| Background:Lung cancer is a huge growing public health problem and is the most common cancer in human worldwide.Along with the enhancement and development of therapeutic methods and detection strategies,the prognosis of lung cancer patients in clinic is still poor,with a five-year survival rate of 20%.The late diagnosis and tumor metastasis persists as the main barrier to successful cancer therapy and as a significant contributor to death in patients with lung cancer.Thus,identification of new diagnostic and prognosis biomarkers and figuring out the mechanisms underlying the occurrence and development of lung cancer are vital in improving survival of lung cancer patients.The C2 domain is independently folded modules,of about 130 residues,found in a large and diverse set of eukaryotic proteins.This domain is originally identified as cellular Ca2+ effectors and is found in various signaling molecules and proteins involved in vesicular tracking.The following study manifests that the C2 domains of a variety of proteins also play a pivotal role in cellular signal transduction,protein-protein interactions and tumorigenesis.For example,the C2 domain of Smurf1 directly interacts with the kinase domain of PIPKIγ and regulates cell growth and migration of lung cancer.Myoferlin is involved in regulation of cellular lipid metabolism and promotion of metastases in triple-negative breast cancer.NEDD4 L is underexpressed in colorectal cancer and suppresses Wnt signaling pathway.TC2N is a putative C2 domain-containing protein that belongs to the carboxyl-terminal type(C-type)tandem C2 protein family.For years,the role of TC2 N in cancer remains completely unexplored.Until recently,we have identified TC2 N as an overexpressed gene in lung cancer through bioinformatics and tissue microarray analysis.Due to the fact that a number of proteins containing C2 domain have a key role in signal transduction and cancer,we intend to further explore the precise role of TC2 N in cancer development and progression.Contents:1.Detection of TC2 N expression in lung cancer tissues and adjacent normal tissues.The mRNA expression of TC2 N between lung tumor tissues and non-tumor tissues were analyzed by the published datasets,Oncomine.Further,the protein expression of TC2 N between human lung tumor specimens and paired normal specimens were verified by IHC.2.Analysis of the clinical characteristics and prognostic significance of TC2 N in lung cancer.The prognostic significance of TC2 N expression was analyzed by two public datasets,GSE3141 and GSE31210.Further,the clinical significance and prognostic value of TC2 N were evaluated by using a tissue microarray containing 272 human lung tumor tissues and 265 adjacent normal tissues.3.Evaluation of the biological properties of TC2 N in lung cancer cells.The effects of in vitro ectopic expression of TC2 N on lung cancer cell proliferation,apoptosis,migration and invasion were analyzed by MTS,colony formation,cell cycle,cell apoptosis,wound healing,transwell assays.The effects of in vivo ectopic expression of TC2 N on tumor growth and metastasis were analyzed by nude mice xenograft model.4.Investigation of molecular mechanisms of TC2N’s biological function in lung cancer cells.The downstream signaling pathways by which TC2 N regulates tumor growth and metastasis phenotype in lung cancer were investigated by bioinformatics,and were verified by luciferase reporter assay and WB.The specific molecular mechanisms of TC2 N on p53 and NF-κB signaling pathway were determined by WB,Co-IP and immunofluorescence assays.Results:1.Elevated expression of TC2 N is associated with progression and poor outcome in human lung cancer.(1)We first assessed the expression of TC2 N between tumor and non-tumor tissues by analyzing the published datasets,Oncomine.Based on data from Oncomine,we found that TC2 N mRNA expression was significantly higher in lung cancer tissues than in normal lung specimens.Subsequently,these findings were further supported by detecting TC2 N protein expression on a tissue microarray containing samples of 272 lung cancer specimens and 265 matched para-carcinoma specimens.Based on IHC results,the protein expression of TC2 N was markedly upregulated in tumor tissues compared to adjacent non-tumor tissues.(2)Analysis of TC2 N expression and clinicopathological parameters reveal a significant association of TC2 N protein expression with clinical stage(P=0.006),histological grade(P<0.001)and lymph node metastasis(N)(P<0.001).(3)To further assess the prognostic significance of TC2 N in lung cancer,univariate Kaplan-Meier analysis and multivariate Cox proportional hazards regression analysis were performed to examine the effect of TC2 N expression on lung cancer prognosis.The results showed that high TC2 N expression exhibited poor outcome of lung cancer patients and was an independent prognostic factor for patient’s overall survival.Subsequently,the association of TC2 N expression with different clinical stages and histological grades were analyzed by cox multivariate regression analysis.Results indicated that high TC2 N expression is an independent prognostic marker for patients with advanced stage or middle and low grade instead of early stage or high grade.2.TC2 N promotes lung tumor growth and metastasis in vitro and in vivo.(1)To explore the biological function of TC2 N in lung cancer cells,we evaluated the proliferation,colony formation,apoptosis,migration and invasion changes of lung cancer cells after TC2 N ectopic expression.We observed that knockdown of TC2 N increased apoptosis and inhibited proliferation,colony formation,migration and invasion of lung cancer cells.In parallel,overexpression of TC2 N decreased apoptosis and promoted proliferation,colony formation,migration and invasion of lung cancer cells.(2)Furthermore,the effect of TC2 N overexpression on tumorigenesis and distant metastasis were examined in vivo.Noticeably,high TC2 N expression promoted tumor growth and resulted in a significant increase in the number and size of lung and liver metastasis loci.3.Upregulation of TC2 N represses p53 signaling pathway to promote cell proliferation and inhibit apoptosis in lung cancer cells by inhibiting Cdk5-induced p53 phosphorylation via inducing Cdk5 degradation or preventing the interaction of Cdk5 with p53.(1)Based on data from TCGA,GO analysis was performed and revealed that co-expressed genes of TC2 N were enriched in p53 signaling in human lung cancer.To explore the possible involvement of TC2 N in regulating p53 signaling pathway,we monitored p53 signaling activity and its downstream target gene expression after ectopic expression of TC2 N.Indeed,TC2 N markedly inhibited p53 signaling and changed the expression of p53 target genes instead of p53 expression.(2)Further,we demonstrated that TC2 N represses p53 signaling by suppressing Cdk5-induced p53 phosphorylation in lung cancer cells which promote cell proliferation and inhibit cell apoptosis in two ways: a.TC2 N facilitated Cdk5 protein degradation via ubiquitination.b.TC2 N interacted with p53 to block the interaction of Cdk5 with p53.4.Upregulation of TC2 N activates NF-κB signaling pathway to promote cell migration and invasion in lung cancer cells by increasing the translocation of NF-κB to the nucleus via promoting phosphorylation of IκB.(1)Based on data from TCGA,GSEA revealed that TC2 N expression is associated with NF-κB signaling in human lung cancer.To explore the possible involvement of TC2 N in regulating NF-κB signaling pathway,we monitored NF-κB signaling activity and its downstream target gene expression after ectopic expression of TC2 N.Indeed,TC2 N markedly upregulated NF-κB signaling activity and its downstream target gene expression.(2)Further,we demonstrated that TC2 N activates NF-κB signaling to promote cell migration and invasion through facilitating NF-κB transportation to the nucleus of lung cancer cells.Due to the fact that IκB acts as a key suppressor of NF-κB by limiting NF-κB migration into the nucleus and masking its DNA-binding and nuclear localization domains,we next to investigate whether TC2 N regulates the phosphorylation and degradation of IκB.Indeed,the results indicated that overexpression of TC2 N enhanced the phosphorylation level of IκB,with decreased total protein level of IκB in lung cancer cells.Conclusion:In summary,we here provide evidence that TC2 N is a novel oncogene and its high expression promotes tumor growth and metastasis and correlates with a worse prognosis in lung cancer.Mechanistically,TC2 N suppresses p53 signaling to promote cell proliferation and inhibit cell apoptosis and activates NF-κB signaling to accelerate tumor metastasis. |
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