Connective tissue growth factor enhances the ... - Wiley Online Library

Connective tissue growth factor enhances the migration of gastric cancer through downregulation of E-cadherin via the NF-jB pathway Zhengfa Mao,1,5 Xiaoyan Ma,2,5 Yefei Rong,1,5 Lei Cui,1 Xuqing Wang,3 Wenchuan Wu,1 Jianxin Zhang3,4 and Dayong Jin1,4 1Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai; 2College of Medicine, Jiangsu University, and 3Department of General Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu province, China

(Received June 21, 2010 ⁄ Revised August 29, 2010 ⁄ Accepted September 2, 2010 ⁄ Accepted manuscript online September 11, 2010 ⁄ Article first published online October 6, 2010)

Local invasion and distant metastasis are difficult problems for surgical intervention and treatment in gastric cancer. Connective tissue growth factor (CTGF/CCN2) was considered to have an important role in this process. In this study, we demonstrated that expression of CTGF was significantly upregulated in clinical tissue samples of gastric carcinoma (GC) samples. Forced expression of CTGF in AGS GC cells promoted their migration in culture and significantly increased tumor metastasis in nude mice, whereas RNA interference-mediated knockdown of CTGF in GC cells significantly inhibited cell migration in vitro. We disclose that CTGF downregulated the expression of E-cadherin through activation of the nuclear factor-jappa B (NF-jB) pathway. The effects of CTGF in GC cells were abolished by dominant negative IjappaB. Collectively, these data reported here demonstrate CTGF could modulate the NF-jappaB pathway and perhaps be a promising therapeutic target for gastric cancer invasion and metastasis. (Cancer Sci 2011; 102: 104–110)

G

astric cancer (GC) is one of the most common malignancies in the world, ranking as the second most common cause of death worldwide.(1) The only way for cure is surgery combined with chemotherapy. However, approximately 65% of patients with gastric carcinoma have regional or distant metastases at the time of diagnosis, limiting the chance for complete excision of these tumors and thus making treatment extremely difficult. The 5-year survival rate remains poor for this type of cancer,(2,3) especially in patients with metastatic and advanced stages, ranging 5–15%.(4) Although we have made great progress in perioperation, recurrence and metastasis of disease are almost uniform and contribute to the main cause of death. Failure of traditional therapeutic strategy to treat this disease might be due to our limited understanding of the underlying mechanism facilitating the migration and invasion of gastric cancer cells. Connective tissue growth factor (CTGF) is a cysteine-rich protein. It belongs to the CCN family, which consists of six members: Cyr61 (cysteine-rich protein 61, CCN1), CTGF ⁄ CCN2, Nov (nephroblastoma overexpressed gene, CCN3), WISP-1 (Wnt-1-induced secreted protein 1, CCN4), WISP-2 (CCN5) and WISP-3 (CCN6).(5,6) Connective tissue growth factor has versatile functions associated with the modular domain structure of itself. It has four structural domains as follow: NH2-terminal signal peptide and four conserved domains with sequence similarities to insulin-like growth factorbinding proteins; von Willebrand type C factor; thrombospondin 1; and a cysteine knot characteristic of other growth factors, including platelet-derived growth factor, nerve growth factor and transforming growth factor (TGF)-b,(7) each of which is thought to have a distinct biological function.(8) Cancer Sci | January 2011 | vol. 102 | no. 1 | 104–110

Although the specific receptor of CTGF has not yet been identified, CTGF is reported to interact with several growth factor signaling pathways, including TGF-b, bone morphogenetic protein (BMP) and Wnt.(9) It can also bind to integrins on the cell surface. It is thought to be involved in extracellular matrix production, desmoplasia, tumor cell proliferation, adhesion, migration, angiogenesis and metastasis.(5,10) Increasing evidence suggests that CTGF plays a role in tumorigenesis. Its overexpression is observed in prostate cancers,(11) gliomas,(12) breast cancers(13) and esophageal adenocarcinoma.(14) Conversely, downregulation of CTGF was found in lung cancer(15) and colon cancer,(16) in which forced expression of CTGF inhibits invasion and metastasis of cancer cells both in vitro and in vivo. Also, CCN2 expression levels correlated with increased survival in chondrosarcoma patients.(17) It is proposed that this disparity in expression in different types of tumors implies that regulation of CTGF expression might be cell-type specific and CTGF might have versatile roles in different cellular contexts. In this study, we demonstrated that the expression of CTGF was significantly increased in GC. Furthermore, we demonstrated that the CTGF activated the NF-jB signaling pathway in GC cells, which enhanced the migration and thus facilitated the metastasis of cancer cells. Our study strongly highlights the significance of CTGF in the migration and invasion of GC, and therefore provides a potential drug target in gastric cancer therapy. Material and Methods Cell culture, cDNA and tissue samples. The GC cell line AGS was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; PAA Laboratories, Pasching, Austria), 10 units ⁄ mL penicillin-G and 10 mg ⁄ mL streptomycin. 293T cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 10 units ⁄ mL penicillin-G and 10 mg ⁄ mL streptomycin. All cells were incubated at 37C in 5% CO2 humidified air. Seventy-three pairs of primary GC and their corresponding adjacent normal tissues, which were at least 5 cm away from the cancer, were obtained from GC patients treated at Zhongshan Hospital of Fudan University (Shanghai, China) from 2007 to 2008. The entire specimen was stored in liquid nitrogen. The GC cDNA was prepared from the samples collected above. Whole cDNA samples were stored at )80C until analysis. Our work was approved by the Institutional Review Board of 4To whom correspondence should be addressed. E-mail: [email protected]; [email protected] 5These authors equally contributed to this work.

doi: 10.1111/j.1349-7006.2010.01746.x ª 2010 Japanese Cancer Association

Table 1. Association between CTGF expression and clinicopathological characteristics of patients with GC (n = 73) Characteristic Sex Male Female Age (years) £62 >62 Size (cm) 0.05) (Fig. 2C). Furthermore, real-time PCR and western analysis showed that cell adhesion-related protein E-cadherin was downregulated in the two stable clones compared with the AGS ⁄ V control cells (Fig. 2D,E). These results indicate that the forced expression of CTGF in GC cells enhanced their cell migration. Knockdown of CTGF inhibited cell migration. To further determine the effect of CTGF on cell migration, we used RNAi mediated knockdown lentiviral vector to decrease the basal level of CTGF in MKN45 cells. After being infected with lentiviral vector for 8 h in condition media, the cells were cultured in DMEM containing 10% FBS for 72 h and then harvested. Cell sorting was performed with the FACS and the GFP-positive cells were collected for further study. Western blot results showed that two sequences markedly decreased the expression of CTGF compared with the control sequence (Fig. 3A). The RNAi dramatically inhibited cell migration of these cells in liquid culture (P < 0.05) (Fig. 3B). To investigate whether E-cadherin also contributes to the decreased cell migration induced by silencing of CTGF, we detected the expression of E-cadherin in MKN45 cells. As expected, E-cadherin was markedly increased after knocking 106

Fig. 1. Levels of connective tissue growth factor (CTGF) are increased in gastric carcinoma (GC) clinical samples. (A) Quantitative real-time PCR results of the relative expression level of CTGF in 42 pairs of GC and normal gastric tissue samples. All of the reactions were performed in triplicate and results represent the mean ± SD (P < 0.05). The expression of CTGF is normalized to beta-actin. (B) Expression of CTGF and p65 protein in four randomly selected paired GC samples was analyzed using western blot. N, normal; T, primary GC. (C) immunohistochemistry (IHC) results of CTGF expression in paired GC tissue samples.

down CTGF (Fig. 3C). These results suggest that downregulation of CTGF in GC cells inhibited the migration of MKN45 cells. Involvement of NF-kB in CTGF-induced cell migration via Ecadherin suppression. NF-jB, a well-known mediator in cell

migration, has been implicated in cancer carcinogenesis.(22,23) We assumed that CTGF could enhance cell migration by activating NF-jB. We first examined the activation of NF-jB by using the luciferase reporter assay. These reporter constructs were transfected into either AGS or 293T cells together with either the CTGF expression construct or pcDNA3.1, respectively, and luciferase activity was determined. In both AGS cells and 293T cells, the cellular transcriptional activity of NF-jB was significantly increased on CTGF overexpression (Fig. 4A). In most cell types, NF-jB is found in the cytoplasm as an inactive dimer bound to one of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IjB) proteins that mask its nuclear localization signal. However, as assessed by immunofluorescence, marked nuclear localization of p65 was observed in AGS ⁄ CTGF cells compared with control cells (Fig. 4B). To investigate whether overexpression of CTGF could alter NF-jB signaling, western blot was performed to examine the expression of nuclear p65. We found that the expression levels of nuclear p65 were elevated significantly in AGS ⁄ CTGF stables cells compared with AGS ⁄ V control cells (Fig. 4C). Meanwhile, expression of p65 coincided with the expression of CTGF in the GC samples (Fig. 1B). To further examine these results, AGS ⁄ CTGF cells were transfected transiently with a dominant negative IjappaB (DN IjappaB). Real-time PCR and western analysis showed that the


(A)

(A)

(B) (B)

(C)

(C)

(D)

(E)

Fig. 2. Effects of connective tissue growth factor (CTGF) on cell migration. (A) Expression of CTGF in AGS ⁄ CTGF stable cell lines was evaluated by western blot. Equal loading was ascertained using b-actin as an internal control. (B) AGS cell migration detected with Boyden chamber assay. Each experiment was performed in triplicate, and results represent the mean ± SD (P < 0.05). (C) Cell growth rates of AGS ⁄ V, AGS ⁄ CTGF3# and -5# in 1% FBS were measured using an MTT assay. Results are representative of three independent experiments (P > 0.05). (D) The level of E-cadherin in AGS/pcDNA3.1, AGS/CTGF3# and AGS/ CTGF5# transfected cells was examined by western blot. (E) Expression of the E-cadherin mRNA level was examined by RT-PCR.

upregulation of nuclear p65 and downregulation of E-cadherin in AGS ⁄ CTGF cells was abolished by DN -IjappaB (Fig. 4D–F). Expression and subcellular localization of E-cadherin, NF-jB p65 subunit with CTGF expression in the sample were compared. There was more nucleus p65 localization and less membrane E-cadherin with CTGF expression in diffuse-type adenocarcinomas than in intestinal-type adenocarcinomas (P < 0.05) (Table 2). These results suggested that CTGF stimulated NF-jB signaling causing downregulation of the E-cadherin. Mao et al.

Fig. 3. Inhibition of MKN45 cell migration by connective tissue growth factor (CTGF) knockdown. MKN45 cells were infected with either CTGF siRNA or CTGF siRNA control (con) for 8 h in condition media, cultured in DMEM containing 10% FBS for 72 h, then harvested for further study. Cell sorting was performed with the FACS and the GFP-positive cells were collected for further study. (A) Western blot analysis to show RNAi-mediated knockdown of CTGF. (B) Migration assay detected with Boyden chamber showed the effect of CTGF gene silencing on cell migration of MKN45 cells. The results are expressed as mean number (±SD) of migrated cells within 6 h of three separate experiments (P < 0.05). (C) Knockdown of CTGF enhanced the expression of E-cadherin in MKN45.

It has been demonstrated that CTGF could affect cell function through ILK ⁄ PI3K ⁄ AKT signaling,(24,25) so we determined whether overexpression of CTGF in AGS ⁄ CTGF cells had an effect on this pathway. We measured the level of ILK, Akt and activated Akt by western blot analysis and ILK activity by ILK kinase assay. As shown in Figure 4G, ILK activity and phosphorylation Akt in both AGS ⁄ CTGF sublines were increased dramatically compared with those in the AGS ⁄ V control cells, while protein levels of ILK and total Akt in AGS ⁄ CTGF cells had no significant difference. Furthermore, downregulated expression of ILK with siRNA dramatically decreased the level of activated Akt in CTGF-expressing cells (Fig. 4H). It is reported that NFjB can be regulated by AKT signaling pathway, we asked if AKT pathway is also involved in CTGF-dependent regulation of NF-jB. Indeed, treatment of Cancer Sci | January 2011 | vol. 102 | no. 1 | 107 ª 2010 Japanese Cancer Association

Fig. 4. Connective tissue growth factor (CTGF) stimulated NF-j B signaling pathway. (A) NF-jB reporter activity in AGS and 293T cells. Cells were co-transfected with NF-jB reporter plasmid as well as either pcDNA3.1 ⁄ CTGF or pcDNA3.1. Reporter activities were normalized to internal control Renilla. Data represent results from triplicate dishes in two separate experiments (P < 0.05). (B) NF-jB nuclear translocation enhanced by CTGF was evaluated by immunofluorescence of p65. (C) The expression of nuclear p65 in AGS ⁄ V and AGS ⁄ CTGF was examined by specific antibodies and nucleoporin 62 served as a loading control. Dominant negative IjappaB (DN IjappaB) blocked downregulation of E-cadherin in AGS ⁄ CTGF cells at the mRNA level (D) and protein level (E) and inhibited the translocation of p65 to the nucleus (F). (G) ILK kinase activity and active Akt were examined in AGS ⁄ V and AGS ⁄ CTGF stable cell lines. (H) Western blot assay demonstrated that knockdown of ILK by siRNA abolished the up-regulation of AKT phosphorylation induced by CTGF overexpression. (I) AGS vector control and CTGF overexpressing cells were treated as follow: no treat (control), LY294002 for 24 h, or transfected with dominant negative Akt-expressing constructs, and then the NF-jB activity was tested by dual luciferase assay.

(A)

(B)

Table 2. Subcellular localization of p65 and E-cadherin with CTGF in the histological type in patients with GC (n = 73) Intestinal CTGF

(C)

(D) p65 Nucleus Cytoplasm Total E-cadherin Normal Aberrant Total

(E)

(G)

(I)

108

(F)

(H)

Diffuse Total

High

Low

5 7 12

9 13 22

5 7 12

18 4 22

Total

CTGF High

Low

14 20 34

21* 6 27

7 5 12

28 11 39

23 11 34

2 25* 27

5 7 12

7 32 39

P < 0.05. CTGF, connective tissue growth factor; GC, gastric carcinoma. —, incidence of nucleus p65 and abberrant E-cadherin with high CTGF in intestinal versus diffuse GC.

AKT inhibitor LY294002, or overexpression of dominantnegative AKT, abolished CTGF-induced activation of NF-jB, respectively. CTGF-induced activation of NF-jB was blocked by LY294002, dominant-negative Akt, respectively (Fig. 4I). These results indicate that the ILK ⁄ Akt pathway is involved in the modulation of NF-jB activity stimulated by CTGF. CTGF promotes metastasis of AGS cells in vivo. To examine the role of CTGF in metastasis in vivo, AGS ⁄ V cells and AGS ⁄ CTGF cells were injected into the heart of 6-week-old nude mice and monitored by in vivo luminescence imaging every week. Once the same amount of cancer cells were injected, luminescence intensity could be detected immediately in the two groups after the luciferin was injected intraperitoneally and there was no difference. Four weeks post-injection, the luminescence intensity detected in the AGS ⁄ CTGF tumor group was higher than the luminescence in the AGS tumor group. In 6 weeks, the luminescence intensity in AGS ⁄ CTGF was more significantly higher than in the control. This suggested that CTGF dramatically enhanced the capability of AGS cells to develop tumors in distant organs (Fig. 5A). To exclude the possibility that the signal examined might be artificial, after the mice were killed we found the metastasis lesions according to the previously obtained data (Fig. 5B). Proteins from the metastasis were extracted and the expression of CTGF, p65 and E-cadherin was examined. As shown in Figure 5C, the level of CTGF and p65 in the AGS ⁄ CTGF metastasis was higher than that in the AGS ⁄ V metastasis, while E-cadherin was lower (Fig. 5C). These data thus suggest that CTGF upregulation also enhanced the ability of metastasis of AGS cells in vivo.


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(B)

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Fig. 5. Overexpression of connective tissue growth factor (CTGF) promotes gastric cell growth in distant organs. (A) AGS vector control and CTGF overexpressing cells, each 2 · 105, both labeled with luciferase, were injected into the left ventricle of nude mice, and metastasis was measured by luciferase activity through intraperitoneal injection of the luciferin every week (five mice for each group). The metastasis events were detected weekly by inspecting the intensity of luminescence with imaging via IVIS systems, which is demonstrated as the color depth in the image. (B) The metastasis lesion was picked out to confirm that the bioluminescence signaling was not artificial. (C) Metastasis lesions were taken out after the mice were killed. CTGF, p65 and E-cadherin expression levels were detected by western blot.

Discussion

In the present study, our results showed that CTGF was highly expressed in GC tissues compared with matched normal gastric tissue from patients. We subsequently evaluated the function of CTGF in AGS, a GC cell line. Forced expression of CTGF in AGS promoted cell migration in vitro and stimulated tumor metastasis in nude mice, whereas knockdown of CTGF in AGS inhibited cell migration in vitro. These results suggest that CTGF might be implicated in the metastasis of GC. We also found that expression of E-cadherin decreased with overexpression of CTGF. E-cadherin is one of the important elements involved in adhesion, which explains in part how CTGF enhances migration of the GC cells. The involvement of CTGF in migration has been mentioned in several previous investigations. Early studies demonstrated that overexpression of CTGF promoted migration in chondrosarcoma cancer(26) and stimulated drug resistance and metastasis in glioblastoma.(27) The level of CTGF showed a significant correlation with tumor stage and survival in breast cancers.(28) Mao et al.

However, some other studies found that CTGF inhibited tumor metastasis and invasion in ovarian carcinoma(29) and lung cancers.(24) Altogether, the data suggest that the role of CTGF in tumor invasion is dependent on the tumor type and microenvironment. An important finding of the present study is the characterization of CTGF as a metastatic enhancer by stimulating the NF-jB signaling pathway then downregulated expression of E-cadherin. NF-jB is one member of the NF-jB ⁄ Rel family, and p65 is the most common subunit of NF-jB translocated into the nuclei, which stimulates the transcription of a set of target genes involved in cell migration.(30) The nuclear localization of p65 is controlled by the bind state regulated by IjB, and the latter is inhibited by IKK through direct phosphorylation. Previous studies have documented that CTGF binds to various integrins such as a6b1(31) and a5b1.(32) Moreover, Cyr61, another CCN member, has been shown to bind with integrins stimulating the activity of integrin-linked kinase and AKT;(33) the latter is considered to directly activate the NF-jB pathway. Because the p65 signaling pathway has been reported to be Cancer Sci | January 2011 | vol. 102 | no. 1 | 109 ª 2010 Japanese Cancer Association

involved in the development of human cancers,(34) and dysfunction of NF-kappaB signaling in cancer of the stomach has been recently reported,(35) regulation of the p65 pathway by CTGF might further explain the mechanism by which CTGF stimulates the migration of GC cells. Besides CTGF, several other CCN molecules function through the p65 pathway in cancers. For example, Cyr61 promotes breast cancer cell growth through activating NF-jB signaling(36). Considering the similarity of the two proteins, we speculate that they may function in similar signaling pathways in the development and progression of tumors. In summary, our results suggest that CTGF can stimulate NF-jB signaling and thus contribute to GC malignancy. These findings recapitulated the involvement of the NF-jB pathway in cancer metastasis. Given the diverse function of CTGF, the NF-jB pathway seems to be involved in more important physiological and pathophysiological processes than earlier conceived. Taken together, our work lays the foundation for novel exploration of the mechanisms and functions of CTGF and the NF-jB signaling pathway in the metastasis and invasion of GC.

Acknowledgments

References

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This work was supported by The Foundation for Young Scientists of Zhongshan Hospital, Fudan University (Grant numbers: 2008-S-61). The authors thank Dr Yuezhen Deng, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for expert technical assistance and Dr YeHe, Department of Neuroscience, Mount Sinai School of Medicine for helpful discussions, suggestions and critical reading of this manuscript.

Disclosure Statement The authors have no conflict of interest.

Abbreviations CTGF GC TNM

connective tissue growth factor gastric carcinoma tumor–node–metastasis
