| BackgroundEsophageal cancer is the eighth most common cancer and the sixth leading cause of cancer death worldwide. The incidence of esophageal cancer has remarkable regional differences. In Asian countries, the predominant histological type is squamous cell carcinoma (ESCC) while in western countries adenocarcinoma is the predominant type. Although great advances in diagnostic methods and treatments have been achieved, the mortality of esophageal cancer remains high and the five-year survival is less than 14%. The reasons of poor prognosis include diagnosed at advanced stage, high recurrence rate after therapy, especially lack of liable markers to predict response to therapy. So it is urgent to identify effective and independent markers for clinical prognostic prediction.RACK1, termed for Receptor for Activated C Kinase 1, is a 36-kDa scaffolding protein with seven conserved WD-40 motifs. The conserved seven blade propeller structure allows RACK1 to function as a signaling hub through binding to diverse signaling and structural proteins, RACK1 can interact with multiple signal pathways and then affect cell motility, cell growth, and so on. Currently more and more reports focus on correlation between RACK1 and malignant tumors. Many studies have shown that RACK1 was associated with tumor progression in multiple malignant tumors, such as hepatocellular carcinoma, breast cancer, prostate cancer, and so on. RACK1 was found to be associated with tumor stage, tumor size and lymph node metastasis. Previous studies have demonstrated that RACK1 function as an independent prognostic factor for patients with malignant tumors including oral squamous cell carcinoma, breast carcinoma, and pulmonary adenocarcinoma. However, there is limited information regarding the predictive significance of RACK 1 in ESCC.Objectives1. To evaluate the expression of RACK1 in human ESCC tissue;2. To investigate the correlation of RACK1 expression with long-term prognosis in ESCC patients.Methods1. Patients and tissue samplesWe enrolled 100 patients who underwent esophagectomy at Qilu Hospital of Shandong University in Jinan (China) from January 2007 to December 2007. Patients who received no preoperative adjunctive therapy and underwent an esophagectomy were included. All patients included in our study had histologic documentation of EAC or ESCC histology and computed tomography scans of the chest and abdomen. Stored information regarding gender, age, tumor location, tumor size(length and diameter), TNM stage, histological grade and treatment regimen. Exclusion criteria:patients with non-squamous cell subtype; patients without histologic documentation; patients who received preoperative adjunctive therapy; patients who died within 30 days after the surgery; uncooperative patients who refused to answer questions. The selected cases involved 79 men and 21 women, aged 42 to 78 years (median,60 years). The data were procured from surgical pathology files maintained in the Pathology Department. All the specimens had been routinely formalin-fixed, paraffin-embedded, and serially sectioned at 5μm in thickness.2. Follow-upAfter esophagectomy, these patients were closely followed up until January 31 2013.3. Immunohistochemistry staining and evaluation of immunostaining intensityAfter immunohistochemical staining, immunohistochemical signals of all the specimens were scored by two independent observers. Five spots of each stained section were calculated and the results were averaged. Two-way scoring system was used for the analysis of RACK1 results in the present study. The final score was achieved by multiplying the staining intensity and the percentage of staining cells. Staining intensity was scored as follows:0, no staining; 1, weak; 2, moderate; 3, strong. Percentage of staining cells was scored as follows:0,0%; 1, 1%-24%; 2, 25%-49%; 3,50%-74%; 4,75%-100%. The samples were judged as follows: negative (final score,≤4) and positive (final score,>4).Results1. RACKl expression in ESCC patientsWe observed RACK1 immunoreactivity was readily detected in the cytoplasm and occasionally in the nucleus. We divided the enrolled patients into positive and negative groups by RACK1 immunoreactivity. We systematically analyzed RACK1 expression in 100 ESCC, among which 59 were RACK1 positive expressed and 41 was negative. The male to female ratio in RACK1 positive group and negative group was 47:12,32:9 respectively. The mean age in positive and negative group was 61.9 (ranged 42-78),60.4 (ranged 45-74) respectively. The expression was not significantly correlated with patients’gender (P=0.846) or age (P=0.416). We found no significant differences in distribution according to tumor location, N stage, TNM stage, histological grade and treatment regimen between the two groups. However, the expression levels of RACK1 were significantly associated with the tumor length(P=0.012), diameter< 3cm (P=0.047), T stage (P=0.032), and lymph node metastasis (P= 0.038), respectively.2. Association of RACK1 expression with poor prognosis in ESCC patientsKaplan-Meier survival analysis revealed that OS rates were 27.1% in the RACK1 positive group and 56.1% in the RACK1 negative group. The DFS rates were 37.3% in the RACK1 positive group and 61.0% in the RACK1 negative group. Both overall and disease-free survival in patients positive for RACK1 expression were significantly shorter than those in patients who were negative (OS, P=0.002; DFS, P=0.001). Univariate and multivariate analyses showed RACK1 expression was an independent predictor for OS (P=0.030) and DFS (P=0.027).Conclusions1. RACK1 was differential expressed in ESCC patients.2. Both OS and DFS in RACK1 positive group were lower than those in negative group, and patients with RACK1 overexpressed showed poorer prognosis.BackgroundThere are many reasons for poor prognosis of esophageal cancer, including imperfect screening and diagnosed methods. Besides, faster tumor progression is one of the important factors. High invasive and metastatic characteristic of esophageal cells lead to tumor progression. It is urgent to look for new target spot by detecting the mechanism of invasion and metastasis. In our country esophageal squamous cell carcinoma is the predominant type, and we focus on it in the present study.As one kind of adapter proteins, RACK1 can recruit various factors or proteins, and regulate signaling pathway, resulting in cell cycle redistribution and cell apoptosis. RACK1 can control tumor progression in this way. Previous studies found that RACK1 promoted the proliferation of breast cancer cells by activating PI3K/Akt signaling pathway. They also found that it activated RhoA/Rho kinase pathway by interacting with RhoA and promoted breast cancer cells migration. Besides, RACK1 showed effect on cell morphology by Ki-Ras pathway. However RACK1 can recruit various kinds of proteins which result in its different effect on tumor growth and migration. In colonic carcinoma, RACK1 inhibited phosphorylation of cyclin dependent kinase (CDK), induced cell cycle arrest and inhibited colonic cells proliferation.We found RACK1 expression was related with T stage and lymph node metastasis in the first part. So we speculate RACK1 was associated with the progression of esophageal squamous cell carcinoma, and RACK1 expression could affect the proliferation, migration and invasion of ESCC.Objectives1. To build esophageal cancer cell line with RACK1 gene stable downregulated;2. To detect the ability of ESCC cell proliferation, invasion and migration after RACK1 downregulated;3. To detect changes in ESCC cell proliferation, invasion and migration after treated with PKC activator and inhibitor.Methods1. Cell culture and transfectionHuman esophageal squamous carcinoma Eca109 and EC9706 cell lines were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum and incubated at 37℃ in a humidified atmosphere containing 5% CO2. pGPU6/GFP/Neo plasmid targeting RACK1(shRACK1) was generated by Shanghai JIMA Biologic Company China. Cells were transfected with Lipofectamine 2000 reagent (Invitrogen). Stably transfected cells were selected with 600μg/mL of G418.2. Drug treatmentEca109 and EC9706 cell lines were treated with 100nM PMA/TPA for 20h, which was considered as PKC activated group. Some other Eca109 cells and EC9706 were treated with 100nM staurosporine for 20h, which was considered as PKC suppressed group.3. Proteins extraction and Western BlotCells were lysed and proteins were extracted. Protein was quantified by the use of a BCA protein quantification kit. The extracts were separated by SDS-PAGE gel electrophoresis. Then the separated bands were blotted onto polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat dry milk in Tris buffered saline (TBS, pH 7.4) and incubated with primary antibody (RACK 1,1:1000, BD Biosciences; Vimentin and E-cadherin,1:1000, DAKO) overnight at 4℃. Then peroxidase-conjugated IgG was used as a secondary antibody. Immunoreactivity was detected with an enhanced chemiluminescence reaction kit (Thermo Scientific Pierce).4. Colony formation assayTransfected cells were treated by Trypsin into single cell suspension. The cell suspension was diluted and cells were plated in dishes. 1×103 cells per dish were cultured at 37℃, in 5% CO2 and saturated humidity environment for 2 to 3 weeks. Stop the assay once clones were visible to the naked eye. The colonies were stained with 0.1% Crystal violet (Sigma) for 1h 37℃, and then we counted clones by microscope.5. Heterotopic nude mouse modelTransfected cells were treated by Trypsin into single cell suspension. After centrifugation and resuspension the cells were transferred into 1.0×107/ml. Each nude mouse was injected subcutaneously into the lateral flank by 0.2ml cell suspension. Tumor growth was measured every five days. Tumor volume was calculated using the formula:tumor volume (mm3)= a × b2× 0.5, where a represents the longest diameter, b is the shortest diameter. The mice were sacrificed after 4 weeks and the tumors were removed, weighted and measured. After weighing and measuring they were fixed in 10% formalin for histopathological analyses.6. Transwell invasion and migration assayWe coated a transwell membrane with Matrigel. Then we resuspended ESCC cell lines in serum-free medium and added the cells to the upper chamber of precoated transwells at a density of 2.0×105/ml. The lower chamber contained normal medium with 10% FBS. After incubation for 24 h, a cotton-tipped swab was used to swab the cells on the upper chamber. The invasive cells, which were attached to the lower surface of the membrane, were fixed with methanol, stained with 0.1% Crystal violet (Sigma) and counted. The experimental procedures of transwell migration assay were similar to the transwell invasion assay except that the membrane was not coated with matrigel and the time of incubation was shortened to 12 h. Results1. Down-regulation of RACK1 protein expression in ESCC cell linesRACK1 protein expression in Eca109 and EC9706 cells was detected by Western Blot. We found that the expression of RACK1 protein was down-regulated after shRACK1 transfection. We established a stable down-regulated cell strain by G418 selection.2. Changes of cell proliferation, migration and invasion after down-regulation of RACK1Down-regulation of RACK1 inhibited cell proliferation of ESCC cells in vitro and vivo. Colony formation assay revealed that stable knockdown cell strains formed fewer colonies than the negative control group in Eca109 (P=0.0044) and EC9706 (P=0.0008). The result was clarified in nude mice model. Tumor weight from the down-regulated cells were significantly lower than the negative control group (P=0.0003, Eca109; P=0.0029, EC9706). Tumor volume from the down-regulated cells were significantly lower than the negative control group (P=0.0119, Eca109; P=0.0192,EC9706).Results from cell invasion and migration assay showed RACK1 down-regulated cell invasion and migration. The number of invaded cells and migrated cells from the RACK1 knockdown group were significantly smaller than those of the negative control (P=0.0014, P=0.0108, Eca109; P=0.0133, P=0.0037, EC9706).3. Changes of cell proliferation, migration and invasion after PKC activated and suppressedCell proliferation was significantly increased by PMA, and decreased by staurosporine (SP). Compared with the control group, PMA treated cells formed more colonies in Eca109 (P=0.0012) and EC9706 (P=0.0015), while SP treated cells formed less colonies in Eca109 (P=0.0016) and EC9706 (P=0.0059). In nude mice model, tumor weight(both P< 0.0001) and tumor volume (P=0.0288, Eca109; P=0.0405,EC9706) from the PKC activated cells were significantly higher than the negative control group; while tumor weight from the PKC suppressed cells were significantly lower than the negative control group (P=0.0001, Eca109; P=0.0002, EC9706), and tumor volume from the suppressed group also showed reverse effect (P=0.0308, Eca109; P=0.0103, EC9706).The number of invaded cells and migrated cells from the activated group were significantly larger than those of the control (P=0.0448, P=0.0040, Eca109; P=0.0138, P=0.0015, EC9706), and these number of suppressed group were smaller (P=0.0049, P=0.0012, Eca109; P=0.0007, P=0.0006, EC9706).Conclusions1. Down-regulation of RACK1 inhibited cell proliferation of ESCC cells, and its formed tumors were smaller and lighter. Down-regulation of RACK1 suppressed the invasion and migration of ESCC cell line.2. Activation of protein kinase C by PMA/TPA promoted cell proliferation, invasion, migration and tumor growth while PKC suppression showed the opposite effect.BackgroundThe process of ESCC progression is continuous including adhesive falling, matrix degradation, migration enhanced and neovascularization. It is clarified that tumor cells acquire the ability of invasion and migration mainly through epithelial-mesenchymal transition (EMT). This is a process in which cells transit from an epithelial phenotype to a mesenchymal phenotype under physiological and pathological circumstance. Tumor cells become less adhesive, more invasive and migrated during this process. Then cells can infiltrate into the peripheral tissue by breaking through basement membrane, migrate to distal organs in lymphatic and blood vessel. New tumors are formed in distal organs by mesenchymal-epithelial transition (MET). Results from experiments in vitro and vivo confirmed cancer cells undergo EMT before metastasis and invasion. All of these evidences implied that EMT is an important part during the process of early tumor metastasis, and blocking EMT may inhibit tumor metastasis.The various proteins-binding surface of RACK1 makes itself functional diversity. RACK1 can regulate tumor progression via modulating the ability of cell motility and migration, invasion, proliferation and anti-apoptosis. RACK1 was already demonstrated to be associated with tumor growth, invasion and metastasis in our second part. As one of the important pathways of tumor progression, whether EMT is involved in RACK1 regulating tumor invasion and migration has not been studied yet.It has been clarified that RACK1 overexpressed can increase the expression of Vimentin and a-SMA, while the expression of E-Cadherin and CK-19 decreased. Whether it is involved in EMT still remains unclear. No reports about RACK1 regulating tumor invasion and migration through EMT come out yet.Objectives To detect whether EMT is involved when RACK1 regulating tumor progression.Methods1. Proteins extraction and Western Blot Procedures are the same as Part Ⅱ Methods 4.8 and 4.9.2. Immunohistochemistry staining and evaluation of immunostaining intensity Procedures are the same as Part Ⅰ Methods 3.Result1. Association of RACK1 with EMT-related proteins in ESCC cell linesThe levels of Vimentin was reduced after RACK1 down-regulated, meanwhile E-cadherin was increased.2. Association of RACK1 with EMT markers in ESCC tissueWe found there was a significant positive correlation between RACK1 expression and Vimentin (P=0.0190), and an inverse correlation between RACK1 expression and E-cadherin (P=0.0047).ConclusionsRACK1 was associated with the expression of EMT markers in ESCC cell lines and ESCC cases. A significant positive correlation was found between RACK1 expression and Vimentin, and an inverse correlation between RACK1 expression and E-cadherin, which demonstrated that RACK1 was involved with EMT. |
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