δ-catenin Promotes Malignant Phenotype Of Non-small Cell Lung Cancer By Non-competitive Binding To E-cadherin With P120ctn In Cytoplasm

IntroductionPrevious studies indicated that p120ctn andβ-catenin, two members of catenin family, were implicated in invasion and metastasis of lung cancer. However, it is not clear that whether 8-catenin, another member of this family, is also correlated with invasion and metastasis of lung cancer. Similar to p120ctn,δ-catenin can not only bind to the juxtamembrane domain (JMD) of E-cadherin, but also bind to Kaiso, a transcriptional repressor, in nucleus. Above data suggestδ-catenin probably play an important role in malignant phenotype of tumors. Burger et al. demonstrated that the transcription ofδ-catenin was up-regulated in prostate cancer, compared with benign prostate hyperplasia; its protein expression was also increased. However, the expression profile ofδ-catenin in lung cancer is not clear; the relationship between its expression and clinicopathological factors in NSCLC is poorly understood; it also needs to define whetherδ-catenin competitively binds to E-cadherin with p120ctn.Initially, the expression ofδ-catenin was limited to brain neurons, and its binding to presenilin 1 was involved in the progression of Alzheimer's disease. Later, it was found thatδ-catenin was critical for the maintenance of synaptic function;δ-catenin was also implicated in transmission of signals to downstream as a scaffold protein. It was reported thatδ-catenin overexpression changed morphology of MDCK cells, including the elaboration of lamellipodia. Moreover,δ-catenin could regulate small GTPases activity, therefore induced dendritic protrusions of cells. Small GTPases are critical mediator of cytoskeletal reorganization, signal transduction, and gene expression pathways; p120ctn also affects invasion and metastasis of lung cancer cells through altering small GTPases activity. So, we speculate that 8-catenin is probably associated with invasion and metastasis of tumors.In present study, we examined the expression of 8-catenin, p120ctn, E-cadherin in 115 cases of NSCLC specimens and analyzed the correlation between their expression and clinicopathological factors. Meanwhile, we explored that whether 8-catenin competitively bound to E-cadherin with p120ctn in lung cancer cells with overexpression (or knockdown) of 8-catenin and p120ctn, and investigated the effect of 8-catenin on small GTPases activity and biological behavior of lung cancer cells.Materials and methods1. Tissue samples and patient data115 cases of NSCLC specimens and 20 cases of normal lung tissue specimens were obtained from 1998 to 2005 following surgical resection at the First Affiliated Hospital of China Medical University. Among the 115 cases,65 cases had complete follow-up records, another 50 cases'paired lymph node metastases lesions were available. All patients had not received radiotherapy or chemotherapy before the operation. Of the patients,61 are male and 54 are female, with a median age of 58 years (from 33 to 80 years). The pTNM staging system of UICC was used to classify specimens as:stageⅠ(n=18),Ⅱ(n=39),Ⅲ(n=56),Ⅳ(n=2). According to the classification of lung cancer by WHO:54 cases of squamous cell carcinoma and 61 cases of adenocarcinoma. In order to verify the results of IHC,30 fresh samples including both lung cancer tissues and corresponding normal lung tissues were obtained for detecting mRNA and protein expression of 8-catenin.2. Immunohistochemical stainingImmunostaining was performed by the Streptavidin-Peroxidase (SP) method. We examined the expression of 8-catenin, p120ctn, E-cadherin in 115 cases of NSCLC specimens and analyzed the correlation between their expression and clinicopathological factors. Moreover, we also detected the expression difference of 8-catenin in 50 cases of paired specimens and the correlation between its expression and prognosis in 65 specimens with follow-up records.3. Cell culture, plasmid construction and transfectionHuman lung adenocarcinoma cell lines A549 and SPC-A-1 (hereafter SPC) were cultured in RPMI 1640 medium, containing 10%fetal calf serum.8-catenin plasmid (pCMV5-FLAG/8-catenin) was transfected into SPC cell line which expresses relative low levels of 8-catenin. A pair of siRNA sequence of higher effectiveness was transfected into A549 cell line which expresses relative high levels of 8-catenin.p120ctn isoforms 1A and 3A cDNA were transfected into A549 cell lines which express moderate levels of p120ctn and SPC cell lines with knock-downing p120ctn stably; By use of our reported p120ctn-siRNA plasmid and methods, we transfected the p120ctn-siRNA plasmid into A549 and SPC cell lines.4. Western Blotting and RT-PCRThe protein and mRNA expression of 8-catenin were examined by WB and RT-PCR in lung cancer tissues and corresponding normal lung tissues.5. Co-immunoprecipitation assayIn order to define whether 8-catenin competitively binds to the same binding site of E-cadherin with p120ctn by co-IP, we over-expressed 8-catenin in SPC cells or knock-downed 8-catenin in A549 cells, also over-expressed or knock-downed p120ctn in SPC and A549 cells. As described by Dai et al., the same amount of total protein was incubated with E-cadherin antibody overnight at 4℃. The immunocomplexes were captured by protein G beads, and then detected by WB.6. Immunofluorescent stainingAfter fixed and blocked, cells were incubated withδ-catenin, p120ctn or E-cadherin antibody overnight at 4℃. The primary antibodies was followed by incubation with secondary antibodies conjugated to rhodamine/fluorescein isothiocyanate (FITC)-labeled. The nuclei were counterstained with Hoechst 33258.7. RhoA, Cdc42 and Racl activity assayIn order to illuminate the mechanism of 8-catenin affecting malignant phenotype of lung cancer cells, we detected the activities of small GTPases after over-expressing or knock-downingδ-catenin by Pull-Down assay and Colorimetry.8. Cell cycle, proliferation and invasive abilityIn order to observe the effect of 8-catenin on biological behavior of lung cancer cells, we examined the changes of cell cycle, proliferation, invasive ability and cytoskeleton after over-expressing or knock-downing 8-catenin, by means of Flow Cytometry, MTT, Matrigel invasive assay and immunofluorescence (F-actin staining).Results1.δ-catenin expression was increased in lung cancer tissues and was associated with poor prognosis of patients8-catenin was expressed weakly in cytoplasm of normal bronchial epithelial cells and submucosal glands (negative expression). In lung cancer tissues, the positive expression of 8-catenin localized in cytoplasm, and its expression rate was 62.61% (72/115), which was significantly higher than that in normal lung tissues (P<0.001). The expression rate of 8-catenin in adenocarcinoma was higher than that in squamous carcinoma (P<0.05); the expression rate of 8-catenin in stages III-IV was also higher than that in stages I-II (P<0.05); the expression rate of 8-catenin in cases with lymph node metastases was higher than that in cases without (P<0.05). In 50 paired specimens, the expression rate of 8-catenin in lymph node metastases lesions was higher than that in corresponding primary tumors (P<0.05). Immunostaining in serial sections showed that, p120ctn and E-cadherin primarily localized in cytoplasm of lung cancer tissues (defined as abnormal expression), compared with primarily membrane staining in normal bronchial epithelial cells. Meanwhile, abnormal expression rate of p120ctn and E-cadherin were higher in tumors with high-stage, poor differentiation, and nodal metastasis (P<0.05).Kaplan-Meier survival curves showed the average survival time of patients with 8-catenin positive expression was shorter than that of negative expression (P=0.019). The mRNA and protein expression of 8-catenin in normal lung tissues was lower than that in paired lung cancer tissues (P<0.05).2. Bothδ-catenin and p120ctn could bind to E-cadherin in cytoplasm of lung cancer cells, but there was no competition between 8-catenin and p120ctnWhen over-expressing (or knock-downing) 8-catenin, the amount of 8-catenin binding to E-cadherin were increased (or decreased), but the amount of p120ctn binding to E-cadherin were unchanged. Similarly, the amount of p120ctn binding to E-cadherin were increased (or decreased) when over-expressing (or knock-downing) p120ctn, while the amount ofδ-catenin binding to E-cadherin were unchanged. In addition, immunofluorescent staining showed that E-cadherin,8-catenin, and p120ctn protein primarily localized in cytoplasm of lung cancer cells.3.δ-catenin overexpression down-regulated RhoA activity, while up-regulated Cdc42 and Racl activityImmunoblotting showed total Cdc42 and Racl protein levels were unchanged after over-expressing 8-catenin,, but the activity of Cdc42 and Racl increased significantly (P<0.05), and RhoA activity decreased significantly (P<0.05). On the contrary, total Cdc42 and Racl protein levels were also unchanged after knock-downing endogenous 8-catenin, but the activity of Cdc42 and Racl decreased significantly (P<0.05), and RhoA activity increased significantly(P<0.05).4.δ-catenin overexpression changed cell cycle, promoted proliferation and invasion of lung cancer cells FCM showed the average percentage of cells in G1 stage were decreased after over-expressingδ-catenin (P<0.05); while the average percentage of cells in S and G2/M stage were increased (P<0.05). On the contrary, the average percentage of cells in G1 stage were increased after knock-downing endogenousδ-catenin (P<0.05); while the average percentage of cells in S and G2/M stage were decreased (P<0.05).MTT showed the proliferation ability of lung cancer cells was obviously increased after over-expressingδ-catenin (all P<0.05). While the proliferation ability of lung cancer cells was obviously decreased after knock-downing endogenousδ-catenin (all P<0.05).Immunofluorescence and invasive assay showed obvious filopodia appeared on the surface of tumor cells after over-expressingδ-catenin, and the average numbers of invasive cells were increased (P<0.01); while actin filaments rearranged evenly in cytoplasm after knock-downingδ-catenin, and the average numbers of invasive cells were decreased (P<0.01).Conclusion1.δ-catenin expression was increased in lung cancer tissues and was associated with poor prognosis of patients.2. There was no competition betweenδ-catenin and p120ctn for binding to E-cadherin in cytoplasm of lung cancer cells.3.δ-catenin could promote proliferation and invasion of lung cancer cells through down-regulating RhoA activity, up-regulating Cdc42 and Racl activity, and changing cell cycle.