Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis - PubMed (original) (raw)

. 2010 Jun 16;102(12):866-80.

doi: 10.1093/jnci/djq153. Epub 2010 May 18.

Dawn Maynard, Patrick Pauwels, Geert Braems, Hannelore Denys, Rudy Van den Broecke, Jo Lambert, Simon Van Belle, Veronique Cocquyt, Christian Gespach, Marc Bracke, Miguel C Seabra, William A Gahl, Olivier De Wever, Wendy Westbroek

Affiliations

Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis

An Hendrix et al. J Natl Cancer Inst. 2010.

Abstract

BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.

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Figures

Figure 1

Figure 1

Effect of ectopic expression of Rab3D, Rab27A, or Rab27B on the formation of cellular extensions and invasiveness. A) Morphology of MCF-7 cells transiently transfected with green fluorescent protein (GFP), GFP-Rab3D, GFP-Rab27A, or GFP-Rab27B-expressing plasmids. Twenty-four hours after transfection, cells were fixed and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Laser scanning confocal images show punctuate GFP signal (green) that is indicative of localization of GFP fusion protein to vesicles. Scale bar, 20 μm. B) Matrigel invasion assay with GFP-Rab–transfected MCF-7 cells. Twenty-four hours after transient transfection with GFP-Rab–expressing plasmids, 105 MCF-7 cells were seeded on top of a Matrigel-coated filter, and their migration toward medium containing serum was quantified by microscopic evaluation (total magnification ×400). The mean total number of invading cells counted after 72 hours from 10 different fields is shown with the upper 95% confidence intervals from the means of three independent experiments performed in triplicate. P values were calculated using two-sided Student t tests. Statistically significant P values are indicated. C and D) Morphology and invasiveness of GFP-Rab–transfected MCF-7 and T47D breast cancer cells. In (C), phase-contrast images are shown of cells seeded on type I collagen matrix 24 hours after transient transfection. In (D), the invasion index was calculated by counting the number of invading and noninvading cells into type I collagen matrix in 10 fields. Invasion indices are means and upper 95% confidence intervals derived from the means of three independent experiments performed in triplicate. P values were calculated using χ2 tests; statistically significant P values are indicated. Scale bar, 50 μm. In (A) and (C), arrows indicate cellular extensions and local spreading.

Figure 2

Figure 2

Rab27B GTP- and geranylgeranyl-dependent cancer cell invasion and cell cycle progression in vitro. A) Phase-contrast images showing morphology of MCF-7 cells stably transfected to express green fluorescent protein (GFP), GFP-Rab27A, GFP-Rab27B (wild type, WT), or GFP-Rab27B mutants. GFP-Rab27B-Q78L (constitutive active), -N133I (dominant negative), and -GER (impaired geranylgeranylation and vesicle targeting) were the mutants used. Arrows indicate cellular extensions and local spreading. Scale bar, 50 μm. B) Quantification of type I collagen invasion by the cells shown in (A). Invasion assays were performed as in Figure 1, D. Invasion indices are means and upper 95% confidence intervals derived from the means of three independent experiments performed in triplicate. P values were calculated using χ2 tests. Statistically significant P values are indicated. C) Laser scanning confocal images of the F-actin cytoskeleton (phalloidin–tetramethyl rhodamine isothiocyanate) (red) and GFP (green) localization in MCF-7 GFP and GFP-Rab27B cells cultured for 24 hours on a collagen type I matrix. Arrow indicates cortical F-actin, and arrowhead indicates membrane blebs. Scale bar, 20 μm. D) Invasion by Rab27B-expressing MCF-7 cells in which Rab27B was depleted. MCF-7 cells that expressed GFP-Rab27B, with or without transfection of control small interfering RNA (siRNA) (siCON) or Rab27B siRNAs (siRab27B-1 and/or -2), were seeded on a Matrigel-coated filter. The inset panel shows the impact of the Rab27B siRNAs on Rab27B expression in these cells by immunoblotting. The numbers of invasive cells were counted after 72 hours in 10 different fields and are expressed as the mean with upper 95% confidence intervals of three independent experiments performed in triplicate. P values shown are for comparisons with the siCON transfection using two-sided Student t tests. E) Effect of Rab27B on cell cycle progression. MCF-7 GFP and GFP-Rab27B cells were grown to 50% confluence, followed by 24 hours serum starvation, and 24 hours serum-induced (0.5%) cell cycle progression. Percentages of MCF-7 GFP and GFP-Rab27B cells in G1, S, and G2 stage of the cell cycle, as measured by flow cytometry, are represented as the means with upper 95% confidence intervals of two independent experiments. F) Western blot analysis in mutant Rab27B-transfected MCF-7 cells of the positive (cyclin A and E) and negative (p27) G1 to S phase cell cycle regulators. Protein levels were quantified as immunostaining intensity relative to tubulin. G) Measurement of cell proliferation rates of MCF-7 cells stably expressing GFP, GFP-Rab27B, or GFP-Rab27B mutants as in (A). A total of 10 000 cells were plated into each well of a total of 15 wells on day 1 to establish one growth curve under each condition in triplicate. The total number of cells per well was manually counted every 2 days until day 8. Mean number of cells is plotted with upper 95% confidence intervals. P values were calculated using the two-way repeated measures analysis of variance test. Statistically significant P values are indicated; data were compared with the GFP control. H) Measurement of cell proliferation rates of MCF-7 GFP-Rab27B cells transiently transfected with control (siCON) or pooled Rab27B siRNAs (siRab27B-1 and -2). The experiment was performed as in (G). An inset panel shows the effect of this siRNA on cyclin A expression in MCF-7 GFP-Rab27B cells. Tubulin was used as loading control.

Figure 3

Figure 3

Effect of Rab27B on invasive tumor growth in vivo. Nude mice were injected in the mammary fat pad with MCF-7 cells expressing green fluorescent protein (GFP), GFP-Rab27A, GFP-Rab27B (wild type, WT), or mutant GFP-Rab27B proteins (Q78L, T23N, N133I, and GER). A) Tumorigenesis in nude mice with MCF-7 GFP-Rab27B xenografts vs controls. Mice with MCF-7 GFP-Rab27B xenografts (lower panel) developed hemorrhagic ascites (blue and swollen appearance of the ventral side) and tumor aggregates (arrows) in the peritoneal cavity and attached to organs such as the ovary. MCF-7 GFP xenografts (upper panel) developed no hemorrhagic ascites. Inset: Pelleted tumor aggregates from the peritoneal fluid of one mouse. Scale bar, 13 mm. B) Effect of Rab27B expression on survival of mice with xenografts. Kaplan–Meier curves with 95% confidence intervals and log-rank testing (P = .031) are shown for nude mice injected with MCF-7 GFP cells (n = 10) vs MCF-7 GFP-Rab27B cells (n = 40; four different clones with 10 mice per group). C) Expression of GFP-Rab27B in tumor aggregates. A western blot loaded with 60 μg peritoneal tumor aggregate and immunostained with primary Rab27B and tubulin antibodies is shown. D) Hematoxylin and eosin (H&E) staining of a peritoneal tumor aggregate. Scale bar, 100 μm. E) H&E staining of MCF-7 GFP (upper panel) and GFP-Rab27B (lower panel) xenografts. Arrowheads indicate striated muscle tissue; arrows indicate areas of muscular invasion by cancer cells to the peritoneal side (P). Scale bar, 100 μm. F) Relative invasiveness of xenografts expressing WT and mutant Rab27B proteins. Percentage of invasive tumors was determined by the total number of mice with an invasive xenograft in the peritoneal wall as assessed by macroscopic observation and immunohistochemistry (n = 10 mice per group). Precise percentages for a single experiment are shown. G) Cellular localization of Rab27B in MCF-7 GFP-Rab27B xenografts. Arrow indicates peripheral Rab27B distribution; arrowheads indicate Rab27B vesicle clustering appearing in the cytoplasm and at cell–cell contact. Scale bar, 25 μm. H) Mean tumor volume in nude mice bearing xenografts that expressed WT or mutant Rab27B proteins (n = 10 mice per group). Tumor size was assessed weekly by measurement of the external diameter of the xenografts for 10 weeks. GFP expression was maintained in the xenografts throughout this time period (data not shown). Error bars represent 95% confidence intervals. I) Mean tumor weight after surgical resection of xenografts expressing WT or mutant Rab27B proteins. Mice were killed at variable time points (ie, the ethical endpoint that limits hemorrhagic ascites formation or the experimental endpoint at 10 weeks) after injection of stably transfected MCF-7 cells (n = 10 mice per group). Error bars represent upper 95% confidence intervals. P values were calculated using two-sided Student t tests; statistically significant P values are indicated. J) Immunohistochemical staining of MCF-7 GFP and GFP-Rab27B xenografts to detect Ki67, a proliferation marker. The mean number of proliferating MCF-7 GFP-Rab27B cells, calculated from 18 images of three primary tumors per cell line, was 85.50 ± 4.04 vs 32.56 ± 2.68 proliferating control cells (two-sided Student t test, P < .001). Scale bar, 50 μm.

Figure 4

Figure 4

Selective stimulation of heat-shock protein (HSP)90α secretion by Rab27B through GTP- and geranylgeranyl-dependent mechanisms. A) Secretome profiling of invasive MCF-7 green fluorescent protein (GFP)-Rab27B cancer cells identified HSP90α and HSP90β. The number of matched peptides and the percentage of sequence coverage are indicated for both proteins. The mass spectrometry/mass spectrometry spectrum recorded on a [M+2H]2+ ion at m/z 618.69 corresponds to a unique peptide [DQVANSAFVER], derived from HSP90α. Peptides fragment along the amide backbone to produce sequence-specific fragment ions; ions containing the C-terminal fragment are known as “y” ions, whereas ions containing the N-terminal fragment are known as “b” ions. The search engine Mascot uses this information to report probability-based scores for each peptide. See “Materials and Methods” for more details. B) Quantification of HSP90α levels in conditioned medium (CM) of GFP- vs GFP-Rab27B-expressing (wild type, WT) MCF-7 cells using enzyme-linked immunosorbent assay. Results are means with upper 95% confidence intervals of two independent experiments with three replicates. C) Western blot analysis of HSP90α and HSP90β in CM (upper panel) and in total protein lysates (lower panel) of transfected MCF-7 cells. Relative intensity was quantified with HSP90β or tubulin as a loading control. D) Impact of Rab27B silencing (siRab27B-1 and -2) vs control silencing (siCON) on the expression of GFP-Rab27B protein (lower panel) and secretion of HSP90α and HSP90β (upper panel) in the CM of MCF-7 GFP-Rab27B cells. Protein levels were quantified as immunostaining intensity relative to tubulin and HSP90β, respectively.

Figure 5

Figure 5

The role of heat-shock protein (HSP)90α and matrix metalloproteinase (MMP)-2 in Rab27B-dependent invasion. A) Phase-contrast images showing morphology (upper panels) and quantification of collagen type I invasion by MCF-7 green fluorescent protein (GFP)-Rab27B cells (lower panel) treated with the HSP90α inhibitors 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and geldanamycin (GA) (1 μM) for 24 hours or left untreated (Control, Con). B) Morphology (upper panels) and quantification (lower panel) of the invasive phenotype induced by GFP-Rab27B in MCF-7 cells cultured on collagen type I matrix treated for 6 hours with HSP90α neutralizing antibody (1 μg/mL) or the control IgG isotype. C) Morphology (upper panel) and quantification (lower panel) of the invasive phenotype induced by HSP90 in MCF-7 GFP cells cultured on collagen type I matrix and treated for 24 hours in the presence or absence (Control, Con) of recombinant (rec) HSP90α protein (1, 5, and 10 μg/mL) or recombinant HSP90β protein (10 μg/mL). In (A), (B), and (C), arrows indicate cellular extensions and local spreading. Scale bar, 100 μm. Invasion indices are means and upper 95% confidence intervals derived from the means of three independent experiments performed in triplicate. P values are calculated using the χ2 test; statistically significant P values are indicated. D) Measurement of cell proliferation rates of MCF-7 GFP cells treated with recombinant HSP90α (10 μg/mL) or left untreated (Con) and of MCF-7 GFP-Rab27B cells challenged with a HSP90α neutralizing antibody (5 μg/mL) or control immunoglobulin (Con IgG). Proliferation assay was performed as in Figure 2, G. Mean number of cells is plotted with upper 95% confidence intervals. P values are calculated using the two-way repeated measures analysis of variance test. E) Cyclin A expression was evaluated in MCF-7 GFP cells treated with recombinant HSP90α (10μg/mL) or left untreated (Con) and in MCF-7 GFP-Rab27B cells challenged with HSP90α neutralizing or control antibody. Intensity was quantified relative to tubulin. F) Analysis of MMP-2 activity in conditioned medium (CM) from cultured MCF-7 cells expressing GFP, GFP-Rab27B (wild type, WT), or the GFP-Rab27B mutants (Q78L, N133I, and GER) by gelatin zymography. G) Gelatin zymography of MMP-2 activity in CM from MCF-7 GFP-Rab27B cells that were preincubated with exogenously added proMMP-2 (100 ng/mL) in serum-free medium for 24 hours. In (F) and (G), arrowhead indicates 72 kDa proMMP-2 and arrow indicates 68 kDa active protease.

Figure 6

Figure 6

Rab27B expression in clinical breast cancer specimens. A) Representative Rab27B stained primary breast cancer samples that illustrate immunohistochemical scores of 0, 1, and 2. Scale bar, 100 μm. B) Associations of Rab27B immunohistochemical scores with estrogen receptor (ER) status and other clinicopathological data for 59 primary breast tumors. The χ2 test was used to test for differences between categorical variables. C) Relative levels of Rab3D, Rab27A, and Rab27B mRNA expression in normal tissue (N, n = 5) vs primary breast carcinoma (T, n = 20). D) Expression of Rab27B mRNA in five normal tissues vs 20 primary breast carcinomas. Tumor samples were divided into three groups according to ER status and lymph node (LN) involvement. In (C) and (D), mRNA expression was measured by quantitative real-time polymerase chain reaction in triplicate. Horizontal bars represent median for each group (two-sided Mann–Whitney test).

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