Functional And Clinical Research On A Novel Tumor-Suppressor Gene CHL1 At 3p26.3 In The Development Of ESCC

Background and objective:Esophageal squamous cell carcinoma(ESCC)is one of the most common cancer in the world and the fourth most common cancer in China.As the most common type of EC,esophageal squamous cell carcinoma shows high mortality and regional variation in incidence.Despite advances in multimodality therapy,such as surgical treatment,radiotherapy,chemotherapy and immunotherapy and other comprehensive treatment,the prognosis of ESCC remains poor and the overall 5-year survival is less than 15%.Early esophageal squamous cell carcinoma is not easy to be found,most patients in the treatment has been late,as invasion and metastasis is still the main cause of death.So far,the exact cellular and molecular mechanisms leading to ESCC have not yet been systematically confirmed.Therefore,it is important to explore the mechanism of the occurrence and development of ESCC at the molecular gene level,which provides the molecular basis for the early diagnosis,individual treatment and prevention of tumor.Like many of other solid tumors,the development of ESCC is also believed a multiple-step process caused by the accumulation of abnormal expression of oncogenes and tumor suppressor genes(TSG).Deletion of 3p is one of the most common genetic alterations in many solid tumors including ESCC.These results suggest that 3p may harbor one or more TSGs responsible for the tumor development and progression.Several frequently deleted regions,including 3p26.3,3p25.3,3p24.1,3p23,and 3p21.1,were found.No known TSG within 3p26.3 has been associated with ESCC development.It has found in our previous studies that cell adhesion molecule L1 like(CHL1)at 3p26.3 is screened by gene microarray and is initially identified as a new esophageal cancer-associated TSG.So we believe that CHL1 in the occurrence and development of ESCC may play an important role.CHL1 is a transmembrane protein composed of six Ig-like domains followed by five fibronectin type Ⅲ repeats,a transmembrane peptide,and a 105aa cytoplasmic domain.A recent study finds that CHL1 is able to recruit ezrin,a member of the ezrin-radixin-moesin(ERM)family of filamentous actin binding proteins to the plasma membrane.ERM proteins can bind to diverse CAM proteins such as L1,ICAMs,VCAMs,L-selectin,and CD44,and are involved in the linkage of membranes to the actin filament(F-actin)cytoskeleton.Therefore,the CHL1/ERM complex may represent a crucial component in the link between the cell matrix and the cytoskeleton.These findings suggest that CHL1 may play an important role in tumor invasion and metastasis.In this study,we explored the relationship between CHL1 expression and clinicopathological features,and assessed its prognostic values in ESCC patients.Then further studies were carried out to investigate the causes of down-regulation of CHL1 expression.The biological function of CHL1 gene was investigated by in vivo and in vitro experiments.We investigated the mechanism of CHL1 in the development and progression of esophageal squamous cell carcinoma by protein spectroscopy,immunoprecipitation(Co-IP),immunofluorescence(IF)and Western-blot.Part Ⅰ Expression and Significance of CHL1 in Esophageal Squamous Cell CarcinomaMethods1)The expression of CHL1 mRNA in esophageal carcinoma tissues and adjacent normal esophageal mucosal tissues was detected by Real time PCR.2)The expression of CHL1 protein in esophageal carcinoma tissues and adjacent normal esophageal mucosal tissues was detected by immunohistochemical staining(IHC).3)The relationship between CHL1 protein expression and clinicopathological features was analyzed.4)The correlation between CHL1 protein expression and survival prognosis was assessed.Result1)In 42 patients with esophageal squamous cell carcinoma,the expression level of CHL1 mRNA in cancerous tissues was significantly lower than that in adjacent normal tissues(t=3.085,P=0.0035).When compared with their normal counterparts,the relative expression level of CHL1 was significantly down-regulated in 23 tumor tissues(57.1%).2)The positive rate of CHL1 expression in esophageal squamous cell carcinoma was 53.5%(154/288),while the positive expression rate of CHL1 protein in adjacent normal esophageal mucosa was 92.7%(267/288).The difference between the two groups was statistically significant(χ2=1.127,P<0.001).3)CHL1 protein down-regulation was significantly associated with tumor cell differentiation(χ2=14.419,P=0.001),invasion(χ2=3.965,P=0.049),lymph node metastasis(χ2=10.883,P=0.001)and advanced clinical stage(χ2=27.007,P=0.000).4)The expression level of CHL1 protein was significantly correlated with the survival prognosis of patients(Log Rank=57.839,P=0.000).In down CHL1 expression group,the 5-years disease-specific survival is poorer than that in normal expression group(P<0.001).Cox proportional hazard regression analysis also showed that down-regulation of CHL1 could be used as an independent prognostic factor for overall survival of ESCC patients(P=0.000).Summary1)In comparison with none-tumor tissues,lower expression of CHL1 was shown in ESCC tissues.And down-regulation of CHL1 protein was significantly correlated with tumor cell differentiation,invasion,lymph node metastasis and advanced clinical stage.All these results suggest that CHL1 may play an important role in the development and progression of ESCC.2)The expression level of CHL1 protein was significantly correlated with the survival of ESSC patients and was an independent risk factor for predicting the prognosis of ESCC.Part II Effect of CHL1 Expression on Biological Behavior of Esophageal Cancer CellsMethods1)RT-PCR and Western-blot were used to detect the expression of CHL1 in ESCC cell lines and normal esophageal immortalized cells.2)Construction of CHL1 over-expression ESCC cell lines and silencing endogenous CHL1 ESCC cell lines.3)The effect of CHL1 gene on the proliferation of ESCC cells was detected in vitro,such as XTT test,Foci formation experiment and Soft agar test.4)The effect of CHL1 gene on tumorigenicity of ESCC cells in vivo was observed by subcutaneous tumorigenesis in nude mice.5)The effect of CHL1 gene on the cell cycle of esophageal carcinoma was detected by flow cytometric analysis.6)Wound-healing assay and transwell migration assay were used to detect the effect of CHL1 on the migration of ESCC cells.7)Cell invasion assay was adopted to test the effect of CHL1 on invasion of ESCC cells.8)The effect of CHL1 on migration and metastasis in vivo was investigated by constructing the model of lymph node metastasis of the popliteal fossa in SCID mice.Results1)Down-regulation of CHL1 was also detected in 7 of 9 ESCC cell lines including HKESC1,EC18,KYSE 30,KYSE140,KYSE180,KYSE510,and KYSE520.KYSE30 and KYSE180 were selected to construct CHL1 stable over-expression ESCC cell lines.The lentiviral-mediated short hairpin RNA(shRNA)interference was used to knockdown CHL1 in cell line KYSE410.2)Real time PCR and Western blot were used to verify the over-expression efficiency and silencing efficiency of CHL1.3)It is shown that the expression of CHL1 gene significantly inhibited the proliferation of ESCC cells in vitro experiments.4)In the subcutaneous tumor model of nude mice,the expression of CHL1 gene inhibited the formation of tumor in nude mice in vivo,and the volume of tumor formed was reduced.5)CHL1 gene expression can arrest the cell cycle of ESCC cells at G1/S checkpoint.6)Wound-healing assay,transwell cell migration assay and cell invasion experiments showed that CHL1 gene inhibited esophageal cancer cell migration and invasion in vitro.7)NOD/SCID mice model of tumor-bearing popliteal lymph node metastasis,CHL1 gene in vivo inhibition of esophageal cancer cell migration and metastasis.Summary1)Successfully constructed CHL1 overexpression esophageal cancer cell lines and CHL1 knockdown esophageal cancer cell lines.2)CHL1 over-expression plays a role in inhibiting tumor proliferation in vitro experiments and in vivo nude mice subcutaneous tumor model.3)CHL1 significantly inhibited the proliferation of ESCC cells through arrestting cell cycle at G1/S checkpoint.4)The expression of CHL1 could inhibit cell migration and invasion in ESCC cells in vitro and in vivo.Part Ⅲ Molecular Mechanism of CHL1 Inhibiting Proliferation and Metastasis of Esophageal Cancer CellsMethods1)Western blot was adopted to detect the expression of p53,p21 and cyclin.2)Western blot was used to detect the effect of CHL1 on epithelial-mesenchymal transition(EMT)in ESCC.3)Rhodamin staining was used to detect the effect of CHL1 expression on F-actin in esophageal cancer cytoskeleton.The effect of CHL1 expression on the activation of Racl was detected by Co-IP and Western blot.4)Immuno-coprecipitation and immunofluorescent staining were used to transiently inoculate CHL1 esophageal cancer cell lines to detect the recruitment of CHL1 to Merlin.5)Immunofluorescence double staining was used to examine the recruitment of CHL1 to Merlin.6)Effect of CHLl/Npl complex on Akt phosphorylation by Co-IP and Western blot.Results1)The expression of CHL1 up-regulates the expression of p21 and p53,and down-regulates the expression of the cyclin proteins cyclin D1 and cyclin E.2)CHL1 inhibits epithelial-to-mesenchymal transition(EMT)transformation and inhibits F-actin formation by decreasing the level of active Racl.3)CHL1 inhibits metastasis by recruiting Merlin to cell membrane.4)Npl and AKT could be specifically co-immunoprecipitated by CHL1 antibody.The expression of p-AktS473 was also enhanced by knockdown CHL1 or Npl individually or simultaneously.Summary1)CHL1 may block the esophageal cancer cells at the G1/S checkpoint by up-regulating the expression of p21 and p53 and down-regulating the expressions of the cycling proteins cyclingD1 and cyclingE2)The expression of CHL1 gene can inhibit the metastasis of esophageal cancer cells by inhibiting the EMT transformation of esophageal cancer cells,recruiting the expression of Merlin in the cell membrane and inhibiting the activation of Rac1 family member F-actin..3)CHL1/Np1 complex is necessary to inhibit Akt phosphorylation in the tumor suppressor gene SEMA3B pathway.Conclusion1)CHL1 is significantly down-regulated in ESCC tissues,and is positively correlated with lymph node metastasis,tumor invasion,differentiation advanced TNM stage and survival prognosis.All these indicate that CHL1 plays an essential role in the carcinogenesis and metastasis of ESCC.2)CHL1 inhibited ESCC cells proliferation,migration and invasion,arrests cell cycle at G1/S checkpoint by down-regulating p53 and p21.3)Functional studies using lentiviral-based overexpression and knockdown systems provided direct support of CHL1 to function as an important tumor suppressor with both anti-proliferation and anti-metastasis abilities,through Merlin and SEMA3B-Np1 mediated inhibition of AKT signaling pathway.