The Mechanisms Of MELK Involved In The Development And Chemoresistance In Uterine Leiomyosarcoma

BackgroundUterine leiomyosarcoma(ULMS)is the most common uterine sarcoma,accounting for about 1-2%of all uterine malignancies1.Despite low incidence of the disease,it is the most lethal gynecologic malignancy,and the 5-year survival rate is quite low at about 15?25%.Even for the patients in early stages(FIGO ?-?)of the disease,the recurrence rate is as high as 53?71%and the overall survival(OS)is below 40%2 which is due to its high invasive capacity and frequent insensitivity to chemotherapy.Poor treatment of uterine leiomyosarcoma remains a global challenge.The main reason is that the mechanism of its occurrence and development is still unclear and the insensitivity of chemotherapeutic drugs has not been elucidated.MELK,a well-known oncogenic gene,was first depicted by analyzing the gene landscape of progenitor cells in pediatric brain tumors.Overexpression of MELK has been detected in many kinds of tumors including colorectal cancer,gastric cancer,prostate cancer,glioblastomas,melanoma,and breast cancer.In addition,high MELK expression level is associated with shorter survival in breast cancer patients.Previous studies have shown that MELK may affect cell cycle regulation,cell proliferation,apoptosis and metastasis.As a well-known molecule in tumor studies,the reason for the abnormal high expression of MELK in uterine leiomyosarcoma and the molecular mechanism involved in the regulation are still unclear.The question of which molecules MELK interacts with in uterine leiomyosarcoma and which downstream target molecules are involved in tumorigenesis and drug resistance remains to be explored.Therefore,elucidating the molecular mechanism of MELK in uterine leiomyosarcoma and its tumorigenesis,development and drug resistance will play a crucial role in the diagnosis,treatment and prognosis of this disease.This study intends to systematically investigate the molecular mechanism of MELK in the regulation of uterine leiomyosarcoma,and explore the relationship between MELK and the occurrence,development and drug resistance of uterine leiomyosarcoma,which Specifically including the following three parts:Part I:The genomic features and global gene expression analysis of uterine leiomyosarcomaObjectUterine leiomyosarcoma is the most common uterine sarcoma and it is the most lethal gynecologic malignancy.Genomic instability is one of the most important characteristics of tumors,which is the early event of tumor development,and tumorigenesis is the continuation of genomic instability.There are two main types of genomic instability:nucleotide instability and chromosomal instability(CIN),which occur by different but overlapping mechanisms.How to fully analyze the "genomic instability" of uterine leiomyosarcoma from the genome level is the key to identify the "genomic instability" of ULMS.The purpose of this part is to comprehensively analyze the characteristics of uterine leiomyosarcoma genomics by means of whole genome sequencing and expression profiling,so as to provide a basis for exploring the pathogenesis of uterine leiomyosarcoma.Contents and methods1.Whole Genomic Sequencing(WGS)was performed on 15 fresh couples of ULMS and normal controls.Target Region Sequencing(TRS)was performed on 50 ULMS fresh tissues.2.12 frozen ULMS tissues and 12 matched control tissues(6 uterine leiomyomas and 6 myometrium)were analyzed for genome-wide expression profile(Illumina Human ht-12 v4 Expression BeadChip).3.27 cases of ULMS Tissue microarray(TMA)were investigated for immunohistochemical staining of UBE2C,TOP2A,PRC1,AURKA,CDC20,BUB1 and other molecules to evaluate the expression of proteins.Results1.The genetic instability of uterine leiomyosarcoma was mainly manifested in chromosome instability.Results of WGS and TRS showed that the frequency of somatic gene mutation in the ULMS exon region was only 1.4±0.09/Mb,which was significantly lower than that of ovarian cancer,endometrial serous cancer,liver cancer,lung cancer,gastric cancer,colorectal cancer,etc.Secondly,P53 mutation was only found in 27.6%(8/29)of ULMS,and few mutations were found in BRCA1/2,ATM,MUTYH and other genes related to DNA repair(<5%).Furthermore,14 cases of ULMS presented ploidy abnormalities,among which 8 cases presented significant ploidy amplification and 6 cases presented significant ploidy deletion.One case of ULMS(ULMS17-T)presented normal ploidy,possibly due to sequencing error caused by tumor purity of only 28%.Finally,the analysis of somatic copy number variation indicated that 10q,12q,2p and 16p were significantly absent,and no significantly different amplification regions were found,which may be due to the small sample size.2.The results of expression profile analysis showed that a number of mitotic proteins including UBE2C,TOP2A,PRC1,AURKA,CDC20,BUB1 and PTTG3P were significantly elevated.The GO pathway enrichment analysis revealed that 88 genes with significant differences were enriched in each mitotic pathway.MELK was the most differentially expressed gene(Fold Change=13.60437,P=5.56E-9).3.Immunohistochemical results confirmed that the expressions of UBE2C,TOP2A,PRC1,AURKA and CDC20 were significantly increased in ULMS compared with normal uterine leiomyomas and myometrium.Conclusion1.The genome characteristics of uterine leiomyosarcoma were mainly characterized by chromosome instability rather than nucleotide instability,and DNA repair related gene mutations were not the mechanism of ULMS genome instability.2.Mitotic related proteins were significantly increased in ULMS,and mitotic abnormalities may be the pathogenesis of ULMS genomic instability.In addition,MELK was the most differential gene.Part ?:MELK promotes occurrence,development and chromosomal instability in uterine leiomyosarcoma by phosphorylating RB1 and MAD2L1ObjectMELK has been demonstrated to be a very strong oncogene in breast cancer,colorectal cancer and many other kinds of tumors.It is involved in many important processes such as cell proliferation,invasion,migration and cell differentiation in malignant tumors.This study has performed exon sequencing in uterine leiomyosarcomas and normal myometriums and found that MELK was highly expressed in uterine leiomyosarcoma.This part aimed to explore the influence of MELK on the biological function of uterine leiomyosarcoma in vitro and in vivo,and to explore the mechanism of MELK promoting the occurrence and development of uterine leiomyosarcoma.Contents and methods1.Vectors with MELK overexpression and suppression were constructed,and uterine leiomyosarcoma cell lines with MELK overexpression and suppression were established.EdU assay,MTT proliferation assay,clonogenic assay,cell cycle assay,migration and invasion assays to investigate the effect of MELK on the metastasis,proliferation and cell cycle of uterine leiomyosarcoma cell lines.In addition,western blot was used to detect the impact of MELK on the expression of important pathways,epithelial-mesenchymal transition(EMT)and cell cycle proteins.2.Immunofluorescence co-location,co-immunoprecipitation and immunohistochemical correlation were used to investigate the interaction of MELK and its target molecules.The close relationship between MELK and the occurrence and development of uterine leiomyosarcoma was explored in combination with the molecular pathways involved in target molecules and their effects on tumor development.3.To investigate whether MELK triggers the occurrence of uterine leiomyosarcoma,MELK overexpressed myometrium cell line and leiomyoma cell line were established.We then explored whether MELK could promote the biological function of malignant tumors in normal cell lines by the use of MTT proliferation assay,clonogenic assay and migration assay.Additionally,the the established cell lines were used in vivo.Nuclear division and atypia were observed after subcutaneous or subcapsular tumor formation.Results1.MELK is upregulated in ULMS and is a poor-prognosis marker of aggressive ULMS.According to data obtained from GEO and the immunohistochemistry of ULMS patients,we found that MELK expression was significantly upregulated in ULMS compared to ULM and MM.Moreover,data from GEO and immunohistochemistry also revealed that the expression of MELK is higher in ULMS with poor prognosis.2.MELK can promote cell proliferation and cell cycle,invasion and migration of ULMS,and affect the expression of proteins involved in cell cycle and EMT.MELK overexpression and suppression ULMS cell lines were established.Then functional experiments in vitro proved that MELK could promote cell cycle,cell proliferation,clone formation,invasion and migration of ULMS cell lines.Meanwhile,western blot showed that with the overexpression of MELK,the expression levels of cell cycle proteins CCND1,CCNE1,CKD4 and CDK6 also increased,and the expression levels of p21 and p27 decreased.As for EMT proteins,the expression of n-cadherin,Slug,Snail and Vimentin also increased,and the expression of e-cadherin decreased.It was demonstrated that the MELK inhibitor,OTSSP167,could significantly inhibit the growth of ULMS with the subcutaneous tumor formation and oral feeding drug experiment in vivo.3.MELK can phosphorylate RB1 and MAD2L1,which could lead to the chromosomal instability(CIN).By means of co-immunoprecipitation,it was proved that both MAD2 and RB1 could bind to MELK,and the phosphorylation levels of MAD2 and RB1 increased after MELK was overexpressed.With the phosphorylation of RBI 3 the expression level of E2F increased,and the expression of cell cycle-related proteins also increased,which then accelerated the cell cycle.,we can find that the expression levels of MAD2 and MELK have a strong correlation through the immunohistochemical correlation analysis of ULMS patients.Moreover,the co-localization of MAD2 and MELK was demonstrated by immunofluorescence.When MAD2 was phosphorylated,it is converted from the active form of C-MAD2 to the inactive form of O-MAD2,which can not only improve the mitosis index of cells,but also lead to the loss of mitosis checkpoint activity,leading to the release of CDC20 and the activation of APC/C.And then unequal mitosis occurs,resulting in CIN.4.MELK could promote the malignant transformation in myometrium and leiomyoma cells.MELK overexpressed myometrium cell lines and leiomyoma cell lines were constructed.The functional experiments showed that MELK could promote the proliferation and migration of myometrium cell lines and leiomyoma cell lines.And meanwhile,the tumorigenesis ability of leiomyoma cell lines and myometrium cell lines was obviously enhanced.And the uclear division and atypia were increased after the overexpression of MELK in myometrium cell lines and leiomyoma cell lines.subcutaneous or subcapsular tumor formation.Conclusion1.MELK is highly expressed in uterine leiomyosarcoma and is associated with poor prognosis.2.MELK can promote the proliferation,cell cycle,invasion,migration and other biological functions of ULMS.3.MELK caused mitotic checkpoint dysfunction and mitotic abnormalities by phosphorylating MAD2,and MELK could also phosphorylate RB1,which leaded to the acceleration of cell cycle.With the combination of these,an abnormal number of chromosomes occurred,resulting in chromosome instability.4.MELK can promote the malignant transformation of myometrium cells and leiomyoma cells,and result in the occurrence and development of uterine leiomyosarcoma.Part III MELK leads to doxorubicin chemoresistance via the miR-34a/JAK2/STAT3 pathway in uterine leiomyosarcomaObjectULMS is commonly insensitive to chemotherapy.In the National Comprehensive Cancer Network(NCCN)2019 treatment guidelines for uterine neoplasms,doxorubicin and gemcitabine/docetaxel remain the most effective regimensin for treating advanced or recurrent disease.However,currently available cytotoxic regimens remain inadequate with a 5-year disease-specific survival of<30%.In a phase III trial containing women with uterine leiomyosarcoma,administering doxorubicin(60 mg/m2)showed objective response in 25%among those with uterine leiomyosarcoma.In the same study,the addition of dimethyl triazenoimidazole carboxamide did not significantly improve treatment response rates.The response rates of current regimens are low.Other therapeutic options have not seemed to yield promising results.Adjuvant radiotherapy(RT)also suggested no appreciable or consistent improvement in the overall survival of ULMS patients.Therefore,this part aims to explore the relevant mechanism of MELK's participation in the chemotherapy resistance of ULMS,and to provide new targets and ideas for the treatment of ULMS.Contents and methods1.Uterine leiomyosarcoma cell lines with MELK overexpression and suppression were established.The effects of MELK on drug resistance to doxorubicin in ULMS were investigated by doxorubicin cytotoxic assay,clonogenic assay with different concentrations of doxorubicin,cell proliferation assay with low concentrations of doxorubicin and western blot assay.2.We compared the differences of mRNA and miRNA expression in MELK overexpressed and negative control cell lines with or without doxorubicin by mRNA sequencing and miRNA sequencing.And then we analyzed molecular pathways that might be involved in doxorubicin resistance.Apoptosis was detected by flow detection assay after doxorubicin was added.Western blot and qRT-PCR were used to investigate the molecular pathways and miRNAs involved in MELK affecting doxorubicin resistance in ULMS.3.Western blot was used to investigate the effect of MELK on the polarization of tumor associate macrophages and the molecular pathways involved.MiRNA with GFP transfection experiments,exosome qRT-PCR and other experiments were used to explore the ways that MELK and the downstream molecule mir-34a participate in the regulation of macrophage polarization,and luciferase reporter experiment was used to explore the target genes of mir-34a.ELISA was used to detect the difference of IL6 secreted by m2-type and m0-type tumor-related macrophages,and the effect of m2-type tumor-related macrophages on drug resistance of ULMS to doxorubicin was investigated by doxorubicin cytotoxicity.Results1.MELK can lead to doxorubicin chemoresistance in ULMS cells.Cytotoxic assay revealed that at various final concentrations,the relative cell viability of cells with MELK knockdown declined much more rapidly than NC cells.Additionally,the relative viability of cells with MELK overexpression declined much more slowly than NC cells.Moreover,in the proliferation assay,relative viability of ULMS cells overexpressing MELK was significantly higher than NC cells.Similarly,MELK expression remarkably increased colony formation in ULMS cell lines.And conversely,MELK suppression decreased colony formation.Also,significantly increased MELK expression in SK-UT-1 cells was seen simultaneously with increasing doxorubicin concentrations.we found that the tumor size and weight of doxorubicin combined with the MELK inhibitor OTSSP167 were significantly smaller than those of doxorubicin alone(0.1618±0.01732g,0.2900±0.02153g,P<0.01)in vivo.2.mRNA sequencing showed that the differentially expressed genes were concentrated in the Interleukin-signaling pathway(Signaling by Interleukins,P=7.15E-5).Interleukin 4 and 13 signaling(P=3.1E-6),Cytokine-Cytokine receptor interaction(P=3.9E-4),etc.IL6 was involved in the above pathways,and the expression difference was significant(P=8.76E-6).miRNA sequencing showed that mirna-34a was significantly reduced in both MELK overexpression and doxorubicin treated cells.3.The effect of MELK on chemoresistance in ULMS cells was anti-apoptosis via the JAK2/STAT3 pathway and the decline of miR-34a.The result of qRT-PCR revealed that MELK could significantly down-regulate miRNA-34a expression in ULMS cells.Western blot showed that MELK overexpression promoted higher levels of phosphor-JAK2 and phosphor-STAT3,Conversely,MELK inhibition reduced JAK2 and STAT3 phosphorylation.Additionally,results showed an increase of BCL2 in MELK overexpressing cells and a decrease of BCL2 in MELK suppressed cells.Finally,the apoptosis assay was conducted,and results indicated that MELK could restrain the apoptosis that is usually induced by doxorubicin treatment.4.MELK induced M2 macrophage polarization through the exosome via miR-34a/JAK2/STAT3 pathway.Macrophages incubated with the medium of MELK overexpressing SK-UT-1 and SK-UT-1B cells showed significantly increased expression of CD206 and Argl(M2 macrophage markers),as well as markedly decreased expression of INOS(Ml macrophage marker).MiRNA with GFP transfection experiments indicated that miR-34a could be transported from ULMS cells to macrophages through exosome.Luciferase was conducted to prove that IL6 is the target gene of miR-34a.Western blots also showed remarkably higher phosphorylation of JAK2 and STAT3 in macrophages cultured with conditioned medium of ULMS cells overexpressing MELK and significantly lower phosphorylation of JAK2 and STAT3 in the macrophages cultured with conditioned medium of ULMS cells suppressing MELK.5.M2 macrophages could secrete IL6 and promote the resistance of uterine smooth muscle cells to doxorubicin.ELISA assessment of IL-6 concentrations revealed that the IL-6 was significantly higher in cultured medium of M2 macrophages.And additionally,ELISA also showed that IL-6 concentration was significantly higher in macrophages treated with conditioned medium of SK-UT-1 cells overexpressing MELK than in macrophages treated with conditioned medium of NC cells.The cytotoxic assay results showed that the relative cell viability of SK-UT-1 cells treated with IL-6 declined much more slowly than that of the untreated cells.Moreover,the results also showed that viability of SK-UT-1 cells treated with conditioned medium of M2 macrophages declined much more slowly than cells treated with conditioned medium of normal macrophages.Conclusion1.MELK induces chemoresistance through an anti-apoptotic mechanism via the JAK2/STAT3 pathway in ULMS cells.2.MELK overexpression could induce M2 macrophage polarization via the miR-34a/JAK2/STAT3 pathway;contributing to doxorubicin chemoresistance in the tumor microenvironment(TME).Conclusion1.The genome characteristics of uterine leiomyosarcoma are mainly characterized by chromosome instability rather than nucleotide instability.2.MELK caused mitotic abnormalities by phosphorylating MAD2,and acceleration of cell cycle by phosphorylating RB1.With the combination of these,an abnormal number of chromosomes occurred,resulting in chromosome instability.3.MELK induces doxorubicin chemoresistance through anti-apoptotic mechanism and M2 macrophage polarization via the miR-34a/JAK2/STAT3 pathway in ULMS.The innovations of this study1.For the first time,whole genome sequencing was performed using fresh tissue of uterine leiomyosarcoma to explore the genomics characteristics of uterine leiomyosarcoma in our country,providing strong basic information for the study of the pathogenesis of uterine leiomyosarcoma.2.Exon sequencing was performed for the first time in a large number of samples of uterine leiomyosarcoma tissues to compare the differential genes and the physiological processes involved in the differential genes,providing strong evidence for the exploration of the pathogenesis of uterine leiomyosarcoma.3.For the first time,MELK inactivates mitotic checkpoint function by phosphorylation of MAD2,leading to chromosomal instability.And additionally,MELK has been shown to accelerate the cell cycle by phosphorylating RB1.All of these lead to the occurrence development of uterus leiomyosarcoma.It provides a new idea for the treatment of uterine leiomyosarcoma.4.It was first demonstrated that MELK inhibited cell apoptosis by activating JAK2/STAT3/mir-34a/IL6R,a circular feedback pathway,and promoted M2 polarization of tumor-associated macrophages,leading to uterine leiomyosarcoma resistance to doxorubicin.It provides a new sensitizing target for chemotherapy resistance of uterine leiomyosarcoma.The defects of this study1.This study is a pre-clinical basic research,and it still needs to go through several research stages to achieve clinical application transformation.2.There are many mechanisms of MELK,and the molecules interacting with it are involved in multiple processes of cell metabolism.In this study,only a small part of the involved pathways were involved,and no other possible molecular pathways were explored.3.Patient-Derived tumor Xenograft(PDX)model is not introduced in this study.If PDX model of several patients can be established,the research results can be further proved and the reliability of the study can be improved.