Coordinate Expression Of δ-catenin And Small GTPase Is Significantly Associated With Malignant Phenotype Of Human Lung Cancer

Introductionδ-catenin, as an important member of p120 subfamily, has similar structure and function of p120. It has ten Arm repetitive sequences, and has the conjunct JMD (juxtamembrane domain) binding sites with p120 on the E-cadherin of epicyte. 8-catenin can adjust the intercellular adhesion by directly combining with E-cadherin(E-cad) to form E-cadherin/catenin complex. Similarly,δ-catenin can also go into the nucleus and combine with kaiso.It is reported that in neurons,δ-catenin is involved in signal transduction, also influence the growth of the cytoskeleton by regulating SmallGTPase (such as RhoA, Cdc42, Rac1) activity. SmallGTPase is the small GTP-binding protein in the Ras superfamily.δ-catenin has activated form (combined with GTP) and inactivated form (combined with GDP) in metabolism. Both of the two forms seem to be the molecular switches which can dynamically adjust the assembly of cytoskeleton, thereby causing a variety of reactions, such as Cell morphology, chemotaxis and cell motility, etc. Therefore, we infer that in lung cancer tissue,δ-catenin and SmallGTPase may be connected with each other.In this study, we tested the expression ofδ-catenin and SmallGTPase in 135 NSCLC samples and probe into the relationship between their expression and clinical pathology. At the same time, in lung cancer cell lines,δ-catenin and SmallGTPase over express or interfereδ-catenin and lung cancer cell thus influence the invasion and metastasis of lung cancer. Materials and methods1. Tissue samples and patient data135 cases with follow-up information of intrinsic Squamous cell carcinoma, adenocarcinoma (from 1998 to 2005, Surgical resection specimens of paraffin blocks, The First Hospital of China Medical University). There are 71 males,64 females, and mean age is 58 years old. According to WHO in 2004 of lung cancer histological classification:squamous cell carcinoma 61 cases; adenocarcinoma 74 cases. Highly differentiated 32 cases, Moderately differentiated 64 cases, poorly differentiated 39 cases. In accordance with the International Union Against Cancer (UICC) in 2002 revised tumor pTNM staging standard:22 patients with stageⅠ,Ⅱof 46 cases,Ⅲof 59 cases,Ⅳof 8 cases.70 of these cases are conducted postoperative follow-up. Survival time is calculated from the date of surgery to recurrence or metastasis date (or last follow-up date) as the date of death.Moreover, for the extraction of protein and RNA, the other 30 samples of fresh lung carcinoma and adjacent normal lung tissue are obtained.2. Immunohistochemical staining135 cases of NSCLC samples are dealt with the method of Streptavidin-Peroxidase (SP) immunohistochemical to detect the expression ofδ-catenin and SmallGTPase and the relationship with clinicopathological factors, and to detect the relationship between the expression ofδ-catenin and SmallGTPase and prognosis in the follow-up samples of 70 patients. Observeδ-catenin expression in tumor tissue and count 400 tumor cells and calculate the percentage of positively stained cells.δ-catenin Rating criteria:immunostaining intensity (0=negative, 1=weak, 2=moderate,3=strong); immunostaining positive tumor cells (0=absent,1=1～25%, 2=26～50%,3=51～75%,4=≥76%). The final score for each specimen will be obtained by multiplying the two ratings. Negative expression＜2; positive expression≥2. RhoA,Cdc42 and Rac1 Cytoplasm expression (-)～(+) are defined as normal expression, (++)～(+++) are defined as overexpression. The circumstance in whichδ-catenin positive expression and SmallGTPase overexpression occur simultaneously is called coordinate expression.3. Cell culture, plasmid construction and transfectionHuman lung adenocarcinoma cell line A549,SPC-A-1 (Manassas, VA, USA) are cultured with 10% inactivated fetal bovine serum,2.3g/L NaHCO3, 100U/ml streptomycin in RPMI 1640 Green or high glucose DMEM (GBICO Inc., Los Angeles, CA, USA) culture medium.(1)δ-catenin cDNA Plasmid Construction and Transfection:Containing human-derived fullδ-catenin cDNA plasmid (pCMV5-FLAG/δ-catenin), using liposome Lipofectamine 2000 (Invitrogen, USA) to leadδ-catenin cDNA Plasmid into the lowerδ-catenin expression in SPC cells, with empty vector as negative control.(2)δ-catenin siRNA Plasmid Construction and Transfection:the previously designedδ-catenin siRNA (5'-CUACGUUGACUUCUACUCAUU-3', 5'-UGAGUAGAAGUCAACGUAGUU-3'), using liposome Lipofectamine 2000 to leadδ-catenin siRNA Plasmid intoδ-catenin expression in high A549 cell lines, while using non-specific siRNA as a negative control.4. Western Blotting and RT-PCRUsing Western Blotting and RT-PCR to detectδ-catenin, SmallGTPase and mRNA expression in lung cancer and the adjacent normal lung tissue.5. RhoA, Cdc42 and Racl activity assayIn lung cancer cell lines, after overexpressing or knocking-outδ-catenin, detecting small GTPases (RhoA, Cdc42, Racl) activity changes by Pull-Down and colorimetry.6. Cell invasion of determinationAfter overexpressing or knocking-outδ-catenin, using Matrigel invasion to test cell invasion. Results1. In lung cancer, positive expression ofδ-catenin and the over-expression of RhoA, Cdc42 and Racl has a good consistency and relevanceIn 135 non-small cell lung cancer cases,86 cases of positive expression,49 cases inδ-catenin expression is negative, the positive rate is 63.70%(86/135); RhoA, Cdc42 and Racl expression in lung cancer is stronger, and the overexpression rate was 63.70% (86/135),67.41%(91/135) and 65.19%(88/135). And their over-expression and positive expression ofδ-catenin has a good correlation (p＜0.001; p＜0.001; p＜0.001). After the analysis of the relationship of these four indicators of positive expression or over-expression (synergistic expression) and clinical pathological factors, the author has found that:Coordinated expression of lung adenocarcinoma was 52.70%(39/74), significantly higher than in squamous cell carcinoma 34.43%(21/61)(P＜0.05);Ⅲ-Ⅳexpression rate of 58.21% synergy (39/67), significantly higher than theⅠ-Ⅱperiod, 30.88%(21/68)(P＜0.05); expression is 52.75%(48/91) in cases of collaborative with LN metastasis, higher than the 27.27%(12/44) without LN metastasis(P＜0.05). But the cooperative expression shows no correlation with age, sex and degree of differentiation(P＞0.05).2. The correlation of positive expression ofδ-catenin and RhoA, Cdc42 and Racl in the overexpression and poor prognosis of patientswhenδ-catenin expression and RhoA, Cdc42 and Rac1 overexpression, the average survival time of patients and the 5-year survival rate is significantly lower than the average survival time and 5-year survival rate (P＜0.05, P＜0.05, P＜0.05, P＜0.05) when they are expressed in normal patients. Therefore,δ-catenin positive expression and RhoA, Cdc42 and Rac1 in the overexpression and poor prognosis of patients are significantly correlated.3. Compared with normal lung tissue, the expression ofδ-catenin and SmallGTP enzyme lung tissue has significantly increasedRT-PCR results shows:in the adjacent normal lung tissue shows normal expression ofδ-catenin RhoA, Cdc42 and Rac1, in addition, in lung cancer tissue their expression has significantly increased.Western Blot results also confirms what we have observed in RT-PCR results. In lung cancer tissueδ-catenin expression has significantly increased, while RhoA, Cdc42 and Rac1 expression has significantly increased as well.4. Overexpression ofδ-catenin makes RhoA activity decrease while at the same time increases the activity of Cdc42 and Rac1; while theδ-catenin silence has the opposite influenceRhoA, Cdc42, Rac1 activity changes are detected after the detection of overexpression andδ-catenin silence. It is shown that after the SPC cells overexpressingδ-catenin, the total level of Cdc42 and Rac1 protein has no change, but the pull-down assay showed Cdc42/Rac1 activity has significantly increased, and the G-LISA analysis showed that RhoA activity has significantly decreased (P＜0.001). After silence ofδ-catenin in the A549 cell line, the total Cdc42 and Rac1 protein dose not change, but GTP-Cdc42/Rac1 has significantly reduced, RhoA activity increased (P＜0.05)5. Overexpression ofδ-catenin accelerate lung cancer cell invasion, while the reduction ofδ-catenin can inhibit invasionChange of invasion ability in lung cancer cells after overexpression or silencingδ-catenin is detected by serum stimulated matrigel invasion assay. After the overexpression ofδ-catenin, the average number of invasive cells is significantly higher than control group; after the silence ofδ-catenin, the average number of invasive cells is significantly lower than control group. These data reveal the upregulation ofδ-catenin can promote cancer cell invasion; the down regulation of endogenousδ-catenin significantly inhabits invasive ability of tumor cells.Conclusionδ-catenin positive expression in lung cancer and RhoA, Cdc42 and Rac1 expression have obvious relevance and consistency, besides this coordinated expression and lung cancer staging, differentiation, and lymph node metastasis and poor prognosis in patients have significant correlation.When theδ-catenin increase, changing smallGTPase activity can promote tumor invasion and metastasis. However, in lung cancer the specific regulatory mechanism between them has yet to be confirmed.