RNF112-mediated FOXM1 Ubiquitination Suppresses The Proliferation And Invasion Of Gastric Cancer

Background and purpose:Gastric cancer is the fifth most common malignancy worldwide and the fourth leading cause of cancer-related deaths.Although there has been many therapeutic options including surgical intervention,radiation therapy,chemotherapeutic treatment,and immunological therapy,the therapeutic efficacy of gastric cancer remains unsatisfactory due to the unclear molecular mechanisms involved.Forkhead Box M1(FOXM1)is one member of the evolutionally conserved forkhead box family,which is characterized by a winged-helix DNA-binding domain.FOXM1 contributes to the occurrence and progression in various types of human malignancies,and it is significantly related to the poor clinical prognosis of cancer sufferers.Literature reports suggested that FOXM1 controls G1/S phase and G2/M phase transition by transcriptionally activating cell cycle-related genes such as CCNB1,CENPA,and CENPB.Recent studies have shown that hyperactivation of FOXM1 can maintain low levels of reactive oxygen species in gastric cancer,which ensures the survival of gastric cancer stem cells,and induces chemotherapy resistance in gastric cancer cells.Moreover,FOXM1 can also mediate the ferroptotic cell death resistance of melanoma cells,ensuring the survival of tumor cells.Conversely,FOXM1 inhibition was found to result in a concomitant reduction in proliferation and glucose metabolism in liver cancer cells.Given the critical role of FOXM1 in the initiation and progression of tumors,it is considered a promising target for cancer therapy.Due to the absence of precisely defined drug binding sites and the large interaction interface,transcription factors such as FOXM1 are often considered "undruggable." Previous research has primarily centered on inhibiting the interaction between FOXM1 and its target genes to impede its regulation of downstream transcription networks.For instance,FID-6 acts as a FOXM1 inhibitor by disrupting the interaction between FOXM1 and DNA,although its effectiveness is limited to high concentrations.Alternatively,STL427944 has been shown to promote the autophagic degradation of FOXM1.While these inhibitors have potential for cancer treatment,their pharmacological specificity remains a concern.Moreover,the lack of a complete crystal structure for FOXM1 presents a challenge for developing FOXM1-specific inhibitors.FOXM1 is a key transcription factor in cancer,and an in-depth investigation of its regulatory network can provide important potential strategies for pharmacological inhibition of FOXM1.Literature reports suggested that Gli1,STAT3,and LXRα can directly bind to the promoter region of FOXM1,regulating its promoter activity.In addition,non-coding RNAs,such as mi R-320 a,lnc RNA NNT-AS1,and circ CLK3,have been identified as regulators of FOXM1 expression at the transcriptional level by directly or indirectly targeting the 3’-UTR region of FOXM1.Moreover,FOXM1 undergoes various post-translational modifications,including phosphorylation,ubiquitination,SUMOylation,and acetylation,which can alter its subcellular localization,expression level,transcriptional activity,and protein stability.Notably,ubiquitin-mediated protein degradation,due to its high specificity,provides an attractive approach for the targeted inhibition,especially for "undruggable" proteins like FOXM1.Although several E3 ubiquitin ligases targeting FOXM1 have been reported,further investigation is needed to explore the potential of FOXM1 as an anti-cancer target in gastric cancer,particularly with respect to its degradation via the ubiquitin-proteasome system.In summary,our research demonstrates that RNF112 exhibits promising anti-tumor effects in gastric cancer by inducing ubiquitination and subsequent degradation of the oncoprotein FOXM1.The small molecule compound RCM-1 can mediate the cytoplasmic localization of FOXM1 and significantly enhance the interaction between FOXM1 and RNF112.The RNF112-FOXM1 axis has the potential to serve not only as a potential biomarker for predicting the prognosis of gastric cancer patients,but also as a therapeutic target against gastric cancer.Methods:1.ON-TARGET plus si RNA library was used to knockdown the expression of E3 ligases by si RNA transfection.Western blot analysis was then performed to screen the E3 ligase targeting FOXM1 for degradation.2.Western blot analysis,immunohistochemistry(IHC),and q RT-PCR were employed to detect the expression of RNF112,FOXM1 and its target genes in cell lines and tissues.3.CRISPR/Cas9 technology was adopted to generate the RNF112-/-gastric cancer cell lines,and Sanger sequencing was then used to validate the CRISPR/Cas9-induced RNF112 depletion.4.RNA sequencing(RNA-seq)together with Gene Ontology(GO)analysis was used to investigate the potential signaling pathways that RNF112 might be involved in in gastric cancer cell lines.5.The luciferase reporter assay was taken to detect the activities of 6×FKH report plasmids,which is indicative of the transcriptional activities of FOXM1.6.Colony formation assay and invasion assay were performed to evaluate the effects of RNF112 on proliferative and invasive ability of gastric cancer cell lines.7.Immunofluorescence(IF)was performed to determine the subcellular localization and colocalization of FOXM1 and RNF112 in cell lines and tissues.8.Co-Immunoprecipitation(Co-IP)and truncation mutation experiment were used to explore the interaction between FOXM1 and RNF112.9.In vitro ubiquitination assay was employed to examine the ubiquitination levels of FOXM1.10.Network pharmacology-based analysis together with GO analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were adopted to predict the possible signaling pathways RCM-1 could involve in.11.Molecular docking and molecular dynamics simulation was performed to explore the potential binding modes of FOXM1,RNF112 and RCM-1,and calculate the binding free energy between FOXM1 and RNF112 in the presence and absence of RCM-1.12.Subcutaneous xenograft tumor model and tail vein injection models of nude mice were established to evaluate the cell proliferation and invasion of gastric cancer in vivo.13.Multiplexed immunofluorescence staining was used to detect the expression of FOXM1 and RNF112 in gastric cancer tissue microarray(TMA)containing 110 cases of gastric cancer and paired adjacent non-cancerous tissue.Kaplan–Meier survival analysis was employed to investigate the correlation of FOXM1 and RNF112 expression with the prognosis of gastric cancer patients.Results:1.RNF112 was identified as a potential E3 ubiquitin ligase targeting FOXM1 for degradation in gastric cancer.The gain of function studies showed that ectopic RNF112 significantly decreases FOXM1 abundance.The analysis based on TCGA and GEO gastric cancer database revealed that RNF112 was negatively correlated with FOXM1 in gastric cancer tissues and exhibited significantly suppressed in gastric cancer compared with adjacent para-cancer tissues.2.RNA-sequencing analysis revealed that both RNF112 and FOXM1 were associated with the signaling pathways associated with proliferation,migration and cell cycle,which implies RNF112 may serve as a potential E3 ubiquitin ligase negatively regulating FOXM1 transcriptional network.3.Western blot and q RT-PCR analysis suggested that ectopic expression of RNF112 dramatically downregulated FOXM1 protein abundance in gastric cancer cells coupled with reduced expression of FOXM1 downstream genes related with cell proliferation and invasion.While,the expression of FOXM1 and its target genes were significantly increased upon RNF112 depletion via a CRISPR/Cas9-based editing approach.4.The luciferase reporter assay illustrated that the transcriptional activity of FOXM1 was significantly downregulated upon ectopic expression of RNF112.5.Colony formation assay,invasion assay and animal experiments indicated that the overexpression of RNF112 significantly suppressed the proliferation and invasion of gastric cancer cells in vitro and in vivo.6.Co-IP and truncation mutation experiment verified the interaction between FOXM1 and RNF112.Immunofluorescence analysis further showed that FOXM1 and RNF112 mainly colocalized in cytoplasm with little in nucleus.7.In vitro ubiquitination assay confirmed a readily increased FOXM1 ubiquitination level after RNF112 overexpression,while catalytic dead RNF112,RNF112-Mut,failed to ubiquitinate FOXM1.8.Network pharmacology-based analysis found that RCM-1 could involve in "Regulation of protein stability" and "Ubiquitin-like protein ligase binding" pathway in gastric cancer.9.Molecular docking and molecular dynamics simulation depicted the potential binding modes of FOXM1,RNF112 and RCM-1 and demonstrated that RCM-1 could significantly promote potential binding affinity between FOXM1 and RNF112.10.Multicolor immunofluorescence analysis revealed that FOXM1 expressed higher in cancer tissues,accompanied with lower RNF112 expression.Kaplan–Meier survival analysis revealed that gastric cancer patients with high FOXM1/low RNF112 displayed the shortest survival time.Conclusions:Taken together,we demonstrate that RNF112 plays a promising antitumor role in gastric cancer by inducing the ubiquitination and degradation of oncoprotein FOXM1.Small-molecule compound,RCM-1,could mediate the cytoplamsic localization of FOXM1 and significantly enhance the interaction between FOXM1 and RNF112.The RNF112-FOXM1 axis could not only be considered a potential biomarker for predicting patients’ prognosis but also could be developed as a therapeutic target to combat gastric cancer.