?.The Function And Mechanism Of YAP Inducing Focal Adhesion Formation And Tumor Invasion In Breast Cancer ?.The Therapeutic Effect Of Laparoscopic Gastrectomy(D2+CME) In Locally Advanced Gastric Cancer

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Evaluation of the correlation between YAP and breast cancer invasiveness in clinical specimensObjective: To determine the relationship between YAP expression and the breast cancer invasiveness in clinical specimens.Methods: Firstly,immunohistochemistry(IHC)examination was performed in a breast cancer tissue microarray(US Biomax),which contained 104 cases of paired primary lesion/lymphatic metastasis to evaluate the expression level of YAP in primary and metastatic tissue.The result was quantified via IHC scoring,in which cytoplasm expression of YAP was evaluated by a cytoplasmic score and the nucleus accommodation of YAP was evaluated by a nucleus score.Then,a gene Set enrichment analysis(GSEA)was performed in the expression dataset which was obtained from GEO database and contained the expression profiling of purified tumor cells from primary breast cancer tissue and lymphatic metastasis.Furthermore,the prognostic analysis of TCGA breast invasive carcinoma dataset was performed by Surv Express program.Results: IHC analysis of tissue microarray revealed that YAP expression and nucleus accommodation were relatively higher in lymphatic metastasis.GSEA of GSE dataset unveiled that YAP conserved signature was enriched in metastatic lymph nodes with statistical significance.Surv Express analysis of TCGA dataset indicated that breast cancer patients with poor prognosis presented a significantly higher expression of YAP and its downstream genes.Conclusion: YAP overexpression and activation were supposed to be a bio-marker of tumor invasiveness and poor prognosis in breast cancer.Part ? Evaluation of YAP inducing focal adhesion formation and tumor invasive in breast cancer cell linesObjectives: To determine whether YAP could induce cell migration,invasion and focal adhesions in breast cancer cell lines.Methods: First,we evaluated the endogenous expression level of YAP in breast cancer cell lines and verified the transfect efficiency of YAP overexpression plasmid and siRNAs via western blot.Consequently,Transwell migration and invasion assay was performed to determine whether YAP expression could induce cell migration/invasion abilities.Then,we used cell adhesion assay to verify the promoting effect YAP in cell adhesion.Finally,through immunofluorescence,we detected focal adhesions in the cells transfected with YAP overexpression plasmid or siRNAs by measuring the co-locations of Actin and Paxillin.Results: Western blot assay revealed that,compared with MCF7 and T47 D,the expression level of YAP was relatively higher in MDA-MB-231 and MDA-MB-468 breast cell lines.Knockdown YAP expression in MDA-MB-231 could significantly reduce cell migration,invasion and adhesion abilities.Contrarily,overexpression of YAP in MCF7 could obvious promote cell adhesion,migration and invasiveness.Furthermore,through immunofluorescence,we observed that the expression level of YAP was positively correlated to focal adhesions both in MDA-MB-231 and MCF7 cell lines.Conclusions: YAP could significantly promote cell adhesion,migration,invasion and focal adhesion formation in breast cancer lines.Part ? The molecular mechanism of YAP-inducing focal adhesionObjective: To investigate the mechanism how YAP regulated focal adhesions.Methods: Firstly,we transfected the nucleus accommodation mutant(S127A)or TEA domain mutant(S94A)of YAP into MCF7 cell line.After evaluating the transfect efficiency and the downstream genes expression,we used Transwell assay and cell adhesion assay to determine whether the migration and adhesion abilities could be affected by different mutants of YAP protein.Moreover,focal adhesions were measured via immunofluorescence.Next,we evaluate whether the phosphorylation level of Tyr397-FAK,a potential upstream regulator of focal adhesion,could be regulated by YAP mutants in MCF7 cell lines or could be down-regulated by knockdown YAP in MDA-MB-231 cell line.Subsequently,Verteporfin was used to verify whether inhibiting YAP-TEADs interaction could reverse cell migration and focal adhesions formation in MCF7-YAP-S127 A cell line or MDA-MB-231 cell line.Meanwhile,Western blot assay was performed to assess whether Verteporfin could affect FAK phosphorylation.Finally,we used Defactinib,a specific inhibitor of FAK-Tyr397 phosphorylation,to verify whether FAK was the regulatory component of YAP induced focal adhesions.Results: Compared with YAP-S94 A,nucleus accommodation mutant(YAP-S127A)could significantly induced cell migration,adhesion and focal adhesion formation.Similarly,using Verteporfin to inhibit YAP-TEAD interaction could significantly reduce cell migration,adhesion abilities and focal adhesions both in MDA-MB-231 and MCF7-YAP-S127 A cell lines.Therefore,we indicated that the interaction between YAP and TEADs was essential for YAP induced focal adhesion.Through Western blot,we found that YAP-S127 A mutant could significantly promote phosphorylation of FAK at 397 tyrosine in MCF7 cell lines,while either knockdown endogenous YAP expression or treating with Verteporfin could down-regulate FAK phosphorylation level.Furthermore,inhibiting FAK with Defactinib could significantly reduce focal adhesion both in MCF7-YAP-S127 A and MDA-MB-231 cell lines.These results revealed that YAP-TEADs promoted focal adhesion formation by inducing FAK phosphorylation.Conclusions: YAP could regulate focal adhesions through FAK phosphorylation by the interaction with TEADs.Part ? The molecular mechanism of YAP regulating FAK phosphorylationObjectives: To investigate the mechanism of YAP inducing FAK phosphorylation in breast cancer cell lines.Methods: The Ch IP-sequence data of MCF7 cell line was downloaded from ENCODE database and analyzed via Ch IP-Seek software.Gene expression profiling was performed to reveal the differently expressed genes in MCF7 cell line transfected YAP-S127 A.Gene ontology enrichment was performed by DAVID software.Protein interaction analysis was used to determine the potential upstream of FAK.The m RNA level of the FAK regulator were verified via RT-q PCR.The protein level of the FAK regulator and phosphorylation level of Tyr397-FAK were evaluated by Western blot assay.The promoter activity was measured by dual luciferase assay.Chromatin immunoprecipitation was performed to verify whether YAP could bind to the promoter region.Gene expression correlation of the clinical specimens in TCGA database was analyzed via R2: Genomics Analysis and Visualization Platform.Transwell assay,cell adhesion assay and immunofluorescence was used to evaluate cell invasive,adhesion and focal adhesion formation.Results: Through analysis of the Ch IP sequence data,we found 192 genes whose promoter was potential combined with TEAD4.Subsequently,gene expression profiling was performed to evaluate the expression level of these 192 genes in MCF7 transfected with YAP-S127 A.Among them,30 genes were significantly up-regulated by YAP-S127 A mutant.Gene ontology enrichment revealed that CTGF,CYR61,BCAM,L1 CAM and HABP2 was classified into the “cell adhesion” category and selected into the further study.Through String analysis,we found that THBS1 was the direct upstream of FAK.RT-q PCR and Western blot assay demonstrated that the mRNA and protein level of THBS1 could be significantly up-regulated by YAP.Ch IP assay showed that YAP could bind the promoter region of THBS1.Dual luciferase assay revealed that YAP-S127 A could significantly induce THBS1-promoter activity.Analysis of TCGA database revealed that THBS1 expression level was positively associated with YAP expression in clinical breast cancer specimens.Finally,through Western blot,Transwell,cell adhesion assay and immunofluorescence,we verified that knockdown THBS1 expression could reverse YAP-inducing FAK phosphorylation,cell invasion,adhesion and focal adhesion formation.Conclusions: YAP induced FAK phosphorylation and focal adhesion through transcriptionally activating THBS1 expression.Part ? Prospective randomized controlled trial to compare laparoscopic distal gastrectomy(D2 + CME)with conventional D2 lymphadenectomy for locally advanced gastric cancerObjective: In previous research,we have demonstrated that D2 lymphadenectomy plus completed mesogastrium excision(D2+CME)appeared to be an optimal surgical approach for locally advanced gastric cancer(AGC).However,the oncologic efficiency of this procedure is still unclear.Therefore,we conduct a single-center,prospective,randomized controlled trial to compare D2+CME procedure with conventional D2 lymphadenectomy in locally AGC.Method: A single-center,paralleled group,randomized controlled trial was performed to compare D2+CME with conventional D2 in locally AGC.Patients` inclusion criteria were as follows:(1)18 to 75 years old;(2)body mass index less than 30;(3)pathologically proven locally AGC with c T2-4,N0-3,M0;(4)expected curative resection via laparoscope-assisted distal gastrectomy(LADG);(5)without severe comorbidities;(6)ASA score was I,II or III,and Zubrod-ECOG-WHO(ZPS)less than 2;(7)written informed consent.The exclusion criteria of this trial included:(1)pregnant or breastfeeding women;(2)severe mental disorder;(3)previous neoadjuvant chemotherapy or radiotherapy;(4)previous upper abdominal surgery;(5)combined with other malignant diseases;(6)total gastrectomy.The primary end-point was 3-year disease-free survival and the secondary end-points were overall survival,recurrence pattern,complications,postoperative recovery course,etc.The short-term outcomes were compared to evaluate the safety and feasibility of this study.Our trial was registered on Clinical Trials.gov,registration number: NCT01978444.Results: Between September,2014 and June,2018,a total of 338 patients were randomly assigned to either D2+CME or D2 group(D2+CME,169 patients;D2,169 patients).There were no significant differences in age,gender,ASA score,and comorbidities.The D2+CME procedure exhibited advantages in intra-laparoscopic bleeding(median 15.0 [interquartile range 23.0] vs.median 37.0 [interquartile range 33.5] ml,p<0.01)and lymph nodes harvesting(median 34 [interquartile range 16] vs.median 27 [interquartile range 13],p<0.01).The morbidity of the D2 group and D2+CME group were 16.0%(27/169)and 20.1%(34/169),respectively(p=0.322).Severe complications were occurred in 2 cases in D2 group.Logistic regression analysis revealed that age and total operation duration were independent risk factors for postoperative morbidities.The mortality rate was 0% in both groups.In postoperative recovery course,flatus appeared earlier in D2+CME group compared with D2 group(p=0.009).Conclusion: The LADG with D2+CME procedure showed advantages in intraoperative bleeding,lymph nodes harvesting and postoperative recovery course.Meanwhile,no significance difference was observed in the complications between the two groups.Therefore LADG with D2+CME appeared safety and feasibility for patients with AGC.Part ? Safety and efficacy of laparoscopic distal gastrostomy(D2+CME)with posterior gastric mesenteric excision in middle-third gastric cancerObjective: The optimal extent of gastrectomy is still controversial in middle-third gastric cancer(MGC).Several studies recommended total gastrectomy in MGC to completely remove potential tumor residual,however,patients with this procedure may suffer from severe complications and bad quality of life.Previously,we first described the morphology of the posterior gastric mesentery(PGM)and put forward D2 plus complete mesogastrium excision(D2+CME)in gastric cancer.In MGC,distal gastrectomy(D2+CME)with PGM excision appeared to be sufficient for radical resection.In order to validate the curative effect of D2+CME procedure,we propose this retrospective study,aiming to investigate its safety,feasibility and oncologic efficacy for middle-third gastric cancer.Method: We retrospectively collected clinic-pathologic and follow-up data from MGC patients(pathology stage Ib-IIIc)who have received laparoscopic radical gastrectomy at the department of GI surgery,Tongji Hospital,Huazhong University of Science and Technology from January,2015 to December,2016.Among them,patients receiving either distal gastrectomy(D2+CME)with posterior gastric mesenteric excision or total gastrectomy with conventional D2 lymphadenectomy were selected and analyzed in this study.Results: The total of 76 middle-third gastric cancer patients were collected.Among them,36 underwent laparoscopic distal gastrostomy(D2+CME)with PGM excision,and the remaining 40 underwent laparoscopic total gastrostomy(D2).No significant difference was found in baseline analysis.Furthermore D2+CME procedure exhibited advantages in lesser intra-laparoscopic bleeding(median: 14.50 ml in D2+CME vs.38.00 ml in D2,p < 0.01) and more lymph nodes harvesting(median: 38 in D2+CME vs.19 ml in D2,p < 0.01).The postoperative morbidity of the D2+CME group and D2 group were 11.1%(4/36)and 7.5%(3/40),respectively(p=0.884).Compared with D2 group,D2+CME group suffered shorter first flatus time(p < 0.01).Through postoperative specimen examination,27 out of 36(75%)patients in D2+CME were found the existence of post mesogastric lymph node(PGLN)and the median number of PGLN in these cases was 2(ranged 0-13).Moreover,metastatic PGLN was found in 3 patients.The median follow-up time of D2 and D2+CME group was 25 months and 24 months respectively.The 3-year cumulative overall survival in D2 and D2+CME group was 63.5% and 81.5%,respectively(p = 0.078).Univariate analysis unveiled that T stage,N stage and surgical procedure were the risk factors of MGC patients(p<0.05).Multivariate analysis revealed that N stage was the independent risk factor(p<0.05).Conclusion: Distal gastrostomy(D2+CME)with PGM excision is safety,feasibility and oncologic efficacy for MGC patients.