Vacuolar H1 ATPase expression and activity is required for Rab27B-dependent invasive growth and metastasis of breast cancer An Hendrix 1,2 , Raija Sormunen 3 , Wendy Westbroek 4 , Kathleen Lambein 5 , Hannelore Denys 2 , Gwen Sys 6 , Geert Braems 7 , Rudy Van den Broecke 7 , Veronique Cocquyt 2 , Christian Gespach 8* , Marc Bracke 1* and Olivier De Wever 1 1 Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium 2 Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium 3 Department of Pathology, Electron Microscopy Laboratory, Biocenter Oulu, University of Oulu, Oulu, Finland 4 Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD 5 Department of Pathology, Ghent University Hospital, Ghent, Belgium 6 Department of Orthopedic Surgery, Ghent University Hospital, Ghent, Belgium 7 Department of Gynecology, Ghent University Hospital, Ghent, Belgium 8 INSERM U938 Molecular and Clinical Oncology, H^ opital Saint-Antoine, Facult e de M edecine, Paris Cedex 12 and Universit e Pierre et Marie Curie-Paris (UMPC Paris 6), France The secretory Rab27B small GTPase promotes invasive growth and metastasis in estrogen receptor (ER) a-positive breast can- cer cells by orchestrating the peripheral targeting of vesicles secreting proinvasive growth regulators. Increased Rab27B expression is associated with poor prognosis in breast cancer patients. The molecular mechanisms of peripheral Rab27B se- cretory vesicle distribution are poorly understood. Mass spectrometry analysis on green fluorescent protein (GFP)-Rab27B vesicles prepared from GFP-Rab27B transfected MCF-7 human breast cancer cells detected eight subunits of the vacuolar H(1)-ATPase (V-ATPase) and the presence of V 0 a1 and V 0 d1 subunits was confirmed by Western blot analysis. Reversible inhi- bition of V-ATPase activity by bafilomycin A1 or transient silencing of V 0 a1 or V 0 d1 subunits demonstrated that V-ATPase con- trols peripheral localization and size of Rab27B vesicles. V-ATPase expression and activity further controls Rab27B-induced collagen type I invasion, cell-cycle progression and invasive growth in the chorioallantoic membrane assay. In agreement, Rab27B-dependent extracellular heat shock protein90a release and matrix metalloprotease-2 activation is markedly reduced by bafilomycin A1 and transient silencing of V 0 a1 and V 0 d1 subunits. Poor prognosis ERa-positive primary breast tumors expressing high levels of Rab27B also expressed multiple V-ATPase subunits and showed a strong cytoplasmic and peripheral V-ATPase V 1 E expression. In conclusion, inhibiting V-ATPase activity by interfering agents and drugs might be an effective strategy for blocking Rab27B-dependent proinvasive secretory vesicle trafficking in ERa-positive breast cancer patients. Regulated exocytosis of secretory vesicles may offer cancer cells a selective advantage for invasive growth. Secretory vesi- cle trafficking is regulated by the combined actions of actin filaments and microtubules using the molecular motor pro- teins dyneins, kinesins and/or members of the myosin family and Rab GTPases as critical regulators of vesicle transport. 1 Key words: proton pump, vesicle transport, exocytosis, Rab27, heat shock protein, invasion Abbreviations: CAM: chorioallantoic membrane; CM: conditioned medium; ER: estrogen receptor; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSP: heat shock protein; MMP: matrix metalloprotease; PBS: phosphate-buffered saline; PPI: proton pump inhibitor; V-ATPase: vacuolar H1 ATPase Additional Supporting Information may be found in the online version of this article. *C.G. and M.B. contributed equally to this work. Grant sponsors: Fund for Scientific Spearheads of the Ghent University Hospital, Stichting tegen Kanker, The Intramural Research Program of the National Human Genome Research Institute, Fund for Scientific Research-Flanders, Research Council of Ghent University DOI: 10.1002/ijc.28079 History: Received 22 Aug 2012; Accepted 2 Jan 2013; Online 7 Feb 2013 Correspondence to: An Hendrix, Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Tel.: 32-9-332 3008, Fax: 132-9-332 4991, E-mail: an.hendrix@ugent.be Cancer Cell Biology Int. J. Cancer: 00, 000–000 (2013) V C 2013 UICC International Journal of Cancer IJC