| Background and objectives:Gastric cancer（GC）is one of the common malignant tumors worldwide.The occurrence of gastric cancer is related to various factors such as genetics,environment,and lifestyle.Its incidence is high globally,especially in Asia,where the incidence of gastric cancer is particularly high.Currently,surgery,chemotherapy,and radiation therapy are still important methods for treating gastric cancer,but these methods are not effective for the treatment of advanced gastric cancer.Therefore,finding new treatment strategies is crucial for the treatment of gastric cancer.With the continuous development of molecular biology and genomics,more and more tumor-targeted therapies and immunotherapeutic strategies have been discovered,bringing new hope for the treatment of gastric cancer.It is worth noting that the molecular subtypes of gastric cancer exhibit significant heterogeneity,so understanding the molecular mechanisms of gastric cancer occurrence and development is crucial for the prevention,diagnosis,and treatment of gastric cancer.FOXM1（Forkhead box M1）is a typical proliferation-associated transcription factor belonging to the forkhead box protein superfamily.In the human genome,the FOXM1 gene is located at the telomeric region of chromosome 12p13-3 and spans approximately 25 kb,consisting of ten exons.Four FOXM1 isoforms have been identified:FOXM1a has an inhibitory effect,while FOXM1b and FOXM1c are transcriptional activation factors.The cytoplasmic FOXM1d does not directly participate in transcriptional regulation.Notably,among these isoforms,FOXM1b exhibits the highest transcriptional activity.As an important transcription factor,FOXM1 regulates key biological processes such as cell cycle G/S and G2/M transition and mitotic progression.It is also regulated by Hedgehog,p53 and Ras signaling pathways and activates downstream targets such as cyclin B1（CCNB1）.Moreover,in humans,overexpression of FOXM1 has been confirmed as an oncogene associated with basal cell carcinoma mediated by Hedgehog signaling pathway.Additionally,it can cooperate with CENPF to promote prostate cancer progression;their co-expression serves as a poor prognostic indicator for short survival time and high metastatic potential.Besides acting as a transcription factor,FOXM1 can also exert carcinogenic effects through interaction with other proteins such asβ-catenin or SMAD3 via Wnt or TGF-βsignaling pathways.hTERT（Human telomerase reverse transcriptase）is the catalytic subunit of telomerase that regulates its activity and maintains telomere integrity.Increased h TERT activity has been observed in human colon cancer cells,lymphomas and myeloma cells;this represents a repair response to radiation-induced DNA damage.Upregulation of h TERT in gastric cancer cells suppresses the expression of mi R-29a and enhances the expression of Integrin subunit beta 1（ITGB1）,significantly increasing the invasive and metastatic ability of gastric cancer cells.Long non-coding RNA（lnc RNA）BC032469 promotes gastric cancer cell proliferation by upregulating h TERT expression.Therefore,understanding the molecular mechanisms that regulate h TERT is crucial.Many studies on h TERT regulation have focused on transcriptional level,while post-translational modifications such as ubiquitination remain poorly explored.Methods:1.The expression levels of FOXM1 and h TERT were analyzed in 34 types of cancer and corresponding normal tissues using the TCGA pan-cancer dataset.GO and KEGG enrichment analyses were performed on differentially expressed genes that were commonly upregulated for FOXM1 and h TERT using the Xiantao academic platform（https://www.xiantao.love/）.2.In vitro ubiquitination experiments were conducted to examine the ubiquitination degradation status of h TERT after overexpression of wild-type or mutant（transcriptional activity site mutation）or truncated forms of FOXM1（N-terminal deletion,DBD deletion,and C-terminal deletion）.3.Dual-luciferase assays were used to analyze the effect of overexpression of wild-type or mutant forms of FOXM1 on the activity of p GL4.26-6×DB reporter plasmid and p GL3-CCNB1 promoter in HEK293T cells.4.Immunoprecipitation experiments were performed to investigate the impact of wild-type or mutant forms of FOXM1 on MKRN1-h TERT or MDM2-h TERT binding.5.Colony formation assays were employed to explore the effect of h TERT knockdown on mutant FOXM1-promoted cell proliferation as well as the impact of DBD domain deletion in mutant FOXM1 on gastric cancer cell proliferation.6.Multiplex immunohistochemistry（m IHC）experiments were carried out for ROC curve analysis and survival analysis on gastric cancer tissue microarray regarding FOXM1and h TERT expression levels.Results:1.FOXM1 was significantly upregulated in 33 cancer types,while h TERT was markedly increased in 29 cancer types compared with normal tissues.The Venn diagram showed that30 upregulated differentially expressed genes were collectively upregulated between the FOXM1 and h TERT groups.GO term and KEGG pathway enrichment analyses of the overlapping genes revealed that the FOXM1 and h TERT-upregulated genes are involved in the cell cycle and meiosis process.2.Overexpression of wild-type FOXM1 dramatically inhibited h TERT ubiquitination.Additionally,transcriptional activity mutant FOXM1 as wild-type FOXM1,strongly inhibited the ubiquitination of h TERT,implying that FOXM1 could suppress h TERT ubiquitination and increase h TERT protein levels independent of its canonical transcriptional activity.Besides,only the DBD-truncated construct remarkably increased the ubiquitination of h TERT and promoted the its protein degradation,which means FOXM1 interacted with h TERT through its DBD region to inhibits h TERT ubiquitination and the ubiquitin-mediated degradation.3.Compared with FOXM1-WT,the transcriptional activity of p GL4.26-6×DB reporter plasmid was significantly reduced in FOXM1-Mut.In addition,the promoter activity of CCNB1,a target gene of FOXM1,was also suppressed by FOXM1-Mut,indicating that this transcriptional activity mutant construct was successfully generated.4.Overexpression of wild-type FOXM1 and mutated FOXM1 did not affect the interaction between MDM2 and h TERT,but they prevented MKRN1 from binding h TERT proteins.5.We found that FOXM1-Mut remarkably promoted the colony formation of gastric cancer cells,while silencing h TERT attenuated the proliferative ability of FOXM1-Mut.The deletion of DBD regions disrupted the FOXM1-h TERT interaction,thereby reducing cell proliferation and clone formation ability.6.In 90 patients with gastric cancer,the ROC curve suggested that FOXM1 and h TERT were both effective predictors of prognosis in gastric cancer patients.In addition,survival analysis showed that high expression of either FOXM1 or h TERT was associated with poor prognosis in gastric cancer patients.Importantly,simultaneous high expression of FOXM1and h TERT suggested the worst prognosis,while simultaneous low expression suggested the best prognosis.Conclusion:Taken together,our results established a novel link between the two important oncogenes in gastric cancers.FOXM1 could non-canonically increase h TERT protein levels by impeding the ubiquitination by E3 ligase MKRN1 and further repressing h TERT degradation.FOXM1 and h TERT were both increased in human gastric cancer samples and their simultaneous upregulation predicted a poor prognosis.Notably,although both FOXM1and h TERT have been shown to activate the Wnt/β-catenin signaling[1,2],we demonstrated that h TERT played an important role in FOXM1-mediated activation of Wnt/β-catenin pathway genes.In brief,our study provides novel rationale for gastric cancer treatment by targeting the FOXM1-h TERT axis.Background and objectives:Gastric cancer（GC）is one of the common malignant tumors in China,and its incidence and mortality rates rank high globally.According to the latest Chinese Cancer Report,the incidence and mortality rates of gastric cancer in China are ranked third among all malignant tumors,with about 480,000 people diagnosed with gastric cancer every year and approximately 370,000 deaths from it annually.Due to a lack of screening awareness,early-stage cases are rare in clinical practice;as a result,most patients have progressed to advanced stages when they are diagnosed with gastric cancer.The five-year survival rate for late-stage gastric cancer patients is less than 40%.Currently,tumor immune escape is one of the ten characteristics of tumor occurrence and development.Tumor cells can evade surveillance by the host’s immune system under various factors’ effects to promote tumor proliferation,invasion,and metastasis through multiple mechanisms such as downregulating tumor antigen expression or abnormal activation of oncogenic signals or inducing immunosuppressive tumor microenvironment（TME）,as well as dysbiosis of host intestinal flora.In recent years significant success has been achieved using immunotherapy targeting immune escape pathways in GC patients;however primary or acquired resistance still occurs frequently among many patients.Therefore elucidating the immune escape mechanism underlying GC holds important significance for expanding beneficiary groups while broadening avenues for immunotherapy against this disease.With the in-depth study of tumor immune escape mechanisms,it has been confirmed that Programmed Death Ligand 1（PD-L1）can interact with the tumor microenvironment,leading to immune escape in gastric cancer.Recent studies have shown that PD-L1 expression is positively correlated with poor prognosis in gastric cancer and serves as an important biomarker for effective immune checkpoint blockade therapy（ICB）.With the development of personalized precision treatment strategies,immunotherapy based on PD-1/PD-L1 targets has become an increasingly important treatment option for gastric cancer.However,single-agent ICB therapy has a low clinical response rate.Therefore,finding combination ICB treatment options is key to improving the effectiveness of gastric cancer immunotherapy,especially exploring dual-immune checkpoint blockade inhibitors which have great potential.Therefore,further exploration of molecular mechanisms regulating PD-L1 expression,development of novel targeted drugs and evaluation of their synergistic effects with ICB therapy will have a significant impact on clinical treatment for gastric cancer.Orlistat is a long-acting and potent specific gastrointestinal lipase inhibitor that directly blocks the absorption of fat in food by the human body.It is the first small molecule compound approved by both FDA and SFDA for treating obesity.Previous studies have shown that it exerts anti-tumor effects in various cancers,including inhibition of tumor cell proliferation,invasion,migration,angiogenesis and induction of apoptosis through direct inhibition of fatty acid synthase（FASN）activity[1-3].Additionally,Orlistat can stimulate host immune system.Some studies have demonstrated that its use enhances macrophage-mediated anti-tumor activity,promotes T-cell proliferation and activation to strengthen the immune surveillance and clearance against tumor cells[4,5].Therefore,Orlistat has broad prospects for application in immunotherapy;however,its exact mechanism of action requires further investigation.Immunotherapy has made significant progress in cancer treatment,but its response rate still needs to be improved.We report a drug,Orlistat,which is FDA-approved for obesity treatment and enhances the efficacy of CTLA-4 blockade immunotherapy by inhibiting PD-L1 expression.Specifically,Orlistat suppresses PD-L1 gene transcription by inhibiting the AKT-FOXO3a-FOXM1 pathway.In addition,we also found that FOXO3 a and FOXM1 mutually inhibit each other: FOXO3 a can suppress FOXM1 transcription while FOXM1 reduces FOXO3a protein expression by activating AKT through promoting interaction between AKT and upstream PDK1.These results demonstrate that FOXM1 plays a critical role in promoting immune escape and support the combined use of Orlistat with CTLA-4blockade to enhance immunotherapy efficacy.Methods:1.Investigating the binding regions of FOXO3 a and FOXM1 promoter,as well as FOXM1 and PD-L1 promoter,using chromatin immunoprecipitation（Ch IP）assay.2.Detect the effect of FOXM1 on AKT-PDK1 binding and explore the binding regions of FOXM1 with AKT and PDK1 through immunoprecipitation experiments.3.Constructing stable MGC803 gastric cancer cell line with FOXM1 knockout using CRISPR/Cas9 technology,and exploring the regulation of PD-L1 by FOXM1 depletion upon treatment with Orlistat.4.Analyzing the upstream signaling pathways of PD-L1 expression inhibition by Orlistat using gene set enrichment analysis（GSEA）.5.Using Auto Dock software for molecular docking to investigate the interaction between AKT protein and Orlistat,further confirmed by cellular thermal shift assay（CETSA）experiment.6.Investigating the effects of Orlistat and/or anti-CTLA-4 antibody on syngeneic mice carrying MC38 tumors,evaluating the proportion of CD8+T cells and CD4+T cells isolated from tumor tissue using flow cytometry（n=5）.7.Analyzing the correlation between PD-L1 expression and FOXO3 a or FOXM1 expression in clinical tissues of gastric cancer patients using multiplex immunohistochemistry techniques.Results:1.Through Chromatin Immunoprecipitation（Ch IP）experiments,it was found that FOXO3 a binds to the（-475-844 bp）region of the FOXM1 promoter,and FOXM1 binds to the（+222 +450 bp）region of the PD-L1 promoter.2.Co-Immunoprecipitation（Co-IP）experiments showed that overexpression of FOXM1 significantly promoted the interaction between AKT and PDK1.In addition,the C-terminus of FOXM1 binds separately to the N-terminus of PDK1 and the PH domain of AKT.3.Western blot experiments revealed that knockdown of FOXM1 significantly reduced the inhibitory effect of Orlistat on PD-L1,indicating that FOXM1 plays a critical role in the downregulation of PD-L1 by Orlistat.4.GSEA analysis showed significant enrichment of PI3K-AKT signaling pathway.FOXO3 a is a downstream molecule of AKT,and AKT can phosphorylate FOXO3 a at Thr32 and Ser253.Phosphorylated FOXO3 a can be degraded in the cytoplasm,and it has been reported that FOXO3 a can inhibit the transcription of FOXM1 in various tumors.Therefore,our next step is to explore whether the inhibition of PD-L1 transcription by Orlistat is mediated by the AKT-FOXO3a-FOXM1 pathway.5.Auto Dock molecular docking showed that Orlistat directly interacts with AKT Tyr38 and Tyr326 through hydrogen bonds with atomic distances of 1.9 （?） and 3.4 （?）,respectively.Consistent with this,cell thermal shift assays showed that Orlistat can enhance the thermal stability of AKT protein,supporting the existence of a direct interaction between AKT and Orlistat.6.Combination therapy with Orlistat and CTLA-4 monoclonal antibody significantly slowed tumor progression,exhibited a better overall survival rate compared to the use of either drug alone,and had smaller tumor size and weight.In addition,the combination therapy group had more CD8+ T cells infiltrating the tumor,but no significant difference in the proportion of CD4+ T cells.7.According to the analysis of multiplex immunohistochemistry results,FOXO3 a is lowly expressed in tumors,while PD-L1 and FOXM1 are highly expressed in tumors.In addition,there is a negative correlation between FOXO3 a and PD-L1 expression,while there is a positive correlation between FOXM1 expression and PD-L1 expression.Conclusion:Targeting both CTLA-4 and PD-1/PD-L1 is currently the most effective cancer immunotherapy,as CTLA-4 and PD-1 have different functions at different stages of T cell activation[6,7].Our results indicate that Orlistat can enhance the efficacy of CTLA-4 antibody by downregulating the m RNA and protein levels of PD-L1 on tumor cells.Meanwhile,we also expanded our understanding of the AKT-FOXO3a-FOXM1 pathway.On one hand,FOXO3 a directly interacts with FOXM 1 promoter to transcriptionally regulate FOXM1;on the other hand,FOXM1 activates AKT by promoting PDK1-AKT interaction,thus facilitating FOXO3 a phosphorylation and subsequent degradation.Overall,targeting the pathway through which FOXM1 promotes tumor immune evasion could be an important target for developing new anti-cancer drugs.Combining Orlistat with CTLA-4 antibodies is a potential novel strategy for treating tumors. |
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