Fig 2: mTOR is required for MDM4 activity on cell migration.a Upper panel: representative pictures of cell migration evaluated through wound-healing assay of Empty Vector- or SK-OV-3 cells expressing wt or MDM4 deletion mutants+GFP as indicated. Pictures were taken at time 0 and 24 h after the scratch. Scale bar = 200 Mμ b quantification of cell migration (n = 4, one-way ANOVA for multiple comparisons, Empty vector vs wt-MDM4, p = 0.0135; Empty vector vs BD, p = 0.015; Empty vector vs BD + RF, p = 0.04). BD = p53-binding domain, RF = Ring Finger domain, Central = Central domain- lacking both BD and RF domains. c WB of MDM4 deletion mutants used in a. d Representative images of cell migration of Empty Vector and MDM4-SK-OV-3 cells using Rapamycin (Rapa) or vehicle (DMSO). Representative micrographs show the analysis performed with Cell Profiler 3.1.9, 15 h after scratching. Colors are randomly assigned by the software. e Quantification of cell migration shown in c using Fiji (n = 4, one-way ANOVA for multiple comparisons, Empty Vector+DMSO vs MDM4 + DMSO, p = 0.004; Empty Vector+DMSO vs Empty vector+Rapa, p = 0.01; Empty Vector+DMSO vs MDM4 + Rapa, p = 0.0002). f Quantification of cell confluency shown in c (one-way ANOVA for multiple comparisons, Empty Vector+DMSO vs MDM4 + DMSO, p = 0.04; Empty Vector+DMSO vs Empty vector+Rapa, p = 0.001; Empty Vector+DMSO vs MDM4 + Rapa, p = 0.0003). g Cell velocity analysis of cell migration shown in c (n = 392 cells for Empty Vector+DMSO, n = 229 cells for MDM4 + DMSO, n = 170 cells for Empty Vector+Rapa, n = 232 for MDM4 + Rapa, one-way ANOVA for multiple comparisons, Empty Vector+DMSO vs MDM4 + DMSO, p < 0.0001; Empty Vector+DMSO vs Empty vector+Rapa, p < 0.0001).

Fig 3: MDM4 decreases ovarian cancer nodules dissemination and improves EOC patients’ overall survival probability.a Number of nodules in peritoneal organs of Empty Vector or MDM4-SK-OV-3-injected mice (n = 6, two-tailed Student’s t test, p = 0.018). b Number of peritoneum membranes nodules as in c (n = 6, two-tailed Student’s t test, p = 0.0016). c Total number of nodules present in both peritoneal organs and peritoneum membranes (n = 6, two-tailed Student’s t test, p = 0.0016). d Percentage of mice showing ascites at the final time point (n = 6, χ2 test, p < 0.0001). e Total number of nodules in Empty Vector or Mdm4-ID8 cells (n = 6 for Empty Vector, n = 7 for Mdm4, two-tailed Student’s t test, p = 0.0042). Nodules were counted blindly by two independent observers. f ID8 ascites analysis as in d (n = 15, χ2 test p < 0.0001). g OS in mice injected with Empty Vector or MDM4-SK-OV-3 cells (n = 5, log-rank test, p < 0.001). h Correlation of MDM4 expression with OS in 1656 patients with epithelial ovarian cancer followed for 15 years (log-rank test, p = 0.0004).

Fig 4: MDM4 expression affects mTOR activation.a Pathway analysis of mTOR signaling in SK-OV-3 cells. Red and green colors underlie proteins differently expressed in MDM4- compared with Empty vector-SK-OV-3 cells (red = upregulated, green = downregulated proteins). b WB of Empty Vector or MDM4-SK-OV-3 cells showing S6K1 and p70S6K1T389 expression (left panel) and quantification (right panel) (n = 3, one-sample t test p < 0.001). c WB of indicated proteins in tumor nodules (one per mouse) generated by IP injection of Empty Vector or MDM4-SK-OV-3 cells in mice; lower panels show quantification (n = 7 mice, two-sided Student’s t test, ***p < 0.001, *p = 0.048). d SunSet assay of Empty Vector- and MDM4-SK-OV-3 cells treated with rapamycin for 6 hours; quantification of two independent assays (right panel). e WB of indicated proteins in whole-cell extract (WCE) and co-immunocomplex from SK-OV-3 cells grown in EBSS for 1 hour and then in absence or presence of complete medium for additional 1 hour (CM). 1 mg of WCE was immunoprecipitated with anti-MDM4 antibody (IPαMDM4) or control Ig (IPCTL). Right panel shows the analysis of 1/100 of WCE.

Fig 5: Cell spreading is impaired by MDM4 in a 3D-bioprinting assay.a Rendering of the 3D-bioprinting geometry used. b Representative pictures of bioprinted constructs carrying MCTS and HUVEC at day 0 (upper panels, black arrow points to the MCTS) and day 20 after bioprinting (lower panels); red and green arrows point to cancer cells spreading and to vessels-like structures, respectively. Right panels show the rendering of day 0 bioprinting strategy (upper panel) and cell invasion at day 20 (lower panel); scale bars = 200 mM. c Confocal microscopy of constructs showing the vessels-like structures (stained with sheep anti-Von Willebrand Factor AbCam Cat# ab11713, green signal), cancer cells expressing mCherry (red signal), and MDM4 (stained with Mouse anti-MDM4 Ab OriGene Cat# TA505706, yellow signal), DAPI stains nuclei. White arrows point to migrating cancer cells. The drawing shows the rendering of cancer cells escaping from the MCTS and entering vessels-like structures.