The inhibition of SRC family kinase suppresses pancreatic cancer cell proliferation, migration, and invasion.

ORIGINAL ARTICLE

The Inhibition of Src Family Kinase Suppresses Pancreatic Cancer Cell Proliferation, Migration, and Invasion Dong Wook Je, MD, MS,* Young Moon O, BS,* Young Geon Ji, MD, PhD,Þ Yunkyung Cho, MD, PhD,þ and Dong Hyeon Lee, MD, PhD*

Objectives: Src is considered a rising therapeutic target for the treatment of solid tumors, and Src family kinases (SFKs) participate in cancer cell proliferation and survival. The role of SFK suppression was investigated in the proliferation, migration, and invasion of pancreatic cancer cells. Methods: Knockdown of the SFKs in pancreatic cancer cells was achieved by transfecting small interfering RNAs, and its effects were investigated using proliferation, wound, and invasion assays. Results: The SFK inhibitors suppressed proliferation and induced cell cycle arrest in pancreatic cancer cells. The SFK messenger RNA profiles showed that Yes1, Lyn, Fyn, Frk, Hck, and Src were expressed. Specific small interfering RNA transfection suppressed the messenger RNA expressions of Yes1, Lyn, Fyn, Frk, and Src, and the knockdown suppressed cell proliferation by 16.7% to 47.3% in PANC-1 cells. Knockdown of any of these 5 SFKs suppressed proliferation in other pancreatic cancer cell lines by 3.0% to 40.5%. The knockdowns significantly reduced pancreatic cancer cell migration by 24.9% to 66.7% and completely inhibited invasion. Conclusions: These results suggest that the knockdown of Yes1, Lyn, Fyn, Frk, or Src reduce human pancreatic cancer cell proliferation, migration, and invasion, and that SFKs should be viewed as critical therapeutic targets of pancreatic cancer. Key Words: Src family kinase, siRNA, pancreatic cancer, proliferation, migration, invasion (Pancreas 2014;43: 768Y776)

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ancreatic cancer is one of the major causes of death associated with cancer. It is an aggressive malignancy with a poor prognosis and survival rates with an overall 5-year survival rate of less than 5% because of late diagnosis, low resection rates, and a lack of effective treatment.1 Conditions of most patients are diagnosed with advanced disease, and patients do not receive the benefits of curative surgery. Furthermore, current adjuvant and neoadjuvant chemotherapies are ineffective in pancreatic cancer.2 Although many studies have evaluated various chemotherapeutic agents, only a few have demonstrated significant effectiveness and improved survival.3 Accordingly, the discovery of antitumor agents and potential targets for new therapeutic strategies is desperately needed for pancreatic cancer treatment. Intracellular signal transducers can promote the proliferation, migration, metastasis, and invasion of cancer cells.2,4 The signal transducer Src is a potential therapeutic target for antitumor therapy and has been intensively investigated in many solid cancers. Src kinase is a nonreceptor protein tyrosine kinase that mediates a From the Departments of *Physiology, †Preventive Medicine, and ‡Internal Medicine, School of Medicine, CHA University, Seongnam, Gyeonggi, South Korea. Received for publication April 26, 2013; accepted January 12, 2014. Reprints: Dong Hyeon Lee, MD, PhD, Department of Physiology, School of Medicine, CHA University, 222 Yatap, Bundang-gu, Sungnam-si, Gyeonggi-do 463-836, South Korea (e-tubulin was from Sigma-Aldrich (St Louis, Mo).

Cell Culture The human pancreatic cancer cell lines, PANC-1 derived from pancreatic epithelioid carcinoma,14 MIA PaCa-2 derived Pancreas

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Volume 43, Number 5, July 2014

Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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Inhibition of SFKs Exerts Anticancer Effects

from undifferentiated pancreatic carcinoma,15 Capan-1 derived from metastatic pancreatic adenocarcinoma obtained from liver, and Capan-2 derived from pancreatic adenocarcinoma, were obtained from the Korean Cell Line Bank (Seoul, South Korea). HPAC, derived from pancreatic adenocarcinoma, was obtained from the American Type Culture Collection (Manassas, Va). The PANC-1 and MIA PaCa-2 cells were maintained in DMEM supplemented with 10% FBS and 1% antimycotic-antibiotics. The Capan-1 and Capan-2 cells were maintained in RPMI with same supplements. The HPAC cells were maintained in DMEM/Ham’s F12 combined medium (1:1) containing 5% FBS, 2 Hg/mL insulin, 5 Hg/mL transferrin, 40 ng/mL hydrocortisone, and 10 ng/mL epidermal growth factor. The cells were grown at 37-C in a humidified atmosphere of 95% air and 5% carbon dioxide. The culture media were exchanged every 2 days.

Cell Proliferation Assay Cell proliferation was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described.16 Briefly, pancreatic cancer cells were diluted with culture medium to the seeding density of 3 103 cells/well, plated on 96-well flat bottom plates, and incubated at 37-C overnight. The cells were treated with SFK inhibitors or were transfected with siRNA. After incubation for 48 hours, 10 HL of MTT solution (5 mg/mL) (Amresco, Solon, Ohio) was added to each well, and the plates were incubated for another 4 hours. After incubation, 100 HL of dimethyl sulfoxide (Amresco) was added to each well to solubilize the MTT formazan product under gentle shaking. Luminescence was quantified using a microplate reader (VICTOR3, PerkinElmer, Waltham, Mass). Absorbance values measured at 560 nm with reference values measured at 670 nm represent the rate of mitochondrial metabolism and correspond to the number of viable cells. Growth inhibition was calculated as the percentage of viable cells compared with untreated cells. Each experiment assay was performed in triplicate. Cells were monitored by phasecontrast microscopy (Olympus, Tokyo, Japan), and digital images were taken after 48 hour posttreatment.

Cell Cycle Analysis Cell cycle distribution was analyzed by flow cytometry. Cells (1 106) were incubated with Src inhibitor for 48 hours. They were collected and washed with phosphate-buffered saline (PBS). After fixing in 70% ethanol at 4-C for 2 hours, they were washed and resuspended in staining solution (PBS containing 50 Hg/mL propidium iodide and 50 Hg/mL RNase A) at room temperature for 30 minutes in the dark for DNA staining. The cells were then analyzed using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ). The cell debris and fixation artifacts were gated out, and the percentages of subG0, G0/G1, S, and G2/M populations were measured. At least 20,000 events were evaluated.

Quantitative Reverse TranscriptionYPolymerase Chain Reaction Analysis Total cellular RNA was isolated and purified from pancreatic cancer cells and was reverse transcriptased as previously described.17 Briefly, each sample was homogenized and lysed in TRIzol reagent (Invitrogen, Carlsbad, Calif ). Total RNA was extracted with chloroform and precipitated with 80% (vol/vol) isopropanol. After removal of the supernatant, the RNA pellet was washed with 75% (vol/vol) ethanol, air-dried, and dissolved in 0.1% (vol/vol) diethyl pyrocarbonate-treated water. For eliminating contaminated DNA, RNA samples were treated with DNase (Qiagen, Hilden, Germany) and cleaned up using RNeasy Mini Kit (Qiagen). Complementary DNA (cDNA) was generated by reverse transcription of RNA using Maxime RT PreMix with Oligo(dT) primers (Intron Biotechnology, Seongnam, South Korea). Primer sequences for Src, Fyn, Yes1, Lck, Lyn, Hck, Fgr, Blk, and Frk (Bioneer, Daejeon, South Korea) are listed in Table 1. Primer for housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was adopted from previous studies.17 Quantitative RT-PCR was performed using a SYBR Green I Master Mix (Roche Applied Science, Mannheim, Germany) in a LightCycler 480 II System (Roche Applied Science). The amplification program included preincubation at 95-C for 5 minutes, followed by 45 cycles of PCR with denaturation at 95-C for

TABLE 1. Quantitative RT-PCR Primer Sequences Gene

Accession

Direction

Sequence

Position

Src

NM_005417.3

Fyn

NM_002037.5

Yes1

NM_005433.3

Lck

NM_001042771.1

Lyn

NM_002350.3

Hck

NM_002110.3

Fgr

NM_005248.2

Blk

NM_001715.2

Frk

NM_002031.2

+ Y + Y + Y + Y + Y + Y + Y + Y + Y

5¶-TGAGTACACGGCGCGGCAAG-3¶ 5¶-GGTCGTGCAGGGACTCGGGA-3¶ 5¶-GGGGCCAAGGACTCACCGTC-3¶ 5¶-GGAGCGGGCTTCCCACCAAT-3¶ 5¶-GGGGTAACGCCTTTTGGAGGTGC-3¶ 5¶-TGCTTCCCACCAATCTCCTTCCGT-3¶ 5¶-AAGGGCACGCTGCTCATCCG-3¶ 5¶-CACCACTCGCCGCTCTGCTC-3¶ 5¶-ATTCACCGGGACCTGCGAGC-3¶ 5¶-CAGGGCGGTCATCACGTCGG-3¶ 5¶-CAGCCGGAAGGACGCAGAGC-3¶ 5¶-AGCCCCCGTTGTCCAGGGTC-3¶ 5¶-CAACCTGCTCATCGCGCCCT-3¶ 5¶-GGCCTTCGGGGACATGGTGC-3¶ 5¶-ACTGCCGCCCCTGGTTGTCTT-3¶ 5¶-GCTCTCCACTCGGGCCACAAA-3¶ 5¶-TGCCGGTGAAGTGGACTGCG-3¶ 5¶-TGTGGACAGTTGGATGGTTGCGG-3¶

1700Y1719 1938Y1919 777Y796 979Y960 420Y442 614Y591 218Y237 414Y395 1372Y1391 1650Y1631 684Y703 859Y840 930Y949 1134Y1115 692Y712 935Y915 1631Y1650 1826Y1804

All nucleic acid sequences are from GenBank.

* 2014 Lippincott Williams & Wilkins

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10 seconds, annealing at 60-C for 10 seconds, and extension at 72-C for 10 seconds. The negative control, no cDNA template, was run simultaneously with every assay, and PCR from each cDNA sample was run in triplicate. Constitutively expressed GAPDH was used as an endogenous control. Results are presented as relative fold changes using the GAPDH reference and applying the formula 2j$$Ct. Fyn was used as a calibrator to illustrate the expression of SFKs relative to each other.

siRNA Inhibition Assay The knockdown of SFKs was performed using siRNAs as previously described.18 Briefly, the siRNAs (Bioneer) against human SFKs (Src, Yes1, Lyn, Fyn, Hck, and Frk) were used. The cells were grown in 96-well plates, 24-well plates, or 60-mm dishes at 70% to 80% confluence overnight and then transfected with predesigned siRNA (100 nM) using Lipofectamine RNAiMAX transfection reagent (Invitrogen) as instructed by the manufacturer. A no-target siRNA (scrambled siRNA) was used as a negative control. Forty-eight hours later, cells were assayed. The SFK expressions were detected by quantitative RT-PCR and/or Western blotting to demonstrate successful silencing of targets. The siRNA decreased messenger RNA (mRNA) expression of SFKs more than 80% and protein expression of SFKs more than 70% in pancreatic cancer cells.

Western Blotting Pancreatic cancer cells were lysed in M-PER Mammalian Protein Extract Reagent (Thermo Scientific, Rockford, Ill) containing protease inhibitor cocktail (Amresco). After sonication, the samples were centrifuged at 12,000g for 10 minutes at 4-C. The proteins were mixed with sample buffer, boiled for 10 minutes, and separated by 10% sodium dodecyl sulfateYpolyacrylamide gel electrophoresis. The gel was transferred to a polyvinylidene difluoride membrane and blocked with Tris-buffered saline with Tween 20 (TBST, 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20) containing 3% bovine serum albumin for 1 hour. The membrane was incubated with primary antibodies against Yes1, Lyn, Fyn, Frk, or Src (1:1000) at 4-C overnight, washed with TBST 3 times, and incubated with antiIgG secondary antibodies (1:2000) in TBST for 2 hours. After the membrane had been washed with TBST 3 times, protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Protein loading was compared by probing the blots with antiY>-tubulin antibody.

Cell Migration Assay Cell migration was measured by wound assay as previously described.19 Briefly, the siRNA-transfected pancreatic cancer cells were grown on 60-mm tissue culture dishes until they became confluent. Then using a sterile plastic micropipette tip, separate wounds were generated by scratching of the monolayer of cells. Cells were rinsed gently with PBS. Wound closure was monitored by phase-contrast microscopy, and digital images were taken after 24 and 48 hour after wounding.

Invasion Assay The invasiveness of pancreatic cancer cells was tested after siRNA transfection using synthetic basement membrane. Invasion assays were performed using a cell invasion assay kit (Millipore, Temecula, Calif ). PANC-1 cells were harvested and plated at a concentration of 1 106 cells/mL in serum-free DMEM into the inserts of a cell invasion assay plate. Dulbecco’s Modified Eagle’s Medium with 10% FBS was added to the lower chamber. Plates were incubated at 37-C in 5% carbon dioxide for 48 hours. Although noninvading cells in the interior of the inserts were mechanically removed by swabbing, the cells that had invaded

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into the exterior of the inserts were stained for evidence of cell invasion through the synthetic basement membrane. Stained cells were solubilized in extraction buffer, and the intensity of staining was quantified by transfer of 100-HL aliquots to a 96-well plate for microplate reader at a wavelength of 560 nm.

Data Analysis Results are presented as the mean values T SEM, and n indicates the number of independent experiments. Differences between groups were analyzed using one-way analysis of variance. P G 0.05 was considered significant.

RESULTS Effects of Src Inhibitors on Pancreatic Cancer Cell Proliferation and on Cell Cycle The antiproliferative roles of Src inhibitors (also known as SFK inhibitors) in pancreatic cancer cells were investigated. Two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2 (both derived from primary pancreatic carcinoma), were treated with the inhibitors AZM475271, A419259, or PP2. The wellknown Src inhibitor PP2 decreases kinase activity of Src, Lck, and Fyn.20 A419259 inhibits more potently Lck and Lyn than Src,21 and AZM475271 also inhibits Src.22 Cell proliferations were measured for 48 hours of treatment with inhibitors using MTT assays. These inhibitors showed antiproliferative activities at micromolar concentrations in the 2 cell lines (Fig. 1). The effects of inhibitors on cell proliferation were concentration dependent (0.1Y300 HM). The concentration of AZM475271 that elicited a half-maximal response (IC50) for the inhibition of PANC-1 cell proliferation was 44.2 T 1.1 HM, that of A419259 was 6.1 T 1.0 HM, and that of PP2 was 28.1 T 1.1 HM (n = 6). In MIA PaCa-2 cells, the IC50 of AZM475271 was 48.8 T 1.1 HM, that of A419259 was 5.3 T 1.2 HM, and that of PP2 was 40.0 T 1.4 HM (n = 6). The 2 cancer cell lines showed similar sensitivities to the 3 Src inhibitors, and A419259 was the more potent inhibitor. To evaluate cell cycle distribution and cell death, cellular DNA contents were analyzed by flow cytometry. PANC-1 cells were treated with 10-HM A419259 (Fig. 2; n = 4), and it reduced the percentages of cells in the S and G2/M phases (P G 0.05) and increased the percentages of cells in the subG0 and G0/G1 phases (P G 0.001 and P G 0.05, respectively). These results are consistent with blockade at the G1 to S transition and the induction of apoptosis23 and indicate that Src inhibition attenuates proliferation, induces cell cycle arrest, and enhances the apoptosis of pancreatic cancer cells.

Expression of SFKs in Pancreatic Cancer Cells The results from the previously mentioned proliferation experiments suggested that PANC-1 and MIA PaCa-2 cells express functional SFKs. To quantify the expressions of Src, Fyn, Yes1, Lck, Lyn, Hck, Fgr, Blk, and Frk, quantitative RT-PCR was used to measure mRNAs in PANC-1 and MIA PaCa-2 cells. Six SFKs, Src, Fyn, Yes1, Lyn, Hck, and Frk, were detected in both cell lines (Fig. 3; n = 4), and Lyn and Yes1 were expressed at significantly higher levels than the others (P G 0.001 and P G 0.001, respectively). The presence of the mRNAs of these SFKs in pancreatic cancer cells is in agreement with the findings of previous studies.9,24,25

Knockdown of SFKs in Pancreatic Cancer Cells To investigate the antiproliferative effects of inhibiting Src, Fyn, Yes1, Lyn, Hck, and Frk, their expression levels were transiently inhibited in PANC-1 and MIA PaCa-2 cells using siRNAs. Figure 4A demonstrates that knockdown of Yes1, Lyn, Fyn, Src, and Frk expression significantly reduced the proliferation by * 2014 Lippincott Williams & Wilkins


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FIGURE 1. The effect of Src inhibitors on the proliferation of human pancreatic cancer cells. PANC-1 (A) and MIA PaCa-2 (B) cells were exposed to AZM475271, A419259, and PP2 at different concentrations (0.1-300 HM; n = 6) for 48 hours then cell proliferation was determined by MTT assay. Representative microscopic images show pancreatic cancer cells treated with AZM475271 (30 HM), A419259 (10 HM), and PP2 (30 HM). Values are reported as mean T SEM. The response curve of nonlinear regression analysis was fitted to the data using GraphPad Prism 5.

16.7% T 2.0%, 43.9% T 0.8%, 30.2% T 0.5%, 47.3% T 2.6%, and 31.4% T 1.5% versus treatment-naive control PANC-1 cells (P G 0.001, n = 4). In MIA PaCa-2 cells, transfection of 5 siRNAs significantly suppressed cell proliferation by 14.9% T 0.9%, 25.9% T 0.4%, 13.5% T 0.8%, 11.5% T 0.8% (P G 0.001), and 3.0% T 0.3% (P G 0.05, n = 4). However, inhibition of Hck did not decrease proliferation. Knockdown of Lyn and Src was most effective in PANC-1 cells, and knockdown of Lyn was most effective in MIA PaCa-2 cells. Five siRNAs inhibited PANC-1 cell growth more than MIA PaCa-2 cell growth.12 Quantitative RT-PCR and Western blotting demonstrated the successful silencing of Yes1, Lyn, Fyn, Src, Frk, and Hck (Figs. 4B, C). The molecular inhibition of SFKs suppressed pancreatic carcinoma cell proliferation. To examine the effects of molecular inhibition of SFKs in different pancreatic cancer cells as primary and metastatic pancreatic adenocarcinoma cells, Capan-2, Capan-1, and HPAC cells were transfected with 5 siRNAs. Transfection of siRNAs targeting Yes1, Lyn, Fyn, Src, and Frk decreased the proliferation by 40.5% T 2.6%, 17.6% T 1.6% (P G 0.001), 10.7% T 0.6% (P G 0.05), 24.1% T 2.1% (P G 0.001), and 1.6% T 2.5% in Capan-2 cells; by 27.7% T 1.8%, 23.3% T 1.0%, 33.9% T 0.7%, 31.7% T 0.6%, and 34.0% T 1.5% (P G 0.001) in Capan-1 cells; and by 28.4% T 0.7%, 18.7% T 1.0% (P G 0.001), 6.1% T 2.3% * 2014 Lippincott Williams & Wilkins

FIGURE 2. The effect of Src inhibition on cell cycle regulation. After the treatment of A419259 (10 HM) to PANC-1 cells, cell cycle was analyzed using flow cytometry. Values are reported as mean T SEM. *P G 0.05 and ***P G 0.001 compared with control group. www.pancreasjournal.com


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FIGURE 3. Quantitative analysis of SFK family expression. Relative expressions of the SFK genes (Src, Fyn, Yes1, Lck, Lyn, Hck, Fgr, Blk, and Frk) in PANC-1 and MIA PaCa-2 cells are presented, which were measured by quantitative RT-PCR (n = 4). Expression levels were normalized to GAPDH and calibrated to Fyn. Values are reported as the mean T SEM. ***P G 0.001 when compared with the other SFK family.

(P G 0.05), 18.8% T 1.0%, and 17.4% T 0.7% (P G 0.001) in HPAC cells (Fig. 5; n = 4). However, siRNA against Frk did not affect the proliferation of Capan-2 cells. Knockdown of Yes1 was most effective in Capan-2 and HPAC cells, and the effects of knockdown of Fyn, Src, and Frk were similar in Capan-1 cells. These results further demonstrate that 5 SFK knockdowns inhibit the proliferation of pancreatic carcinoma and adenocarcinoma cells.

Inhibition of SFK Attenuated the Metastatic Potential of Pancreatic Cancer Cells To evaluate the effects of Yes1, Lyn, Fyn, Src, and Frk knockdown on the migration of pancreatic cancer cells, wounding assays were performed (Fig. 6). Scratch widths were recovered by 70.9% T 1.5% in PANC-1 and 32.7% T 3.0% in MIA PaCa-2 after 2 days (n = 7). Inhibition of Yes1, Lyn, Fyn, Src, or Frk with each

FIGURE 4. The effect of siRNA-targeting SFKs on pancreatic cancer cell proliferation. A, After the transfection with scrambled siRNA (SC) and siRNA (100 nmol/L) against Yes1, Lyn, Fyn, Src, Frk, or Hck (siYes1, siLyn, siFyn, siSrc, siFrk, or siHck, respectively), the proliferation of PANC-1 and MIA PaCa-2 cells for 48 hours was measured (n = 4). The SC was used as control. B, The target SFK gene inhibition percent is presented, which was measured by quantitative RT-PCR (n = 7). C, Western blotting shows that the siRNAs reduced the protein expression of Yes1, Lyn, Fyn, Src, Frk, and Hck. Values are reported as mean T SEM. *P G 0.05 and ***P G 0.001 compared with the SC-treated group.

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FIGURE 5. The effect of siRNA-targeting SFKs on cell proliferation of pancreatic adenocarcinoma cell lines. A, After transfection with scrambled siRNA and siRNA (100 nmol/L) against Yes1, Lyn, Fyn, Src, or Frk, the proliferation of Capan-2, Capan-1, and HPAC cells for 48 hours were measured (n = 4). The SC was used as control. Values are reported as mean T SEM. *P G 0.05 and ***P G 0.001 compared with the SC-treated group.

FIGURE 6. The effect of siRNA-targeting SFKs on migration of pancreatic cancer cells. After transfection with scrambled siRNA and siRNA (100 nmol/L) against Yes1, Lyn, Fyn, Src, or Frk, the migration capacities of PANC-1 and MIA PaCa-2 cells were monitored using wound healing assay and were expressed as the percent of scratched closed (n = 7). Microscope images represent healing results in PANC-1 cells. Values are reported as mean T SEM. *P G 0.05, **P G 0.01, and ***P G 0.001 compared with the SC-treated group. * 2014 Lippincott Williams & Wilkins



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FIGURE 7. The effect of siRNA-targeting SFKs on migration of pancreatic adenocarcinoma cell lines. After the transfection with scrambled siRNA and siRNA (100 nmol/L) against Yes1, Lyn, Fyn, Src, or Frk, the migration capacities of Capan-2, Capan-1, and HPAC cells were measured (n = 7). Values are reported as mean T SEM. *P G 0.05, **P G 0.01, and ***P G 0.001 compared with the SC-treated group.

siRNAs effectively reduced the migratory activities of PANC-1 cells to 37.3% T 3.8%, 40.0% T 5.7%, 40.3% T 4.9%, 53.4% T 6.0%, and 33.0% T 6.3% (P G 0.001), and MIA PaCa-2 cells to 46.0% T 5.0% (P G 0.001), 56.6% T 6.6% (P G 0.01), 66.7% T 4.7% (P G 0.05), 29.9% T 6.1%, and 28.0% T 8.3% (P G 0.001), respectively (Fig. 6). PANC-1 cells covered scratches faster than MIA PaCa-2 cells under control and knockdown conditions. In addition, significant reductions in wound healing were observed in 3 pancreatic adenocarcinoma cell lines, that is, Capan-2 cells to 40.0% T 5.3%, 29.8% T 3.5%, 33.8% T 5.7%, 27.4% T 7.1%, and 37.8% T 10.7% (P G 0.001); Capan-1 cells to 54.1% T 7.3%, 46.9% T 8.6%, 48.2% T 7.5%, 37.2% T 2.4%, and 48.5% T 6.7% (P G 0.001); and HPAC cells to 65.3% T 5.1% (P G 0.05), 41.9% T 3.8%, 27.1% T 6.9% (P G 0.001), 57.8% T 12.4% (P G 0.01), and 24.9% T 6.9% (P G 0.001), respectively (Fig. 7). Inhibition of SFKs significantly suppressed the migrations of pancreatic carcinoma and adenocarcinoma cells. In addition, a decrease in cell invasion was caused by 5 SFK knockdowns in PANC-1 cells (Fig. 8). Cell invasion was significantly inhibited by transfecting PANC-1 cells with 5 siRNAs to 21.3% T 7.0%, 9.9% T 8.6%, 10.5% T 5.6%, 11.6% T 12.0%, and 15.5% T 7.4% (P G 0.001). Cell migration and invasion assay results supported observed decrease in proliferation caused by SFK knockdown.

the pancreatic cancer cell growth, cellular growth rate in monolayer cultures, and the progression, wound healing, and synthetic membrane invasion. To examine the specific role of each SFK in pancreatic cancer cells, we used a siRNA approach whereby SFKs were specifically knockdown in pancreatic cancer cells. Proliferation and metastatic activity were much higher in controls than in siRNA or Src inhibitor-treated groups. The siRNA-induced inhibitions of 5 SFKs, Yes1, Lyn, Fyn, Src, or Frk, decreased the growth, migration, and invasion of pancreatic cancer cells. These results suggest that the expression and/or activation of SFK members Yes1, Lyn, Fyn, Src, and Frk contribute directly to cancer cell growth and metastatic potential and that their suppressions have anticancer effects in human pancreatic cancer cells. This finding broadens the applications of siRNAs in pancreatic cancer, pancreatic adenocarcinoma and carcinoma. As far as we are aware, this is the first report to describe the expression profile of

DISCUSSION Pancreatic cancer is one of the most aggressive forms of cancer and its prognosis remains poor. Its high mortality is in part due to its lower responsiveness to therapeutic agents and the untreatability of metastatic disease at diagnosis. Thus, research on the important therapeutic targets and the impact of these targets on the spread of cancer cells are critical to develop a successful therapy for pancreatic cancer. Because inhibitors targeting Src override chemoresistance in chronic myelogenous leukemia,26 several inhibitors of Src have been developed and used in studies of various cancer treatments.5,7 Studies indicate that activated Src is important for pancreatic tumor growth and metastases and suggest that Src is an attractive therapeutic target in pancreatic cancer.13,27 However, despite emphasis on the therapeutic role of Src inhibition, other SFK members have received little attention in the context of pancreatic cancer. This study described the expression profiles of SFK members in human pancreatic cancer cell lines and showed that inhibition of SFK, Yes1, Lyn, Fyn, Src, or Frk, significantly affected

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FIGURE 8. The effect of SFKs inhibition using siRNA on cell invasion. After transfection with scrambled siRNA and siRNA (100 nmol/L) against Yes1, Lyn, Fyn, Src, or Frk, the invasion potency of PANC-1 cells were measured (n = 4). Values are reported as mean T SEM. ***P G 0.001 compared with the SC-treated group. * 2014 Lippincott Williams & Wilkins


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SFK members and the anticancer effects of siRNA-targeting SFK members in pancreatic cancer cells. Src is an upstream of several important pathways that have been implicated in the neoplastic phenotype.28 It promotes cancer cellYspecific functions, such as, migration, cell growth, and survival. Src is overexpressed, and its activity is increased in a variety of malignancies,5,6 including pancreatic cancer,9 and its expression is associated with malignant cellular behavior,29 which suggests that Src is more active during the invasion and metastasis of advanced cancer.30 Src activity plays a role in the control of cell adhesion and cytoskeletal changes, which increase the metastatic potentials of tumor cells.31 Src regulates angiogenesis and vascular endothelial growth factor secretion30 and induces metalloprotease production in pancreatic cancer cell lines.12 These properties provide the evidence of regulating the effect of Src on tumor progression and metastasis. The treatment with Src inhibitors and the down-regulation of Src expression using siRNA significantly reduced tumor growth and the incidence of metastases in an orthotopic implantation model using human pancreatic tumor cells.13,32 Our results indicate that the knockdown of Src using siRNA in human pancreatic cancer cells significantly affected cell growth and migration. Src inhibition has also been reported to be associated with cell cycle arrest and increased apoptosis.13,23 Members of the SFK participate in the control of a variety of cellular functions. The SFKs are activated by a number of receptor tyrosine kinases in a variety of cell types, which promote DNA synthesis, mitogenesis, and cell proliferation. They regulate receptor turnover and synaptic transmission and plasticity. In addition, the cytoskeletal-linked events are modulated by SFKs to promote cell-to-cell adhesion, migration, and cell motility. Although SFK members have similar structure and aforementioned functions, they show a variety of expression patterns. Src, Fyn, and Yes are expressed in a broad range of tissues, whereas Lyn is expressed in B-cells, myeloid cells, and the brain; Lck in T-cells, NK cells, and the brain; Hck in myeloid cells; and Frk in epithelial tissues.5Y7,33,34 Theses expression pattern means each SFK can have different roles as tissues. Src is involved in bone formation and synapses, and Fyn is involved in hippocampal development and memory.33 Lck and Fyn participate in T-cell development and activation, and Lyn participate in B-cell development and signaling.33 Frk controls migration and invasion of glioma cells,35 and Hck regulates activation of monocytes/macrophages.36 Pharmacological inhibitors and siRNA against SFKs have shown combinatorial effects on cancer cell growth and metastasis.13 Src inhibitors act like siRNAs and reduce the activities of SFK members with respect to proliferation, migration, and invasion in pancreatic cancer cells. This is because Src inhibitors decrease the phosphorylations of Src, Lck, Yes, or so.13,37 Furthermore, these findings suggest that the functions of SFK members are required for pancreatic cancer cell growth and progression. However, it should be noted that the treatment with high concentration of Src inhibitors abolished the pancreatic cancer cell proliferation, whereas siRNAs reduced cell proliferation by 20%È40%. The abolishment of cancer cell proliferation is probably due to the inhibition of most SFKs expressed in pancreatic cancer cells by Src inhibitors. These findings suggest that Src inhibitors should be tested with respect to their abilities to inhibit major SFK members. In this study, the expression profiles of SFK mRNAs were measured by quantitative RT-PCR. Three SFKs, Lyn, Yes1, and Fyn, were expressed abundantly, and Src, Frk, and Hck were expressed at lower levels than formers in pancreatic carcinoma PANC-1 and MIA PaCa-2 cells. Previous studies have reported that Src, Lyn, Yes1, and Fyn are expressed in pancreatic cancer cells.24,25 The SFKs expressed in cancer cells may have cancerspecific roles, and effect proliferation, regulate gene expression, * 2014 Lippincott Williams & Wilkins


adhesion, migration, and angiogenesis.38 Lyn kinase is involved in cell invasion and is negatively regulated by Csk homologous kinase.24 Fyn kinase regulates apoptosis with HnRNPA2B1 and Sam68 synergistically in pancreatic cancer.25 However, SFK expression patterns are not correlated with anticancer effects. In the inhibition of proliferation, siRNA-induced Lyn knockdown was most effective in pancreatic carcinoma, and that of Yes1 was the most effective in pancreatic adenocarcinoma. Regarding the inhibition of migration, the siRNA-induced suppression of Frk was most effective in carcinoma, and knockdowns of 5 SFKs were similarly effective in adenocarcinoma. Even Hck was expressed in carcinoma cells, but siRNA against Hck was not found to have an anticancer effect in this study. The siRNA is an important tool for the suppression of expressed target genes in mammalian cells and cancers, and its use could result in cancer therapies based on targeting specific proteins and genes that contribute to malignancies. Small interfering RNA uses the RNA interference pathway to degrade specific cellular mRNAs involved in the malignant process and thus modify protein expression levels.39 Three features of siRNAs offer advantages over other cancer therapies, namely, as high degree of specificity, the facility to use multiple forms to target a single protein, and the ability to simultaneously target 2 or more gene products.40 Small interfering RNAs have been used in studies on specific disease conditions in cultured cells and in animals.41 In humans, the systemic administration of siRNA by direct intravenous administration to patients with solid cancers supports the usefulness of the siRNA approach to knockdown target proteins in cancers.42 It is believed that the siRNA concept can prove to be a powerful therapeutic method for the treatment of various human cancers including pancreatic cancer. The data presented in this study show that the inhibition of SFK members in pancreatic cancer cells using chemical inhibitors or siRNAs inhibits cell proliferation in carcinoma and adenocarcinoma cell lines. Furthermore, siRNAs targeting SFK members were found to inhibit the metastatic potential of pancreatic cancer cells by inhibiting cell migration and invasion. The inhibition of SFKs may serve the dual function of reduced cancer cell proliferation and inhibiting the ability of cancer cells to migrate and invade tissues, which is a critical step during the disease progression and metastasis in pancreatic cancer. Furthermore, the results from this study suggest that Yes1, Lyn, Fyn, Src, and Frk are important candidates for targeted therapy in pancreatic cancer. ACKNOWLEDGMENTS The authors would like to thank Ji Hun Choi for the technical assistance with the collection of data.

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MicroRNA-145 suppresses cell proliferation, invasion and migration in pancreatic cancer cells by targeting NEDD9.

Enterolactone alters FAK-Src signaling and suppresses migration and invasion of lung cancer cell lines.

Inhibition of Aurora-B suppresses osteosarcoma cell migration and invasion.

Kif20a inhibition reduces migration and invasion of pancreatic cancer cells.

Epigenetic modification suppresses proliferation, migration and invasion of urothelial cancer cell lines.

MiRNA-203 suppresses cell proliferation, migration and invasion in colorectal cancer via targeting of EIF5A2.

Knockdown of ZFR suppresses cell proliferation and invasion of human pancreatic cancer.

SOX2 enhances the migration and invasion of ovarian cancer cells via Src kinase.

miR-132 targeting E2F5 suppresses cell proliferation, invasion, migration in ovarian cancer cells.

Small interfering RNA-induced inhibition of epithelial cell transforming sequence 2 suppresses the proliferation, migration and invasion of osteosarcoma cells.

miR-203 inhibition of renal cancer cell proliferation, migration and invasion by targeting of FGF2.

MicroRNA-326 suppresses the proliferation, migration and invasion of cervical cancer cells by targeting ELK1.

miR-140-5p suppresses the proliferation, migration and invasion of gastric cancer by regulating YES1.

ZKSCAN3 promotes bladder cancer cell proliferation, migration, and invasion.

Progesterone and Src family inhibitor PP1 synergistically inhibit cell migration and invasion of human basal phenotype breast cancer cells.

mTOR signaling pathway in breast cancer.

microRNA-218 suppresses the proliferation, invasion and promotes apoptosis of pancreatic cancer cells by targeting HMGB1.

invasion via PRKA kinase anchor protein 9.

Gga-miR-219b targeting BCL11B suppresses proliferation, migration and invasion of Marek's disease tumor cell MSB1.

Silencing of SOX12 by shRNA suppresses migration, invasion and proliferation of breast cancer cells.

Inhibition of REST Suppresses Proliferation and Migration in Glioblastoma Cells.

Restoration of miR-20a expression suppresses cell proliferation, migration, and invasion in HepG2 cells.

miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1.

miR-203 suppresses the proliferation and migration and promotes the apoptosis of lung cancer cells by targeting SRC.