Fig 2: Effect of selected genes targeted by gCISs on tumorigenesis and cell migration.a Incidence of microscopic mammary tumors in MMTV-Cre:Rbf/f:Fbxw7f/f female mice relative to single mutant mice. Left, whole mount stained mammary gland from a representative MMTV-Cre:Rbf/f:Fbxw7f/f mouse with multiple microscopic tumors (arrows). Right, quantification of mammary tumors in indicated mice. P value calculated by one-way ANOVA, Tukey’s multiple comparison test. b Incidence of microscopic lung lesions in MMTV-Cre:Rbf/f:Ptenf/f mice relative to single mutant mice. Left, cross section through a representative lung; arrow points to a large lung lesion. Right, number of lung mets in indicated mouse strains. Error bars represent SD; P value calculated by one-way ANOVA, Tukey’s multiple comparison test. c Kaplan–Meier Disease-free survival curves for breast cancer patients with high RBKO pathway (loss; left), low PTEN mRNA level (center), or RBKO high (loss) plus PTEN mRNA low (right) compared to all other genotypes. HR hazard ratio. P values calculated by Log-rank test. d Western blot analysis for expression of indicated M-targets in different TNBC cell lines: MDA-MB-231; MDA-MB-436; MDA-MB-468; Hs578t; and HER2-enriched SKBR3. e Effect of shRNA-mediated FBXW7, CDC42BPA, SRGAP2 or MTMR3 depletion on cell migration (scratch-wound assays) in indicated TNBC lines. P values calculated by unpaired, two-tailed student t-test. * denotes P = 0.0313 by one-sided t test. f CDC42BPA depletion counteracts the effect of Rho-kinase inhibitor, fasudil, on pMLC2 phosphorylation and cell migration. Top, MDA-MB-231 cells stably transduced with empty or CDC42BPA lenti-shRNA virus were treated with fasudil (25uM) or vehicle alone followed by western blots for CDC42BPA, anti-pMLC2-Thr18/Ser19 or total MLC2 with tubulin as loading control. Middle, statistical analysis on three independent biological replicates (see supplemental Fig. S7d). Bottom, statistical analysis on 9 independent scratch assays (see supplemental Fig. S7e). P values by unpaired two-tailed student t-tests. g Effect of RB depletion on TNBC cell migration in indicated cell lines. Error bars represent SD. h Effect of FBXW7 depletion on primary tumor formation of MDA-MB-436 cells following orthotopic transplantation into immune-deficient NSG mice (≥6 mice per group). P values calculated by one-way ANOVA, Tukey’s multiple comparison test. i, j. Effects of CDC42BPA or MTMR3 depletion on primary tumor formation and metastases of MDA-MB-436 cells following orthotopic transplantation into NSG mice (≥6 mice per group). Shown are primary tumor weights at end point (left) and number of lung mets (right) from multiple lung sections. P values calculated by one-way ANOVA, Tukey’s multiple comparison test. Source data are provided as a Source data file.

Fig 3: Sleeping Beauty (SB) mutagenesis screens on Rb-deficient background identify overlapping oncogenic alterations in mammary and lung tumors.a Setup of SB screens using MMTV-Cre:Rbf/f:T2/Onc3a:R26-lsl-SB11 and MMTV-Cre:Rbf/f:T2/Onc3b:R26-lsl-SB11 mice to identify primary mammary tumors and lung metastases driven by SB transposons on Rb-deficient background. b Macroscopic lung tumors detected by stereomicroscope were dissected and subjected to ligation-mediated PCR and deep sequencing. c PCR-based analysis of primary tumors to identify the 283 bp Rb-floxed allele (lanes 1–3) and 260 Rb deletion product following Cre-mediated recombination (lanes 1 and 3, but not 2). Source data are provided as a Source data file. Representative histology (d) and pie distribution (e) of primary and lung lesions. Pleomorphic/Squamous cell carcinoma (P/SCC), Adenosquamous carcinoma (ASC), Papillary/ Micropapillary (P/MP), poorly differentiation adenocarcinoma (PDA), Cribriform (CF). Scale bar, 100 μm. f Top gene-centered Common Integration Sites (gCIS) identified in primary (top) and lung (bottom) tumors following SB screens on Rb-deficient background. All gCISs are listed in Supplementary Fig. 1A and shown in Supplementary Data 1 and 2. S-specific gCIS are highlighted in red, and percentage in primary and lung lesions is tabulated. Representative gCIS functionally analyzed herein are marked in green (top – Fbxw7) and blue (bottom – Wdr33, Srgap2, Fbxw4, Pten, Cdc42bpa, and Mtmr3). g Venn diagram for significant gCISs identified in primary and lung lesions and the 7 S-drivers. Arrows point to direction of transposons. h Schematic structure of the NF1 gene locus and relative location of CIS in primary tumors and lung metastasis. > denotes SB integration in the 5′ to 3′ direction of the gene;