The Rho GTPase Rnd1 suppresses mammary tumorigenesis and EMT by restraining Ras-MAPK signalling (original) (raw)

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Acknowledgements

We thank X. Zhang for advice, reagents, and for sharing the structure of the Plexin-GAP–Rap1 complex before its publication, R. A. Weinberg, J. S. Brugge, M. Overholtzer, A. Ridley, B. Park, L. Tamagnome, M. Resh, H. Djaballah, T. Kataoka, M. Negishi and D. Medina for reagents, K. Manova for assistance with confocal microscopy, R. Khanin and G. P. Gupta for help with multivariate analysis, M. Buck for comments on the manuscript, and members of the Giancotti laboratory for discussions. We thank the Geoffrey Beene Translational Oncology Core, the Genomic Core, and the HTG Core of MSKCC for technical assistance. This work was supported by grants from the National Institutes of Health (P01 CA094060 Project 4 to F.G.G. and P30 CA08748 to MSKCC) and the Geoffrey Beene Cancer Center at MSKCC.

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Author notes

1. Tomoyo Okada, Gaia Schiavon, Miguel A. López-Lago, Christine A. Pratilas, Adriana Heguy & Giorgio Inghirami
   Present address: Present addresses: Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York 10065, USA (T.O.); Breast Unit, Royal Marsden Hospital, London SW3 6JJ, UK (G.S.); Department of Surgery and Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York 10065, USA (M.A.L-L.); The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA (C.A.P.); Department of Pathology, Office for Collaborative Science, New York University, New York 10016, USA (A.H.); Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York 10065, USA (G.I.).,
2. Tomoyo Okada, Surajit Sinha and Ilaria Esposito: These authors contributed equally to this work.

Authors and Affiliations

1. Cell Biology Program, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
   Tomoyo Okada, Surajit Sinha, Ilaria Esposito, Gaia Schiavon, Miguel A. López-Lago, Wenjing Su, Jonathan M. Hernandez & Filippo G. Giancotti
2. Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
   Christine A. Pratilas & Neal Rosen
3. Department of Pediatrics, Memorial Hospital, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
   Christine A. Pratilas
4. Department of Biomedical Sciences and Human Oncology, Center of Experimental Medicine and Research, University of Torino, Torino 10126, Italy
   Cristina Abele & Giorgio Inghirami
5. Department of Surgery, Memorial Hospital, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
   Jonathan M. Hernandez
6. Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan
   Masahiro Ohara & Morihito Okada
7. Genomics Core Facility, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
   Agnes Viale
8. Geoffrey Beene Translational Oncology Core Facility, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
   Adriana Heguy
9. Bioinformatics Core Facility, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
   Nicholas D. Socci
10. Department of Medical Sciences, Center of Experimental Medicine and Research, University of Torino, Torino 10126, Italy
    Anna Sapino
11. Department of Epidemiology and Biostatistics, Memorial Hospital, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
    Venkatraman E. Seshan
12. Structural Biology Program, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
    Stephen Long
13. Department of Medicine, Memorial Hospital, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
    Neal Rosen

Authors

1. Tomoyo Okada
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2. Surajit Sinha
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3. Ilaria Esposito
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4. Gaia Schiavon
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5. Miguel A. López-Lago
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6. Wenjing Su
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7. Christine A. Pratilas
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8. Cristina Abele
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9. Jonathan M. Hernandez
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10. Masahiro Ohara
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11. Morihito Okada
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12. Agnes Viale
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13. Adriana Heguy
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14. Nicholas D. Socci
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15. Anna Sapino
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16. Venkatraman E. Seshan
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17. Stephen Long
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18. Giorgio Inghirami
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19. Neal Rosen
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20. Filippo G. Giancotti
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Contributions

T.O., S.S., I.E., G.S., M.A.L-L., W.S., C.A.P., J.M.H. and C.A. performed and interpreted experiments; M.Ohara, M.Okada and A.S. provided annotated breast tumour samples, A.V. designed and performed genomic analyses; G.S. and V.E.S. performed bioinformatic analyses; S.L. provided structural insight; G.I. examined the results of FISH experiments; N.R. and G.I. supervised some of the experiments; F.G.G. supervised the entire study and wrote the paper.

Corresponding author

Correspondence toFilippo G. Giancotti.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 8 Silencing of Rnd1 induces EMT in MCF-10A, HMLE, and NMuMG mammary epithelial cells and RND1 is downregulated in aggressive and metastatic breast cancers.

(a) Quantification of scattering in control and Rnd1-silenced MCF-10A cells from Fig. 1c. (b–e) Ectopic expression of Rnd1 rescues Rnd1-silenced cells from EMT. Q-PCR analysis of RND1 in control and Rnd1-silenced MCF-10A cells transduced with a control vector or one driving re-expression of Rnd1 (b). Phase Contrast images (top) or immunostaining (bottom) for E-cadherin (Red), Phalloidin (actin, Green), and DAPI (Scale bar, 50 μm for top panels and 15 μm for middle and bottom panels) (c). Quantification of scattering (d) and immunoblotting for E-cadherin and Rho-GDI (e). (f–h) Silencing of Rnd1 induces EMT traits in NMuMG cells. Control and Rnd1-silenced NMuMG cells were subjected to Q-PCR for mouse RND1 (f), immunoblotting for E-cadherin, N-cadherin and Rho-GDI (g), and immunostaining for E-cadherin (Red), β-catenin (Green) and DAPI (top) or Zo-1 (Green) and DAPI (bottom). Scale bar, 15 μm (h). (i,j) Silencing of Rnd1 induces EMT traits in HMLE cells. Control and Rnd1-silenced HMLE cells were subjected to immunoblotting for E-cadherin, Vimentin, and Rho-GDI (i) or to immunostaining for E-cadherin (Red) and DAPI (top) or β-catenin (Green) and DAPI (bottom). Scale bar, 15 μm (j). (k,l) MCF-10A cells were transduced with a control shRNA (sh-Co.) or two shRNAs targeting RhoD (sh #1 and #2) and subjected to Q-PCR for RHOD (k) or photographed under Phase Contrast. The arrows point to apoptotic cells in the field. Scale bar, 50 μm (l). (m) Correlation between RND1 mRNA levels and the 70 Genes or the Lung Metastasis poor prognosis signature (LMS) in the MSKCC dataset (n = 117). (n) Patients from the MSKCC dataset were divided according to the Z-score for RND1 expression as indicated. (o) Kaplan–Meier analysis of the correlation between expression of RND1 and metastasis-free survival (left), lung metastasis-free survival (middle) and bone metastasis-free survival (right) in the MSKCC cohort. See Methods for statistical analysis. (p) Q-PCR for RND1 and GATA3 in a panel of breast cancer cell lines of the indicated transcriptomic subgroups. Panels a and d show averages and SD from n = 3 independent experiments. Panels b, c, e, g, and h show one representative experiment out of three performed, whereas panels, f, i, j, k, I, and p show one representative experiment out of two performed. Error bars indicate SD. P values by the Student’s _t_-test are: ∗, P = < 0.05; ∗∗, P = < 0.01; ∗∗∗, P < 0.001. For source data, see Supplementary Table 8. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 9 Rnd1 is upregulated by cytostatic stimuli and inhibits cell cycle progression.

(a) MCF-10A cells were incubated with mitogens (5 ng/ml EGF, 10 μg/ml Insulin, 0.5 μg/ml Hydrocortisone, and Bovine Pituitary Extract) or TGF-β (10 ng/ml), plated under either sparse or confluent conditions, or exposed to UV (100 Joules/cm2) for the indicated times, and subjected to Q-PCR for RND1 mRNA. The graphs show average values from one experiment in which triplicate samples were assessed in parallel. The experiments on the left and right panels were repeated 2 times and the one in the middle panel once. (b,c) MCF-10A cells were infected with a retrovirus encoding HA-Rnd1 or with empty vector and subjected to immunoblotting as indicated (b) or to MTT assay at the indicated times. The graph shows the averages and SD of n = 6 technical replicates. The experiment was repeated 2 times (c). (d) MCF-10A cells transduced with either a control shRNA or 2 shRNAs targeting Rnd1 were cultured for 3 days and subjected to DNA microarray analysis. Genes concomitantly upregulated or downregulated by >1.5 fold were subjected to unsupervised hierarchical clustering with Partek. 59 were downregulated and 117 upregulated (left). Ingenuity Pathways Analysis (IPA) was conducted to assign the genes to the functional categories of Biofunctions and Canonical Pathways. The graphs show the Biofunctions and Canonical Pathways with _P_-value < 0.05 (right-tailed Fisher Exact Test). Genes in red are upregulated and those in blue downregulated. See Supplementary Table 3 for the gene list. (e) Control (Co.) and Rnd1-silenced (#1 and #2) MCF-10A cells were cultured for 5 days and subjected to Q-PCR for indicated cell cycle genes or (f) immunoblotting as indicated. (g) Control (sh-Co.) and Rnd1-silenced (sh #1 and #2) MCF-10A cells were subjected to immunoblotting with antibodies to p27, p21 and p53 at the indicated times after infection. Panels b, f, and g show representative results from one of three independent experiments, whereas panels a, c and e show results from one of two independent experiments. Biological replicates yielded similar results. For source data, see Supplementary Table 8. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 10 Expression of Myc or loss of p53 and Rb induces neoplastic transformation of Rnd1-silenced mammary epithelial cells.

(a–c) Rnd1-silenced MCF-10A cells, which are maintained in culture over a 1-month period, undergo spontaneous immortalization accompanied by overexpression of c-Myc. Phase Contrast pictures of control and Rnd1-silenced MCF-10A cells that have been cultured over a 1-month period (a). Control (sh-Contr.) and spontaneously immortalized Rnd1-silenced (sh-Rnd1 #2) MCF-10A cells were subjected to soft agar assay (b) and immunoblotting as indicated (c). Scale bars, 50 μm. (d–f) Expression of Myc enables neoplastic conversion of Rnd1-silenced MCF-10A cells. MCF-10A cells, which were transduced with c-Myc or left uninfected, were infected with a control shRNA or one targeting Rnd1. (d) Cells were deprived of growth factors, stimulated or not with 10% FBS, and subjected to immunoblotting as indicated (top) or Q-PCR for RND1 (bottom). Graph shows one of two representative experiments. (e) Cells were subjected to SA-βtGalactosidase staining at 12 days after infection. (f) Cells were subjected to soft agar assay. The images show representative pictures at 2 weeks. Scale bar is 50 μm. (g,h) Silencing of Rnd1 induces anchorage-independent growth of HMLE cells. (g) Cells were transduced with a control shRNA or two shRNAs targeting Rnd1 and cultured in soft agar for 2 weeks. The images show representative pictures (scale bar, 50 μm). (h) The graph shows the average number of colonies and SD from one experiment performed of n = 3 technical replicates. The experiment was repeated 2 times. (i-l) Silencing of Rnd1 induces hyperproliferation but not suppression of apoptosis in 3D Matrigel. Parental MCF-10A cells and MCF-10 cells expressing c-Myc were infected with a control shRNA or two shRNAs targeting Rnd1, cultured in 3D Matrigel for 8 days, and subjected to immunofluorescent staining with anti-Ki-67 followed by DAPI. Representative images (scale bar, 50 μm) are shown (i). The graph shows the average percentage of Ki-67-positive cells and SD from n = 3 independent experiments (j). The graph shows the average percentage of cleaved caspase 3-positive cells and SD from n = 3 independent experiments (k). The pictures show representative images of pseudoacinar structures stained with anti-cleaved caspase 3 and DAPI. The arrows point to cells that have lost their apical positioning of Golgi apparatus and invaded inot the Matrigel. Note that they possess intact nuclei. Scale bar, 15 μm (l). P values are: ∗, P = < 0.05; ∗∗, P = < 0.01; ∗∗∗, P < 0.001 by the Student’s _t_-test. Panels b-f show one representative experiment out of three and panels a and g show one representative out of two performed. Biological replicates yielded similar results. Source data is given in Supplementary Table 8. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 11 Loss of Rnd1 promotes hyperproliferation and EMT through activation of the Raf-ERK cascade.

(a) Control (sh-Co.) and Rnd1-silenced (sh #1 and #2) MCF-10A cells were subjected to GST-pull down assay to measure activated Cdc42, Rac1, and Rho (A, B, and C). (b) MCF-10A cells expressing either a control shRNA (sh-Co.) or one targeting Rnd1 (sh #1) were starved for 24 hours, stimulated with growth factors for the indicated times, and subjected to GST-RBD pull down assay to measure GTP-Ras. (c) HMLE (top) and HEK 293T (middle) cells expressing a control shRNA (sh-Co.) or two sh-RNAs targeting Rnd1 (sh #1 and #2) were starved for 24 h, and HUVECs (bottom) untransfected or transfected with a control si-RNA or one targeting Rnd1 (si-Rnd1) were subjected to pulldown assay to measure GTP-Ras. (d) MCF-10A cells carrying either a control shRNA or one targeting Rnd1 (sh #1) were cultured for 24 h with PD98059 (MEK inhibitor) or vehicle, and subjected to immunofluorescent staining as indicated. Scale bar, 15 μm. (e) MCF-10A cells expressing either a control shRNA (sh-Co.) or one targeting Rnd1 (sh #2) were cultured for 3 days and treated with Rho kinase inhibitor (Y27632), myosin light chain kinase inhibitor (Blebbistatin) or DMSO for 24 h. Scale bar, 15 μm. (f) MCF-10A cells expressing either a control shRNA or 2 shRNAs targeting Rnd1 (sh #1 and #2) were cultured for 2 days and treated with PD98059 (MEKi), Wortmannin (PI-3Ki), or vehicle for 24 h. Total lysates were subjected to immunoblotting with the indicated antibodies. (g) Gene set enrichment analysis plots showing that low RND1 mRNA levels do not correlate with the expression of a Src or a β-catenin signature in the 3 breast cancer datasets. (h) The levels of mRNAs encoding Myc and Rnd1 exhibit an inverse correlation in 3 breast cancer DNA microarray datasets. Spearman correlation = − 0.46 for EMC192, − 0.36 for EMC286 and − 0.43 for MSKCC99; P < 0.001. See Method for analysis. Data in a, b, c and e are from one of two independent experiments, whereas d and f are from one of three independent experiments. Biological replicates yielded similar results. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 12 Plexin B1 does not modulate MET or HER2 signaling in breast epithelial cells.

(a) Total lysates from the indicated normal mammary epithelial cells and breast cancer cells were subjected to immunoblotting to assess the expression of Plexin B1, Plexin A1, and Semaphorin 4D. (b) Phase Contrast images of control and Plexin B1-silenced MCF10A cells. Scale bar, 50 μm. (c) HMLE cells were starved in growth factor-deprived medium supplemented with 0.2% serum for 15 h and incubated with HGF with or without Semaphorin 4D for the indicated times. Total lysates were subjected to immunoblotting with the indicated antibodies. (d) ZR751 cells were starved as in c and incubated with HRG with or without Semaphorin 4D for the indicated times. Total lysates were subjected to immunoblotting with the indicated antibodies. Representative data from two independent experiments are shown. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 13 Rnd1-PlexinB1-Rasa1 signaling axis is altered in breast cancer.

(a) Analysis of mRNA levels of RND1, PLXNB1 and major RasGAPs in the PAM50 basal-like breast tumor dataset from TCGA using cbioportal. (b) Immunoblotting of MAPK signaling pathway components in p120RasGAP-silenced MCF10A cells. Blots are representative images from two independent experiments. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 14 Silencing of Rnd1 accelerates tumorigenesis, whereas ectopic expression of Rnd1 exerts the opposite effect.

(a-g) Comma-D cells infected with a control shRNA (sh-Co.) or two shRNA targeting Rnd1 (sh #4 and #5) were subjected to Q-PCR to verify depletion of Rnd1. Data are averages and SD from n = 3 independent experiments (a). Cells were photographed under Phase Contrast. Scale bar, 50 μm (b). Cells were subjected to double immunofluorescent staining for β-catenin (Green) and E-cadherin (Red) followed by DAPI. Scale bar, 15 μm (c). Cells were subjected to MTT assay. Data are from one experiment shown as averages of five technical repeats (n = 2 biological replicates) (d). Cells were subjected to immunoblotting as indicated (e, f). Cells were cultured in soft agar for 2 weeks. The graph indicates the average number of colonies from five technical repeats (n = 2 biological replicates) (left) and the pictures representative images (right) (g). (h) 4T1 and ErbB2 cells infected with control vector or one encoding HA-Rnd1 were subjected to immunoblotting. (i,j) 4T1 cells infected with retroviral vector encoding Rnd1 or control vector were inoculated intravenously. Lung metastasis was measured by bioluminescent imaging. The panels shows representative images at the indicated times (i). The graph shows individual data points for normalized photon flux (lung, dotted area in i) at different time points after tail-vein injection from n = 5 mice in each cohort (j). (k) Quantification of bioluminescence from lungs from the spontaneous metastasis experiment of Fig. 7f. n = 3 mice and individual data points are shown. Panels b and c show one representative experiment out of three independent experiments. Error bars are SD in a, d and g and SEM in j and k. P values are: ∗, P = < 0.05; ∗∗, P = < 0.01; ∗∗∗, P < 0.001 by the Student’s _t_-test. Biological replicates yielded similar results. For source data, see Supplementary Table 8. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 15 Genetic and epigenetic inactivation of RND1 in human breast cancer cell lines.

(a) Box plots depicting the distribution of RND1 copy number loss defined as the ratio of signal observed for the _RND1_-specific and centromeric chr 12 probes in wild type (blue dots) and deleted (red dots) tumor cell subpopulations. Black bars indicate the mean values for each subpopulation. Samples are indicated with Arabic numbers, N stands for normal cells. (b) The indicated breast cancer cells were treated with SAHA alone (24 h), 5-AZA alone (72 h) or a combination of SAHA and 5-AZA (72 h) and subjected to Q-PCR for RND1. Data are from one experiment and are shown as averages of three technical replicates (n = 2 biological replicates). (c) Subcellular localization of wild type Rnd1. Rnd1 is localized at the plasma membrane and on the Golgi. HUVECs were transfected with a plasmid encoding HA-tagged Rnd1 and a Farnesylated CAAX motif fused to GFP and subjected to immunostaining with anti-HA (Red), anti-Giantin (Cyan) and DAPI. (d) Co-localization of Rnd1 with H-Ras and K-Ras. HUVECs transfected with plasmids encoding HA-tagged Rnd1 and GFP-H-Ras (Top) or GFP-K-Ras4B (Bottom) were subjected to immunostaining with anti-HA (Red), anti-Giantin (Cyan) and DAPI (Blue). Images in (c) and (d) are representative of three independent experiments. Scale bar, 15 μm. (e) EpiTYPER was carried out to analyse the methylation state of CpGs islands in the RND1 gene promoter in selected breast tumour cell lines. Each circle indicates a CpG dinucleotide. WGA: negative control. IVD: positive control. (f) HCC1428 cells infected with a control shRNA (Co.) or 3 shRNAs targeting EZH2 (#1, #2, #3) were subjected to immunoblotting with the indicated antibodies. A representative blot from two independent experiments is shown. (g) The indicated cell lines were infected with a shRNA targeting EZH2 (#1) or not, treated with SAHA (6 h) or vehicle control, and then subjected to Q-PCR for RND1 (left). HCC1428 cells expressing a control shRNA or 2 shRNAs targeting EZH2 (#2 and #3) were treated with SAHA (24 h) or vehicle control and subjected to Q-PCR for RND1 (right). One of two independent experiments is shown. For source data, see Supplementary Table 8. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Table 1 Metastasis-Free Survival analysis for all RhoGTPases

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Supplementary Table 2 Univariate and Multivariate analysis for survival

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Supplementary Table 3 list of genes up- or down-regulated in RND1 knocked down cells

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Supplementary Table 4 Cases positive for RND1 deletion by FISH related to Fig. 8a and Supplementary Fig. 8a

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Supplementary Table 5 Solid sequencing results for each mutation of _RND1_related to Fig. 8

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Supplementary Table 6 Antibody list used in this study

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Supplementary Table 7 Q-PCR Primer list

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Okada, T., Sinha, S., Esposito, I. et al. The Rho GTPase Rnd1 suppresses mammary tumorigenesis and EMT by restraining Ras-MAPK signalling.Nat Cell Biol 17, 81–94 (2015). https://doi.org/10.1038/ncb3082

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• Received: 24 April 2014
• Accepted: 10 November 2014
• Published: 22 December 2014
• Issue Date: January 2015
• DOI: https://doi.org/10.1038/ncb3082