Role And Molecular Mechanism Of TUSC3 Gene In Colorectal Carcinogenesis And Development

BACKGROUND&OBJECTIVEColorectal cancer (CRC) is a common malignant tumor of digestive system harmful to human health. In our country, CRC is of top priority in common malignant tumors and ranks the forth and third leading cause of cancer death in male and female patients. With the rapid development of economy in our country, people’s diet composition and life style have largely changed and the incidence rate of CRC has been gradually increasing. The age of CRC onset of has become younger, with remarkably elevated incidence rate among 30- to 40-year-old men and women. Although great progress has been achieved in the diagnosis and treatment of CRC. there’s still no significant decline in the death rate. Studies have shown that tumor metastasis is a pivotal factor affecting the therapeutic effectiveness and prognosis in patients suffering from CRC. Therefore, it is an urgent task to investigate the key genetic factors and underlying mechanisms of CRC Carcinogenesis and metastasis and find effective methods in the prevention and treatment of CRC, which is of great significance to contemporary oncology.TUSC3 (tumor suppressor candidate 3), also called N33, OST3A, is located at the human chromosomal band 8p22. The total length of TUSC3 is 224 kbp. The gene contains 11 exons with a CpG island existing between the promoter and the first exon. TUSC3 is predicted to encode a protein containing 348 amino acids with four transmembrane domains and one N-terminal signal peptide which was sheared during the process of protein maturation (http://www.cbs.dtu.dk/services/signalP). TUSC3 is located in endoplasmic reticulum and is one of the oligosaccharides transferase complex subunits with redox enzyme activity. TUSC3 plays a very important role in the catalyzing of N-glycosylation of proteins. Aberrant function of TUSC3 may lead to changes in the N-glycosylation process, which is closely related to the initiation and progression (invasion and metastasis) of cancer. In addition, TUSC3 is an indispensable member involving in the plasma membrane transport of Mg2+. Knock out of TUSC3 expression results in a decrease in the concentration of the total Mg2+ and free Mg2+, thus affects the absorption of Mg2+ and impedes development of zebrafish.TUSC3 is originally identified as a tumor suppressor gene within a homozygous-deleted region in metastatic carcinoma of the prostate. Although no mutation in the nucleotide sequence of TUSC3 has been reported before, homozygous deletion of TUSC3 is frequently found in pancreatic cancer, prostate cancer, and ovarian cancer. Up to now, studies about TUSC3 in CRC mainly focused on aberrant methylation of promoter CpG island. Few studies have investigated the expression and function of TUSC3 protein. Therefore, the role and underlying mechanism of TUSC3 in the initiation and metastasis of CRC are still largely unknown. It is worth further in-depth study and investigation.In this study, we aim to reveal the role and molecular mechanism of TUSC3 in the carcinogenesis and development of CRC and explore the potential signaling pathways TUSC3 may involve in. Furthermore, our study provided the experimental basis for TUSC3 as a potential diagnostic biomarker and therapeutic target of CRC.MethodsResearch on the function of TUSC3 in the initiation and development of CRC were conducted in three main aspects:clinical pathological analysis, cell functional experiments, and investigations of signaling pathway and molecular mechanism.1. Clinical pathological analysis of differential TUSC3 expression in colorectal tumor tissue and para-carcinoma normal tissue.The expression levels of TUSC3 protein in tumor tissue and para-carcinoma normal colorectal tissue were detected by immunohistochemical assay. A statistical analysis was performed to detect the significant differences between the TUSC3 expression and the age, gender, TNM stage, and differentiation degree of all the examined patients. Using western blotting analyses, the expression levels of TUSC3 in colorectal tumor tissues and paired normal tissues were examined and the expression differences were compared. In addition, TUSC3 expression at the mRNA level was detected in colorectal tumor tissue and paired normal tissue using qRT-PCR. TUSC3 expression in eight CRC cell lines were examined using western blotting and qRT-PCR analyses. Two CRC cell lines with the highest TUSC3 expression and other two cancer cell lines with the lowest TUSC3 expression were selected for TUSC3 silencing and overexpression treatment.2. Detection of TUSC3 expression in CRC cells and cell function assay.Overexpression and shRNA-induced down-regulation of TUSC3 were achieved using lentiviral vectors and HEK293T packaging cell line. Three shRNA fragments targeting the TUSC3 gene were designed and synthesized. Silencing efficiencies of these shRNA sequences were tested and the shRNA with the highest silencing efficiency was selected for lentiviral transduction. The silencing and overexpression efficiencies were examined using western blotting and qRT-PCR analyses. The cell proliferation ability, plate colony formation ability, invasive ability, and migration ability of CRC cells before and after TUSC3 silencing or overexpression were tested using the MTT assay, transwell migration assay, wound healing assay, and plate colony formation assay, respectively. Meanwhile, the tumorigenesis ability, proliferation ability of CRC cells in nude mice before and after TUSC3 silencing or overexpression were tested using in vivo tumourigenesis assay.3. Investigation of TUSC3-reIated signaling pathways in the initiation and development of CRC.Potential signaling pathways relating to TUSC3 function were searched from literature and public database. Proteins directly interact with TUSC3 were screened using the co-immunoprecipitation method. Expression differences of these proteins related to the TUSC3 protein is examined using western blotting analysis, thus validating the preliminary relationships between TUSC3 and the Wnt/β-catenin signaling pathways.4. Statistical analysisStatistical differences for all data were performed by SPSS. A threshold value was set at 0.05 (two tailed).Results1. Correlation analysis between TUSC3 expression and clinical pathological factorsQuantitative real-time PCR analyses showed higher levels of TUSC3 mRNA in 10 cases of surgical CRC tissues compared with the paired normal colorectal samples. Results of western blotting further revealed higher expression levels of TUSC3 in 20 cases of CRC tissues. Immunohistochemical of results showed that TUSC3 expression were positive in 95 cases of CRC samples of the total 120 cases. The positive rate is 79.17%. In the 120 paired normal colorectal samples, TUSC3 was undetectable in 102 (85%) cases and weakly expressed in 18 (15%)cases. Expression of TUSC3 protein in CRC tissues was significantly higher than in normal tissues (p < 0.05). The expression level of TUSC3 in CRC patient was significantly associated with Dukes stage (p< 0.05), lymph node metastasis (p< 0.05), tumor dimension (p < 0.05), and distant metastasis (p< 0.05). No significant correlation was detected between TUSC3 protein expression and other clinicopathological parameters including age, gender, and tumour histology.2. Expression of TUSC3 in CRC cell linesReal-time PCR and Western blot revealed different expression levels of TUSC3 in eight colorectal carcinoma cell lines (HCT116, LS174T, RKO, LOVO, SW480, HT29, SW620, and M5). The mRNA and protein expression levels of TUSC3 were the lowest in LS174T and HCT16 cells and the highest in SW480 and HT29 cells.3. TUSC3 overexpression promotes the proliferation, invasion and migration abilities of LS174T and HCT 116 cellsTUSC3 cDNA was transfected into LS174T and HCT116 CRC cells by retrovirus infection. Overexpression of TUSC3 in both cell lines were confirmed by qRT-PCR and western blot analyses.Meanwhile, LS174T-TUSC3 and HCT116-TUSC3 cells showed significantly enhanced proliferation, invasion, and migration capabilities compared with the control cells as determined by MTT assay (p< 0.05), plate colony formation assay (p < 0.05),and transwell migration assay (p< 0.05). In vivo tumourigenesis assay showed that TUSC3 overexpression significantly enhanced the tumorigenesis ability of LS174T-TUSC3 cells in nude mice (p< 0.05).4. Down regulation of TUSC3 represses the proliferation, invasion and migration abilities of SW480 and HT29 cellsWe transfected human CRC cell lines SW480 and HT29 with TUSC3-shRNA. Decreased expression of TUSC3 in both cell lines were confirmed by qRT-PCR and western blot analyses. SW480-shRNA-TUSC3 and HT29-shRNA-TUSC3 cells showed significantly reduced proliferation, invasion, and migration capabilities compared with the control cells as determined by MTT assay (p< 0.05), plate colony formation assay (p< 0.05), and transwell migration assay (p< 0.05). In vivo tumourigenesis assay showed that TUSC3 silencing significantly reduced the tumorigenesis ability of SW480-shRNA-TUSC3 cells in nude mice (p< 0.05).5. TUSC3 is involved in the MAPK, PI3K/Akt, and Wnt/p-catenin signaling pathwaysTUSC3 overexpression induced the epithelial-mesenchymal transition (EMT) in LS174T-TUSC3 cells. Western blot analyses showed that overexpression of TUSC3 in LS174T-TUSC3 cells led to a remarkable decrease in the expression of the epithelial marker E-cadherin and increase in the expression of mesenchymal marker Vimentin as compared with the control group.We next investigated the possible oncogenic pathways that may involve the function of TUSC3. Using western blot analyses, we investigated the downstream activation of P13K/Akt, MAPK, and Wnt/β-catenin signaling pathways in TUSC3 overexpressed and silenced CRC cells. We observed increased β-catenin and decreased GSK3P abundance in TUSC3 overexpressed cells with or without serum stimulation, while opposite effects in the expression level changes of β-catenin and GSK3β were observed in TUSC3-silenced cells. Co-immunoprecipitation analysis revealed protein-protein interaction between TUSC3 and β-catenin, indicating that endogenous human TUSC3 was physically associated with P-catenin. In addition, Down-regulation of TUSC3 in SW480-shRNA-TUSC3 cells caused significantly decreased phosphorylation level of AKT and ERK1/2 as compared with the control group. On the contrary, TUSC3 overexpression in LS174T-TUSC3 cells led to sustained phosphorylation of AKT and ERK1/2. Meanwhile, the expression level of AKT and ERK1/2 remained unchanged. Our results showed that TUSC3 activates the MAPK and PI3K/Akt pathways and induces the EMT, thus promotes the invasive and migratory properties of CRC cells.CONCLUSIONTUSC3 promotes initiation, progression and EMT of CRC through PI3K/AKT, MAPK signaling pathway and the classical Wnt/b-catenin signaling pathway. TUSC3 is not only a valuable biomarker for CRC diagnosis and prognostic evaluation, but also a potential target for CRC treatment.INNOVATION1. Our study confirms the function of TUSC3 gene in CRC initiation and development.2. Our study reveals the underlying mechanisms of TUSC3 in CRC. TUSC3 promotes CRC progression via PI3K/Akt and MAPK signaling pathways, the classic and Wnt/ β -catenin signaling pathwayssignaling pathways.