The Roles And Mechanisms Of CDP138 And LZTS2 In Tumor Metastasis

Non-communicable diseases(NCDs)are now responsible for the majority of global deaths,and cancer is expected to rank as the leading cause of death and the single most important barrier to increasing life expectancy in every country of the world in the 21 st century.According to estimation from the World Health Organization(WHO)in 2015,cancer is the first or second leading cause of death before age 70 years in 91 of 172 countries,and it ranks third or fourth in an additional 22 countries.The incidence and mortality of lung cancer and liver cancer are extremely high.Although several different types of treatments are applied,such as surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy,distant metastasis is still one of the main causes leading to the failure of cancer treatment.Therefore,elucidating the molecular mechanism of tumor metastasis and identifying new therapeutic targets are crucial for establishing effective treatment for cancer.This paper contains two parts,which clarified the roles and mechanisms of CDP138 and LZTS2 in tumor metastasis respectively.We aim to identify the new metastatic targets in lung cancer and liver cancer.We find that CDP138 acts as an oncoprotein and LZTS2 acts as a tumor suppressor protein,which are expected to be new therapeutic targets for patients,especially in patients with tumor metastasis.Part 1.CDP138 silencing inhibits TGF-?/Smad signaling to impair metastasis via GDF15 in lung cancer Objective: Lung cancer is the most common malignancy and the leading cause of cancer death worldwide.To date,surgery,radiotherapy,chemotherapy,personalized targeted therapy and immunotherapy remain the main treatment for this type of cancer.However,tumor metastasis is one of the important facors leading to treatment failure.Therefore,intense efforts are needed to better understand the oncogenesis of lung cancer and to identify novel therapeutic targets.CDP138,a CDK5 binding partner,regulates cell proliferation and migration.However,the mechanisms by which CDP138 functions in these processes remain unclear.Methods: To evaluate the clinical significance of CDP138 in lung cancer,we first detected the expression of CDP138 protein level in lung cancer cells and the normal human bronchial epithelial cell line.Meanwhile,we performed immunohistochemical analysis of CDP138 expression in a human lung cancer tissue microarray,which containing 88 carcinoma tissues and paired paracarcinoma tissues.Kaplan-Meier analysis showed the relationship between expression of CDP138 and overall survival of lung cancer patients.Using siRNA or shRNA,cell growth,clone formation assay,transwell migration and invasion assay were performed to investigate the biological function of CDP138.To elucidate the underlying mechanisms by which CDP138 functioned in lung cancer,we performed gene microarray analysis to compare the genomic expression profiles of H1299 cells transfected with control or CDP138-targeting siRNAs.Finally,a series of rescue experiments were conducted to determine whether GDF15 was indeed required for the observed phenotypes induced by CDP138 kncokdown in lung cancer cells.Results: We found the protein level of CDP138 was obviously higher in five lung cancer cell lines(H1299,HCC827,H292,A549 and H1975)compared to the normal human bronchial epithelial cell line HBE.CDP138 was primarily located in the cytoplasm and was highly expressed in lung cancer tissues.Furthermore,there was a significant correlation between CDP138 expression and lymph node metastasis.However,there was no significant correlation between CDP138 expression and overall survival in lung cancer patients as determined using Kaplan-Meier analysis.CDP138 silencing suppressed cell proliferation,migration and invasion in lung cancer cells.According to gene expression microarrays,GDF15 was a key downstream effector of CDP138.CDP138 silencing attenuated TGF-?/Smad signaling activation at least in part through the downregulation of GDF15.More importantly,the observed phenotypes caused by CDP138 knockdown were partially dependent on GDF15 inhibition.Conclusions: Our findings demonstrated that CDP138 positively modulated the TGF-?/Smad signaling pathway via GDF15 to promote cell growth and metastasis in lung cancer,suggesting CDP138 as a potential oncogenic biomarker and a promising therapeutic target in the treatment of lung cancer.Part 2.?-Trcp and CK1?-dependent degradation of LZTS2 activates the PI3K/AKT signaling pathway to drive hepatocellular carcinoma progression and metastasisObjective: Liver cancer is one of the most frequent malignancies and the forth leading cause of cancer-related deaths worldwide.Hepatocellular carcinoma(HCC)represents approximately 90% of all cases of primary liver cancer.Surgical resection,radiofrequency ablation,chemoembolization and personalized targeted therapy are beneficial for the patients with HCC.The overall prognosis of patients remains poor due to its aggressive nature,including an invasive phenotype that is mediated by factors yet to be elucidated.Leucine zipper tumor suppressor 2(LZTS2),also called LAPSER1,functions as a tumor suppressor protein.Many studies have shown that LZTS2 plays a key role in tumor occurrence and development.Our previous study demonstrated that LZTS2 functioned as an upstream inhibitor of PI3K/AKT signaling and suppressed tumorigenesis and radioresistance in nasopharyngeal carcinoma by its interaction with p85.Further research showed that LZTS2 silencing increased cell proliferation,migration and invasion in HCC.However,the underlying mechanisms of LZTS2 deregulation in tumorigenesis and metastasis remain largely unknown.Methods: We performed immunohistochemical analysis of LZTS2 expression in HCC tissues and paired para-carcinoma tissues.Kaplan-Meier method was used to analyze the survival curve of patients with HCC.Co-immunoprecipitation(Co-IP)was performed to validate the interaction between LZTS2 and ?-Trcp,and to identify the potential kinases.Through ubiquitination assay,we explored whether LZTS2 could be degraded by ?-Trcp and CK1?.The effect of ?-Trcp on the half-life of LZTS2 was tested by protein half-life experiment.To further investigate whether the phosphodegron DSGXXS of LZTS2 was associated with ?-Trcp,we generated three LZTS2 mutant plasmids.Western blot was used to detect the downstream signaling pathway of LZTS2.Using si RNA or sh RNA,we investigated the biological function of LZTS2 in vivo and in vitro.Rescue experiments were performed to explore whether the functions of LZTS2 in HCC depended on its ubiquitination.Results: LZTS2 positivity was significantly higher in the adjacent para-carcinoma tissues than in the HCC tissues.Kaplan-Meier curve analysis demonstrated that patients with low LZTS2 expression levels had a shorter overall survival than HCC patients with high LZTS2 expression levels.We demonstrated that LZTS2 binded to and was ubiquitinated by ?-Trcp ubiquitin ligase and CK1? kinase for degradation.The half-life of LZTS2 was markedly extended in ?-Trcp depleted cells.What's more,we found that the Ser220,Ser224,Ser273 and Ser277 sites in conserved motifs were critical for the degradation of LZTS2 by ?-Trcp and CK1?.Moreover,LZTS2 negatively regulated PI3K/AKT signaling pathway.Thus,LZTS2 silencing promoted the tumorigenesis and metastasis of HCC in vitro and in vivo.Importantly,?-Trcp and CK1?-mediated degradation of LZTS2 promotes HCC progression and metastasis by activating the PI3K/AKT signaling pathway in a p85-dependent manner.Conclusions: In this study,we present evidences that LZTS2 is downregulated and correlated with poor prognosis in HCC.We further uncover that ?-Trcp and CK1?-mediated destruction of LZTS2 contribute to tumorigenesis and metastasis in vitro and in vivo by activating the PI3K/AKT signaling pathway through a p85-dependent manner in HCC.Our study reveals a previously unknown antimetastatic role and a novel posttranslational modification of LZTS2,indicating that LZTS2 may be an attractive therapeutic target for HCC.