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Cancer Research 67, 9637, October 15, 2007. doi: 10.1158/0008-5472.CAN-07-1489
© 2007 American Association for Cancer Research
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Priority Reports

Nek2 as an Effective Target for Inhibition of Tumorigenic Growth and Peritoneal Dissemination of Cholangiocarcinoma

Toshio Kokuryo1,2, Takeshi Senga2, Yukihiro Yokoyama1, Masato Nagino1, Yuji Nimura1 and Michinari Hamaguchi2

Divisions of 1 Surgical Oncology and 2 Cancer Biology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan

Requests for reprints: Michinari Hamaguchi, Division of Cancer Biology, Nagoya University Graduate School of Medicine, 65-Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Phone: 81-52-744-2462; Fax: 81-52-744-2464; E-mail: mhamagu{at}med.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We investigated the role of Nek2, a member of the serine/threonine kinase family, Nek, in the tumorigenic growth of cholangiocarcinoma cells. Expression of Nek2 is elevated in cholangiocarcinoma in a tumor-specific manner as compared with that of normal fibroblast cells. Expression of exogenous Nek2 did not perturb the growth of cholangiocarcinoma cells, whereas suppression of the Nek2 expression with siRNA resulted in the inhibition of cell proliferation and induced cell death. In xenograft-nude mouse model, s.c. injection of Nek2 siRNA around the tumor nodules resulted in reduction of tumor size as compared with those of control siRNA injection. In peritoneal dissemination model, Nek2 siRNA–treated mice showed statistically longer survival periods in comparison with those of the control siRNA–treated mice. Taken together, our data indicate a pivotal role of Nek2 in tumorigenic growth of cholangiocarcinoma. [Cancer Res 2007;67(20):9637–42]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Cholangiocarcinoma, one of the most common liver tumors in Southeast Asian countries (1, 2), is a primary malignancy derived from the bile duct epithelium. Despite the progress of the combined therapy (3, 4), its prognosis is still extremely miserable among all malignancies (5). To search for the molecular targets for the treatment of cholangiocarcinoma, we compared the gene expression pattern of cholangiocarcinoma with that of a normal liver using cDNA expression array containing 1,176 unique genes (6). Among these explored genes, we identified Nek2 as a candidate that showed high expression in a tumor-specific manner.

Nek2 is a member of the serine/threonine kinase family, Nek, structurally related to the essential mitotic regulator NIMA (3). Functional studies have implicated Nek2 in regulation of centrosome separation and spindle formation (4, 7). Moreover, increased expression of Nek2 has been reported in cell lines derived from breast, cervical, and prostate carcinomas, as well as lymphomas (8), suggesting the involvement of Nek2 in tumorigenesis. Indeed, forced expression of Drosophila Nek2 leads to abnormalities in cleavage furrow formation and cytokinesis failure (9), which can cause chromosome instability and aneuploidy (10). Despite its importance, however, the role of Nek2 in tumorigenesis remains largely unclear.

To obtain clues, we investigated the role of Nek2 in cholangiocarcinoma by use of siRNA against Nek2. We show for the first time that Nek2 depletion in cholangicarcinoma cell lines causes growth suppression and cell death. Moreover, we show that administration of siRNA against Nek2 into tumor-bearing mice induces substantial elongation of survival length.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell culture and plasmid construction. The cell lines HuCCT1, TFK1, HuH28, ETK1, RBE, and IHGGK were obtained from Tohoku University and Riken Cell Bank. CCKS1 is kindly supplied by Y. Nakanuma (Department of Human Pathology, Kanazawa University Graduate School of Medicine, Takaramachi, Kanazawa, Japan). HA-Dsred Nek2 plasmid was made by ligation of COOH-terminally hemagglutinin (HA)-tagged Nek2 gene into a pDsred express vector (Clontech).

Preparation of siRNAs. The sense and antisense strands of Nek2 siRNAs obtained from Qiagen were as follows: Nek2 siRNA 19, 5'-GGAGGGGAUCUGGCUAGUGdTdT3'(sense), 5'CACUAGCCAGAUCCCCUCCdTdT-3' (antisense); Nek2 siRNA 27, 5'-GGAAUGCCACAGACGAAGUdTdT-3' (sense), 5'-ACUUCGUCUGUGGCAUUCCdTdT-3' (antisense); and Nek2 siRNA 65, 5'-GAGGGCGACAAUUAGGAGAdTdT-3'(sense), 5'-UCUCCUAAUUGUCGCCCUCdTdT-3' (antisense).

Luciferase GL2 siRNA (Dharmacon) and negative control siRNA (Qiagen) were used as controls. Transfection of siRNA in vitro was carried out with GenePORTER (Genlantis).

Semiquantitative reverse transcription-PCR. Semiquantitative PCR in a GeneAmp PCR system 9700 (Applied Biosystems) was previously described (11). Primer sequences were as follows: Nek2, 5'-AAGGAATGCCACAGACGAAG-3'(sense), 5'-TGCGATTCATTTGTTCAGGA-3' (antisense); ß-actin, 5'-CTCTGTGTTGGCGTACAGGT-3'(sense), 5'-TCATCACCATTGGCAATGAG-3'(antisense).

Immunoblotting and immunocytochemistry. SDS-PAGE and immunoblotting analysis were done as previously described (12). Mouse monoclonal anti-Nek2 (Transduction laboratories), mouse monoclonal anti–{gamma}-actin (Sigma-Aldrich), and rabbit anti-mouse immunoglobulin Gs (Biosource) were used for the analysis. Immunocytochemistry was done as previously described (13). Nuclear DNA was counterstained with 4',6-diamidino-2-phenylindole (DAPI; Biosource).

Cell proliferation assay and dye exclusion test. Cell proliferation was determined by a colormined assay of cell viability that is based on the cleavage of tetrazolium salt by mitochondrial dehydrogenases (Tetra Color One, Seikagaku Corp.). The absorbance of the formazan dye was measured at 450 and 630 nm 1.5 h after adding the reagent. To assess the viability of cells after siRNA treatment, dye exclusion test was done.

Live cell imaging. Cells, grown on a glass-base dish (Iwaki), were mounted in the CO2 microincubator regulated at 37°C and placed on the microscope stage. Images were captured at intervals of 2 or 5 min with inverted microscope IX81 (Olympus) and the MetaMorph software (Universal Imaging Corp.).

Animal efficacy studies. Male nude mice, 8 weeks old and 20 to 25 g in weight (CREA) Japan, were used. HUCCT1 cells (1 x 107) were inoculated s.c. into the femoral area of mice. After 4 weeks when the tumor reached 150 to 200 mm, Nek2 siRNA 27 (20 µmol/L) dissolved in 100 µL of cell matrix was administered directly into cancer once a week, thrice. PBS or luciferase pGL2 siRNA was used as a control. Tumors were removed 4 weeks after the final siRNA treatment. Tumor growth was assessed by its volume. Apoptosis was quantified by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) analysis (DeadEnd TUNEL system, Promega). For the analysis of survival rate, mice were inoculated with HUCCT1 cells (1 x 107) into the abdominal cavity. Nek2 siRNA 27 (20 µmol/L) dissolved in 100-µL cell matrix was directly injected into abdominal cavity once a week, thrice. Animal experiments were done in compliance with the guideline of the Institute for Laboratory Animal Research, Nagoya University Graduate School of Medicine.

Statistical analysis. All data were presented as means ± SE. The statistical differences were analyzed by the Student's t test, the repeated measures ANOVA, or the Kaplan-Meier method with Statview for Windows. The results were considered significant when P < 0.05.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
We compared the gene expression pattern of cholangiocarcinoma with that of a normal liver using cDNA expression array containing 1,176 unique genes. Among these genes, the expression of Nek2 was substantially increased in cholangiocarcinoma in a tumor-specific manner (data not shown). We therefore compared the expression levels of Nek2 in cholangiocarcinoma cell lines, HuCCT1, TFK1, HuH28, CCKS1, ETK1, RBE, and IHGGK, with that in HFF, a cell line derived from normal human fibroblast. Cholangiocarcinoma cells expressed higher levels of Nek2 mRNA and protein than did HFF cells (Fig. 1A and B ). Nek2 has three spliced variants, Nek2A, Nek2B, and Nek2A-T (14). The predominant isoform shown in Fig. 1B corresponds to Nek2A/Nek2A-T and the smaller band is Nek2B (14). Cholangiocarcinoma cells expressed relatively larger amounts of Nek2A/Nek2A-T and Nek2B, as compared with those of HFF. In HuCCT1, we confirmed the localization of Nek2 to the centrosomes (Fig. 1C).


Figure 1
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  		Figure 1. Expression of Nek2 in cholangiocarcinoma. A, detection of Nek2 expression in cholangiocarcinoma cell lines (HuCCT1, TFK1, HuH28, and CCKS1) and a normal fibroblast cell line (HFF) by semiquantitative PCR. The quantity of cDNA was assessed by amplification of ß-actin. B, detection of Nek2 expression in cholangiocarcinoma cell lines (HuCCT1, TFK1, HuH28, CCKS1, ETK1, RBE, and IHGKK) and HFF by immunoblotting analysis. ß-Actin was used as a control. Top, Nek2A and Nek2B; bottom, ß-actin. C, detection of Nek2 expression in HuCCT1 by immunocytochemistry. {gamma}-Tubulin was used as a marker of centrosome. DNAs were counterstained with DAPI. Left, Nek2 (arrowhead); middle, {gamma}-tubulin (asterisk); right, DAPI. D, proliferation of HuCCT1 stably transfected with Nek2 (HuCCT1/Nek2). Left, expression of HA-Dsred Nek2 in HuCCT1/Nek2 stable clone by immunoblotting with anti-Nek2. Right, cell proliferations with 0.5% or 10% serum that were quantified by cell proliferation assays (see Materials and Methods). Similar results were obtained in three independent experiments. P < 0.05, Student's t test.

 
To assess the role of Nek2 in the growth of cholangiocarcinoma cells, we examined the effect of exogenous Nek2 expression in HuCCT1. HA-tagged Nek2 ligated with Dsred vector (HA-Dsred Nek2) was transfected into HuCCT1 (HuCCT1/Nek2). Immunoblotting with anti-Nek2 showed that substantial amount of HA-Dsred Nek2 was expressed in HuCCT1/Nek2 to the level similar to that of endogenous Nek2 (Fig. 1D, left). Expression of HA-Dsred Nek2 did not perturb the growth of HUCCT1 but rather stimulated it (Fig. 1D, right). With the medium containing 0.5% serum, the growth-promoting effect of HA-Dsred Nek2 became more evident.

To study the function of Nek2, we next treated the cholangiocarcinoma cells with double-stranded siRNA oligonucleotides against Nek2. Three types of siRNA, Nek2 siRNA 65, Nek2 siRNA 27, and Nek2 siRNA 19, were designed and transfected into HuCCT1 cells. As shown in Fig. 2A , the expression of Nek2 in HuCCT1 was substantially suppressed by treatment with Nek2 siRNA, but not with control siRNA. Surprisingly, we found that treatment of HuCCT1 with Nek2 siRNA caused concomitant suppression of cell growth to the level of ~50% of control siRNA–treated cells (Fig. 2B). In contrast to HuCCT1, the effect of Nek2 siRNA on the growth of HFF cells was limited.


Figure 2
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  		Figure 2. Intracellular consequences of Nek2 siRNA. A, a Western blot showing Nek2 levels in HuCCT1 collected 48 h after transfection with Nek2 siRNAs (Nek2 siRNA 65, Nek2 siRNA 27, and Nek2 siRNA 19). Control siRNA and luciferase pGL2 siRNA were used as nonsilencing siRNA. ß-Actin was used as a control. Top, Nek2A and Nek2B; bottom, ß-actin. B, proliferation of cholangiocarcinoma cell by Nek2 siRNA treatment. Graphs show the proliferation levels in HuCCT1, TFK1, and HFF 48 h after transfection with Nek2 siRNAs (Nek2 siRNA 65, Nek2 siRNA 27, and Nek2 siRNA 19). Control siRNA was used as nonsilencing siRNA. Cell proliferations were quantified by cell proliferation assays (see Materials and Methods). Similar results were obtained in three independent experiments. P < 0.05, Student's t test. C, selected images from a time-lapse movie of HuCCT1 were transfected with either rhodamine-labeled Nek2 siRNA 27 or FITC-labeled negative control siRNA. The cells were transfected with either rhodamine-labeled Nek2 siRNA or FITC-labeled control siRNA, and then were cocultured. A time-lapse movie was captured for 12 h. D, histograms indicate cell death of cholangiocarcinoma (HuCCT1 and TFK1) and HFF treated with Nek2 siRNAs by dye exclusion test using trypan blue. Similar results were obtained in three independent experiments. P < 0.05, Student's t test.

 
To study further the effect of Nek2 siRNA on cell growth, we studied the living siRNA-transfected cells by time-lapse microscopy. The cells were transfected with either rhodamine-labeled Nek2 siRNA or FITC-labeled control siRNA, and both cells were subsequently incubated for 12 h. As shown in Fig. 2C, Nek2 siRNA, but not control siRNA, caused suppression of cell division and induced cell death. We confirmed the Nek2 siRNA–dependent cell death by trypan blue dye exclusion test. As shown in Fig. 2D, all three types of Nek2 siRNA could induce cell death in HuCCT1 and TFK1 cells. In contrast, the effect of Nek2 siRNA on the survival of HFF cells was at limited levels.

We next examined the effect of Nek2 siRNA treatment on the growth of cholangiocarcinoma tumor in xenograft-nude mouse model. Mice were s.c. inoculated with HUCCT1. Four weeks after inoculation, 20 µmol/L of either Nek2 siRNA in cell matrix or control siRNA in cell matrix, as well as cell matrix alone, was s.c. injected around the tumor nodules of mice once a week for 3 weeks. After the treatment, tumor size was measured once a week until 12 weeks after tumor inoculation. Surprisingly, mean tumor volumes in Nek2 siRNA–treated mice were substantially reduced in comparison with those of the control mice (Fig. 3A and B ). The Nek2 expression in the tumor tissues was dramatically suppressed in Nek2 siRNA–treated tumors as compared with control siRNA–treated tumors (Fig. 3C).


Figure 3
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  		Figure 3. In vivo consequences of Nek2 siRNA. A, photos of HuCCT1 xenograft. Four weeks after tumor inoculation, nude mice bearing HuCCT1 tumors were treated with Nek2 siRNA 27 (20 µmol/L) once a week for 3 wk (arrows). On the 28th day after the final dose of Nek2 siRNA, HuCCT1 tumors were photographed. Control was used as placebo and control siRNA was used as nonsilencing siRNA. B, the volume of HuCCT1 tumors shown in A. C, detection of Nek2 expression in established HuCCT1 tumors after Nek2 siRNA treatment. Immunoblot analysis confirmed the expression of Nek2 in tumors. Four weeks after tumor inoculation, nude mice bearing tumors were treated with Nek2 siRNA (20 µmol/L) once a week for 3 wk and, 48 h after final administration of siRNA, tumors were resected. Control was used as placebo and luciferase pGL2 siRNA was used as nonsilencing siRNA. Top, Nek2; bottom, ß-actin. D, right, H&E staining (top) and TUNEL staining (bottom) of resected tumor sections from HuCCT1 xenografts in A. Left, analysis of siRNA induction in HuCCT1 xenografts by direct injection of siRNA. Fluorescent images were captured in 48 h after injection of rhodamine-conjugated siRNA. DNAs were counterstained with DAPI. Control siRNA was used as nonsilencing siRNA.

 
Tumor sections of the xenografts were analyzed by H&E staining followed by TUNEL staining. Multiple empty spaces occupied with debris were observed in the Nek2 siRNA–treated tumor tissues (Fig. 3D, right). Surprisingly, debris inside the space was strongly TUNEL positive, suggesting that Nek2 siRNA treatment of the xenograft specifically induced apoptosis in the tumor. Delivery of siRNA to cancer cells in xenograft models was confirmed by rhodamine-labeled siRNA (Fig. 3D, left).

To further evaluate the effects of Nek2 siRNA treatment, we challenged siRNA in the peritoneal dissemination model in which HuCCT1 was injected i.p. It should be noted that peritoneal dissemination is one of the major undesirable complications frequently associated with inoperable cases (15). To confirm the incorporation of siRNA into cancer cells in the peritoneal cavity, rhodamine-labeled Nek2 and control siRNA in atelocollagen complexes were administered i.p. into mice after the injection of HuCCT1. As shown in Fig. 4A , similar levels of both siRNAs were incorporated into cancer cells. Nek2 siRNA or control siRNA (20 µmol/L) was administrated once a week i.p. for 3 weeks. All controlled mice (n = 5) died within 40 days (mostly within 14 days) following the final treatment. Mice treated with control siRNA showed similar lengths of survival with control mice. In contrast, the Nek2 siRNA–treated mice showed statistically longer survival periods as with those of the control mice (Fig. 4B). Survival rates of the Nek2 siRNA–treated mice were ~1.5 times higher than those of the control siRNA–treated mice. It should be noted that these mice tolerated the siRNA treatment well and showed no decrease in their body weight. We observed no sign of toxicity in this treatment during the experimental period of up to 12 weeks.


Figure 4
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  		Figure 4. Consequences of Nek2 siRNA in peritoneal dissemination model. A, analysis of siRNA induction in cancer cells in peritoneal cavity after administration of siRNA. Nude mice bearing established HuCCT1 peritoneal dissemination were treated with Nek2 siRNA 20 µmol/L. Rhodamine-labeled siRNAs were administered a week after the cancer inoculation. Twenty-four hours after injection of siRNAs, mice were sacrificed and cancer cells obtained by lavage were resuspended in RPMI medium with 10% fetal bovine serum. Fluorescent images were captured 3 h after resuspension. DNAs were counterstained with DAPI. Control siRNA was used as nonsilencing siRNA. B, survival curves of mice inoculated with HuCCT1 and treated with Nek2 siRNA 27, control siRNA, or PBS. PBS was used as placebo and control siRNA was used as nonsilencing siRNA.

 
Among all malignancies, the prognosis of cholangiocarcinoma is extremely miserable. For the improvement of its therapy, identification of specific molecular targets critical for its tumorigenicity is an important issue. In this report, we showed that Nek2 is abundantly expressed in cholangiocarcinoma and required for the tumorigenic growth and survival of the cells. In normal adult mouse, expression of Nek2 is specifically strong in the testis but weak in other tissues, suggesting that expression of Nek2 in cholangiocarcinoma is abnormally controlled. Surprisingly, suppression of Nek2 expression in cholangiocarcinoma subsequently caused apoptotic cell death. Although the precise mechanism by which suppression of Nek2 expression causes cell death remains to be clarified, our results strongly suggest that Nek2 is required for the survival of cholangiocarcinoma cells. Nek2 siRNA did not induce massive cell death in normal fibroblasts, suggesting that cholangiocarcinoma cells have specific dependence on Nek2 for their growth and survival, which is called by Weinstein (16) as "oncogene addiction."

Many reports suggest that delivery, especially in vivo delivery, is the most important obstacle for siRNA-based gene therapy. In this report, we showed that cell matrix was an effective delivery system for siRNA treatment. The synthetic siRNA resisted biodegradation for at least 48 h in tumor. A significant number of cancer cells in the peritoneal cavity have accepted the siRNAs. Direct injection with cell matrix is less insensitive to nuclease than an i.v. injection (17) and is insufficiently immunogenic to stimulate IFN expression in vivo (18).

In summary, this is the first demonstration that Nek2 plays a critical role in tumorigenic growth of cholangiocarcinoma, and inhibition of Nek2 expression with its siRNA causes suppression of cholangiocarcinoma growth and survival. Our data in vivo provide a strong rationale for the further investigation of Nek2 inhibitors as a new cancer therapy, holding the potential to provide regression of multiple human malignancies.


   Acknowledgments
 
Grant support: Grant-in-Aid for the Center of Excellence Research from The Ministry of Education, Science, Sports, and Culture of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the members of Hamaguchi Laboratory for their helpful discussions.

Received 4/27/07. Revised 6/25/07. Accepted 7/26/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Nimura Y, Kamiya J, Nagino M, et al. Aggressive surgical treatment of hilar cholangiocarcinoma. J Hepatobiliary Pancreat Surg 1998;5:52–61.[CrossRef][Medline]
  2. Capdeville R, Buchdunger E, Zimmermann J, Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov 2002;1:493–502.[CrossRef][Medline]
  3. Fry AM, Meraldi P, Nigg EA. A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators. EMBO J 1998;17:470–481.[CrossRef][Medline]
  4. Fry AM. The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene 2002;21:6184–94.[CrossRef][Medline]
  5. Nagino M, Kamiya J, Arai T, Nishio H, Ebata T, Nimura Y. One hundred consecutive hepatobiliary resections for biliary hilar malignancy: preoperative blood donation, blood loss, transfusion, and outcome. Surgery 2005;137:148–55.[CrossRef][Medline]
  6. Kokuryo T, Yamamoto T, Oda K, et al. Profiling of gene expression associated with hepatolithiasis by complementary DNA expression array. Int J Oncol 2003;22:175–9.[Medline]
  7. Fletcher L, Cerniglia GJ, Nigg EA, Yend TJ, Muschel RJ. Inhibition of centrosome separation after DNA damage: a role for Nek2. Radiat Res 2004;162:128–35.[CrossRef][Medline]
  8. Hayward DG, Clarke RB, Faragher AJ, Pillai MR, Hagan IM, Fry AM. The centrosomal kinase Nek2 displays elevated levels of protein expression in human breast cancer. Cancer Res 2004;64:7370–6.[Abstract/Free Full Text]
  9. Prigent C, Glover DM, Giet R. Drosophila Nek2 protein kinase knockdown leads to centrosome maturation defects while overexpression causes centrosome fragmentation and cytokinesis failure. Exp Cell Res 2005;303:1–13.[Medline]
  10. Hayward DG, Fry AM. Nek2 kinase in chromosome instability and cancer. Cancer Lett 2006;237:155–66.[CrossRef][Medline]
  11. Machida K, Matsuda S, Yamaki K, et al. v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells. Oncogene 2000;19:1710–8.[CrossRef][Medline]
  12. Hamaguchi M, Matsuyoshi N, Ohnishi Y, Gotoh B, Takeichi M, Nagai Y. p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin cell adhesion system. EMBO J 1993;12:307–14.[Medline]
  13. Mon NN, Ito S, Senga T, Hamaguchi M. FAK signaling in neoplastic disorders: a linkage between inflammation and cancer. Ann N Y Acad Sci 2006;1086:199–212.[CrossRef][Medline]
  14. Fardilha M, Wu W, Sa R, et al. Alternatively spliced protein variants as potential therapeutic targets for male infertility and contraception. Ann N Y Acad Sci 2004;1030:468–78.[CrossRef][Medline]
  15. Weber SM, DeMatteo RP, Fong Y, Blumgart LH, Jarnagin WR. Staging laparoscopy in patients with extrahepatic biliary carcinoma. Analysis of 100 patients. Ann Surg 2002;235:392–9.[CrossRef][Medline]
  16. Weinstein IB. Addiction to oncogenes—the Achilles heal of cancer. Science 2002;297:63–4.[Free Full Text]
  17. Takei Y, Kadomatsu K, Yuzawa Y, Matsuo S, Muramatsu T. A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics. Cancer Res 2004;64:3365–70.[Abstract/Free Full Text]
  18. Heidel JD, Hu S, Liu XF, Triche TJ, Davis ME. Lack of interferon response in animals to naked siRNAs. Nat Biotechnol 2004;22:1579–82.[CrossRef][Medline]



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Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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