Fig 2: Modulators of TNBC cell migration are related to BC metastasis-free survival. a Prognostic gene signatures were used to generate minimum interaction PPI networks and compared to our candidate TNBC cell migration gene networks. Candidate genes affecting cell migration feed into similar networks essential for BC progression and metastasis formation. b Hierarchical clustering (Euclidean distance, complete linkage) of genetic modifications (mutations, deletions and amplifications combined) of 43 candidate genes in 29 cancer types. Data was derived from The Cancer Genome Atlas. Annotation shows the expression of the candidates in relation to BC metastasis-free survival in different BC subtypes. P-values were calculated using Cox proportional hazards regression analysis, with gene expression values as continuous variable and metastasis-free survival as end point. Genes marked in red and blue are highlighted in c. Red genes were selected for further analysis. c Contribution of different genetic modifications to the rate in several highly mutated or amplified candidates. d Kaplan–Meier curves of for expression of PRPF4B, BUD31, and BPTF and relation to metastasis-free survival in ERpos or TNBC breast cancer. Gene expression data of lymph-node-negative BC patient cohort without prior treatment using optimal split was used to obtain the curves

Fig 3: PRP4K restricts Yki nuclear localization and inhibits Hpo target gene expression. a–h Late third instar wing imaginal discs of control (a, c, e, g) or expressing UAS-PRP4K-RNAi with hh-Gal4 (b, d, f, h) were immunostained to show the expression of Ci (red) and ex-lacZ (green in a, b), diap-GFP3.5 (green in c, d), ban-lacZ (green in e, f), or Wg (green in g, h). Arrows indicate posterior compartments marked by the lack of Ci expression. Of note, hh-gal4 is expressed in posterior compartment cells. i–j Late third instar wing imaginal discs of control (i) or expressing UAS-PRP4K-RNAi with hh-Gal4 (j) were immunostained to show the expression Yki (red), Ci (green), and a nuclear dye Hoechst (blue). Arrows indicate the enlarged regions shown in the right most panels. k–l Late third instar wing imaginal discs bearing wts mutant clones that express either UAS-GFP (k) or UAS-PRP4K-GFP (l) under the control of tub-Gal4 were immunostained for Yki and GFP. wts mutant clones are marked by GFP expression (green in the right panels). Of note, GFP and PRP4-GFP mark the cytoplasm and nucleus, respectively

Fig 4: PRPF4B is essential for breast cancer metastasis formation in vivo. a mRNA and b protein expression of PRPF4B in shPRPF4B MDA-MB-417.5 cell line compared to shCtrl#1 and shCrtl#2 MDA-MB-417.5 cell lines. Mean + s.d. of three biological replicates. Statistical significance was determined using one-way ANOVA correcting for multiple testing. *p < 0.05, **p < 0.01. c Immunofluorescent staining for PRPF4B (green) and DNA (DAPI; blue) in shCtrl#1, shCtrl#2 and shPRPF4B MDA-MB-417.5 cell lines. Scale bar = 50 μm. d Tumor growth of shPRPF4B, shCtrl#1, and shCtrl#2 MDA-MB-417.5 cells engrafted in the mammary fat pad of Rag2–/– Il2rg–/– mice (n = 7–8 animals per group). e Dissemination of shPRPF4B, shCtrl#1 and shCtrl#2 MDA-MB-417.5 cells in the lung, liver, spleen, kidney, heart, uterus, and axillar lymph node as determined by ex vivo bioluminescent analysis (total flux (p/s)) of different target tissues (n = 7–8 animals per group). f Number of lung metastases for shPRPF4B, shCtrl#1, and shCtrl#2 injected mice as determined by macroscopic evaluation of lungs injected with ink (n = 7–8 animals per group). g Images of ink injected lungs (left) and bioluminescent signal in the lungs (right) of all individual mice. Blanc indicates mice that did not receive MDA-MD-417.5 cells. Groups were compared by Kruskal–Wallis test with Dunn’s post correction test (luminescence) or ANOVA (metastasis count). *p < 0.05, **p < 0.01

Fig 5: PRP4K regulates organ size through the Hpo pathway. a–d Side (top) or dorsal view (middle) of Drosophila adult eyes or late third instar eye imaginal discs expressing diap-GFP3.5 (bottom) of the following genotypes: GRM-Gal4 (a), GMR-Gal4 UAS-Yki (b), GMR-Gal4 UAS-Yki UAS-PRP4K-RNAi (c), and GMR-Gal4 UAS-Yki UAS-PRP4K (d). e Quantification of eye size for the indicated genotypes. f Quantification of GFP signal in the indicated eye discs in anterior and posterior regions relative to the morphogenetic furrow. g–j Drosophila adult eyes containing control clones without (g; white color) or with (i) GMR>Yki, or PRP4K mutant clones without (h; orange color) or with (j) GMR>Yki. (k) Quantification of eye size for the indicated genotypes. l–m Drosophila adult eyes expressing UAS-Sd-GA either alone (l) or in conjunction with UAS-PRP4K-RNAi (m) under the control of GMR-Gal4. n–o Drosophila adult eyes expressing UAS-Wts-RNAi either alone (n) or in conjunction with UAS-PRP4K (o) under the control of GMR-Gal4. p–q Quantification of eye size for the indicated genotypes. Data are means ± s.d. from three independent experiments. N ≥ 5 for each genotype