Total RNA was extracted from cells using an RNeasy kit (QIAGEN, Valencia, CA).
Real-time RT-PCR and cDNA microarray analysis were performed as described by Kapoun et al. (2006). Sequences of primers and probes for real-time RT-PCR can be found in Table 1.
TABLE 1 Primers and probes used for real-time RT-PCR
Invasion Assays. Cell invasion was analyzed in 24-well Matrigel-coated invasion chambers (BD Biosciences Discovery Labware, Bedford, MA) according to manufacturer's directions with the following modifications: hydrated chambers were transferred to a new 24-well plate containing DMEM with 10% serum plus TGF1 (2 ng/ml) and/or SD-208 (1 µM) or DMSO (0.1%). Cells (5 x 105) were added to each chamber and incubated for 20 h. Cells that did not pass through the filter were removed with a cotton swab before processing the filter with a Hema-3 staining kit (Fisher, Pasadena, CA) and mounting the filter on a microscope slide. Cells in five fields on the filter were photographed at 40x magnification and counted manually for each treatment.
Tumor Implantation and Imaging. All of the animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Scios. Tumors from PANC-1 luciferase were grown subcutaneously in SCID mice to generate trocar fragments. For the orthotopic implantation, 6- to 8-week-old male nude mice were anesthetized with ketamine/xylazine cocktail before implanting one piece of tumor fragment in the tail region of the pancreas using 6-0 vinyl sutures. Tumor growth was monitored weekly with the Xenogen Living Image System (Alameda, CA). When tumors were palpable (at day 10 after implantation), animals were randomized and assigned to vehicle and treatment groups (n = 12) based on the intensity of luminescence. SD-208 was administered orally at 20 or 60 mg/kg twice daily. At study termination (day 56), animals were sacrificed, and primary tumors were resected and weighed. Organs harboring metastatic lesions were manually visualized and counted. Metastatic lesions were confirmed with ex vivo Xenogen imaging.
Statistical Analysis. Statistical analysis was performed using one-way analysis of variance with Bonferroni correction using Prism version 4.02 (GraphPad Software Inc., San Diego, CA) unless otherwise stated.
Inhibition of TGFRI Reduces Smad Signaling and Levels of Secreted TGF-Inducible Proteins. We observed previously that SD-208 blocks signaling from TGFRI in fibroblasts (Kapoun et al., 2006), smooth muscle cells, and various tumor cell lines (Uhl et al., 2004; N. Gaspar and G. Li, unpublished data). To assess the potency of SD-208 on TGFRI inhibition in PANC-1 cells, we measured inhibition of Smad2 phosphorylation by SD-208 after induction with TGF. Maximum phosphorylation of Smad2 occurred at approximately 1 h after induction with TGF1 (data not shown). Pretreatment of cells with SD-208 before TGF1 induction inhibited phosphorylation in a dose-dependent manner (Fig. 1). IC50 was achieved at 62.5 to 125 nM SD-208. To confirm inhibition of TGF signaling, downstream events were measured. These included production of CTGF, PAI-1, and VEGF, as well as TGF1 and TGF2, which are regulated in an autocrine manner by TGF1. Levels of the secreted proteins increased after TGF treatment except when cells were cotreated with SD-208 (Fig. 2). Similar profiles were observed when we analyzed Smad phosphorylation and levels of secreted proteins from luciferase-expressing cells, indicating that introduction of the gene did not affect TGF signaling (data not shown). In agreement with a previous report (Uhl et al., 2004), these results demonstrate that SD-208 inhibits signaling from the TGF receptor complex. Furthermore, they demonstrate that SD-208 inhibits expression of proteins that regulate matrix remodeling and angiogenesis.
Fig. 1. Dose-response of SD-208 on SMAD2 phosphorylation. Western analysis of lysates from cells treated with 0.1% DMSO (vehicle) in lanes 1 and 2 or with increasing concentrations of SD-208 in lanes 3 to 8 (31.25, 62.5, 125, 250, and 500 nM, and 1 µM) for 15 min before treatment with 2 ng/ml TGF (lanes 2-8) for 65 min. Sequential probing with a vimentin antibody verified equal loading. The IC50 value for SD-208 is 62.5 to 125 nM.
Fig. 2. Effect of TGF1 and SD-208 on production of TGF1, TGF2, CTGF, PAI-1, and VEGF. Quantitation of secreted proteins after culturing PANC-1 for 48 h in the presence of 0.1% DMSO (vehicle), 400 nM SD-208, 5 ng/ml TGF1, or TGF1 and SD-208. A representative study from three independent studies is shown. Mean ± S.D. values were determined for triplicate biological replicates. Protein levels in vehicle-treated and SD-208-treated samples were compared with levels in TGF1-stimulated samples (*, **, and *** represent p values <0.05, <0.01, and <0.001, respectively). SD-208 inhibited TGF-stimulated induction of these factors. Analysis of levels of TGF1 after stimulation with TGF1 is confounded by residual TGF1 from treatment; nonetheless, SD-208 significantly decreased TGF1 levels.
Inhibition of TGFRI Attenuates Gene Responses Involved in Tumor Processes. As a first step toward understanding the TGF-dependent processes important for tumor progression, we assessed the regulation of gene expression in vitro by TGF. We used gene array analysis of PANC-1 cultures grown in serum-containing medium to survey genes that are regulated by TGF except when cells are cotreated with SD-208. A majority of the TGF-affected genes (82%) were inhibited by SD-208. These genes were grouped into functional categories based on their involvement in the tumor-promoting processes. The gene responses highlight the major cellular processes such as ECM remodeling, cell motility, and adhesion; cell cycle, proliferation, and apoptosis; and angiogenesis (Table 2).
TABLE 2 Effect of SD-208 on TGF-dependent gene responses
The functional gene clusters identify genes that are regulated by TGF and are affected by SD-208 in PANC-1 cells: ECM remodeling, cell motility, and adhesion; cell cycle, proliferation, and apoptosis; and angiogenesis. Mean fold expression of TGF-regulated genes in PANC-1 from quadruplicate hybridizations: control vs. TGF, 24 h (TGF); TGF vs. TGF + SD-208, 24 h (TGF + SD-208); control vs. SD-208, 24 h (SD-208). Gene expression profiles were determined from cDNA microarrays as described previously (Kapoun et al., 2006). Functional groups were classified using Gene Ontology terms and published functions.
Included in the ECM remodeling/cell motility/adhesion functional group are genes encoding extracellular matrix proteins (COL4A2, COL7A1, COL11A1, LAMA4), regulators of matrix synthesis (SERPINE 1, CTGF), accessory proteins in the tight junction (CLDN3, CLDN4), and adhesion proteins (CDH1, CDH5, ITGA2, ITGAV, ITGB2, F2R, ACTN1, CYR61). TGF up-regulated the majority of these genes, whereas it down-regulated a few (CLDN3, CLDN4, CDHI). The addition of SD-208 reversed these trends. Collectively, the data show that SD-208 opposed gene responses that promote matrix remodeling and cell motility.
Another large functional group of genes whose expression was regulated by TGF and reversed by SD-208 includes genes involved in proliferation, cell cycle, or apoptosis. Examples of TGF up-regulated genes in this group are SNF1LK, PDGFA, PDGFB, CDK6, MYC, and IGF2. TGF also down-regulated a number of genes including TP531, TNFRSF1B, PBEF1, NBL1, and PTEN. Because two of four replicates for IGF2 expression (TGF + inhibitor) on the array did not meet the signal/background criteria, we confirmed the regulation through RT-PCR (see below).
Angiogenesis genes comprise the third functional cluster. Included in this cluster are NRP2, JAG1, CYR61, and VEGFC, which are novel TGF-responsive genes for PANC-1. The gene array analysis, therefore, identified a number of genes in PANC-1 that are regulated by TGF and may be important for tumor-associated processes. Furthermore, the analysis revealed that SD-208 inhibited regulation of these genes by TGF.
To confirm the expression patterns observed in our microarray studies, we used real-time RT-PCR to validate gene responses of representative genes in each of the functional groups (Fig. 3). The validated genes include novel "TGF-regulated" genes for PANC-1 and genes reported previously to be involved in tumor progression. The same genes were analyzed in PANC-1 luciferase (data not shown). Real-time RT-PCR results confirm the gene responses seen on the array. Furthermore, they demonstrate that PANC-1 and PANC-1 luciferase respond similarly.
Fig. 3. Validation of TGF-regulated gene responses for PANC-1 by real-time RT-PCR. Expression results for COL7A1, CTGF, CYR61, F2R, ITGAV, IGF2, ITGB5, JAG1, and SERPINE1. Expression levels were normalized to 18S rRNA. For each gene, the control (DMSO) was set to 1. All real-time RT-PCR reactions were performed in triplicate on each of the three biological replicates. TGF positively regulated all genes, whereas SD-208 inhibited induction.
Fig. 4. Effect of SD-208 on TGF-dependent invasion. PANC-1 cells were cultured for 20 h in the presence of 0.1% DMSO (vehicle), SD-208 (1 µM), and TGF1 (2 ng/ml) alone or in combination with SD-208. Cells that crossed chamber membranes were quantified as described under Materials and Methods. Mean ± S.D. values were determined for triplicate wells per condition from a representative study. Responses for vehicle-treated and SD-208-treated samples were compared with the response for the TGF-stimulated sample (* and ** represent p values <0.05 and <0.01, respectively). The analysis reveals that TGF induced invasion and that SD-208 inhibited invasion. The response for the SD-208-treated sample was also compared with the response for the vehicle-treated sample and shows no statistical significance between the two groups.
Inhibition of TGFRI Attenuates TGF-Stimulated Invasion in Vitro. The gene array identified many TGF-responsive genes that may promote metastasis. To test whether SD-208 inhibits metastasis in vitro, we measured invasion using Boyden chambers. Membranes in the chambers are coated with ECM to mimic basement membrane. Movement of cells through the membrane requires not only migration but also degradation of matrix, a critical step in the metastatic process. Treatment of cells with TGF1 stimulated invasion, whereas cotreatment with SD-208 inhibited TGF1-induced invasion (Fig. 4). These results are consistent with gene responses on the array that suggest that TGF signaling promotes cell motility and matrix remodeling.
To confirm that other pancreatic cancer cells are similarly affected by SD-208, we tested the effect of SD-208 on TGFRI signaling and motility in the Smad4-deficient cell line, BxPC3. Similar to PANC-1, the IC50 value of signaling from the receptor was achieved at 62.5 to 125 nM SD-208 (Supplemental Fig. S1A). Likewise, invasion assays demonstrated that TGF induced cell motility and that SD-208 inhibited cell motility (Supplemental Fig. S1B). Together, these results confirm that TGF signaling and TGF-stimulated motility are inhibited in another pancreatic tumor cell line harboring different mutations.
Inhibition of TGFRI Reduces Primary Tumor Growth and Metastasis in an Orthotopic Xenograft Model. The cell studies revealed that SD-208 inhibited TGF-dependent processes in PANC-1. To determine whether SD-208 affected these processes in vivo, we tested this inhibitor in a mouse model of pancreatic cancer. In this model, tumor cells were labeled with luciferase so tumor progression could be followed by luminescent imaging. Oral treatment with SD-208 (20 or 60 mg/kg twice daily) began at day 10 when tumors were established. Weekly monitoring revealed that tumors in the vehicle group continued to increase in luminescence throughout the 56-day period. At day 56, tumors in the SD-208 group had lower luminescence intensity compared with the vehicle group (data not shown). Furthermore, tumors in the SD-208 group were smaller than tumors in the vehicle group: the tumor weight (mean ± S.D.) for animals in the vehicle group was 0.7 ± 0.4 g, whereas the tumor weight for animals treated with 20 or 60 mg/kg SD-208 was only 0.3 ± 0.2 and 0.2 ± 0.2 g, respectively (Fig. 5A). The difference in tumor weight between vehicle and treated animals was significant (p < 0.01 and < 0.001, respectively). Moreover, two tumors completely regressed in animals treated with 60 mg/kg SD-208.
Fig. 5. Effect of SD-208 on tumor growth in an orthotopic xenograft model. Mean tumor weights of vehicle-treated and SD-208-treated tumors from two independent studies (A and B). Tumor weights were measured at study termination on day 56. Mean ± S.D. was determined for 12 mice in each treatment group. Statistical significance was determined using one-way analysis of variance with Bonferroni correction (A) or Student's t test (B). Comparison of tumor weights between vehicle and SD-208 treatment groups indicates that SD-208 reduced tumor growth (** and *** represent p values <0.01 and <0.001, respectively).
Metastatic lesions were less prevalent in treated groups compared with the vehicle group (Table 3, study 1). Nine mice in the vehicle group (n = 12) had metastatic lesions in the lymph nodes, spleen, liver, and peritoneum, whereas only five mice (42%) in the 60 mg/kg treatment group (n = 12) had metastatic lesions. These were found primarily in the lymph node. The 20 mg/kg dose had metastatic lesions in 50% of the mice with several in the spleen and peritoneum, suggesting a dose effect.
TABLE 3 Reduced incidence of metastasis with SD-208 treatment
Incidence and distribution of metastatic lesions from two independent studies. Treatment with SD-208 reduced the incidence and distribution of metastatic lesions in a dose-dependent manner.
A second study using a 60 mg/kg dose confirmed the results of the first study. Examination of tumors revealed that the 60 mg/kg group responded similarly to treated animals in the first study, with a dramatic decrease in mean tumor weight (0.2 ± 0.2 versus 0.6 ± 0.4 g) and reduction in number of metastatic lesions. The mean tumor weight for the treated group was statistically different (p = 0.004) from the mean tumor weight for the vehicle group (Fig. 5B). Once again, two tumors completely regressed. Nine of the 12 mice had metastatic lesions in the vehicle group, whereas only 4 mice had lesions in the treatment group (Table 3, study 2). Lesions in the treated animals resided in the lymph node and spleen but not in distal organs. Together, these studies demonstrate that treatment with SD-208 reduced both tumor growth and invasiveness in an orthotopic model of human pancreatic cancer.
The lack of effective treatment for pancreatic cancer underscores the need to develop new therapies. In the last decade, our understanding of the role of TGF signaling in tumor progression has made the TGF pathway an attractive target for intervention with soluble receptors (Rowland-Goldsmith et al., 2001, 2002), antisense oligonucleotides (Schlingensiepen et al., 2006), and small-molecule inhibitors (Singh et al., 2004; Subramanian et al., 2004; Uhl et al., 2004; Halder et al., 2005). Previously, SD-208 has been shown to reduce tumor progression in an orthotopic syngeneic model of glioblastoma (Uhl et al., 2004). The increased survival of animals treated with SD-208 seems to have been driven by the host mounting an immune response against the tumor. Here we extend these findings and demonstrate that SD-208 can inhibit tumor progression in pancreatic cancer, which, like glioblastoma, has no effective therapy. To our knowledge, this is the first example to demonstrate the efficacy of a small-molecule TGF-signaling inhibitor in an animal model of pancreatic cancer. The observed effect in an immune-compromised animal further suggests that inhibition of TGF signaling can reduce tumor progression and, in some cases, cure animals of tumor in the absence of a normal immune response. This is significant in that it demonstrates an additional mechanistic rationale for inhibiting TGF signaling.
The involvement of the TGF pathway has been established in cancers of many organs including the breast, lung, colon, prostate, and pancreas (Elliott and Blobe, 2005). TGF signaling is frequently attenuated in pancreatic cancer because of alterations in components of the pathway (Jonson et al., 2001). Although PANC-1 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 (Nicolás and Hill, 2003). Its attenuated signaling may be caused by low levels of TGFRI and by high levels of Smad7 (Nicolás and Hill, 2003). Despite these alterations, the phosphorylation status of the Smad2, a substrate of TGFRI, can be used to monitor receptor activity. We observed that phosphorylation of Smad2 increased in PANC-1 after treatment with TGF, whereas nanomolar levels of SD-208 inhibited induction. Similar receptor activation and inhibition profiles were observed when we tested the Smad4-deficient cell line BxPC-3. These results demonstrate that SD-208 is a potent inhibitor of TGFRI signaling in two pancreatic cancer cell lines that differ with respect to tumorigenic mutations and Smad4 activity. We also tested the efficacy of SD-208 inhibition on the expression of proteins regulated by TGFRI in PANC-1 cells. We observed that SD-208 reduced levels of CTGF, PAI-1, and VEGF in the culture medium. It is noteworthy that levels of TGF1 and TGF2 were also reduced, indicating that SD-208 inhibits autocrine induction of TGF. The effect of SD-208 on the production of these TGF-regulated proteins, known modulators of fibrosis, angiogenesis, and metastasis, confirmed that SD-208 is a potent inhibitor of TGFRI signaling.
Using the gene array data, we identified TGF-responsive genes in PANC-1 involved in ECM remodeling, cell motility, adhesion, angiogenesis, cell cycle, proliferation, and apoptosis. Regulation of these genes by TGF is inhibited by SD-208. Some genes, such as SERPINE (PAI-1), CTGF, and CDH1, are known to be regulated by TGF in pancreatic ductal adenocarcinoma (Geng et al., 1999; Halder et al., 2005). Regulation of other genes such as JAG1, CYR61, VEGFC, NRP2, and IGF2 by TGF is novel for PANC-1 and, to our knowledge, pancreatic cancer. An interesting finding in the expression analysis is that MYC was induced instead of repressed by TGF.
The TGF-dependent induction of SERPINE and CTGF, known regulators of matrix remodeling, is consistent with the responses we observed in invasion assays and in cells studies when we measured secreted PAI-1 and CTGF. Negative regulation of E-cadherin (CDH1)byTGF and reversal with TGFRI inhibitor SB-431542 has been observed previously in PANC-1 cultures (Halder et al., 2005). Down-regulation of E-cadherin is required for epithelial-to-mesenchymal transition (EMT) and metastasis (Hay, 1995) and has been associated with lymph node metastasis in high-grade and advanced-stage pancreatic cancer (Pignatelli et al., 1994). The TGF-dependent regulation of JAG1 on the array is interesting because its gene product, the Notch-ligand Jagged1, has been reported to be induced by TGF in cultured epithelial cells, in which it is important for EMT (Zavadil et al., 2004). Thus, two gene responses suggest that SD-208 may inhibit EMT in pancreatic cancer. It is interesting that JAG1 has also been found to play an important role in vascular angiogenic remodeling (Xue et al., 1999).
Several of the other genes affected by TGF (CYR61, VEGFC, and NRP2) regulate angiogenesis or lymphangiogenesis. CYR61 encodes a secreted, matricellular protein that binds to integrin v3 in human umbilical vein endothelial cells and promotes angiogenesis and cell adhesion, migration, and tumor growth (Kireeva et al., 1996; Babic et al., 1998). The gene product of VEGFC (VEGF-C) plays a role in the early development of the vascular system and lymphangiogenesis (Kukk et al., 1996). It is implicated in tumor progression because its expression in pancreatic cancer correlates with increased lymphatic vessel invasion and lymph node metastasis (Tang et al., 2001). The last gene, neuropilin (NRP2), encodes a coreceptor for VEGF-C and VEGF-A and is highly expressed in pancreatic cancer, in which it is believed to promote tumor angiogenesis and progression (Fukahi et al., 2004). Although VEGFA was not on the array, the regulation of VEGFC and NRP2 in PANC-1 is consistent with the TGF induction of total VEGF protein observed in this study and reported previously (Teraoka et al., 2001). The gene analysis, therefore, indicates that SD-208 is a potent inhibitor of TGF-dependent gene responses that support angiogenesis or lymphangiogenesis.
The observation that TGF signaling induced expression of IGF2 and MYC in PANC-1 is notable because both genes are involved in cell proliferation and are frequently dysregulated in tumors. IGF2 has been shown to promote DNA synthesis and cell survival and to reduce apoptosis in various pancreatic cell types (Hogg et al., 1993; Petrik et al., 1998). It is a ligand for the IGF1 receptor (IGFR1) and is overexpressed in most primary tumors (Fürstenberger and Senn, 2002). Regulation of c-Myc by TGF is essential for cell-cycle arrest: G1 arrest requires TGF-mediated down-regulation of c-Myc to relieve transcriptional suppression of p15 and p21Cip1/WAF1 (Donovan and Slingerland, 2000; Adhikary and Eilers, 2005). Tumor cells escape TGF-mediated growth arrest by overexpressing or activating c-Myc (Adhikary and Eilers, 2005). Thus, the induction of these genes by TGF could explain why we have observed (M. Henson and N. Gaspar, unpublished data) and others have reported (Nicolás and Hill, 2003; Subramanian et al., 2004) that PANC-1 is unresponsive or weakly responsive to TGF growth arrest.
When we evaluated TGF-dependent motility of PANC-1, we found that TGF induced migration (G. Li, unpublished data) and invasion (this report), whereas SD-208 inhibited TGF-dependent motility. Similar results were seen when we evaluated motility of the pancreatic adenocarcinoma cell line, BxPC3. Contrary to our study, PANC-1 has been reported to be unresponsive in motility assays to induction with TGF or inhibition with SD-093, another TGFRI inhibitor (Subramanian et al., 2004). BxPC3, on the other hand, was reported to be responsive to induction and inhibition in invasions assays but only inhibition in migration assays (Subramanian et al., 2004). The differences observed in our study and the previous study may reflect differences in assay conditions or assay sensitivity. The responses we observed for PANC-1 in motility assays are consistent with our gene array results, which reveal that motility-promoting gene responses are induced by TGF and inhibited by SD-208. Moreover, they are consistent with the observation that SD-208 inhibits metastasis in the animal model and provide an explanation for the effect.
TGF signaling has been targeted previously in animal models of human pancreatic ductal adenocarcinoma (Rowland-Goldsmith et al., 2001, 2002). The studies demonstrated that when COLO-357 or PANC-1 tumor cells expressing soluble TGFRII receptor (sTRII) were injected into mice, they formed smaller tumors than sham-transfected cells. From these studies, it is not possible to determine whether inhibiting TGF signaling attenuated tumor progression or prevented the establishment of tumors. Because we treated animals with established tumors, our studies demonstrate that inhibiting TGF signaling can in fact attenuate tumor growth and metastasis and, in some cases, cure animals of tumor.
In summary, we have demonstrated that SD-208 inhibits tumor-associated processes in a human pancreatic adenocarcinoma at the cellular level and in an animal model. The study identifies potential mechanisms through which inhibition of TGFRI signaling with a small-molecule inhibitor can reduce tumor progression. Furthermore, it provides hypotheses that can be tested in animals or in the clinical setting. The results of this study are encouraging because they demonstrate that SD-208 not only reduced TGF-mediated gene expression, protein expression, and invasion, but that it also reduced both primary tumor growth and metastasis in vivo. Thus, these findings suggest that molecules similar to SD-208 and, moreover, small-molecule inhibitors of TGFRI, may be effective as new therapies to treat human pancreatic cancer.
Acknowledgements
The contributions of others have made this work possible. We are grateful to Ute Schellenberger for producing CTGF antibodies, Frauke Schimmoller for developing the CTGF ELISA, Maggie Sable and Bijal Patel for helping with graphics, and Bruce Koppelman and Aaron N. Nguyen for critically reviewing the manuscript.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
【参考文献】
Adhikary S and Eilers M (2005) Transcriptional regulation and transformation by Myc proteins. Nat Rev Mol Cell Biol 6: 635-645.
Babic AM, Kireeva ML, Kolesnikova TV, and Lau LF (1998) CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc Natl Acad Sci U S A 95: 6355-6360.[Abstract/Free Full Text]
Derynck R and Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425: 577-584.
Donovan J and Slingerland J (2000) Transforming growth factor-beta and breast cancer: Cell cycle arrest by transforming growth factor-beta and its disruption in cancer. Breast Cancer Res 2: 116-124.
Dumont N, Bakin AV, and Arteaga CL (2003) Autocrine transforming growth factor- signaling mediates Smad-independent motility in human cancer cells. J Biol Chem 278: 3275-3285.[Abstract/Free Full Text]
Eckel F, Schneider G, and Schmid RM (2006) Pancreatic cancer: a review of recent advances. Expert Opin Investig Drugs 15: 1395-1410.
Ellenrieder V, Hendler SF, Boeck W, Seufferlein T, Menke A, Ruhland C, Adler G, and Gress TM (2001) Transforming growth factor beta1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation. Cancer Res 61: 4222-4228.[Abstract/Free Full Text]
Elliott RL and Blobe GC (2005) Role of transforming growth factor Beta in human cancer. J Clin Oncol 23: 2078-2093.[Abstract/Free Full Text]
Friess H, Kleeff J, Korc M, and Buchler MW (1999) Molecular aspects of pancreatic cancer and future perspectives. Dig Surg 16: 281-290.
Fukahi K, Fukasawa M, Neufeld G, Itakura J, and Korc M (2004) Aberrant expression of neuropilin-1 and -2 in human pancreatic cancer cells. Clin Cancer Res 10: 581-590.[Abstract/Free Full Text]
Fürstenberger G and Senn HJ (2002) Insulin-like growth factors and cancer. Lancet Oncol 3: 298-302.
Geng MM, Ellenrieder V, Wallrapp C, Muller-Pillasch F, Sommer G, Adler G, and Gress TM (1999) Use of representational difference analysis to study the effect of TGFB on the expression profile of a pancreatic cancer cell line. Genes Chromosomes Cancer 26: 70-79.
Grau AM, Zhang L, Wang W, Ruan S, Evans DB, Abbruzzese JL, Zhang W, and Chiao PJ (1997) Induction of p21waf1 expression and growth inhibition by transforming growth factor beta involve the tumor suppressor gene DPC4 in human pancreatic adenocarcinoma cells. Cancer Res 57: 3929-3934.[Abstract/Free Full Text]
Halder SK, Beauchamp RD, and Datta PK (2005) A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers. Neoplasia 7: 509-521.
Hay ED (1995) An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154: 8-20.
Heldin CH, Miyazono K, and ten Dijke P (1997) TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390: 465-471.
Hogg J, Han VK, Clemmons DR, and Hill DJ (1993) Interactions of nutrients, insulin-like growth factors (IGFs) and IGF-binding proteins in the regulation of DNA synthesis by isolated fetal rat islets of Langerhans. J Endocrinol 138: 401-412.[Abstract/Free Full Text]
Hruban RH, Goggins M, Parsons J, and Kern SE (2000) Progression model for pancreatic cancer. Clin Cancer Res 6: 2969-2972.[Free Full Text]
Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, and Thun MJ (2006) Cancer statistics, 2006. CA Cancer J Clin 56: 106-130.[Abstract/Free Full Text]
Jonson T, Albrechtsson E, Axelson J, Heidenblad M, Gorunova L, Johansson B, and Hoglund M (2001) Altered expression of TGFB receptors and mitogenic effects of TGFB in pancreatic carcinomas. Int J Oncol 19: 71-81.
Kapoun AM, Gaspar NJ, Wang Y, Damm D, Liu YW, O'Young G, Quon D, Lam A, Munson K, Tran TT, et al. (2006) Transforming growth factor- receptor type 1 (TGFRI) kinase activity but not p38 activation is required for TGFRI-induced myofibroblast differentiation and profibrotic gene expression. Mol Pharmacol 70: 518-531.[Abstract/Free Full Text]
Kireeva ML, Mo FE, Yang GP, and Lau LF (1996) Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion. Mol Cell Biol 16: 1326-1334.
Kukk E, Lymboussaki A, Taira S, Kaipainen A, Jeltsch M, Joukov V, and Alitalo K (1996) VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 122: 3829-3837.
Moore PS, Sipos B, Orlandini S, Sorio C, Real FX, Lemoine NR, Gress T, Bassi C, Kloppel G, Kalthoff H, et al. (2001) Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4. Virchows Arch 439: 798-802.
Naumann M, Savitskaia N, Eilert C, Schramm A, Kalthoff H, and Schmiegel W (1996) Frequent codeletion of p16/MTS1 and p15/MTS2 and genetic alterations in p16/MTS1 in pancreatic tumors. Gastroenterology 110: 1215-1224.
Nicolás FJ and Hill CS (2003) Attenuation of the TGF-beta-Smad signaling pathway in pancreatic tumor cells confers resistance to TGF-beta-induced growth arrest. Oncogene 22: 3698-3711.
Petrik J, Arany E, McDonald TJ, and Hill DJ (1998) Apoptosis in the pancreatic islet cells of the neonatal rat is associated with a reduced expression of insulin-like growth factor II that may act as a survival factor. Endocrinology 139: 2994-3004.[Abstract/Free Full Text]
Pignatelli M, Ansari TW, Gunter P, Liu D, Hirano S, Takeichi M, Kloppel G, and Lemoine NR (1994) Loss of membranous E-cadherin expression in pancreatic cancer: correlation with lymph node metastasis, high grade, and advanced stage. J Pathol 174: 243-248.
Rowland-Goldsmith MA, Maruyama H, Kusama T, Ralli S, and Korc M (2001) Soluble type II transforming growth factor-beta (TGF-beta) receptor inhibits TGF-beta signaling in COLO-357 pancreatic cancer cells in vitro and attenuates tumor formation. Clin Cancer Res 7: 2931-2940.[Abstract/Free Full Text]
Rowland-Goldsmith MA, Maruyama H, Matsuda K, Idezawa T, Ralli M, Ralli S, and Korc M (2002) Soluble type II transforming growth factor-beta receptor attenuates expression of metastasis-associated genes and suppresses pancreatic cancer cell metastasis. Mol Cancer Ther 1: 161-167.[Abstract/Free Full Text]
Schlingensiepen KH, Schlingensiepen R, Steinbrecher A, Hau P, Bogdahn U, Fischer-Blass B, and Jachimczak P (2006) Targeted tumor therapy with the TGF-beta2 antisense compound AP 12009. Cytokine Growth Factor Rev 17: 129-139.
Schutte M, Hruban RH, Hedrick L, Cho KR, Nadasdy GM, Weinstein CL, Bova GS, Isaacs WB, Cairns P, Nawroz H, et al. (1996) DPC4 gene in various tumor types. Cancer Res 56: 2527-2530.[Abstract/Free Full Text]
Singh J, Ling LE, Sawyer JS, Lee WC, Zhang F, and Yingling JM (2004) Transforming the TGFbeta pathway: convergence of distinct lead generation strategies on a novel kinase pharmacophore for TbetaRI (ALK5). Curr Opin Drug Discov Devel 7: 437-445.
Subramanian G, Schwarz RE, Higgins L, McEnroe G, Chakravarty S, Dugar S, and Reiss M (2004) Targeting endogenous transforming growth factor beta receptor signaling in SMAD4-deficient human pancreatic carcinoma cells inhibits their invasive phenotype1. Cancer Res 64: 5200-5211.[Abstract/Free Full Text]
Tang RF, Itakura J, Aikawa T, Matsuda K, Fujii H, Korc M, and Matsumoto Y (2001) Overexpression of lymphangiogenic growth factor VEGF-C in human pancreatic cancer. Pancreas 22: 285-292.
Teraoka H, Sawada T, Nishihara T, Yashiro M, Ohira M, Ishikawa T, Nishino H, and Hirakawa K (2001) Enhanced VEGF production and decreased immunogenicity induced by TGF-beta 1 promote liver metastasis of pancreatic cancer. Br J Cancer 85: 612-617.
Uhl M, Aulwurm S, Wischhusen J, Weiler M, Ma JY, Almirez R, Mangadu R, Liu YW, Platten M, Herrlinger U, et al. (2004) SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo. Cancer Res 64: 7954-7961.[Abstract/Free Full Text]
Villanueva A, Garcia C, Paules AB, Vicente M, Megias M, Reyes G, de Villalonga P, Agell N, Lluis F, Bachs O, et al. (1998) Disruption of the antiproliferative TGF-beta signaling pathways in human pancreatic cancer cells. Oncogene 17: 1969-1978.
Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, Gendron-Maguire M, Rand EB, Weinmaster G, and Gridley T (1999) Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet 8: 723-730.[Abstract/Free Full Text]
Yasutome M, Gunn J, and Korc M (2005) Restoration of Smad4 in BxPC3 pancreatic cancer cells attenuates proliferation without altering angiogenesis. Clin Exp Metastasis 22: 461-473.
Zavadil J, Cermak L, Soto-Nieves N, and Bottinger EP (2004) Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition. EMBO J 23: 1155-1165.
作者单位：Scios Inc., Fremont, California