NRAS destines tumor cells to the lungs
2017; Springer Nature; Volume: 9; Issue: 5 Linguagem: Inglês
10.15252/emmm.201606978
ISSN1757-4684
AutoresAnastasios D. Giannou, Antonia Marazioti, Nikolaos I. Kanellakis, Ioanna Giopanou, Ioannis Lilis, Dimitra E. Zazara, Giannoula Ntaliarda, Danai Kati, Vasileios Armenis, Georgia A. Giotopoulou, Anthi C. Krontira, Marina Lianou, Theodora Agalioti, Malamati Vreka, Maria Papageorgopoulou, Sotirios Fouzas, Dimitrios Kardamakis, Ioannis Psallidas, Μάγδα Σπέλλα, Georgios T. Stathopoulos,
Tópico(s)Cancer Cells and Metastasis
ResumoResearch Article24 March 2017Open Access Source DataTransparent process NRAS destines tumor cells to the lungs Anastasios D Giannou Anastasios D Giannou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Antonia Marazioti Antonia Marazioti Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Nikolaos I Kanellakis Nikolaos I Kanellakis orcid.org/0000-0002-0065-2282 Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Ioanna Giopanou Ioanna Giopanou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Ioannis Lilis Ioannis Lilis Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Dimitra E Zazara Dimitra E Zazara Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Giannoula Ntaliarda Giannoula Ntaliarda Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Danai Kati Danai Kati Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Vasileios Armenis Vasileios Armenis Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Georgia A Giotopoulou Georgia A Giotopoulou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Anthi C Krontira Anthi C Krontira Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Marina Lianou Marina Lianou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Theodora Agalioti Theodora Agalioti Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Malamati Vreka Malamati Vreka Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), Member of the German Center for Lung Research (DZL), University Hospital, Ludwig-Maximilians University and Helmholtz Center Munich, Munich, Germany Search for more papers by this author Maria Papageorgopoulou Maria Papageorgopoulou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Sotirios Fouzas Sotirios Fouzas Pneumology Unit, Department of Pediatrics, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Dimitrios Kardamakis Dimitrios Kardamakis Department of Radiation Oncology and Stereotactic Radiotherapy, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Ioannis Psallidas Ioannis Psallidas Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Oxford Centre for Respiratory Medicine, Oxford University Hospitals NHS Trust, Oxford, UK Search for more papers by this author Magda Spella Corresponding Author Magda Spella [email protected] orcid.org/0000-0003-2505-7778 Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Georgios T Stathopoulos Corresponding Author Georgios T Stathopoulos [email protected] [email protected] orcid.org/0000-0002-9215-6461 Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), Member of the German Center for Lung Research (DZL), University Hospital, Ludwig-Maximilians University and Helmholtz Center Munich, Munich, Germany Search for more papers by this author Anastasios D Giannou Anastasios D Giannou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Antonia Marazioti Antonia Marazioti Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Nikolaos I Kanellakis Nikolaos I Kanellakis orcid.org/0000-0002-0065-2282 Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Ioanna Giopanou Ioanna Giopanou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Ioannis Lilis Ioannis Lilis Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Dimitra E Zazara Dimitra E Zazara Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Giannoula Ntaliarda Giannoula Ntaliarda Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Danai Kati Danai Kati Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Vasileios Armenis Vasileios Armenis Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Georgia A Giotopoulou Georgia A Giotopoulou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Anthi C Krontira Anthi C Krontira Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Marina Lianou Marina Lianou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Theodora Agalioti Theodora Agalioti Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Malamati Vreka Malamati Vreka Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), Member of the German Center for Lung Research (DZL), University Hospital, Ludwig-Maximilians University and Helmholtz Center Munich, Munich, Germany Search for more papers by this author Maria Papageorgopoulou Maria Papageorgopoulou Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Sotirios Fouzas Sotirios Fouzas Pneumology Unit, Department of Pediatrics, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Dimitrios Kardamakis Dimitrios Kardamakis Department of Radiation Oncology and Stereotactic Radiotherapy, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Ioannis Psallidas Ioannis Psallidas Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Oxford Centre for Respiratory Medicine, Oxford University Hospitals NHS Trust, Oxford, UK Search for more papers by this author Magda Spella Corresponding Author Magda Spella [email protected] orcid.org/0000-0003-2505-7778 Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Search for more papers by this author Georgios T Stathopoulos Corresponding Author Georgios T Stathopoulos [email protected] [email protected] orcid.org/0000-0002-9215-6461 Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), Member of the German Center for Lung Research (DZL), University Hospital, Ludwig-Maximilians University and Helmholtz Center Munich, Munich, Germany Search for more papers by this author Author Information Anastasios D Giannou1,‡, Antonia Marazioti1,‡, Nikolaos I Kanellakis1,‡, Ioanna Giopanou1,‡, Ioannis Lilis1, Dimitra E Zazara1, Giannoula Ntaliarda1, Danai Kati1, Vasileios Armenis1, Georgia A Giotopoulou1, Anthi C Krontira1, Marina Lianou1, Theodora Agalioti1, Malamati Vreka1,2, Maria Papageorgopoulou1, Sotirios Fouzas3, Dimitrios Kardamakis4, Ioannis Psallidas1,5,‡, Magda Spella *,1,‡ and Georgios T Stathopoulos *,*,1,2,‡ 1Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece 2Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), Member of the German Center for Lung Research (DZL), University Hospital, Ludwig-Maximilians University and Helmholtz Center Munich, Munich, Germany 3Pneumology Unit, Department of Pediatrics, Faculty of Medicine, University of Patras, Rio, Greece 4Department of Radiation Oncology and Stereotactic Radiotherapy, Faculty of Medicine, University of Patras, Rio, Greece 5Oxford Centre for Respiratory Medicine, Oxford University Hospitals NHS Trust, Oxford, UK ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +30 2610 969154; Fax: +30 2610 969176; E-mail: [email protected] *Corresponding author. Tel: +49 (89) 3187 4846; Fax: +49 (89) 3187 4661; E-mails: [email protected]; [email protected] EMBO Mol Med (2017)9:672-686https://doi.org/10.15252/emmm.201606978 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The lungs are frequently affected by cancer metastasis. Although NRAS mutations have been associated with metastatic potential, their exact role in lung homing is incompletely understood. We cross-examined the genotype of various tumor cells with their ability for automatic pulmonary dissemination, modulated NRAS expression using RNA interference and NRAS overexpression, identified NRAS signaling partners by microarray, and validated them using Cxcr1- and Cxcr2-deficient mice. Mouse models of spontaneous lung metastasis revealed that mutant or overexpressed NRAS promotes lung colonization by regulating interleukin-8-related chemokine expression, thereby initiating interactions between tumor cells, the pulmonary vasculature, and myeloid cells. Our results support a model where NRAS-mutant, chemokine-expressing circulating tumor cells target the CXCR1-expressing lung vasculature and recruit CXCR2-expressing myeloid cells to initiate metastasis. We further describe a clinically relevant approach to prevent NRAS-driven pulmonary metastasis by inhibiting chemokine signaling. In conclusion, NRAS promotes the colonization of the lungs by various tumor types in mouse models. IL-8-related chemokines, NRAS signaling partners in this process, may constitute an important therapeutic target against pulmonary involvement by cancers of other organs. Synopsis Mutations in the NRAS oncogene are shown to promote lung metastasis by regulating chemokine expression in tumor cells and hence their affinity for the pulmonary vasculature and their ability to form metastatic niches. NRAS mutations and/or gain promote lung metastasis of circulating tumor cells. NRAS promotes interleukin-8-related chemokine secretion by tumor cells. Chemokines signal to CXCR1 and CXCR2 receptors on lung endothelial and bone marrow cells to initiate metastatic niches in the lungs. Inhibition of CXCR1 and/or CXCR2 signaling inhibits lung metastasis in mouse models. NRAS gain-of-function is correlated with the presence of pulmonary metastases in a meta-analysis of a large autopsy study using contemporary genomic data from COSMIC. Introduction Metastasis is a central hallmark of cancer and the most common mode of cancer death (Nguyen et al, 2009; Vanharanta & Massague, 2013). Together with the bones, liver, and brain, the lungs are common target organs of metastasis, being affected in 25–40% of cancer patients overall and in even higher proportions of patients with some tumors (i.e. lung, breast, colon, and melanomatous skin cancers) that display predilection for the lungs (Hess et al, 2006; Disibio & French, 2008). Hence preventing or curing lung metastasis would likely lead to reductions in cancer deaths. For this, the mechanisms of multiple steps of the metastatic cascade need to be better understood: the mobilization of malignant cells from the primary tumor and of immune cells from the bone barrow; the homing of these cells to target organs; and the development of clinically evident metastases with structural and functional disruption (Kim et al, 2009; Nguyen et al, 2009; Borovski et al, 2011; Comen et al, 2011). Mounting evidence indicates that metastasis cannot be explained by anatomic and physiologic factors alone and suggests the existence of metastatic traits in tumor cells (Klein, 2003; Edlund et al, 2004; Meuwissen & Berns, 2005; Nguyen et al, 2009). Indeed, multiple investigations support this hypothesis via the identification of gene signatures that drive tropism of a given cancer for a given organ (Gupta et al, 2007; Minn et al, 2007; Padua et al, 2008; Chen et al, 2011; Oskarsson et al, 2011; Acharyya et al, 2012). However, these signatures are hard to target, and direct links between a single cancer mutation and metastatic tropism to a given organ, such as those suggested by observational studies of NRAS and KRAS mutations in pulmonary and hepatic metastasis of colon cancer and melanoma (Tie et al, 2011; Urosevic et al, 2014; Lan et al, 2015; Pereira et al, 2015; Ulivieri et al, 2015), are intriguing and of potential clinical value. Mutations and overexpression of the neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) are found across multiple tumor types and are common in highly metastatic cancers such as tumors of unknown primary, melanomas, and sarcomas that display a striking propensity for lung metastasis (Disibio & French, 2008; Stephen et al, 2014; Forbes et al, 2015). Clinical studies suggest that patients with NRAS-mutant or amplified tumors suffer from more aggressive disease than patients with wild-type NRAS alleles (Jakob et al, 2012; Jang et al, 2014; Ulivieri et al, 2015), and one recent study identified increased frequency of lung metastases in NRAS-mutant patients (Lan et al, 2015). However, the role of the oncogene in pulmonary metastasis has not been functionally studied. We discovered that tumor cells of various tissues of origin that carry NRAS mutations are able to spontaneously metastasize to the lungs of mice from subcutaneous (s.c.) primary sites, while cancer cells with wild-type NRAS cannot. We document that mutant or overexpressed NRAS is required for this capability of tumor cells and that it suffices to transmit it to cancer cells without NRAS mutations or even to benign cells. Importantly, we show that this phenotype of cancer cells that is triggered by NRAS is not due to enhanced growth capacities conferred by the oncogene, but rests on inflammatory chemokine signaling to cognate receptors on host lung endothelial and myeloid cells and can thus be targeted by chemokine receptor inhibition. Results An inflammatory link between NRAS and pulmonary metastasis We initially cross-examined the genetic alterations of eleven murine and human tumor cell lines with their spontaneous growth and dissemination patterns. For this, mouse cellular RNA was Sanger-sequenced for eight common cancer genes and human cell line data were obtained from the catalogue of somatic mutations in cancer (COSMIC) cell lines project (http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/) (Ikediobi et al, 2006; Forbes et al, 2015). In parallel, tumor cells of various tissues of origin were implanted s.c. into appropriate hosts at a titer yielding 100% tumor incidence (0.5 × 106 mouse and 106 human cells) and mice were sacrificed when moribund for lung examination. Three NRAS-mutant (NRASMUT; Lewis lung carcinoma, LLC, and AE17 malignant pleural mesothelioma of C57BL/6 mice carried NrasQ61H; human SKMEL2 skin melanoma carried NRASQ61R) and eight NRAS-wild-type (NRASWT; MC38 colon adenocarcinoma, B16F10 skin melanoma, and PANO2 pancreatic adenocarcinoma of C57BL/6 mice; CT26 colon adenocarcinoma and AB2 malignant pleural mesothelioma of BALB/c mice; as well as human A549 and HCC-827 lung adenocarcinomas and MDA-MB-231 breast carcinoma) tumor cell lines were identified (Fig EV1A). Six cell lines harbored KRAS, four TP53, two EGFR, one BRAF, and one STK11 mutations that coexisted with NRASMUT in a random fashion (Fig 1A). While all cell lines caused flank tumors in all mice injected (155/155; Fig EV1B), they exhibited a dichotomous capacity for automatic lung metastasis: Most mice (43/46) that received NRASMUT cell lines developed more than two bulky pulmonary macrometastases (diameter > 200 μm; clearly visible by the naked eye), as opposed to only 3/109 mice with NRASWT tumor cells, with the remaining exhibiting only tumor emboli and micrometastases (diameter < 200 μm; not visible by the unaided eye; Fig 1B and C). This phenotype co-segregated with NRAS, but not with KRAS mutation status or tissue of origin (Fig 1D and E). NRASMUT cells also displayed overexpression of the oncogene and activation of various downstream signaling pathways (Fig EV1C and E), in accord with what is observed in human KRAS- and NRAS-mutant tumors (Stephen et al, 2014; Pfarr et al, 2016). We next transfected C56BL/6 mouse tumor cells carrying either NRASMUT (LLC and AE17; NrasQ61H) or NRASWT (MC38) with a home-made plasmid encoding firefly luciferase (pCAG.Luc) to noninvasively track them in vivo after s.c. injection to syngeneic C57BL/6 hosts. All mice developed primary tumors emitting comparable bioluminescent signals that were excised after 2 weeks, but only mice with NRASMUT s.c. tumor cells developed bioluminescent lung metastases after additional 2 weeks (Figs 2A and EV2A). In complementary experiments, C57BL/6 recipients were lethally irradiated, reconstituted with bioluminescent bone marrow transplants (BMT) from syngeneic CAG.Luc.eGFP mice (Cao et al, 2004; Marazioti et al, 2013; Giannou et al, 2015), and received non-luminescent tumor cells s.c. After 4 weeks, bioluminescent myeloid cells were detected in all primary tumors, but only in the thoraces of mice with NRASMUT primary tumors and lung metastases (Figs 2B and EV2B). To study early premetastatic niches in the lungs, BMT was also performed using red-fluorescent mT/mG donors (Muzumdar et al, 2007) and s.c. injections of tumor cells harboring NRASMUT (LLC, AE17) or NRASWT (MC38) labeled using lentiviral eGFP plasmid (peGFP). In animals terminated after 2 weeks, that is, prior to frank metastasis, mononuclear and polymorphonuclear mT+ myeloid cells co-segregated in lung niches with GFP+ metastatic LLC and AE17 cells; in contrast, mice with s.c. MC38 tumors displayed no pulmonary GFP+ tumor cells and evenly dispersed mononuclear mT+ myeloid cells (Figs 2C and EV2C). Flow cytometric analysis of naïve and tumor-bearing C57BL/6 mice revealed increased numbers of circulating and splenic myeloid cells in all tumor-bearing mice, but increased pulmonary myeloid cells exclusively in the lungs of mice carrying NRASMUT LLC and AE17 tumors (Figs 2D and EV2D). Taken together with published work (Lyden et al, 2001; Rafii et al, 2002; Kaplan et al, 2005; Yang et al, 2008; Chen et al, 2011; Acharyya et al, 2012), these data suggested that tumor cells with mutant NRAS possess enhanced capability for automatic metastasis to the lungs, being thereby accompanied by myeloid cells to form metastatic niches. Click here to expand this figure. Figure EV1. Nras mutations of murine cancer cell lines A. Representative Sanger sequencing traces of Nras codons 60–63 of some mouse cancer cell lines employed in these studies showing Nras mutations (red font, black arrows). Shown is one representative of three traces. B. Weekly monitored primary tumor volume of C57BL/6, BALB/c, and NOD/SCID host mice after s.c. delivery of 0.5 × 106 mouse or 106 human tumor cells (n for each group is given in Fig 1E, table). C–E. mRNA and protein of mouse and human tumor cell lines harboring wild-type (WT) and mutant NRAS and KRAS alleles were examined by qPCR (C, n = 3) and immunoblotting (D, E; shown are one representative of three experiments). Data information: Cell lines are described in the text. All data are presented as mean ± SEM. P: probability by two-way ANOVA (B) and one-way ANOVA (C). (B, D, E): NRAS-mutant cell lines are in red font. (C): Genes overexpressed specifically by NRAS-mutant cell lines are in bold font. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. NRAS mutations and spontaneous lung metastasis of mouse and human cancer cell lines A. Mutation summary of eight cancer genes sequenced in seven mouse cancer cell lines (top) combined with human cell line mutation data (bottom). Red font indicates three cell lines identified carrying mutant NRAS. B–E. Eleven different mouse and human tumor cell lines with known KRAS, NRAS, HRAS, EGFR, BRAF, PIK3CA, TRP53, and STK11 mutation status were injected s.c. in appropriate host mice (0.5 × 106 mouse and 106 human cells; n of cell lines is given in D and of mice in E). Primary tumor volume was monitored weekly and the animals were killed for macroscopic and microscopic lung examination when terminally ill. Shown are representative images of intravascular tumor emboli, micrometastases (red arrows) and macrometastases (black arrows) (B), representative lung stereoscopic images (C), summary of spontaneous lung metastatic behavior (D), and number (graph) and incidence (table) of macrometastases (E). Note visible B16F10 micrometastases expressing melanin (B). Data information: Cell lines are described in the text. NRAS-mutant cells are in red font. Data are presented as mean ± SEM. P: probability by Fisher's exact test (D), one-way ANOVA (E, graph), or chi-square test (E, table). ***: P < 0.001 for comparison between any NRAS-wild-type and NRAS-mutant cell line by Bonferroni post-test (E, graph) or Fisher's exact test (E, table). Source data are available online for this figure. Source Data for Figure 1 [emmm201606978-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Figure 2. Lung colonization by NRAS-mutant tumor cells is associated with local accumulation of myeloid cells A, B. Bioluminescent images and data summaries of C57BL/6 mice with s.c. tumor cells expressing pCAG.Luc (A; n = 3/group) and of irradiated C57BL/6 chimeras reconstituted with CAG.Luc.eGFP bone marrow (B; n = 5) at 4 weeks after s.c. delivery of 106 tumor cells. Arrows indicate primary tumors; primary tumors of experiment (A) were excised at 2 weeks post-injection. Dashed lines denote the thorax. C. Lung sections of irradiated C57BL/6 mice, reconstituted with mT+ bone marrow and injected s.c. with 106 tumor cells overexpressing peGFP (n = 5/group) at 2 weeks post-tumor cells. Note the association of metastatic GFP+ tumor cells (green arrows) with mT+ bone marrow cells (red arrows) in niches (dashed outlines). Note also that mT+ myeloid cells in LLC- and AE17-colonized lungs were mononuclear and polymorphonuclear, while they were mononuclear in the lungs of mice with MC38 tumors. b, bronchus; a, alveolus. D. Blood, spleen, and lung CD11b+Ly6c+ cells and splenic dimensions of C57BL/6 mice (n = 4/group) which received s.c. saline (naïve) or tumor cells. Similar results were obtained using CD11b+Gr1+ and CD11b+Ly6g+ (not shown). Data information: Cell lines are described in the text. Data are presented as mean ± SEM. P: probability by one-way ANOVA. ns, *, **, and ***: P > 0.05, P < 0.05, P < 0.01, and P < 0.001, respectively, for indicated comparisons by Bonferroni post-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Primary tumor volume of experiments from Figs 2, 3, 4 and 7 A. Primary tumor volume of experiment from Fig 2A (n = 3 mice/cell line). B. Primary tumor volume of experiment from Fig 2B (n = 5 mice/cell line). C. Primary tumor volume of experiment from Fig 2C (n = 5 mice/cell line). D. Primary tumor volume of experiment from Fig 2D (n = 4 mice/cell line). E. Primary tumor volume of experiment from Fig 3A (n = 4 mice/cell line). F. Primary tumor volume of experiment from Fig 3D (n = 8 mice/cell line). G. Primary tumor volume of experiment from Fig 3E (n = 7–8/cell line). H, I. Primary tumor volume of experiments from Fig 4C (n is given in Fig 4C, tables). J, K. Primary tumor volume of experiments from Fig 7A (n is given in Fig 7A, tables). Data information: Cell lines are described in the text. All data are presented as mean ± SEM. P: probability by two-way ANOVA. ** and ***: P < 0.01 and P < 0.001 compared to controls at the time point indicated by Bonferroni post-test. Download figure Download PowerPoint NRAS drives circulating tumor cells to the lungs We next tested whether NRAS mutation and overexpression are functionally involved in pulmonary metastasis and at which step: primary tumor escape or lung homing? For this, peGFP-labeled mouse tumor cells with NRASMUT (LLC, AE17) or NRASWT (MC38) were implanted s.c. in C57BL/6 mice and were chased in the blood and the lungs by flow cytometry. Interestingly, all tumor types gave rise to similar numbers of circulating tumor cells, but only NRASMUT LLC and AE17 cells could home to the lungs (Figs 3A and EV2E). In addition, stable transfection of NRASWT MC38 cells with NRASMUT-encoding vector (pNRASG61K; Khosravi-Far et al, 1996) and also with NRASWT-encoding vector (pNRASWT; Fiordalisi et al, 2001), but not a control (pC; Morgenstern & Land, 1990) vector, rendered them capable of spontaneously colonizing the lungs from ectopic s.c. sites (Figs 3B and D, and EV2F). Even benign HEK293T cells delivered either s.c. or i.v. were rendered metastatic to the lungs of NOD/SCID mice (Blunt et al, 1995) upon NRASG61K transfection, as compared with pC-expressing cells that caused mere emboli and micrometastases (Figs 3B, C and E, and EV2G). In reverse experiments, lentiviral shRNA-mediated stable silencing of Nras (shNras) in NrasQ61H-mutant LLC and AE17 cells was done. Since these also carried KrasG12C mutations, side-by-side silencing of Kras (shKras) and of no known target (control shRNA; shC) using otherwise identical vectors was performed and the efficacy and specificity of this approach were validated (Figs 4A and B, and EV3). shNras exerted specific anti-metastatic effects, since it rendered s.c.-implanted LLC and AE17 cells virtually incapable for spontaneous lung metastasis without impacting primary tumor growth, while shKras reduced both primary tumor growth and pulmonary metastasis compared with shC (Figs 4C and EV2H and I). Moreover, LLC and AE17 cells (NrasQ61H) readily colonized the lungs upon intravenous (i.v.) delivery compared with MC38 cells (NrasWT), a propensity that was also abrogated by shNras (Fig 4D). Collectively, these results show that NRASMUT and/or overexpression of NRASWT promotes lung colonization by circulating tumor cells in mouse models. Figure 3. Mutant NRAS promotes lung colonization by circulating tumor cells A. Serial circulating and lung-homed tumor cells at 4 weeks after s.c. injection of 106 peGFP-expressing tumor cells (n = 4/group). Histogram: flow cytometry of tumor cells. Images: representative lung photographs and biofluorescence at 445- to 49
Referência(s)