Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.
Humans ; Forkhead Box Protein M1/genetics* ; Female ; Macrophages/cytology* ; Endometriosis/genetics* ; Ubiquitin Thiolesterase/genetics* ; Cells, Cultured ; Endometrium/metabolism* ; Ubiquitination ; Adult ; Interleukin-10/metabolism* ; Interleukin-6/metabolism* ; Protein Stability ; Stromal Cells/metabolism*

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.
Humans ; bcl-2-Associated X Protein/metabolism* ; Carcinoma ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Drug Resistance ; Forkhead Box Protein M1/metabolism* ; Gene Expression Regulation, Neoplastic ; MicroRNAs/genetics* ; Proliferating Cell Nuclear Antigen/metabolism* ; Receptor, Notch1/metabolism* ; RNA, Small Interfering/genetics* ; Stomach Neoplasms/genetics*

OBJECTIVETo explore the effects of EGFR-TKI AG1478 on the expression of FoxMl and FOXO3a genes in non-small cell cancer (NSCLC) cell lines, and explore the effect on cell proliferation and drug sensitivity to AG1478 after down-regulation of FOXMl and FOXO3a expression by RNAi technique.

METHODSHuman lung cancer cells were treated with AG1478 at different concentrations. RT-PCR and Western blot were used to examine the expression of P-EGFR, FOXM1, FOXO3a mRNA and protein. After transient transfection of FOXM1 and FOXO3a siRNA, RT-PCR and Western blot were employed to determine the transfection efficiency and expression of the related proteins. CCK-8 assay, colony formation assay and flow cytometry were performed to evaluate the cell proliferation, colony formation ability and the changes in cell cycle distribution.

RESULTSThe expressions of FOXM1 mRNA and protein were inhibited by AG1478 in a dose-dependent manner (both P < 0.05). After transfection with FOXM1 siRNA, the expressions of FOXM1 mRNA and protein, and proteins of cyclin B1, c-Myc, and Bcl-2 were significantly down-regulated, and the expressions of p21 and cleaved-PARP proteins were significantly up-regulated (all P < 0.05). The colony number of FOXM1siRNA transfection group was 37.3 ± 8.6, significantly lower than that of the blank control (135.3 ± 7.0) and negative control group (125.3 ± 7.5, P < 0.05). The colony formation inhibition rate was (7.40 ± 0.94)% in the negative control group and (72.4 ± 6.09)% in the FOXM1 siRNA transfection group. FOXM1siRNA transfection induced cell cycle arrest at G2/M phase with a percentage of (55.6 ± 4.83)%, significantly higher than that of the blank control [(24.30 ± 1.95)%] and negative control group [(21.3 ± 2.06)%, P < 0.05]. Additionally, the FOXM1siRNA transfection significantly increased the chemosensitivity of A549 cells to AG1478 (P < 0.05). Besides, AG1478 induced expression and nuclear relocation of FOXO3a. After the FOXO3a siRNA transfection, the expression of FOXM1 protein was significantly up-regulated, and resulted in a reduction of AG1478-induced inhibition of FOXM1.

CONCLUSIONSThe expression of FOXM1 is down-regulated by AG1478 via FOXO3a in the NSCLC cell lines, and then increases the chemosensitivity of A549 cells to AG1478. It suggests that FOXM1 could be a potential target for the therapy and drug exploitation for NSCLC.

Adenocarcinoma ; metabolism ; pathology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Dose-Response Relationship, Drug ; Down-Regulation ; Forkhead Box Protein M1 ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Quinazolines ; administration & dosage ; pharmacology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; Transfection ; Tyrphostins ; administration & dosage ; pharmacology

Targeted therapies include small-molecule inhibitors and monoclonal antibodies, have made treatment more tumor-specific and less toxic, and have opened new possibilities for tailoring cancer treatment. Nevertheless, there remain several challenges to targeted therapies, including molecular identification, drug resistance, and exploring reliable biomarkers. Here, we present several selected signaling pathways and molecular targets involved in human cancers including Aurora kinases, PI3K/mTOR signaling, FOXO-FOXM1 axis, and MDM2/MDM4-p53 interaction. Understanding the molecular mechanisms for tumorigenesis and development of drug resistance will provide new insights into drug discovery and design of therapeutic strategies for targeted therapies.
Aurora Kinases ; metabolism ; Drug Resistance, Neoplasm ; Forkhead Box Protein M1 ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; metabolism ; Humans ; Molecular Targeted Therapy ; Neoplasms ; metabolism ; therapy ; Nuclear Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

The FOXO3a and FOXM1 forkhead transcription factors are key players in cancer initiation, progression, and drug resistance. Recent research shows that FOXM1 is a direct transcriptional target of FOXO3a, a vital downstream effector of the PI3K-AKT-FOXO signaling cascade. In addition, FOXM1 and FOXO3a also antagonize each other's activity by competitively binding to the same target genes, which are involved in chemotherapeutic drug sensitivity and resistance. Understanding the role and regulation of the FOXO-FOXM1 axis will provide insight into chemotherapeutic drug action and resistance in patients, and help to identify novel therapeutic approaches as well as diagnostic and predictive biomarkers.
Antineoplastic Agents ; pharmacology ; therapeutic use ; Carcinogenesis ; genetics ; metabolism ; Drug Resistance, Neoplasm ; Forkhead Box Protein M1 ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Neoplasms ; drug therapy ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

OBJECTIVE: To investigate the expression of Forkhead Box M1 transcription factor (FoxM1) mRNA and protein in laryngeal squamous cell carcinoma (LSCC) and its clinical significance. METHOD: Immunohistochemistry was used to examine the expression of FoxM1 in 89 LSCC tissues, 89 adjacent normal tissues and 20 laryngeal papilloma (LP) tissues. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine the expression of FoxM1 in 20 LSCC tissues, 20 LP tissues and 20 adjacent normal tissues. The relationship between FoxM1 expression and the clinicopathological parameters of LSCC were analyzed. RESULT: FoxM1 mRNA and protein expression were gradually decreased from LSCC to LP and normal tissues. The difference was significant (P<0.05). There was a significantly correlation between FoxM1 and histologic differentiation, T stage, lymph node metastasis and clinical stage. Tumors with poorer differentiation, in higher T stage, with lymph node metastasis or in higher clinical stage presented higher FoxM1 expression than tumors with better differentiation, in lower T stage, without lymph node metastasis or in lower clinical stage (P<0.05). The positive expression of FoxM1 was not related with the age, gender or primary site of LSCC patients (P>0.05). CONCLUSION The up-regulation of FoxM1 may play an important role in the invasion and metastasis of LSCC.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Forkhead Box Protein M1 ; Forkhead Transcription Factors ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Prognosis

OBJECTIVETo investigate the expression of forkhead box M1 (FOXM1) and its correlation with clinicopathological features and prognosis in patients with non-small cell lung cancer (NSCLC).

METHODSThe expression of FOXM1 in 68 cases of NSCLC was detected by immunohistochemistry. The FOXM1 expression in 6 tumor tissues (3 cases with negative and 3 cases with positive expression of FOXM1) was analyzed by Western blotting to confirm the immunohistochemical results. The correlation of the expression of FOXM1 with clinicopathalogical features and overall survival of the NSCLC patients was analyzed.

RESULTSThe expression of FOXM1 protein was detected in the nuclei or cytoplasms of the tumor cells. The positive expression rate of FOXM1 was 36.8% (25/68). Western blotting confirmed the immunohistochemical results. The expression level of FOXM1 in advanced stage cancer was significantly higher than that in early stage NSCLC (P = 0.001). The median OS was 23.0 months in patients with negative expression of FOXM1 and 13.0 months in those with positive expression (P = 0.001). Univariate analysis revealed that histological grade, lymph nodes status, TNM stage and FOXM1 expression were significantly associated with prognosis in the NSCLC patients (P < 0.05). The Cox multivariate analysis demonstrated that lymph nodes status, TNM stage and FOXM1 expression were independent poor prognostic factors (P < 0.05).

CONCLUSIONThe expression status of FOXM1 in NSCLC is an independent prognostic factor and negatively correlated with prognosis.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Female ; Follow-Up Studies ; Forkhead Box Protein M1 ; Forkhead Transcription Factors ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Grading ; Neoplasm Staging ; Proportional Hazards Models