Overexpression of Axin Downregulates TCF-4 and Inhibits the Development of Lung Cancer

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1 Annals of Surgical Oncology (Ó 2007) DOI: /s Overexpression of Axin Downregulates TCF-4 and Inhibits the Development of Lung Cancer Hong-Tao Xu, MD, PhD, Qiang Wei, MD, PhD, Yang Liu, MD, PhD, Lian-He Yang, MD, Shun-Dong Dai, MD, PhD, Yang Han, MD, PhD, Juan-Han Yu, MD, Nan Liu, MD, and En-Hua Wang, MD, PhD Department of Pathology, College of Basic Medical Sciences, China Medical University and Department of Pathology, the First Affiliated Hospital of China Medical University, Shenyang , China Background: T cell factor 4 (TCF-4) mediates a nuclear response to wingless/int (Wnt) signals by interacting with b-catenin. Axis inhibition protein (axin) is an important negative regulator of the Wnt signaling pathway. Our aims were to examine the relationship between axin and TCF-4 and to explore the effects of axin on the development of lung cancer. Methods: Expression levels of axin and TCF-4 were examined in 107 lung cancer specimens by immunohistochemistry. The axin gene was transfected into lung cancer BE1 cells. The expression levels of axin, b-catenin, and TCF-4 were detected with immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR) experiments. Apoptosis, proliferation, and the invasive ability of lung cancer cells were examined using flow cytometry, 3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), and Matrigel invasive assays. Results: Preserved axin expression correlated negatively with TCF-4 expression (P =.031). Axin expression differed with respect to degree of differentiation (P =.025) and histological tumor type (P =.031). TCF-4 expression differed relative to tumor, node metastasis (TNM) stage (P =.024). BE1 cells transfected with axin (BE1-axin cells) exhibited a significant decrease in TCF-4 expression. The level of apoptosis in BE1-axin cells was significantly increased, while the proliferative and invasive abilities of BE1-axin cells were decreased. Conclusion: These results suggest that reduced expression of axin or augmented expression of TCF-4 is associated with the malignant behavior of lung cancers. Overexpression of axin can downregulate expression of TCF-4 and can inhibit the ability of lung cancer cells to proliferate and invade. Key Words: Axin T cell factor 4 Lung cancer Proliferation Invasion. The wingless/int (Wnt) signaling pathway plays an important role in tumor cell de-differentiation and proliferation. Activation of the Wnt pathway requires nuclear accumulation of b-catenin as well as the binding of b-catenin and T cell factor-4 (TCF-4). Received May 11, 2007; accepted July 10, 2007 Address correspondence and reprint requests to: En-Hua Wang, MD, PhD; wangeh@hotmail.com Published by Springer Science+Business Media, LLC Ó 2007 The Society of Surgical Oncology, Inc. (Fig. 1) TCF-4 induces transcription of target genes, such as c-myc, cyclin D1, vascular endothelial growth factor (vegf) and matrix metalloproteinase-7 (mmp-7). 1,2 An increasing number of studies have shown that abnormal activation of the Wnt pathway plays an important role in tumor progression. 3 6 Axis inhibition protein (axin) is an important scaffolding protein within the cell. One of its key functions is to negatively regulate the activity of the Wnt pathway. Axin forms a multiprotein complex with adenomatous polyposis coli (APC) and glycogen synthase kinase-3b (GSK-3b). This complex regulates

2 H.-T. XU ET AL. MATERIALS AND METHODS FIG. 1. Wnt signaling pathway. The upstream Wnt signals promote the accumulation of b-catenin in cytoplasm. Then b-catenin enters into nucleus and binds with TCF-4. The complex of b- catenin and TCF-4 activates the transcription of Wnt target genes that promote tumor cell proliferation. Axin forms a multiprotein complex with APC and GSK-3b and regulates the degradation of b-catenin, ensuring decreased activity of the Wnt pathway. the phosphorylation and degradation of b-catenin and thereby inhibits activity of the Wnt pathway. When axin levels are low, the axin complex may not function properly, resulting in accumulation of b- catenin. 7,8 Other studies have suggested that axin is involved in regulation of the c-jun NH2-terminal kinase (JNK) and transforming growth factor-b (TGF-b) signaling pathways and may therefore contribute to the responses and apoptosis. 9,10 Decreased axin expression has been reported to correlate with tumor differentiation, 11 whereas high axin levels may induce cell apoptosis. 9,12 Our previous study also found that decreased axin expression correlated positively with the nuclear expression of b-catenin and poor differentiation of lung cancers. 6 As a downstream constituent of the Wnt pathway, TCF-4 plays an important role in regulating target gene transcription. We hypothesized that axin may regulate TCF-4 and induce b-catenin degradation. It has not been resolved whether axin influences transcription or translation of TCF-4, nor whether axin regulation of TCF-4 expression is positive or negative. Furthermore, the question of how axin may be involved in the regulation of development of lung cancer cells still needs to be addressed. Therefore in this study, we examined the expression levels of axin and TCF-4 in lung squamous cell carcinomas (SCCs) and adenocarcinomas. We also transfected axin into lung cancer cells and explored the effect of axin overexpression on b-catenin and TCF-4 expression, cellular proliferation, apoptosis, and the invasive ability of lung cancer cells. Part I: Expression Levels of Axin and TCF-4 in Lung Cancer Specimens Patients The 107 samples examined in this study were selected randomly from patients with lung SCC or adenocarcinoma who underwent surgery in the First Affiliated Hospital of China Medical University between 2000 and The study was conducted according to the regulations of the institutional review boards at China Medical University. Patients were years of age, and the mean age was 58 years (71 males and 36 females). These tumors were diagnosed as SCCs (n = 48) or adenocarcinomas (n = 59) and classified as well (n = 25), moderately (n = 38), or poorly (n = 44) differentiated according to the classification system of the World Health Organization (2004). 13 Sixty-one cases showed lymphatic metastasis. All the patients were classified as stages I IV (n = 42, 16, 44, and 5, respectively) according to the tumor, node metastasis (TNM) classification scheme of the International Union Against Cancer. 14 Immunohistochemistry All the resected specimens were fixed with 10% neutral-buffered formalin and embedded in paraffin blocks. Tissue blocks were cut into 4-lm slides, deparaffinated, rehydrated, and immunostained with polyclonal rabbit anti-axin antibody (1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and polyclonal rabbit anti-tcf-4 antibody (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA ). Binding of primary antibodies was detected by the streptavidin-peroxidase method. Some slides were also stained in the absence of primary antibodies and served as negative controls. All specimens were independently evaluated by two investigators who were unaware of the clinical data. Consistent with previous studies, 6,11 when 40% of the tumor cells showed positive staining, the sample was scored as having preserved axin expression. When <40% of the tumor cells showed positive staining, the sample was scored as having reduced axin expression. In contrast, specimens in which 30% of the tumor cells showed positive staining were defined as having high TCF-4 expression, whereas those with TCF-4 staining in less than 30% of the tumor cells were defined as having low TCF-4 expression. 15 When 10% of the tumor cells per specimen were stained in nuclear regions, the sample

3 AXIN INHIBITS TCF-4 AND LUNG CANCER DEVELOPMENT was scored as having positive nuclear expression of axin or TCF-4. Part II: Effect of Axin Overexpression on TCF-4 and Cellular Activity in Lung Cancer Cells Cell Culture and Transfection The BE1 cell line, derived from the PG cell line, was established from a human pulmonary giant cell carcinoma (a gift from Dr. Jie Zheng, College of Medicine, Beijing University, China). BE1 cells have 100% metastatic frequency and low axin expression. 16 The axin expression vector pflag-cmv-5b-axin was obtained from Professor Mark A. Perrella (Brigham & WomenÕs Hospital, Boston, MA). 17 The BE1 cells were cultured in RPMI 1640 media (GIBCO Inc., Grand Island, NY, USA) with 10% fetal calf serum (GIBCO Inc., Grand Island, NY, USA) at 37 C ina humidified atmosphere (5% CO 2 ; 95% air), then transfected with pflag-cmv-5b-axin or the empty vector pflag-cmv-5b (as control, Sigma, USA) using Lipofectamine 2000 according to the manufacturerõs protocol (Invitrogen, Carlsbad, CA, USA). Immunofluorescence and RT-PCR Analysis Forty-eight hours after transfection, cells transfected with pflag-cmv-5b-axin (BE1-axin cells) were fixed and incubated with anti-axin (1:50, Santa Cruz, CA, USA), anti-flag (1:200, Sigma, USA), anti-bcatenin (1:200, Upstate, USA) or anti-tcf-4 (1:50, Santa Cruz, CA, USA) primary antibody overnight at 4 C. Cells were incubated with FITC or TRITC-conjugated secondary antibody followed by 0.1% 4,6- Diamidino-2-phenylindole (DAPI) for 10 min at 37 C. Nontransfected BE1 cells and cells transfected with empty vector (BE1-FLAG cells) served as controls. Finally, cells were examined by immunofluorescence microscopy (BX60, OLYMPUS, Japan). Meanwhile, total RNA of the BE1-axin, BE1- FLAG, and nontransfected BE1 cells was extracted with Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Axin, b-catenin, and TCF-4 RT-PCRs were performed using a TaKaRa RNA PCR Kit (AMV) Ver. 3.0 (Ta- KaRa, Japan), according to the manufacturerõs protocol. b-actin served as an internal control. The primers used in this study were: axin: forward, 5 -AC- CGAAAGTACATTCTTGATAAC-3 and reverse: 5 -TCCATACCTGAACTCTCTGC-3 ; b-catenin: forward: 5 -GCCAAGTGGGTGGTATAGAG-3 and reverse: 5 -GCTGGGTATCCTGATGTGC-3 ; TCF-4: forward: 5 -CGAGGGTGATGAGAACC TGC-3 and reverse: 5 -CCCATGTGATTCGATG CGT-3 ; b-actin: forward: 5 -AGAGCTACGAGCT GCCTGAC-3 and reverse: 5 -AGTACTTGCGCTC AGGAGGA-3. The PCR product sizes were 452 bp (axin), 330 bp (b-catenin), 444 bp (TCF-4), and 308 bp (b-actin). After electrophoresis, the PCR products were stained with ethidium bromide and analyzed using a BioImaging system (UVP, Upland, CA, USA). Relative band intensities were determined using NIH image software. Flow Cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide (MTT) Assay and Matrigel Invasive Assay The BE1-axin and BE1-FLAG cells were fixed and suspended at a concentration of cells/ml in a propidium iodide solution for 30 minutes, 48 hours after transfection. 18 The nontransfected BE1 cells served as a control. Changes in apoptosis and cell cycle were detected using flow cytometry (FAC- SCalibur, BD Biosciences, San Jose, CA, USA) and analyzed with Modfit LT V3.0 software (Verity Software House, Inc., Topsham, ME, USA). Meanwhile, the BE1-axin, BE1-FLAG and nontransfected BE1 cells were seeded in 96-well plates ( cells/well). Cell proliferation was evaluated each day for 4 days after transfection using the MTT method. The absorbance, which is directly proportional to the number of living cells in culture, was measured at 570 nm using a microplate reader (Model 550, Bio-Rad, Hercules, CA, USA). A blank with dimethyl sulfoxide (DMSO) alone was measured and subtracted from all values. Cell invasive ability was examined using a 24-well Transwell with 8-lm pore polycarbonate membrane inserts (Corning Inc., Corning, NY, USA) according to the manufacturerõs protocol. The Matrigel (100 lg/ ml) was applied to the upper surface of the membranes. After transfection for 24 hours, BE1-axin and BE1-FLAG cells were seeded on the upper chamber ( cells/well) and incubated for 48 hours. Nontransfected BE1 cells served as the control. Cells that had invaded the surface of the membrane were fixed with methanol and stained with hematoxylin. Five random high-magnification microscope fields per filter were counted. Statistical Analysis The Pearson v 2 test was used to assay whether the expression levels of axin and TCF-4 were related to the clinicopathologic alterations of lung cancer. The SpearmanÕs correlation test was used to examine the correlation between the expression levels of axin and

4 H.-T. XU ET AL. FIG. 2. Immunohistochemical staining of axin and TCF-4 in lung squamous cell carcinomas and adenocarcinomas. The expression of axin was negatively correlated with TCF-4 e- xpression. In a squamous cell carcinoma, the expression of axin was preserved in cytoplasm (A), but the expression of TCF-4 was very low in the similar vision of the same sample (B). In an adenocarcinoma, the expression of axin was reduced (C), but the expression of TCF-4 was very high in the similar vision of the same sample (D). In a squamous cell carcinoma (E), axin was expressed in both nuclear and cytoplasmic regions. In an adenocarcinoma (F), TCF-4 was expressed in both nuclear and cytoplasmic regions. (Representative images. O- riginal magnification: A D at 200 ; E and F at 400 ). TCF-4. All of the in vitro experiments were replicated three times. The Mann Whitney U test and the Kruskal Wallis test were used to analyze the results of RT-PCR, flow cytometry, MTT, and the Matrigel invasive assay. P values less than.05 were considered statistically significant. RESULTS Axin Expression Correlated with Reduced Cellular Activity in Lung Cancer Cells In 107 samples of lung SCCs and adenocarcinomas, 59 cases showed preserved axin expression (55.1%), including 14 cases that showed both cytoplasm and nuclear expressions of axin. In contrast, 86 cases showed high TCF-4 expression (80.3%), including 33 cases that showed both cytoplasm and nuclear expression of TCF-4 (Fig. 2). Five cases exhibited expression of both axin and TCF-4 in the nucleus. Interestingly, the SpearmanÕs correlation test revealed that preserved axin expression was negatively correlated with high TCF-4 expression (P =.031, correlation coefficient = )0.209). The nuclear expressions of axin and TCF-4 did not correlate with each other (data not shown). The correlative analysis between axin expression and the clinicopathologic characteristics of patients with SCCs or adenocarcinomas is summarized in Table 1. Axin expression between patients with differing degrees of differentiation differed significantly (P =.025). The preserved expression rate of axin was significantly higher in SCCs than in adenocarcinomas (P =.031). However, axin expression did not differ significantly by age (P =.167), sex (P =.951), TNM stage (P =.239), or lymph node metastasis (P =.886). We did not find any correlation between nuclear expression of axin and clinicopathologic characteristics of patients with SCCs or adenocarcinomas. Meanwhile, there were significant differences in TCF-4 expression level between patients diagnosed to be at differing TNM stages (P =.024). TCF-4 expression did not differ significantly by age (P =.777), sex (P =.973), differentiation degree (P =.305), histological type (P =

5 AXIN INHIBITS TCF-4 AND LUNG CANCER DEVELOPMENT TABLE 1. Preserved Axin expression, High TCF-4 expression and clinicopathologic characteristics in lung SCCs and adenocarcinomas Axin expression Parameter Reduced Preserved P Low High P TCF-4 expression Low 5 16 High Sex Male Female Age 58 years >58 years TNM stage IorII III or IV Differentiation Well or moderate Poor Histological type SCC Adenocarcinoma Lymphatic metastasis No Yes SCC, squamous cell carcinoma. expres- TCF-4 sion.207), or lymph node metastasis (P =.633) (Table 1). Nuclear expression of TCF-4 did not correlate with clinicopathologic characteristics of patients with SCCs or adenocarcinomas. Overexpression of Axin Downregulated Expression of b-catenin and TCF-4 Changes in the protein expression levels of axin, b- catenin, and TCF-4 in BE1-axin, BE1-FLAG or nontransfected BE1 cells were revealed by immunofluorescence staining. In nontransfected BE1 cells, axin expression was generally weak (Fig. 3A, top), and b-catenin expression (Fig. 3C, top), and TCF-4 expression (Fig. 3D, top) were high in the cytoplasm and the nucleus. The reporter FLAG was not observed in nontransfected BE1 cells (Fig. 3B, top). Forty-eight hours after transfection, the expression of FLAG (Fig. 3B, middle) was high in BE1-FLAG cells, although expression levels and patterns of axin (Fig. 3A, middle), b-catenin (Fig. 3C, middle), and TCF-4 (Fig. 3D, middle) were unchanged. In the BE1- axin cells, expression of axin (Fig. 3A, bottom) and FLAG (Fig. 3B, bottom) were increased and localized to the cytoplasm and nucleus. At the same time, protein expression of b-catenin was markedly decreased (Fig. 3C, bottom). Interestingly, the expression of TCF-4 was also decreased (Fig. 3D, bottom). As shown in Fig. 4, RT-PCR experiments showed that mrna expression of axin 48 hours after transfection was significantly increased in BE1-axin cells compared with BE1-FLAG or nontransfected BE1 cells (P =.004). Although immunofluorescence staining revealed a decrease in b-catenin protein in BE1-axin cells, there was no difference in b-catenin mrna levels between BE1-axin, BE1-FLAG, and nontransfected BE1 cells (P >.05). However, TCF-4 mrna levels were decreased significantly in BE1- axin cells compared to BE1-FLAG and nontransfected BE1 cells (P =.003). Overexpression of Axin-Induced Apoptosis, Inhibited Proliferative and Invasive Ability of Lung Cancer Cells As shown in Fig. 5, flow cytometry analysis revealed that the level of apoptosis was significantly increased to 12.83% (n =3,P =.027) in BE1-axin cells. Meanwhile in nontransfected BE1 or BE1- FLAG cells, levels of apoptosis were low (0.01% or 0.10%, respectively, n = 3). MTT assay results showed that after transfection, the level of proliferation in BE1-axin cells was significantly lower than that of BE1 or BE1-FLAG cells [P >.05 (day 1); P <.001 (days 2 4), n = 3] (Fig. 6). Meanwhile, the number of BE1-axin cells (2.20 ± 0.83) that penetrated through the Matrigel and adhered to the Transwell filter was significantly less than in nontransfected BE1 (19.80 ± 2.77, P =.009, n =3)or BE1-FLAG cells (21.20 ± 3.77, P =.009, n =3) (Fig. 7). DISCUSSION Abnormal accumulation of b-catenin plays an important role in the carcinogenesis and development of many tumors. Previous reports have suggested that abnormal b-catenin protein expression is due to mutations of the b-catenin gene However, other studies indicate that mutations of the b-catenin gene are uncommon and do not contribute to lung cancer, 6,22 breast cancer, renal cell carcinoma, cervical and ovarian carcinoma, 22 oral squamous cell carcinoma, 23 gastrointestinal carcinoid tumors, 24 and pancreatic adenocarcinoma. 25 As one of the key molecules regulating the degradation of b-catenin, axin may provide insight into how abnormal b-catenin accumulation may arise independent of gene mutations. Nakajima et al. 11 found that axin expression correlated inversely with depth of invasion, lymph node metastasis, and lymphatic invasion

6 H.-T. XU ET AL. FIG. 3. Expression of axin, b-catenin and TCF-4 in BE1-axin, BE1-FLAG, or nontransfected BE1 cells. In BE1-axin cells, the expression of axin was remarkably enhanced in cytoplasm and nucleus (A). The reporter FLAG expressed outstandingly in BE1-axin and BE1-FLAG cells but not in BE1 cells (B). The expressions of b-catenin (C) and TCF-4 (D) were reduced obviously in BE1-axin cells than in BE1-FLAG or n- ontransfected BE1 cells. FIG. 4. Changes in axin, b-catenin and T- CF-4 mrna levels in BE1-axin, BE1-FL- AG, or BE1 cells. (A) The electrophoresis images of axin, b-catenin and TCF-4 mr- NA in BE1-axin, BE1-FLAG, or nontransfected BE1 cells after RT-PCR. b-actin served as an internal control. (B) The relative expression rate of axin mrna was significantly enhanced, the mrna of b- catenin had not marked change, but the mrna of TCF-4 was significantly reduced in BE1-axin cells than in BE1-FLAG or B- E1 cells. Columns, mean (n = 3); bar, SD. *, P <.01. and suggested that reduced axin expression correlated with tumor progression of esophageal squamous cell carcinoma. Zhou and Gao 26 also concluded that reduced expression of axin might play a pivotal role in the carcinogenesis and metastasis of oral squamous cell carcinoma. Previous studies from our laboratory have also shown that reduced expression of axin is common in lung cancer and that this may account for nuclear accumulation of b-catenin and dedifferentiation of lung cancer. 6 In this study, we confirmed that axin expression was negatively correlated with poor differentiation in 107 cases of lung SCCs and adenocarcinomas. The preserved expression rate of axin was significantly higher in SCCs than in adenocarcinomas. These results indicate that the expression level of axin may provide useful information for estimating malignancy of lung cancers. TCF-4 is a vital member of the Wnt signaling pathway, and its expression and activity of TCF-4 are linked directly with activation of Wnt target genes. Consistent with previous studies suggesting that high TCF-4 expression is associated with tumor progres-

7 AXIN INHIBITS TCF-4 AND LUNG CANCER DEVELOPMENT FIG. 5. Changes in the level of cell proliferation and apoptosis in BE1-axin, BE1-FLAG, or BE1 cells. Forty-eight hours after transfection, the DNA content and apoptosis were examined using flow cytometry. (A) The representative images showed the apoptosis and cell cycle of BE1-axin, BE1-FLAG, or BE1 cells. (B) The apoptosis rate of BE1-axin cells (12.83%) was significantly higher than that of BE1-FLAG (0.10%) or BE1 cells (0.01%). Columns, mean (n = 3); bar, SD. *, P <.05. FIG. 6. Cell proliferation in BE1-axin, BE1- FLAG, and BE1 cells. Differences in cell proliferation were determined using the MTT assay. The absorbance at 570 nm represents cell viability at each time point and is a measure of cell proliferation. The growth curves indicated that the growth rates had not marked difference among BE1-axin, BE1-FLAG, and BE1 cells in the first day after transfection, but in each day of the latter three days, the growth rate was significantly lower in BE1-axin cells than in BE1-FLAG, and BE1 cells. Points, mean (n = 3); bar, SD. *, P <.001. FIG. 7. Invasive ability of BE1-axin, BE1- FLAG, or BE1 cells. The cell invasive ability was examined with Matrigel cell culture chambers. (A), (B), or(c) showed a representative microscope field of filters under t- he Matrigel from BE1-axin, BE1-FLAG or BE1 cells respectively. Bar, 10 lm. (D) The histogram showed that the number of invasive cell was significantly less in BE1-axin cells than BE1-FLAG or BE1 cells. Columns, mean (n = 3); bar, SD. *, P <.01. sion, 15 the present study revealed that TCF-4 was more likely to be highly expressed in stage III IV than in stage I II in lung cancer tumors. Hence these findings support the hypothesis that TCF-4 expression may reflect cellular activity in cancer cells and progression of lung cancer. Furthermore, we investigated the role of axin on TCF-4 expression using immunohistochemistry. Although a role for axin in the degradation of b-catenin has been widely described, the putative relationship between axin and TCF-4 has never been addressed. Our results revealed that axin expression was negatively correlated with TCF-4 expression in lung cancer tissues, which indicated that axin might have some direct or indirect function on TCF-4 expression. To further investigate the role of axin on TCF-4 expression suggested by our immunohistochemistry experiments, we transfected axin gene into BE1 cells and examined changes in b-catenin and TCF-4 expression as well as cellular activity in lung cancer cells. The results showed that although axin overexpression downregulated b-catenin protein levels, the

8 H.-T. XU ET AL. mrna level of b-catenin remained unchanged. These findings confirm that axin induces degradation of b- catenin protein, but does not effect the transcription of b-catenin mrna. It is more likely that the overexpression of axin promotes the formation and activation of the axin-apc-gsk-3b complex that leads to the degradation of b-catenin. Moreover, we report for the first time that axin overexpression downregulates the transcription and expression of TCF-4 in lung cancer cells, which confirmed our results in lung cancer tissues. These findings indicate that axin not only induces the degradation of b-catenin protein, but it is also involved in the regulation of other Wnt pathway signaling molecules at the mrna and/or protein level. Such involvement may contribute further to the inhibition of tumor development. Furuhashi et al. 10 observed that increased levels of axin inhibited the transcription of the Xenopus twin gene promoter, which is induced by b-catenin and TCF-4 in HepG2 cells. However, in these studies the effect of axin overexpression on TCF-4 expression was not examined. Our results indicate that axin may inhibit the target promoter transcription both by inducing b-catenin degradation and downregulating TCF-4 transcription. A mechanism by which axin downregulates the transcription of TCF-4 has never been described. However, it has been shown that axin may stimulate p53 directly or indirectly, 12 and TCF-4 has been identified as a transcriptional target of p53 signaling. 27 In addition, Saegusa et al. revealed that b-catenin could directly induce TCF-4 transcription from the TCF-4 promoter, the effect being enhanced by the p300 coactivator. 28 So it is possible that axin may downregulate the transcription and activity of TCF-4 indirectly by interacting with p53 and/or downregulating b-catenin. It still needs to be determined whether such a mechanism links axin to TCF-4 via p53 and/or b-catenin. Our flow cytometry, MTT and Matrigel invasive studies suggest that axin overexpression inhibited proliferative and invasive ability of lung cancer cells. It has been reported that axin induces apoptosis by stimulating p53 function, activating the JNK pathway and downregulating b-catenin levels. 9,12 The level of apoptosis in the BE1-axin cells used in this study was lower than that observed by Rui et al. 12 in HEK 293 cells. This discrepancy may occur because the BE1 cell line is a highly malignant lung cancer cell line and therefore has more proliferative and antiapoptotic abilities than the HEK 293 cells. The inhibition of invasive ability may occur because axin downregulates the expression and function of b- catenin and TCF-4, resulting in a reduction in the transcription of target genes relevant to invasion, such as MMP-7 and CD44. 1,29,30 In conclusion, we show here that axin is negatively correlated with the expression of TCF-4. Overexpression of axin can promote the degradation of b- catenin, downregulate the expression of TCF-4, and inhibit the proliferative and invasive ability of lung cancer cells. Our findings indicate that axin can inhibit the development of tumors in a number of ways. Future studies on the various anticancer roles of axin will be useful for improving our understanding of carcinogenesis. Moreover, axin is a potential target for gene therapy. In particular, induction of axin overexpression has the potential to be developed into a future treatment for lung cancer patients. ACKNOWLEDGMENTS We thank Dr. Jie Zheng at Medical College of Beijing University, China for providing lung cancer BE1 cell lines. We also thank Professor Mark A. Perrella at Brigham & WomenÕs Hospital, Boston, MA, USA for kindly providing axin expression vector pflag-cmv-5b-axin containing mouse axin1 gene. This study was supported by National Natural Science Foundation of China (Grants and to E.-H. Wang) and Doctoral Fund of Ministry of Education of China (No.[2004]165 to E.- H. Wang). REFERENCES 1. Lustig B, Behrens J. The Wnt signaling pathway and its role in tumor development. J Cancer Res Clin Oncol 2003; 129: Li F, Chong ZZ, Maiese K. Winding through the WNT pathway during cellular development and demise. Histol Histopathol 2006; 21: Ueta T, Ikeguchi M, Hirooka Y, Kaibara N, Terada T. Betacatenin and cyclin D1 expression in human hepatocellular carcinoma. Oncol Rep 2002; 9: Gerstein AV, Almeida TA, Zhao G, et al. APC/CTNNB1 (beta-catenin) pathway alterations in human prostate cancers. Genes Chromosomes Cancer 2002; 34: Cheng XX, Sun Y, Chen XY, Zhang KL, Kong QY, Liu J, Li H. Frequent translocalization of beta-catenin in gastric cancers and its relevance to tumor progression. Oncol Rep 2004; 11: Xu HT, Wang L, Lin D, Liu Y, Liu N, Yuan XM, Wang EH. Abnormal beta-catenin and reduced axin expression are associated with poor differentiation and progression in non-small cell lung cancer. Am J Clin Pathol 2006; 125: Luo W, Lin SC. Axin: a master scaffold for multiple signaling pathways. Neurosignals 2004; 13: Salahshor S, Woodgett JR. The links between axin and carcinogenesis. J Clin Pathol 2005; 58:

9 AXIN INHIBITS TCF-4 AND LUNG CANCER DEVELOPMENT 9. Neo SY, Zhang Y, Yaw LP, Li P, Lin SC. Axin-induced apoptosis depends on the extent of its JNK activation and its ability to down-regulate beta-catenin levels. Biochem Biophys Res Commun 2000; 272: Furuhashi M, Yagi K, Yamamoto H, et al. Axin facilitates Smad3 activation in the transforming growth factor beta signaling pathway. Mol Cell Biol 2001; 21: Nakajima M, Fukuchi M, Miyazaki T, Masuda N, Kato H, Kuwano H. Reduced expression of Axin correlates with tumour progression of oesophageal squamous cell carcinoma. Br J Cancer 2003; 88: Rui Y, Xu Z, Lin S, et al. Axin stimulates p53 functions by activation of HIPK2 kinase through multimeric complex formation. EMBO J 2004; 23: Travis WD, Brambilla E, Muller-Hermelink HK, et al. Pathology and genetics of tumors of the lung, pleura, thymus and heart. World Health Organization Classification of Tumors Lyon: IARC Press; Watanabe Y. TNM classification for lung cancer. Ann Thorac Cardiovasc Surg 2003; 9: Li CY, Wang Y, Cui ZS, Wang EH. Expression of T cell factor-4 in non-small-cell lung cancer. Chin Med J (Engl) 2005; 118: Liu CR, Ma CS, Ning JY, You JF, Liao SL, Zheng J. Differential thymosin beta 10 expression levels and actin filament organization in tumor cell lines with different metastatic potential. Chin Med J (Engl) 2004; 117: Fukumoto S, Hsieh CM, Maemura K, et al. Akt participation in the Wnt signaling pathway through Dishevelled. J Biol Chem 2001; 276: Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991; 139: Sparks AB, Morin PJ, Vogelstein B, Kinzler KW. Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. Cancer Res 1998; 58: Park JY, Park WS, Nam SW, et al. Mutations of beta-catenin and AXIN I genes are a late event in human hepatocellular carcinogenesis. Liver Int 2005; 25: Clements WM, Wang J, Sarnaik A, et al. beta-catenin mutation is a frequent cause of Wnt pathway activation in gastric cancer. Cancer Res 2002; 62: Ueda M, Gemmill RM, West J, et al. Mutations of the betaand gamma-catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas. Br J Cancer 2001; 85: Iwai S, Katagiri W, Kong C, Amekawa S, Nakazawa M, Yura Y. Mutations of the APC, beta-catenin, and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma. J Cancer Res Clin Oncol 2005; 131: Su MC, Wang CC, Chen CC, et al. Nuclear translocation of beta-catenin protein but absence of beta-catenin and APC mutation in gastrointestinal carcinoid tumor. Ann Surg Oncol 2006; 13: Zeng G, Germinaro M, Micsenyi A, et al. Aberrant Wnt/betacatenin signaling in pancreatic adenocarcinoma. Neoplasia 2006; 8: Zhou CX, Gao Y. Frequent genetic alterations and reduced expression of the Axin1 gene in oral squamous cell carcinoma: involvement in tumor progression and metastasis. Oncol Rep 2007; 17: Rother K, Johne C, Spiesbach K, et al. Identification of Tcf-4 as a transcriptional target of p53 signalling. Oncogene 2004; 23: Saegusa M, Hashimura M, Kuwata T, Hamano M, Okayasu I. Upregulation of TCF4 expression as a transcriptional target of beta-catenin/p300 complexes during trans-differentiation of endometrial carcinoma cells. Lab Invest 2005; 85: Wielenga VJ, Smits R, Korinek V, et al. Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway. Am J Pathol 1999; 154: Marhaba R, Zoller M. CD44 in cancer progression: adhesion, migration and growth regulation. J Mol Histol 2004; 35: