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【摘要】
The nonreceptor protein tyrosine kinase Src is overexpressed in 70% of pancreatic adenocarcinomas. Here, we describe the effect of molecular and pharmacological down-regulation of Src on incidence, growth, and metastasis of pancreatic tumor cells in an orthotopic model. Src expression in L3.6pl human pancreatic tumor cells was reduced by stable expression of a plasmid encoding small interfering RNA (siRNA) to c-src. In stable siRNA clones, Src expression was reduced >80%, with no change in expression of the related kinases c-Yes and c-Lyn, and proliferation rates were similar in all clones. Phosphorylation of Akt and p44/42 Erk mitogen-activated protein kinase and production of VEGF and IL-8 in culture supernatants were also reduced (P < 0.005). On orthotopic implantation of varying cell numbers into nude mice, tumor incidence was unchanged; however, in the siRNA clones, large tumors failed to develop, and incidence of metastasis was significantly reduced, suggesting that c-Src activity is critical to tumor progression. To examine this possibility further, animals bearing established wild-type tumors were treated with the Src/Abl-selective inhibitor BMS-354825 (dasatinib). Tumor size was decreased, and incidence of metastases was significantly reduced in treated mice compared with controls. These results demonstrate that Src activation contributes to pancreatic tumor progression in this model, offering Src as a candidate for targeted therapy.
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Adenocarcinoma of the exocrine pancreas is the fourth most common cause of cancer death in developed countries with more than 30,000 estimated deaths in 2004 in the United States alone.1 Of the 5% of patients who present with resectable disease, only 12% survive 1 year after diagnosis and less than 5% survive 5 years.2-4 Metastasis to the lymphatics, liver, and vessel walls leads to widespread disease, resulting in a severe wasting condition that accounts for approximately 80% of deaths in advanced pancreatic cancer.5 Even when potentially curative surgery is performed, approximately 80 to 90% of patients develop disease recurrence with standard chemotherapeutic agents having marginal effect on patient survival. Because of the high mortality associated with pancreatic adenocarcinoma and early systemic disease, it is essential that therapeutic regimens be developed to inhibit tumor progression and metastasis.
The progression of pancreatic adenocarcinoma has been associated with deregulation of several signaling molecules.6 One of the potential therapeutic targets receiving considerable recent attention is activation of c-Src, a nonreceptor protein tyrosine kinase. c-Src is a 60-kd prototype of a nine-member family of structurally related Src family kinases (SFKs). In normal cells, SFKs regulate diverse biological processes by associating with multiple signaling and structural molecules. Overexpression of SFKs occurs in many solid tumors, often at later stages of disease,7 and can be predictive of poor prognosis.8 In addition, Src activation can be associated with chemoresistance. Thus, Gleevec-resistant chronic myeloid leukemia (CML)9 and taxol-resistant ovarian cancer cells10 are frequently associated with increased expression of SFKs.
In pancreatic adenocarcinomas, Src is activated in more than 70% of primary tumors.11 Several recent reports have implicated this activity as important to properties of tumor progression. Ito et al12 demonstrated that inhibition of Src resulted in a 90% decrease in in vitro pancreatic cancer cell invasiveness by inhibiting Src-dependent matrix metalloproteinases MMP 2 and MMP 9. We have recently demonstrated that Src is a critical regulator of pro-angiogenic molecules.13-15 Duxbury et al16 have provided evidence that gemcitabine resistance correlates with increased Src activity, and Src inhibition overcomes this resistance. Recently, Src inhibition with a novel Src family kinase inhibitor has demonstrated significant antitumor and antimetastatic activity in a pancreatic cancer orthotopic nude mouse model.17 These data support a potential role for Src inhibitors in the treatment of pancreatic cancer.
However, signal transduction inhibitors affect multiple targets, and "off-target" inhibition can be responsible for antitumor effects. Additionally, SFKs have overlapping functions in multiple signaling pathways. Therefore, we first used molecular strategies to examine the specific role of c-Src in pancreatic tumor growth in vitro and in vivo. We then determined whether dasatinib, a dual Src/Abl inhibitor,18 would give results similar to those of the molecular approach. The data in this study strongly support a role for activation of c-Src, as opposed to other SFK members, in pancreatic tumor progression in a relevant mouse model and suggest that Src-selective inhibitors could have efficacy in preventing or delaying pancreatic tumor metastasis.
【关键词】 inhibition expression activity inhibits progression metastasis pancreatic adenocarcinoma orthotopic
Materials and Methods
Cell Lines
The L3.6pl pancreatic cancer cell line was obtained from Dr. Lee Ellis (University of Texas-MD Anderson Cancer Center). The L3.6pl cell line was derived from a repeated cycle of injecting COLO-357 cells into the pancreas of nude mice, selecting for liver metastases, and re-injecting into the pancreas.19 The cells were plated on 10-cm tissue culture dishes, grown as monolayer cultures, and maintained in culture in minimal essential media supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 2 mmol/L L-glutamine, and 0.6% penicillin/streptomycin and 5% CO2/95% air at 37??C.
Cell Lysis and Protein Extraction
Cells (1 x 106) were plated in 10-cm dishes and maintained in minimal essential media with 10% FBS. At 70 to 80% confluence, the cells were washed with Dulbecco??s phosphate buffered saline (D-PBS) at 37??C and maintained in serum-free media (10 ml) for 24 hours. The cells and supernatants were harvested at 24 hours. The cells were washed with ice-cold 1x D-PBS, scraped from the plates, lysed, and harvested on ice in radio-immune precipitation assay (RIPA-B) buffer (20 mmol/L sodium phosphate buffer, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, and 0.5% sodium deoxycholate) supplemented with one tablet complete mini-EDTA protease inhibitor cocktail (Roche Diagnostic, Mannheim, Germany) and sodium orthovanadate (1 mmol/L, pH 7.4). Harvested orthotopic pancreatic tumors were homogenized in RIPA-B buffer using a tissue homogenizer. The homogenates were clarified by centrifugation at 15,000 x g for 15 minutes at 4??C and prepared for Western analysis and immunoprecipitation. Metastases were isolated from normal liver, frozen in liquid nitrogen, and lysed in RIPA-B via mortar and pestle.
Creation of Small Interfering RNA (siRNA) Expression Plasmids for Silencing Src Gene Expression
siRNA expression plasmids were created as described elsewhere,13 using the Ambion pSilencer 1.0-U6 (Austin, TX) according to manufacturer??s directions. Briefly, c-Src-specific target sequences were designed using the Ambion siRNA Web design tool. The two target sequences used were (52 to 71 bp) 5'-AACAAGAG CAAGCCCAAGGAT-3' and (226 to 244 bp) 5'-AAGCTGTTCGGAGGCTTCAAC-3'. Oligonucleotides corresponding to these sequences with flanking ApaI (5') and EcoR1 (3') ends were purchased from Invitrogen/Life Technologies (Carlsbad, CA) and ligated into the expression plasmid at compatible sites. Constructs were confirmed by DNA sequencing. L3.6pl cells were then transfected with 0.5 ng of each siRNA plasmid and 10 ng of pcDNA G418 resistance promoterless plasmid for selection of transfectants. Cells were then grown in selective media containing G418 as previously described.20 Negative controls were transfected with empty vector target sequences and pcDNA plasmids at identical concentrations. Total c-Src expression levels in siRNA clones were determined by Western blot analysis.
Cell Proliferation Assay
Cell proliferation was quantified by 3-(4,5-dimethyl-2-thiazol-2-yl) 2,5-diphenyltetrazolium bromide assay (Trivegen, Gaithersburg, MD). Cells were seeded into 96-well plates at 1 x 103 cells per well and allowed to adhere overnight in medium containing 10% FBS. The cells were maintained in standard culture conditions, and cellular proliferation and viability were assayed at different time points. Plates were read using spectrophotometric analysis at a wavelength of 570 nm using the TECAN Genios plate reader (TECAN US, Durham, NC) and Magellan version 4.0 software. Twelve samples were used for each cell clone, and the experiments were performed in triplicate.
Immunoblot Analysis
Total protein concentrations were determined via the Bio-Rad Dc protein assay protocol (Bio-Rad, Hercules, CA) followed by spectrophotometric analysis using the TECAN Genios plate reader and Magellan version 4.0 software. Equal amounts of protein (50 µg) were loaded in each well, separated via 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroblotted onto Immobilon-P membranes (Millipore, Billerica, MA). The membranes were blocked with Tris-buffered saline/Tween (0.1%) + 5% dried milk for 30 minutes and probed with desired primary antibody diluted 1:1000 in blocking buffer overnight at 4??C. Membranes were probed with polyclonal antibodies to phospho-AktS473 (Cell Signaling Technology, Beverly, MA), phospho-p44/42 ErkT202/Y204 (Cell Signaling Technology), and total p44/42 Erk mitogen-activated protein kinase (MAPK) (Ab-2; Calbiochem, San Diego, CA) and monoclonal antibodies to total Src (Calbiochem), c-Yes (Wako Chemicals, Richmond, VA), Lyn (Santa Cruz Biotechnology, Santa Cruz, CA), Akt (5G3; Cell Signaling Technology), and vinculin (Sigma-Aldrich, St. Louis, MO). Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Bio-Rad goat anti-mouse and sheep anti-rabbit) diluted 1:2000 in blocking buffer for 1 hour at room temperature with gentle rocking. Western blot analyses of actin and vinculin expression were performed as a loading control using anti-actin and anti-vinculin monoclonal antibodies (Sigma-Aldrich). Proteins were visualized by incubation with ECL detection reagents (Perkin-Elmer, Boston, MA) and exposed to film (Kodak Biomax MR, Rochester, NY). Membranes were stripped and reprobed.
Immunoprecipitation
For detection of c-Yes expression in tumor samples, 500 µg of the samples in 650 µl of RIPA buffer was incubated by rotation with 6 µl of antibody to total c-Yes overnight at 4??C. Fifty µL of a 1:1 slurry of protein G agarose in RIPA-B buffer was added and incubated with rotation for 1 additional hour at 4??C. Bound proteins were pelleted by centrifugation, washed three times with RIPA-B buffer, and eluted by boiling in 1x Laemmli??s sample buffer with subsequent immunoblotting with antibodies against c-Yes (1:1000).
Determination of Interleukin 8 (IL-8) and Vascular Endothelial Growth Factor (VEGF) Levels
Culture supernatants were centrifuged for 1 minute at 15,000 rpm to pellet debris and transferred to microcentrifuge tubes. Supernatants not assayed immediately were frozen at C80??C. Quantitative measurements of IL-8 and VEGF in the cell supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits (Quantikine Human IL-8 Immunoassay; R&D Systems, Minneapolis, MN; and Human VEGF Immunoassay kit; Biosource International, Inc., Camarillo, CA) following the manufacturers?? instructions. The detection limits of the IL-8 and VEGF ELISAs were 37 and 23.4 pg/ml, respectively. IL-8 and VEGF concentrations (picograms/milliliter) were determined spectrophotometrically at 450 nm using a TECAN Genios plate reader and Magellan version 4.0 software and normalized against total protein levels in the corresponding cell lysate. The results are presented as the means of triplicate determinations (??SD).
Animals
Specific, pathogen-free male nude mice (strain NCR-NU) were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed and maintained in specific, pathogen-free conditions. The facilities have been approved by the American Association for Accreditation of Laboratory Animal Care and meet all current regulations and standards of the U.S. Department of Agriculture, the U.S. Department of Health and Human Services, and the National Institutes of Health. The mice were used between the ages of 8 and 12 weeks, in accordance with institutional guidelines.
Orthotopic Pancreatic Injections
For in vivo injection, cells were harvested from 10-cm tissue culture dish by a 2- to 3-minute treatment with 1x trypsin followed by suspension in a D-PBS solution. Only single-cell suspensions of greater than 90% viability, as determined by trypan blue exclusion, were used for injection. Male nude mice were anesthetized with methoxyflurane. A small left abdominal flank incision was made, and the spleen and pancreas were exteriorized. Tumor cells (5 x 105), including siRNA clones, vector, and wild-type parental controls, in D-PBS were injected subcapsularly into a region of the pancreas just beneath the spleen with a 27-gauge needle and 1-ml disposable syringe. To prevent intraperitoneal leakage, a cotton swab was held for 1 minute over the site of injection. Both layers of the abdominal wound were closed with wound clips (Autoclip; Clay Adams, Parsippany, NJ). A successful subcapsular intrapancreatic injection of tumor cells was identified by the appearance of a fluid bleb without intraperitoneal leakage. Mice were sacrificed via cervical dislocation 6 weeks after orthotopic injections.
Treatment of Established Human Pancreatic Tumors with Dasatinib
For these studies, we used dasatinib, a dual Src/Abl inhibitor currently in clinical trials for CML. Fourteen days after orthotopic injection of wild-type L3.6pl pancreatic tumor cells, the mice were randomized into two groups: treatment and control (n = 8). The treatment group received 15 mg ?
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作者单位：From the Departments of Cancer Biology,* Surgical Oncology, Medical Oncology, Molecular Therapeutics,¶ and GI Medical Oncology,|| The University of Texas M.D. Anderson Cancer Center, Houston, Texas; and Bristol-Meyers Squibb Oncology, Princeton, New Jersey