AMOT Involved In The Pathogenesis Of Diffuse Large B-cell Lymphoma Through DNA Damage Response Pathway

Diffuse large B-cell lymphoma(DLBCL)is a common aggressive tumor with rapid progression and poor prognosis.DLBCL is a highly heterogeneous disease,with gene heterogeneity accounting for different clinical outcomes.In the era of "molecular therapy",the treatment strategy of DLBCL is changing from traditional methods to chemotherapy combined with molecular targeted drugs.Therefore,the research of prognostic stratification and targeted therapy based on new molecular and signaling pathways is the main research strategy at current.Angiomotin(AMOT)was first identified in 2001 as a mediator that bound to angiostatin and regulated angiogenesis or endothelial cell migration.The AMOT protein composed of highly conserved coiled-coil domain and the C-terminal PDZ-binding domain.AMOT is mainly expressed in the endothelial cells and the human placental blood vessels,being located at the tight junctions and the actin cytoskeleton Functionally,AMOT regulates cytoskeletal rearrangement,tubular structure formation,and cell polarity,etc.Recent investigations had shown that aberrant AMOT expression was closely related to tumorigenesis,but the specific mechanism was still controversial.AMOT acted as a tumor suppressor in undifferentiated pleomorphic sarcoma,non-small cell lung cancer,and gastric cancer,but in breast cancer,liver cancer,prostate cancer,and sinonasal tumors,AMOT was overexpressed and promoted tumor cell proliferation or invasion.AMOT participated in the regulation of tumorigenesis mainly through Hippo signaling pathway,acting as the upstream positive or negative regulator of Yes-associated protein 1(YAP1).AMOT could also participate in tumor development through Wnt/catenin,ERK1/2,PI3K-AKT or VEGFR-2 pathways.However,the expression level and clinical significance of AMOT in DLBCL have not been reported yet.This study aimed to analyze the expression of AMOT in DLBCL and its association with the clinical characteristics of DLBCL patients.We further studied the effects of AMOT on the proliferation,cell cycle and other characteristics of DLBCL,and initially discussed the biological role of AMOT in the pathogenesis of DLBCL.Part I The expression of AMOT and its clinicopathological significance in diffuse large B-cell lymphomaObjective:DLBCL is an aggressive NHL with a high degree of heterogeneity.The patient's survival time is short,with about 30-40%of patients are ineffective to first-line immunochemotherapy,or present refractory or relapsed process.The strategies of prognostic judgment and target therapy based on new molecular targets and signal pathways are of great significance.Currently studies showed that AMOT function as a tumor suppressor or promotor in the development of various tumors,but the expression level and clinical significance of AMOT in DLBCL is indefinite.The purpose of the first part study is to detect the expression of AMOT in DLBCL pathological tissues and cell lines,and to preliminarily explore the clinical significance of AMOT for the diagnosis and prognosis of DLBCL.Material and Methods:1.Patient selection and specimen collection;2.Immunohistochemistry(IHC);3.Isolation of Peripheral blood mononuclear cells(PMBCs);4.Cell Culture;5.Real-time quantitative polymerase chain reaction(qRT-PCR);6.Protein isolation and Western blot;7.Statistical analysis.Results:1.We used immunohistochemical staining to detect the expression of AMOT protein in the pathological tissues of 52 newly diagnosed DLBCL patients and 25 patients with reactive lymphoid hyperplasia(RLH).Compared with the control group,the expression of AMOT in the tissue samples of DLBCL patients significantly decreased.2.The results of real-time quantitative PCR and Western blot detection showed that,the mRNA and protein expression levels of AMOT in human DLBCL cell lines LY1,LY8 and Val was significantly decreased compared with the healthy volunteers PBMCs(control group)(p<0.001).3.Statistical analysis of the clinical data of the above 52 DLBCL patients showed that the expression level of AMOT protein was negatively correlated with the serum lactate dehydrogenase(P=0.023),IPI score(P=0.017)and NCCN-IPI score(P=0.005),while was irrelevant with the age(P=0.219),Gender(P=0.991),Ann Arbor staging(P=0.12),B symptoms(P=0.245),disease subtype(P=0.1)and the extranodal invasion(P=0.458)of the patients.We performed Kaplan-Meier analysis on 28 DLBCL patients and found that the survival time of patients with positive AMOT expression was significantly longer than that of patients with negative AMOT expression(p=0.035).Conclusions:This study showed that the expression of AMOT in DLBCL tissues and cell lines was reduced,and low expression of AMOT was associated with the poor prognosis of DLBCL patients.AMOT may serve as a potential biomarker for pathological diagnosis and prognosis stratification of DLBCL.Part ? The biological functions of AMOT in diffuse large B-cell lymphomaObjective:The research of molecular targets is the main strategy to study the pathogenesis of DLBCL and find new stratification and therapeutic targets.Numerous studies had shown that AMOT can participate in the occurrence and development of tumors by converting Hippo/YAP1,Wnt/catenin and other signal pathways,promoting or inhibiting the development of cancers.Therefore,the relationship between AMOT and cancers deserves a deeper discussion.The purpose of the second part study is to explore the effects of AMOT on the proliferation,cell cycle and other characteristics of DLBCL,explain the molecular biological mechanisms and provide new strategies for targeted therapy of DLBCLMaterial and Methods:1.Cell culture;2.Cell transfection by lentivirus vectors either encoding AMOT or empty lentivirus vectors;3.Real-time quantitative polymerase chain reaction(qRT-PCR);4.Protein isolation and Western blotting;5.Cell Counting Kit-8(CCK-8);6.Propidium iodide(PI)staining;7.Annexin V PE/7-aminoactinomycin D(7-AAD)assay;8.Statistical analysis.Results:1.AMOT overexpression lentivirus vectors(LV-AMOT)and empty vector control(LV-Con)was used to stably transfect DLBCL cell lines(LY1 and LY8).CCK8 assay shown that the proliferation activity of DLBCL cells decreased significantly in the LV-AMOT groups.2.CCK8 assay shown that tankyrase inhibitors XAV939 and JW55 could inhibit the proliferation activity of LY1 and LY8 cells in a concentration-dependent and time-dependent manner.Western blot detection shown that after treating with XAV939 and JW55 respectively,the level of AMOT protein in the LY1 and LY8 cells significantly increased.3.Flow cytometry analysis indicated that overexpressed AMOT in DLBCL cells resulted in G1 phase arrest,but the apoptosis rate did not change.Western blot detection revealed that the phosphorylation levels of key proteins ATM(Ser1981),ATR(Ser428),Chkl(Ser345),Chk2(Thr68)and H2A.X(Ser139)in the DNA damage response(DDR)pathway of LV-AMOT group were significantly decreased,but there was no change in the mRNA expression level of related genes.4.Increased sensitivity to cytotoxic drug doxorubicin was observed in LV-AMOT group cells,which was manifested by decreased proliferation activity,G1 phase arrest of the cell cycle,and increased apoptosis rate.Western blot detection revealed that after treatment with doxorubicin,the phosphorylation levels of ATM(Ser1981),ATR(Ser428),Chkl(Ser345),Chk2(Thr68)and H2A.X(Ser139)in the LV-AMOT group were significantly lower than the control group.Conclusions:This study confirmed that AMOT acts as a tumor suppressor in DLBCL by inhibiting the proliferation,inducing G1 arrest,and increasing the sensitivity of DLBCL cells to doxorubicin.AMOT can restrain the phosphorylation activation of key proteins in the DDR pathway of DLBCL cells,thereby suppressing the ability of DLBCL cells to repair DNA damage.AMOT is expected to become a novel point of targeted drug combination chemotherapy for DLBCL patients.