Fig 2: High activation of FoxM1–STMN1 signal axis is a risk of poor prognosis in cancers. a Overall survival and disease-free survival rate of 31 solid tumors (including ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS and UVM) from TCGA database were analyzed using the GEPIA platform; n = 9350. b Overall survival and disease-free survival rate of LIHC from TCGA database were analyzed using the GEPIA platform; n = 364. c Overall survival rate of LIHC from the TCGA database was analyzed according to the mRNA levels of FoxM1 and STMN1. HH, FoxM1High/STMN1High (n = 136). HL, FoxM1High/STMN1Low (n = 42). LH, FoxM1Low/STMN1High (n = 42). LL, FoxM1Low/STMN1High (n = 136). d A working model showing that aberrantly high activation of FoxM1–STMN1 axis contributes to tumorigenesis in cancer cells by promoting cell mitosis, while interruption of FoxM1–STMN1 axis can result in mitotic catastrophe-mediated cell death

Fig 3: SMYD2-mediated ß-catenin methylation is required for expression of Wnt downstream genes and ß-catenin nuclear translocation by FOXM1A. SMYD2 knockdown attenuated transcriptional levels of two pivotal Wnt pathway downstream genes, CCND1 and cMYC in two HCC cell lines, SNU449 (upper left panel ) and SNU475 (upper right panel ), and two colon cancer cell lines, HCT116 (lower left panel) and SW480 cells (lower right panel). Cells were transfected with siNC or siSMYD2 (siSMYD2#1). After 48h-incubation, RNAs were prepared from these cells and transcriptional levels of SMYD2, CCND1 and cMYC were measured by quantitative RT-PCR. Statistical analyses were performed using unpaired Student's t-test (two groups). The asterisks indicate statistical significance; *, **, and *** indicate p-value of < 0.05, < 0.01 and < 0.005, respectively, compared to the corresponding value of the siNC (control) group. Error bars indicate values of one standard deviation (n = 3). B. SMYD2-mediated methylation at K133 of ß-catenin is required for its interaction with FOXM1, BCL9 and a -catenin. 293T cells were co-transfected with FLAG-WT-ß-catenin or FLAG-K133A-substituted ß-catenin, and HA-SMYD2. After 48-h incubation, cell lysates were immunoblotted with anti-FLAG (WT-ß-catenin or K133A-substituted ß-catenin), anti-FOXM1, anti-BCL9, anti-a -catenin, and anti-HA (SMYD2) antibodies before immunoprecipitation (input), and after immunoprecipitation with anti-FLAG M2 agarose. FOXM1 and BCL9 were co-immunoprecipitated with FLAG-WT-ß-catenin, but not with FLAG-K133A-substituted ß-catenin. a -catenin was co-immunoprecipitated more preferentially with FLAG-K133A-substituted ß-catenin than FLAG-WT-ß-catenin. However, HA-SMYD2 was co-immunoprecipitated almost equally with both WT- and K133A-substituted ß-catenin proteins. C. Structural analysis of an a -catenin-binding region of ß-catenin including lysine 133. Without methylation of lysine 133 of ß-catenin, hydrogen bonds are made between lysine 133 (K133) and methionine 98 (M98), and between lysine 133 and glutamic acid 101 (Glu 101) in an a -catenin-binding region of ß-catenin. When K133 is methylated, these hydrogen bonds might be affected and the affinity of ß-catenin to a -catenin could be reduced. The crystal structure of residues 84-682 of ß-catenin is obtained from PDB: 4ONS and 2Z6G.

Fig 4: Coordinated expression of FoxM1 and STMN1 in cancers. a Analysis of the mRNA levels of FoxM1 and STMN1 (cancer vs. normal) in multiple solid cancers from Oncomine Database. b The expression of FoxM1 and STMN1 in hepatocellular carcinoma cohorts (GSE14520, GSE14323), gastric cancer cohorts (GSE13911, GSE19826, GSE27342) and colon cancer cohorts (GSE8671). The data were presented as the mean ± SD of different samples. The significance was analyzed by the Student’s t-test. **P < 0.01, ***P < 0.001, ****P < 0.0001. c The correlation of FoxM1 and STMN1 expression in 31 solid tumors (including ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS and UVM) from the TCGA database was analyzed using the GEPIA platform, and the correlation of FoxM1 and STMN1 expression in LIHC (n = 373), GC (n = 415) and CRC (n = 328) TCGA data. d Protein levels of FoxM1 and STMN1 in LIHC (n = 79), GC (n = 61) and CRC (n = 68) were analyzed by IHC. The correlation of FoxM1 and STMN1 was analyzed by linear regression analysis

Fig 5: FoxM1-mediated cancer cell proliferation requires STMN1 expression. a The hepatocellular carcinoma cell line SMMC-7721, gastric cancer cell line SGC-7901, colorectal cancer cell lines HCT 116 and HT-29 were used to established FoxM1-silenced and STMN1-overexpressed cell lines by lentivirus-mediated system. The protein levels of FoxM1 and STMN1 were detected by Western blot and the mRNA levels were detected by RT-qPCR. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. b Cell viability of cells was detected by cell counting-8 kit. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. c Plate clone assay was performed and the number of clones was measured by ImageJ software. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. d Cell cycle was analyzed by flow cytometry. e Cell mitosis was analyzed by immunofluorescence of a-Tubulin. The nuclei (blue) are stained with DAPI and the a-Tubulin (red) is stained with Alexa Flour 555. The images were captured at 60× magnification, and the scale bar is 10 µm. The yellow arrow points to the normal mitosis cells. The percentage of normal or abnormal cells was calculated by numbers of normal or abnormal nucleus divided by numbers of total nucleus × 100%. The data were presented as the mean ± SD of three different fields of view at low magnification (20×). The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01. f Colorectal cancer cell lines HTC 116 were infected with lentivirus. The mice were separated into four groups, control group (cells infected with pLKO.1-control lentivirus and pLV-GFP lentivirus), FoxM1-knockdown group (cells infected with pLKO.1-shFoxM1 lentivirus and pLV-STMN1-GFPSpark lentivirus), rescue group (cells infected with pLKO.1-shFoxM1 lentivirus and pLV-STMN1-GFPSpark lentivirus) and STMN1-overexpressed group (cells infected with pLKO.1-control lentivirus and pLV--GFP lentivirus). Cells were injected subcutaneously into the back of 5-year-old nude mice for 4 weeks to grow tumors. The mice were killed and the tumors were removed to measure the weight and volume. Tumor volume was measured and calculated by v = 0.5 × Length × Width2; n = 5. The data were presented as the mean ± SD. The significance was analyzed by Student’s t-test. *P < 0.05