Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis.
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.
Hendrix
An
A
Medical Genetics Branch, National Human Genome Research Institute, 10 Center Dr, Bethesda, MD 20892, USA.
Maynard
Dawn
D
Pauwels
Patrick
P
Braems
Geert
G
Denys
Hannelore
H
Van den Broecke
Rudy
R
Lambert
Jo
J
Van Belle
Simon
S
Cocquyt
Veronique
V
Gespach
Christian
C
Bracke
Marc
M
Seabra
Miguel C
MC
Gahl
William A
WA
De Wever
Olivier
O
Westbroek
Wendy
W
D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry / Q000201:enzymology* / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002853:Chromatography, Liquid; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D013058:Mass Spectrometry; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D016133:Polymerase Chain Reaction; D011379:Prognosis; D012333:RNA, Messenger / Q000378:metabolism; D011960:Receptors, Estrogen / Q000032:analysis*; D014183:Transplantation, Heterologous; D015854:Up-Regulation; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism
D014408:Biomarkers, Tumor; D018841:HSP90 Heat-Shock Proteins; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D000075745:rab27 GTP-Binding Proteins; C492134:RAB3D protein, human; C066694:RAB27A protein, human; C512960:Rab27B protein, human; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins