Higher Expression of Rhoc Is Related to Invasiveness in Non-Small Cell Lung Carcinoma

5282 Vol. 9, 5282–5286, November 1, 2003 Clinical Cancer Research

Higher Expression of RhoC Is Related to Invasiveness in Non-Small Cell Lung Carcinoma

Yasunori Shikada, Ichiro Yoshino,1 pulmonary tumors, NSCLC2 accounts for a large portion of the Tatsuro Okamoto, Seiichi Fukuyama, patient population, and advanced NSCLC associated with me- Toshifumi Kameyama, and Yoshihiko Maehara tastasis to the lymph nodes and/or distant organs via vascular permeation remains intractable because adjuvant therapies, in- Department of Surgery and Science, Graduate School of Medical cluding chemoradiotherapy, are less effective. Therefore, more Sciences, Kyushu University, Fukuoka, Japan knowledge regarding the genetic and molecular mechanisms of tumor metastasis in NSCLC has been much desired. ABSTRACT To date, a number of small GTP-binding proteins have Purpose: The activation of Rho proteins has been shown been identified and are thought to be involved in the signal to lead to loss of polarity in cancer cells, as well as reorga- transduction pathway that controls a diverse set of essential nization of the cytoskeleton and facilitation of cell motility, cellular functions such as cell growth, cell differentiation, cy- possibly resulting in their malignant potential. The clinico- toskeletal organization, intracellular vesicle transport, and se- pathological significance of RhoC, however, is not yet well cretion (1). The Rho family, a member of the Ras superfamily of known in the case of non-small cell lung cancer (NSCLC). small GTP-binding proteins, has specifically been shown to be Experimental Design: The intratumor expression level ADP-ribosylated by exo-enzyme C3 from Clostridium botuli- of RhoC mRNA was determined and compared with that in num, resulting in its inactivation (2–4). Activation of Rho adjacent nontumorous lung tissue using quantitative reverse proteins leads to the assembly of actin-myosin contractile fila- transcription-PCR in 49 patients with NSCLC. The relation- ments into focal adhesion complexes that lead to cell polarity ship between the level of RhoC transcript and clinicopath- and facilitate motility (5–8), as well as to the formation of ological factors was examined. RhoC protein expression was lamellipodia and adhesion during directed motility (9–11). confirmed by immunohistochemistry and Western blot anal- Recently, it has been demonstrated that overexpression of ysis in several cases. RhoC enhanced the metastasis, invasion, and cell morphology Results: Tumor tissue of NSCLC patients demonstrated of tumor cells and that the dominant-inhibitory Rho mutant was a copy number of RhoC mRNA that was well correlated capable of inducing metastasis (12). The relationship between with its protein level in each case and was significantly the expression level of RhoC and disease aggressiveness has higher than that found in the corresponding nontumorous also been shown in clinical breast cancer (13–15) and pancreatic -lung tissue (2.73 ؋ 105 versus 1.13 ؋ 104 copies/0.08 ␮g cancer (16); however, clinical information regarding RhoC ex mRNA; P < 0.05). Histopathologically positive cases of lym- pression in NSCLC is sparse. phatic permeation showed a significantly higher copy num- To determine the possible role of RhoC expression in ber of RhoC than negative cases (4.31 ؋ 105 versus 1.93 ؋ NSCLC, the present study quantified the RhoC mRNA of NSCLC 105 copies/0.08 ␮g mRNA; P < 0.05). With regard to venous tissue and examined its significance with respect to clinicopatho- permeation, the RhoC copy number in positive cases tended logical factors, especially the invasiveness of tumor cells. to be higher than that seen in negative cases (3.72 ؋ 105 MATERIALS AND METHODS .(0.06 ؍ versus 2.14 ؋ 105 copies/0.08 ␮g mRNA; P Conclusions: This is the first demonstration that the Patients. Between January 1996 and April 2000, 49 Japa- expression level of RhoC is correlated to vascular perme- nese patients with NSCLC underwent surgical resection at the ation in NSCLC. Department of Surgery II, Kyushu University Hospital (Fukuoka, Japan). No patient had received antitumor treatment before surgery. INTRODUCTION Tumor and normal lung tissue samples were freshly obtained from Lung cancer is currently a major cause of death due to resected specimens, frozen immediately in liquid nitrogen, and malignancies in Japan. Among the histological subtypes of stored at –80°C until the following experiments. Immunohistochemistry. Frozen, nonfixed tissue sections (diameter, 5 ␮m) were fixed with acetone and stored at –20°C. After thawing, the sections were rinsed with 1% hydro- Received 9/30/02; revised 7/2/03; accepted 7/22/03. gen peroxide for 20 min and subsequently rinsed twice with The costs of publication of this article were defrayed in part by the PBS. After an incubation period of 24 h at 4°C with goat payment of page charges. This article must therefore be hereby marked anti-RhoC antibody (diluted 1:400 in PBS; Santa Cruz Biotech- advertisement in accordance with 18 U.S.C. Section 1734 solely to nology), the sections were rinsed twice with PBS, and biotiny- indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan. Phone: 81-92- 642-5466; Fax: 81-92-642-5482; E-mail: [email protected] 2 The abbreviations used are: NCLSC, non-small cell lung cancer; u.ac.jp. RT-PCR, reverse transcription-PCR.

Fig. 1 Immunohistochemical analysis for RhoC protein. Each panel shows an immunohistochemical staining reaction using an anti-RhoC protein antibody for the representative cases. The cytoplasm of tumor cells was highly stained for RhoC protein in both squamous cell carcinoma (b, ϫ20; d, ϫ50) and adenocarcinoma (c, ϫ20; e, ϫ50). RhoC was not detected in the normal lung epithelium (a, ϫ20).

lated secondary antibody (diluted 1:250 in PBS) was added at RNA Extraction and First-Strand cDNA Synthesis. room temperature for 30 min. After application of avidin-biotin Total RNA of cultured cells growing exponentially was ex- staining at room temperature for 30 min, RhoC protein was tracted using ISOGEN (Nippon Gene, Inc., Tokyo, Japan) ac- visualized using diaminobenzidine (17). cording to the manufacturer’s protocol. Total RNA of clinical Western Blot Analysis. The tissue samples of homoge- tissue was isolated using the same method. Synthesis of cDNA nates derived from normal lung or cancer tissue were treated was done with 2.0 ␮g of total RNA using a First-Strand cDNA with detergent solution [62.5 mM EDTA, 50 mM Tris (pH 8.0), Synthesis Kit (Amersham Pharmacia Biotech). 0.4% deoxycholic acid, and 1% NP40] and subjected to SDS- Real-Time Quantitative RT-PCR. Real-time RT-PCR PAGE. Protein on the gel was transformed electrophoretically to is a sensitive, quantitative, and highly reliable method for RNA a nitrocellulose membrane sheet at 280 mA for 30 min using quantification. The theoretical basis of the method has been semi-dry blotting methods. The sheet was then washed inten- described previously (18–20). Gene expression was measured sively with TBS and incubated with the polyclonal antihuman using the ABI Prism 7700 Sequence Detection System (Perkin- RhoC antibody (1:1000 dilution; Santa Cruz Biotechnology) at Elmer Corp., Foster City, CA). Primers and Taqman Probes 4°C for 8 h. After washing three times with TBS, the membrane (Custam Oligonucleotide Factory, Foster City, CA) were de- was incubated with peroxidase-conjugated goat antibodies signed to span exon-intron junctions to prevent amplification of against goat IgG at room temperature for 40 min. After the genomic DNA and also to result in amplicons of fewer than 200 reaction, a semiquantification of RhoC protein was performed bp to enhance the efficiency of PCR amplification. Probes were by densitometry analysis using the NIH Image program. labeled at the 5Ј-end with the reporter dye molecule 6-carboxy- ␭ ϭ Ј Culture Cell Lines. The lung cancer cell lines PC-9 florescein (emission max 582 nm) and at the 3 -end with the (adenocarcinoma), QG95 (squamous cell carcinoma), and A549 quencher dye molecule 6-carboxytetramethyl-rhodamine (emis- ␭ ϭ (bronchioloalveolar carcinoma) were maintained in continuous sion max 582 nm). Upon amplification, probes annealed to culture in RPMI 1640 supplemented with 100 units/ml penicil- the template are cleaved by the 5Ј-nuclease activity of the Taq lin-streptomycin and 10% fetal bovine serum. These cell lines polymerase reaction. This process separates the fluorescent label were washed, and cell pellets were collected, frozen in liquid from the quencher and allows the release of 1 unit of fluores- nitrogen, and stored at –80°C until RNA extraction. cence for each unit of amplification. By determining the amount

Fig. 2 Expression of RhoC mRNA in lung cancer cell lines. RhoC amplicons of 180 bp were detected in all cell lines (A549, PC-9, and QG56).

of fluorescence with each cycle, it is possible to determine the number of cycles necessary to reach a chain amount of fluorescence in a test sample compared with known standard amounts of template provided as a standard curve. DNA strands were generated by PCR amplification of gene products, purification, and quantification by spectrophotometry (absorbance at 260 nm). The number of copies was calculated with the use of the molecular gene’s weight. Real-time PCR of cDNA specimens and DNA strands was conducted in a total volume of 25 ␮lof with 1ϫ Taqman Master Mix (Perkin-Elmer Corp.) and primers ␮ ␮ Fig. 3 Detection and quantification of RhoC mRNA in lung cancer tis- at 15 M and probes at 10 M. The primer sequences were as sues. RhoC mRNA expression in tumor and paired normal tissues from 49 follows: forward primer, 5Ј-TCCTCATCGTCTTCAGCAAG; patients was quantified by quantitative RT-PCR. RhoC mRNA expression reverse primer, 5Ј-CAGGATGACATCAGTGTCCG; and Taq- was found to be significantly higher in tumor tissues than in normal tissues, man probe, CTTTGAGAACTATATTGCGGACATTGAGGT- with mean values of 27.3 ϫ 104 copies/0.08 ␮g mRNA in tumor tissues and ϫ 4 ␮ Ͻ TGG. Thermal cycler parameters included a 2-min incubation at 1.1 10 copies/0.08 g mRNA in normal tissues (P 0.05). 50°C, 10 min at 95°C, and 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C at 10 min. Each sample was analyzed in duplicate, and a calibration curve constructed using a 10-fold serial dilution of a total extracted from A549 cells was run parallel with each analysis. The coefficient of correlation was r ϭ 0.97, and the slope was constant in each experiment. As internal controls, the same samples were tested for ␤-actin mRNA (Perkin-Elmer Corp.) in the same manner. Statistical Analysis. For statistical analyses, StatView-J 5.0 software (SAS Institute Inc.) was used. The relationship between RhoC mRNA expression and clinicopathological factors was examined using Mann-Whitney U tests. Fig. 4 Analysis for detection of RhoC protein in lung cancer tissues. RESULTS Protein lysates derived from tumor tissues (T) and paired normal lung tissues (N) were subjected to analysis. A band of Mr 24,000 was Detection of RhoC Protein in NSCLC Tissues. Cyto- observed in each lane. The ratio of RhoC protein [tumor/normal (T/N) plasm of tumor cells was highly stained for RhoC protein in ratio of RhoC protein] calculated by densitometry is shown. both squamous cell carcinoma (Fig. 1, b and d) and adenocarcinoma (Fig. 1, c and e). RhoC was not detected in the normal lung epithelium (Fig. 1a) or the stromal cells of the tumor tissue. . RhoC amplicon (Fig. 2). To confirm the genome of RhoC, Expression of RhoC mRNA in Cell Lines and Clinical sequence analysis for the amplicon was performed. This result NSCLC Tissues. All lung cancer cell lines tested (PC-9, revealed that lung cancer cells readily express RhoC mRNA. QG95, and A549) expressed the band corresponding to the RhoC mRNA expression of tumor and normal tissues from 49

Table 1 The relationship between the expression level of RhoC mRNA and each clinicopathological factor Copy number Factors (/0.08 ␮g mRNA) P Gender Male (n ϭ 30) 396890.1 0.123 Female (n ϭ 19) 77389.1 ] Pack-year index Heavy (Ͼ40) (n ϭ 20) 260671.4 ] 0.40 Mild (0–40) (n ϭ 15) 49671.3 0.28 0.11 None (0) (n ϭ 14) 57399.6 ] ] Histology Adenocarcinoma (n ϭ 36) 347673.3 0.22 Others (n ϭ 13) 66219.3 ] Pathological stage I/II (n ϭ 31) 263597.2 0.90 III/IV (n ϭ 18) 289198.3 ] Lymphatic permeation Ϫ (n ϭ 31) 193276.9 0.002 ϩ (n ϭ 17) 430284.1 ] Vessel permeation Ϫ (n ϭ 29) 214822.1 0.066 ϩ (n ϭ 19) 372451.3 ] Pleural invasion Ϫ (n ϭ 26) 248057.0 0.22 ϩ (n ϭ 23) 296222.3 ]

patients was quantified by quantitative RT-PCR. RhoC mRNA DISCUSSION expression was significantly higher in tumor tissues than in In the present study, we demonstrated the substantial ex- ␮ normal tissues (Fig. 3). Data are shown as copy number/0.08 g pression of RhoC protein as well as the objective higher expres- ϫ 4 ␮ total mRNA. The mean values were 27.3 10 copies/0.08 g sion of RhoC in NSCLC by quantitative real-time RT-PCR. To ϫ 4 ␮ total mRNA in tumor tissues and 1.1 10 copies/0.08 g total the best of our knowledge, this is the first demonstration of the Ͻ mRNA in normal tissues (P 0.05). We confirmed that the positive correlation of RhoC expression with invasion to lym- ␤ level of -actin expression, which served as the internal control, phatic vessels in tissue samples from patients with NSCLC. was almost the same expression in tumor and normal lung Recent intensive studies have demonstrated several upstream tissues (data not shown). pathways that activate Rho as well as downstream targets of acti- To confirm the expression of RhoC protein, we performed vated Rho (21–25). The activity of Rho forming stress fibers and Western blot analysis. As shown for eight pairs of normal versus focal adhesion is mediated by the phosphorylation and activation of tumor tissues from the lung, the amount of RhoC protein was two of these targets, Rho-kinase/ROK/ROCK (6, 24, 26) and the was determined using densitometry. A band of M 24,000 was r myosin-binding subunit of myosin phosphatase (27, 28). Activated observed in each lane. Lung cancer tissues expressed RhoC Rho kinase directly phosphorylates the myosin light chain, which protein at significantly higher levels than normal tissues in all regulates the formation of stress fibers and focal adhesion contacts cases tested (Fig. 4). The means of the ratio were 4.05 for protein and 80.2 for mRNA, and those level of tumor tissue and leads to cell membrane ruffling and cell motility (29). There- were consistently higher than normal tissue in eight cases. fore, expression of Rho might be expected to be not only a good Relationship between the Expression of RhoC mRNA candidate marker for invasive and proliferative tumor cells, includ- and Clinicopathological Factors. We examined the relation- ing those in NSCLC, but also a molecular target of these cells. ship between the expression level of RhoC mRNA and clinico- Recent studies suggest a possible link between RhoC ex- pathological factors (Table 1). There were no significant differ- pression and tumor progression in both breast cancer (13–15) ences in copy number of RhoC mRNA among subgroups and ductal adenocarcinoma of the pancreas (16). Kleer et al. according to gender, pack-year index, histology, and patholog- (13) have demonstrated the significant correlation of RhoC ical stage. With a special reference to tumor invasiveness, the expression, tumor stage, and lymph node metastasis; interest- mean copy number of RhoC mRNA was 4.31 ϫ 105 copies/0.08 ingly, they suggested that RhoC could be a specific molecular ␮g total mRNA for positive lymphatic permeation and 1.93 ϫ indicator for detecting invasive carcinoma with metastatic po- 105 copies/0.08 ␮g total mRNA for negative lymphatic perme- tential among breast cancers. Additionally, Van Golen et al. (14, ation (P ϭ 0.002), whereas that of RhoC mRNA was 3.72 ϫ 105 15) also reported that the expression level of the RhoC gene was copies/0.08 ␮g total mRNA for positive venous permeation and associated with rapid growth in inflammatory breast cancers; 2.14 ϫ 105 copies/0.08 ␮g total mRNA for negative venous similar findings have also been reported in metastatic adenocar- permeation (P ϭ 0.066). For pleural invasion, the mean copy cinomas of the pancreas (16). These studies thus suggest that the number of RhoC mRNA was 2.9 ϫ 105 copies/0.08 ␮g total expression level of the RhoC gene is associated with the inva- mRNA for positive pleural invasion and 2.4 ϫ 105 copies/0.08 sive potential of cancer cells, and the present study on NSCLC ␮g total mRNA for negative pleural invasion (P ϭ 0.27). also supports these findings. Furthermore, all of these studies,

including ours, also suggest that Rho-related intracellular sig- 12. Clark, E. A., Golub, T. R., Lander, E. S., and Hynes, R. O. Genomic nals might definitively determine the metastatic potential of analysis of metastasis reveals an essential role for RhoC. Nature malignant neoplasms. Further research is therefore critical. (Lond.), 406: 532–535, 2000. In the present study, we first demonstrated that the expression 13. Kleer, C. G., van Golen, K. L., Zhang, Y., Wu, Z. F., Rubin, M. A., and Merajver, S. D. Characterization of RhoC expression in benign and level of RhoC mRNA is positively correlated with the invasiveness malignant breast disease. Am. J. Pathol., 160: 579–584, 2002. of NSCLC, which was histologically detected as lymphatic and 14. Van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W., and Merajver, vascular permeation. Whereas lymphatic permeation and venous S. D. RhoC GTPase, a novel transforming oncogene for human mam- permeation are independent prognostic factors in patients with mary epithelial cells that partially recapitulates the inflammatory breast stage I NSCLC (30, 31), overexpression of RhoC may be a signif- cancer phenotype. Cancer Res., 60: 5832–5838, 2000. icant determinant of the aggressiveness of the disease. The present 15. Van Golen, K. L., Davis, S., Wu, Z. F., Wang, Y., Bucana, C. D., study is limited in that it includes no assessment of the role of RhoC Root, H., Chandrasekharappa, S., Strawderman, M., Ethier, S. P., and expression in the long-term prognosis of patients; such an assess- Merajver, S. D. A novel putative low-affinity insulin-like growth factor- binding protein, LIBC (lost in inflammatory breast cancer), and RhoC ment was impossible to carry out due to the limited availability of GTPase correlate with the inflammatory breast cancer phenotype. Clin. fresh NSCLC tissue stocks in our laboratory. Our plans for future Cancer Res., 9: 2511–2519, 1999. research therefore include an investigation of the patients’ progno- 16. Suwa, H., Oshio, G., Imamura, T., Watanabe, G., Arii, S., Imamura, sis at each stage of NSCLC and its subgroups. M., Hiai, H., and Fukumoto, M. Overexpression of the rhoC gene In summary, this is, to the best of our knowledge, the first correlates with progression of ductal adenocarcinoma of the pancreas. report indicating that RhoC could be both a biological determi- Br. J. Cancer, 77: 147–152, 1998. nant of and a molecular marker for vessel invasiveness of 17. Fritz, G., Just, I., and Kaina, B. Rho GTPase are over-expressed in NSCLC. Because a recent experimental study demonstrated that human tumors. Int. J. Cancer, 81: 682–687, 1999. the molecular targeting of RhoC using retroviruses expressing 18. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. Real time quantitative PCR. Genome Res., 88: 986–994, 1996. dominant-inhibitory Rho mutant diminished invasive activity in 19. Gibson, U. E., Heid, C. A., and Willaims, P. M. A novel method for vitro as well as metastatic potentials in vivo (12), a similar real time quantitative RT-PCR. Genome Res., 6: 995–1000, 1996. strategy, including not only dominant-inhibitory Rho mutant but 20. Bieche, I., Laurendeau, I., Tozlu, S., Olivi, M., Vidaud, D., also RhoC-inhibitory compounds, could provide an efficient Lidereau, R., and Vidaud, M. Quantitation of MYC gene expression in anticancer therapy against RhoC-expressing cancer cells by sporadic breast tumors with a real-time reverse transcription-PCR assay. limiting their invasive potential. Cancer Res., 59: 2759–2765, 1999. 21. Clark, E. A., King, W. G., Brugge, J. S., Symons, M., and Hynes, R. O. Integrin-mediaded signals regulated by members of the Rho REFERENCES family of GTPase. J. Cell Biol., 142: 573–586, 1998. 1. Hall, A. The cellular functions of small GTP-binding proteins. Sci- 22. Fujisawa, K., Madaule, P., Ishizaki, T., Watanabe, G., Bito, H., ence (Wash. DC), 249: 635–640, 1990. Saito, Y., Hall, A., and Narumiya, S. Different regions of Rho determine 2. Aktories, K., Rosener, S., Blashke, U., and Chhatwal, G. S. Botuli- Rho-selective binding of different classes of Rho target molecules. num ADP-ribosyltransferase C3. Purification of the enzyme and char- J. Biol. Chem., 273: 18943–18949, 1998. acterization of the ADP-ribosylation reaction in platelet members. Eur. 23. Maekawa, M., Ishizaki, T., Boku, S., Watanabe, N., Fujita, A., J. Biochem., 172: 445–450, 1988. Iwamatsu, A., Obinata, T., Ohashi, K., Mizuno, K., and Narumiya, S. 3. Chardin, P., Boquet, P., Madaule, P., Popoff, M. R., Rubin, E. J., and Signaling from Rho to the actin cytoskeleton through protein kinases Gill, D. M. The mammalian G protein RhoC is ADP-ribosylated by ROCK and LIM-kinase. Science (Wash. DC), 285: 895–898, 1999. Clostridium botulinum exoenzyme C3 and affects actin microfilaments 24. Fukuta, Y., Oshiro, N., Kinoshita, N., Kawano, Y., Bennet, V., Mat- in Vero cells. EMBO J., 8: 1087–1092, 1989. suura, Y., and Kaibuchi, K. Phosphorylation of adducin by Rho-kinase 4. Rubin, E. J., Gill, D. M., Bouquet, P., and Popoff, M. R. Functional plays a crucial role in cell motility. J. Cell Biol., 145: 347–361, 1999. modification of a 21-kDa G protein when ADP-ribosylated by exoen- 25. Amano, M., Chihara, K., Fukuta, Y., Nakamura, N., Matsuura, Y., zyme C3 of Clostridium botulinum. Mol. Cell. Biol., 8: 418–426, 1988. and Kaibuchi, K. Formation of actin stress fibers and focal adhesions 5. Nobes, C. D., and Hall, A. Rho, rac and cdc42 GTPases regulate the enhanced by Rho-kinase. Science (Wash. DC), 275: 1308–1311, 1997. assembly of multimolecular focal complexes associated with actin stress 26. Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, fibers, lamellipodia and filopodia. Cell, 81: 53–62, 1995. T., Matsuura, Y., and Kaibuchi, K. Phosphorylation and activation of 6. Leung, T., Chen, X. Q., Manser, E., and Lim, L. The p160 RhoA- myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem., 271: binding kinase ROK ␣ is a member of a kinase family and is involved in the 20246–20249, 1996. reorganization of the cytoskelton. Mol. Cell. Biol., 16: 5313–5327, 1996. 27. Apenstrom, P. Effectors for the Rho GTPase. Curr. Opin. Cell Biol., 7. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukuta, Y., Nakafuku, M., 11: 95–102, 1999. Yamamori, B., Feng, J., Nakamoto, T., Okawa, K., Iwamatsu, A., and 28. Koch, A. E., Kunkel, S. L., Burrows, J. C., Evanoff, H. L., Haines, Kaibuchi, K. Regulation of myosin phosphatase by Rho and Rho-associated G. K., Pope, R. M., and Strieter, R. M. Synovial tissue macrophage as a kinase (Rho-kinase). Science (Wash. DC), 273: 245–248, 1996. source of chemotactic cytokine IL-8. J. Immunol., 147: 2187–2195, 1995. 8. Itoh, K., Yoshioka, K., Akedo, H., Uehata, M., Ishizaki, T., and 29. Small, J. V., Kaverina, I., Krylyshkina, O., and Rottner, K. Cy- Narumiya, S. An essential part for Rho-associated kinase in the trans- toskelton cross-talk during cell motility. FEBS Lett., 452: 96–99, 1999. cellular invasion of tumor cells. Nat. Med., 5: 221–225, 1995. 30. Ichinose, Y., Yano, T., Yokoyama, H., Inoue, T., Asoh, H., and 9. Barbacid, M. ras genes. Annu. Rev. Biochem., 56: 779–827, 1987. Katsuda, Y. The correlation between tumor size and lymphatic vessel 10. Morris, J. D., Price, B., Lloyd, A. C., Self, A. J., Marshall, C. J., and invasion in resected peripheral stage I non-small-cell lung cancer. Hall, A. Scrape-loading of Swiss 3T3 cells with ras protein rapidly J. Thorac. Cardiovasc. Surg., 108: 684–686, 1994. activates protein kinase C in the absence of phospholinositide hydroly- 31. Ichinose, Y., Yano, T., Asoh, H., Yokoyama, H., Yoshino, I., and sis. Oncogene, 4: 27–31, 1989. Katsuda, Y. Prognostic factors obtained by a pathologic examination in 11. Nobes, C. D., and Hall, A. Rho GTPase and actin cytoskelton. completely resected non-small-cell lung cancer. An analysis in each patho- Science (Wash. DC), 279: 509–514, 1998. logic stage. J. Thorac. Cardiovasc. Surg., 110: 601–605, 1995.

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Yasunori Shikada, Ichiro Yoshino, Tatsuro Okamoto, et al.

Clin Cancer Res 2003;9:5282-5286.

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