Prognostic Analysis In Molecular Subtypes Of Ovarian Cancer And The Underlying Mechanism Of KIAA0101 In Serous Ovarian Cancer

Backgrounds and objectiveOvarian cancer is one of the three female reproductive system malignancies,and its mortality is the highest among all gynecological malignancies.There are no obvious symptoms in the initial stage of ovarian cancer,about 75%of patients have been treated at advanced stage(? and ?).Serous ovarian cancer is the most common subtype of ovarian cancer,with rapid growth,and it is prone to peritoneal metastasis early and chemotherapy resistance.Patients with ovarian cancer treated with cytoreductive surgery and chemotherapy by platinum combined paclitaxel to achieve complete or partial clinical remission,still face the risk of recurrence,metastasis or death,the 5-year survival rate was only about 30%.The fundamental reason for poor prognosis of ovarian cancer is lack of effective means of early diagnosis,prognostic indicators and the molecular mechanism of development of ovarian cancer is still not clear.Looking for specific markers for early screening of ovarian cancer and new targets for the treatment of ovarian cancer is the focus and difficulty of ovarian cancer,these will be conducive to the early diagnosis and treatment of ovarian cancer so as to improve prognosis and reduce mortality.According to The Cancer Genome Atlas Research Network,HGSOC could be divided into four molecular subtypes:immunoreactive,proliferative,differentiated and mesenchymal.T-cell chemokine ligands and the receptor characterized the immunoreactive subtype,such as CXCL11,CXCL10 and CXCR3.HMGA2,SOX11,MCM2,PCNA were the representatives for the proliferative subtype.The differentiated subtype was associated with MUC1,MUC16,SLPI.HOX,FAP,ANGPTL2,ANGPTL1 characterized the mesenchymal subtype.Molecular subtypes of ovarian cancer by gene expression profiling were related to clinical and pathologic features.On clinics,there are few patients with mesenchymal subtype,and most patients can be divided into immunoreactive,proliferative,and differentiated subtypes.In this study,we aimed to evaluate the prognostic efficacy of the aforementioned three common molecular subtypes of HGSOC-immunoreactive subtype,proliferative subtype and differentiated subtype.We analyzed CXCL11,HMGA2,MUC16 proteins expression by using the immunohistochemistry in an independent tissue microarray(TMA)HGSOC set.We aimed to derive an immunohistochemistry-based multi-protein signature model for clinical risk stratification approaches of HGSOC tissue samples.In this study group,according to studies on molecular subtypes of HGSOC,different subtypes have different prognosis,and proliferation subtype plays a particularly significant role in the prognosis.As an important representative molecule of ovarian cancer proliferation subtype,PCNA can be specifically binded to the conserved binding site by P15PAF(ie,KIAA0101).In this research,through expression microarray technology combined with protein mass spectrometry analysis,we found that the expression of KIAA0101 was significantly higher in patients with poor prognosis.In a short,the expression of KIAA0101 was higher in serous ovarian canceT(SOC)than in normal fallopian tube.Moreover,the expression of KIAA0101 in serous ovarian cancer was directly correlated with the prognosis of patients.The expression of KIAA0101 is higher,the prognosis of SOC is poorer.KIAA0101 is mainly involved in DNA repair,cell proliferation and cell cycle progression.It has been reported in the literature that KIAA0101 plays an oncogenic role in gastric cancer,esophageal cancer,breast cancer,renal cell carcinoma,and hepatocellular carcinoma,and overexpression of KIAA0101 has prognostic significance.Based on high-throughput sequencing results and previous literature studies,we hypothesize that KIAA0101 may play an oncogenic role in SOC and is associated with patient prognosis.PI3K/AKT/mTOR pathway is an important intracellular signaling cascade.This signaling pathway is involved in cell cycle regulation,cell proliferation,apoptosis,and autophagy.Its abnormal activation is related to cell transformation,tumorigenesis,tumor progression and chemotherapy resistance.The PI3K/AKT/mTOR signal pathway is one of the most disordered signal pathways in many human malignancies including ovarian cancer.The PI3K/AKT/mTOR pathway is frequently activated in ovarian cancer and plays a key role in the development of ovarian cancer and is closely related to the poor prognosis of ovarian cancer.This topic mainly discusses the relationship between ovarian cancer molecular subtypes and prognosis,studies the effects of different markers of different molecular subtypes on prognosis,aims to combine markers of different subtypes to build a mathematical model,which could predict prognosis and guide clinical treatment.KIAA0101,a binding molecule of proliferative subtype PCNA,was selected to study its regulation mechanism in serous ovarian cancer.The research mainly includes the following three parts:Part ?:The prognosis of ovarian cancer molecular subtypesPart ?:The underlying molecular mechanism of KIAA0101 for proliferation and metastasis in serous ovarian cancerPart ?:The underlying molecular mechanism of KIAA0101 for chemotherapy resistance in serous ovarian cancerPart ?:The prognosis of ovarian cancer molecular subtypes Backgrounds and objective:Epithelial ovarian cancer is the most lethal gynecologic malignancy,largely because that most patients are diagnosed in advanced stages.Nearly 70%of epithelial ovarian cancer is HGSOC.There are currently no effective screening methods for early diagnosis of ovarian cancer.There are no specific markers that can accurately predict the clinical outcome of HGSOC.According to the cancer genomic map research network,HGSOC can be divided into immunoreactive,proliferative,differentiated and interstitial 4 subtypes.CXCL11 is representative of immunoreactive subtypes,and CXCL11 can bind to CXCR3 receptors.The high expression of CXCL11 is associated with poor prognosis in non-small cell lung cancer,colorectal cancer,renal cell carcinoma and gastric adenocarcinoma.There have been no studies on the effects of CXCL11 in HGSOC.HMGA2 is representative of proliferative subtypes.HMGA2 is a member of the high mobility family A protein family.HMGA2 is a proto-oncogene involved in cell growth,differentiation,apoptosis,and tumorigenesis.HMGA2 plays an important role in the pathogenesis of various human tumors.MUC16 is a representative of differentiated subtypes.CA125 is a repeat peptide epitope of the MUC16 protein and is an important biomarker for monitoring ovarian cancer.MUC16 may promote the occurrence of epithelial ovarian cancer.The relationship between MUC16 expression and the prognosis of ovarian cancer is controversial.In this study,tissue microarray(TMA)was used to perform immunohistochemical staining on representative molecules of the three common subtypes of the HGSOC:immune subtypes,proliferative subtypes,and differentiation subtypes.The staining results were quantitatively analyzed and judged by two pathologists separately.This study aimed to establish a multi-protein model that can be used to assess the clinical outcome of HGSOC.Materials and methods:By the use of TMA,we performed immunohistochemical staining on CXCL11,HMGA2,and MUC16.According to the staining results,they were divided into low-expression group and high-expression group.The clinical information of HGSOC patients was summarized,and the expression of CXCL11,HMGA2,and MUC16 was analyzed.Univariate and multivariate Cox proportional hazards regression analysis was applied to assess the association between biomarker expression and clinical outcome.Based on the correlation between immuno-histochemistry and prognosis of CXCL11,HMGA2,and MUC16,the three markers were combined to generate a highly accurate protein model that can be used for prognostic evaluation.Results:1.Immunohistochemical staining of different molecular subtypes of HGSOCThe immunohistochemical staining results of representative molecules of the three subtypes were divided into four different intensity stainings:negative staining,weak staining,medium staining,and strong staining.Among them,no staining and weak staining were defined as low expression,medium staining and strong staining were defined as high expression.CXCL11 and MUC16 showed cytoplasmic staining,and HMGA2 mainly showed nuclear staining.2.The predictive value of three markers on the prognosis of HGSOCThe expression of CXCL11 in HGSOC was significantly higher than that of the fallopian tube.The overall survival time of the CXCL11 high expression group was significantly lower than that of the CXCL11 low expression group.However,there was no significant difference on progression free survival time between the two groups.The expression of HMGA2 in HGSOC was significantly higher than that of the fallopian tube.The overall survival time and progression-free survival time of the HMGA2 high expression group were significantly lower than those of the HMGA2 low expression group.MUC16 expression was not associated with prognosis.3.Derivation of multi-protein models based on clinical outcomesBased on the results of immunohistochemistry,CXCL11 and HMGA2 were combined to establish a 2-protein signature for predicting the prognosis of HGSOC.The results showed that the 2-protein signature model was an independent predictor of overall and progression-free survival of HGSOC.4.Prediction value of multi-protein signatureTo test the predictive efficacy of the 2-protein signature,we compared the sensitivity,specificity,and area under the curve(AUC)of CXCL11,HMGA2 and 2-protein signature.The sensitivity and specificity of the 2-protein signature was 75.00%,64.50%(P = 0.002),AUC was 0.694.The 2-protein signature has a great predictive value for HGSOC prognosis.Conclusions:1.The expression of CXCL11 and HMGA2 were higher in HGSOC than those in normal fallopian tube,and high expression suggested that poor prognosis of HGSOC.MUC16 is not associated with poor prognosis of HGSOC.2.The 2-protein signature was an independent risk factor for poor prognosis of HGSOC.3.The 2-protein signature would be used clinically to determine the prognosis of HGSOCs and to guide clinically personalized treatment.Part II:The underlying molecular mechanism of KIAA0101 for proliferation and metastasis in serous oyarian cancerBackground and objective:Ovarian cancer is one of the gynecological malignancies that endanger the health of women worldwide,with high mortality and poor prognosis,mainly due to the rapid growth and the strong invasion ability,early metastasis.The underlying reason is that the pathogenesis of ovarian cancer is not yet clear.KIAA0101 is closely related to DNA damage repair and cell proliferation.Although KIAA0101 has been reported to play a role in various malignancies,its role and mechanism in SOC have not yet been clarified.TCGA data revealed that approximately 87%SOC had abnormal FOXM1 pathway activation.Our previous expression microarray sequencing results revealed that FOXM1 was highly expressed in SOC.FOXM1 can promote malignant transformation and participate in tumor progression,such as tumor proliferation,invasion and metastasis,angiogenesis,chemotherapy resistance and many other processes.Lots of studies have demonstrated that FOXM1 played an important role in the development of SOC.FOXM1 is an important transcription factor that can regulate the expression of a series of important downstream genes.Through CHIP-seq combined with bioinformatics analysis,KIAA0101 might be a downstream target gene of FOXM1,but the specific regulation of FOXM1 on KIAA0101 and the regulatory site were not yet clear.This study aimed to investigate the specific mechanism that FOXM1 regulated KIAA0101 by luciferase,rescue experiments.In this study,the results of KIAA0101 immunohistochemical staining in a large number of clinical specimens were analyzed to investigate its relationship with prognosis.KIAA0101 was knocked down or overexpressed in ovarian cancer cell lines to study the proliferation and metastasis of ovarian cancer cells.This study also explored the specific mechanism of FOXM1 regulating the proliferation and metastasis of KIAA0101 through luciferase,rescue,etc.Materials and methods:The expression levels of KIAA0101 in SOC and normal FT were detected by RT-qPCR and western blot.Using the three tissue microarrays(TMA)containing FT and SOC made by this laboratory,KIAAO101 immunohistochemical staining was performed to detect the differences in the expression of KIAA0101 in FT and SOC and relationship between KIAA0101 expression and the prognosis of SOC.Multiple ovarian cancer cell lines(A2780,HO8910,and SKOV3)were selected and KIAA0101 was overexpressed or knocked down with lentiviral packaging system to construct stable cell lines.In order to study the role of KIAA0101 in proliferation,we performed MTT assay to detect the growth curve,plate cloning formation to study the colony formation ability,flow cytometry to investigate the cell cycle distribution.For proliferation,the changes of CCNA2,CCNB1,CCND1,CCNE1,P21,and P27 were studied by western blot.Scratch test was used to detect the change of cell wound healing ability.Transwell chamber was used to detect the migration and invasion of ovarian cancer cell lines.For metastasis,the changes of E-cad,N-cad,ZEB1,MMP2,MMP9,and Snail were detected by western blot.Through immunofluorescence techniques,the intracellular localization of FOXM1 and KIAA0101 was detected.Immunohisto-chemistry was performed with FOXM1 and KIAA0101.According to the immunohistochemistry,there were four subgroups:FOXM1 low expression group,FOXM1 high expression group,KIAA0101 low expression group,and KIAA0101 high expression group.Chi square test was applied to observe the expression correlation between FOXM1 and KIAA0101.To investigate the prognosis of FOXM1 on ovarian cancer,KM plotter was used to analyze the overall survival time and progression-free survival time of FOXM1 on ovarian cancer.FOXM1 siRNA used to downregulate FOXMI in A2780,H08910 and SKOV3,RNA and protein were extracted,and RT-qPCR and western blot were performed to detect the change of KIAA0101 expression after FOXM1 knockdown.Using bioinformatics methods,the correlation of mRNA expression of FOXM1 and KIAA0101 in the TCGA database was analyzed to further verify the regulation of KIAA0101 by FOXM1.The promoter sequence of KIAA0101 was obtained by Ensembl,and potential regulatory sequences of FOXM1 on KIAA0101 was obtained by Gene Regulation combined literatures.The promoter of KIAA0101 was amplified and cloned into pGL4.26 to generate the wild-type(WT)promoter.For mutant(MT)plasmids,construction was performed using overlap extension PCR.Three classical mutations and one non-canonical mutation(?CHR)were constructed.Luciferase activity was measured using the Dual-Glo luciferase assay system and statistical analysis was performed.Through rescue experiments,we observed that whether knockdown of FOXM1 could rescue the promoted effects of KIAA0101 in ovarian cancer cells to verify the regulation of KIAA0101 by FOXM1.Results:1.The expression of KIAA0101 in SOC is higher and correlated with the prognosis of SOC patientsKIAA0101 was highly expressed in ovarian cancer:RT-qPCR detected that the expression of KIAA0101 mRNA in SOC was significantly higher than that in normal controls.Western blot and immunohistochemistry showed that the expression of KIAA0101 protein in SOC was significantly higher than that in normal control.The expression of KIAA0101 correlated with the prognosis of SOC:According to the results of immunohistochemical staining,the expression of KIAA0101 was divided into two groups:high expression group and low expression group.The prognosis of KIAA0101 in high expression group was significantly poorer than KIAA0101 low expression group.The two-year and five-year survival rate of patients with high KIAA0101 expression were shorter than those with low KIAA0101 expression.The expression of KIAA0101 was also associated with the FIGO stage.2.KIAA0101 promoted the proliferation of serous ovarian cancer cellsWhen KIAA0101 overexpressed in ovarian cancer cell lines,MTT assay showed that the growth rate was accelerated,and plate cloning suggested that clone formation ability was enhanced.KIAA0101 knockout in ovarian cancer cell line slowed done the cell growth rate and reduced the colony forming ability.KIAA0101 affected the cell cycle distribution of ovarian cancer cells.Adenosine double-blocks to synchronize A2780 PLKO.1-NC and A2780 PLKO.1-shKIAA0101 cells to observe the influence of KIAA0101 in cell cycle distribution,and cell cycle analysis revealed that knockdown of KIAA0101 caused G1/S arrest in A2780 cell line.Western blot of cell proliferation and cell cycle-related proteins suggested that the expression of CCNA2,CCNB1,CCND1 and CCNE1 was down-regulated and the expression of P27 and P21 was up-regulated after KIAA0101 silencing.KIAA0101 overexpression reversed the results.3.KIAA0101 promoted metastasis of serous ovarian cancer cellsKIAA0101 overexpression accelerated migration and invasion of ovarian cancer cells in transwell chamber.KIAA0101 knockdown decreased migration and invasion of ovarian cancer cells in transwell chamber and prolonged the wound healing time of the' scratch test.Western blot showed that KIAA0101 could regulate epithelial to mesenchymal transition(EMT).The overexpression of KIAA0101 decreased the expression of E-cadherin and increased the interstitial markers such as N-cad,ZEB1,MMP2,MMP9 and Snail.After KIAA0101 knockdown,epithelial markers increased and interstitial markers decreased.4.FOXM1 regulated the expression of KIAA0101Immunofluorescence co-localization showed that both FOXM1 and KIAA0101 were located in the nucleus.Immunohistochemical analysis of 116 HGSOC patients showed that FOXM1 had a certain correlation with KIAA0101 expression.TCGA database analysis showed that the expression levels of FOXM1 and KIAA0101 in TCGA ovarian cancer were correlated.FOXM1 was knockdown with siRNA in A2780,HO8910 and SKOV3,the mRNA and protein levels of FOXM1 were significantly decreased,meanwhile the mRNA and protein expression of KIAA0101 were also significantly downregulated.5.FOXM1 transcriptionally activated the promoter of KIAA0101 and regulated its expressionWe amplified 3 segments of promoter sequences containing potential binding sites for FOXM1,which were above the 5' transcription initiation site:544 bp(-712/-169),519 bp(-2244/-1726),496 bp(-2700/-2205)were ligated into the PGL4.26 plasmid.The luciferase reporter system showed that only 496 bp(-2700/-2205)of the 3 regions were activated by FOXM1.Four possible binding sites of 496 bp(-2700/-2205)were mutated by overlapping PCR.The luciferase reporter system showed that only the-2370 bp sites mutation decreased the luciferase activity.Then-2370 bp was the binding site for FOXM1 transcriptional activating KIAA0101.6.Down-regulation of FOXM1 rescued the overexpression of KIAA0101 in serous ovarian cancerWe transfected si-FOXM1 or si-NC into A2780 cells with KIAA0101 overexpression or negative control.Silencing of FOXM1 could fully abrogated KIAA0101 mediated promotion on metastasis and proliferation of ovarian cancer cells.In agreement,si-KIAA0101 or si-NC was transfected into A2780 cells with FOXM1 overexpression or normal control.We found that KIAA0101 knockdown could significantly decrease the elevated metastasis and proliferation of FOXM1 induced in ovarian cancer cells.These observations support that KIAA0101 was one of the major targets mediating the FOXM1 effects on tumor metastasis and proliferation of human ovarian cancer.Conclusions:1.The expression of KIAA0101 in SOC was significantly higher than that in normal fallopian tube,and high expression of KIAA0101 suggested that poor prognosis of SOC.2.KIAA0101 promoted proliferation and metastasis in serous ovarian cancer.3.FOXM1 promotes the proliferation and metastasis of KIAA0101 by transcriptional regulation.Part ?:The underlying molecular mechanism of KIAA0101 for chemotherapy resistance in serous ovarian cancerBackground and objective:Tumor cytoreductive surgery combined with postoperative chemotherapy with cisplatin and paclitaxel is the standard treatment strategy for ovarian cancer.However,in the past few decades,the 5-year survival rate of ovarian cancer has remained at around 30%,due to its prone to primary or secondary chemotherapy resistance,easy to relapse after complete remission.It has been reported that KIAA0101 is associated with doxorubicin resistance in hepatocellular carcinoma and is associated with cisplatin resistance in esophageal cancer.It is not yet clear whether KIAA0101 plays a role in chemotherapy resistance in ovarian cancer.The PI3K/AKT/mTOR pathway is often abnormally activated in ovarian cancer,and the exact mechanism is unknown.Autophagy is an important metabolic pathway in cells,and it has a certain relationship with the occurrence and development of tumors.This study investigated the effect of KIAA0101 on PI3K/AKT/mTOR pathway and autophagy.At present,there are few studies on KIAA0101 and autophagy.In addition,we performed co-expression analysis through GEPIA and Oncomine database to construct the downstream molecular network regulated by KIAA0101.Chemotherapy resistance and molecular mechanism studies of KIAA0101 would provide important reference value for the role of KIAA0101 in serous ovarian cancer.Materials and Methods:A certain concentration gradient of cisplatin stimulation was applied to ovarian cancer cells.Western blot was used to observe whether the expression of KIAA0101 changed correspondingly with the change of dosing concentration.After stimulation of cells with cisplatin,changes in the morphology of ovarian cancer cells after KIAA0101 knockout were observed.The effects of KIAA0101 on the chemotherapeutic resistance of ovarian cancer cells were detected by drug resistant MTT and resistant plate cloning formation.The PI3K/AKT/mTOR pathway was examined,and whether KIAA0101 affected autophagy and apoptosis of ovarian cancer cells was observed.Flow cytometry was used to detect the apoptosis rate of ovarian cancer cells after cisplatin administration.The effects of KIAA0101 on autophagy were detected by immunofluorescence and observed by laser confocal microscopy.For apoptosis,western blot was used to analyze the changes of PARP,Cleaved PARP,Caspase 9,Cleaved caspase 9,Caspase 7,Cleaved caspase 7,Bax,and Bcl2.For autophagy,changes of Atg16L1,Atg 12,Atg7,Atg5,Beclinl,P62,LC3 were studied by western blot.Co-expression analysis was applied to detect possible downstream molecules regulated by KIAA0101,and western blot was used for preliminary validation.Results:1.KIAA0101 promoted chemo-resistance of serous ovarian cancer cells Cisplatin stimulation in A2780,HO8910 and SKOV3 cells,western blot showed that KIAA0101 expression increased with the increase of drug concentration.Drug resistant MTT found that KIAA0101 overexpressed cells were more tolerant to cisplatin and its IC50 was significantly higher than that of normal control cells.After cisplatin stimulation was applied to the cells,changes in cell morphology were observed.The results showed that the cells with KIAA0101 knockdown were more sensitive to chemotherapeutic drugs and tended to undergo apoptosis.KIAA0101 knockdown attenuated resistant to chemotherapy.As the concentration of cisplatin increased,colony forming ability in cells with KIAA0101 knockdown decreased significantly.2.KIAA0101 activated the PI3K/AKT/mTOR pathwayKIAA0101 overexpression activated PI3K/AKT/mTOR pathway to increase phosphorylated PI3K,AKT,mTOR.While KIAA0101 knockdown inhibited PI3K/AKT/mTOR pathway to decrease phosphorylated PI3K,AKT,mTOR.The activation of this pathway inhibits apoptosis and autophagy and promotes chemotherapy resistance in serous ovarian cancer.3.KIAA0101 inhibited cisplatin-mediated apoptosis in serous ovarian cancer cellsAfter overexpression of KIAA0101,western blot results showed that Cleaved PARP,Cleaved caspase 9,Cleaved caspase 7,Bax decreased,Bcl2 increased.When KIAA0101 was knockdown,the pro-apoptotic protein Bax was elevated,the anti-apoptotic protein Bc1-2 was decreased,and the apoptosis-related proteins Cleaved PARP,Cleaved caspase 9,and Cleaved caspase 7 were all increased.Flow cytometry confirmed that KIAA0101 inhibited serous ovarian cancer cells apoptosis to cisplatin treatment.4.KIAA0101 inhibited autophagy in serous ovarian cancerImmunofluorescence was used to investigate the effects of KIAA0101 on autophagy.After KIAA0101 knockout,confocal laser scanning microscopy showed increased autophagy.Western blot analysis of autophagy-related proteins showed that KIAA0101 overexpression downregulated Atg16L1,Atg12,Atg7,Atg5,Beclinl,and LC3 and upregulated P62,indicating that overexpression of KIAA0101 inhibited the autophagy of serous ovarian cancer cells.5.KIAA0101 regulated the expression of a series of important downstream moleculesOncomine database was applied to find out molecular associated with KIAA0101 through the co-expression analysis in several ovarian cancer datasets.A total of 9 molecular was included in the final analysis.To validate the association of the above mentioned 9 molecular with KIAA0101,western blot was applied in ovarian cancer A2780 and HO8910 cells with KIAA0101 knockdown or overexpression.The protein levels of KIF23,PCNA,PRC1,CDC25C,UBE2C,TOP2A,Rad51,KIF20A and Aurora A were elevated with KIAA0101 increased in A2780 and HO8910 cells.Conversely,these protein expressions were downregulated when KIAA0101 was knockdown in A2780 and HO8910 cells.In short,these results indicated that KIAA0101 regulated many key genes that played essential roles in cell growth,metastasis and chemoresistance.Conclusions:1.KIAA0101 activated PI3K/AKT/mTOR pathway,inhibited cisplatin-mediated apoptosis and autophagy of serous ovarian cancer cells,promoted cisplatin chemotherapy resistance.2.KIAA0101 regulated the expression of a series of downstream important genes such as KIF23,PCNA,PRC1,CDC25C,UBE2C,TOP2A,Rad51,KIF20A and AuroraA.