Poster Sessions

CANCER-33

Wendy Westbroek

W. Westbroek, D. Maynard, A. Hendrix, M.C. Seabra, O. De Wever, W.A. Gahl

The Secretory Small GTPase Rab27B Regulates Invasive Tumor Growth and Metastasis through Extracellular HSP90α

Key players in exocytic and endocytic trafficking include Rab GTPases. Our studies on various secretory Rab GTPases revealed that over-expression of Rab27B in several breast cancer cell lines promoted cell cycle progression, F-actin reorganization, and invasion into matrigel. By establishing MCF-7 cells that stably expressed wild type Rab27B, Rab27B-Q78L, Rab27B-T23N, and the geranylgeranyl mutant Rab27B-GER, we determined that Rab27B-mediated invasion was dependent on geranylation and GTP-binding state. Our xenograft mouse model showed that mice injected with MCF-7-Rab27B or Rab27B-Q78L developed invasive xenografts that were 8-fold larger than the non-invasive MCF-7-Rab27B-T23N or Rab27B-GER xenografts. Proteomic analysis of Rab27B-positive purified vesicles and the secretome of MCF-7 Rab27B identified HSP90α as a key pro-invasive factor. By ELISA and western blotting, we showed that HSP90α secretion was upregulated in the conditioned media prepared from MCF-7-Rab27B and Rab27B-Q78L. Treatment with an HSP90α neutralizing antibody reversed the invasive phenotype of MCF-7-Rab27B cells while a dose-dependent treatment with recombinant HSP90α of MCF-7 cells induced invasion. Finally, we examined endogenous Rab27B expression in 60 clinical samples derived from breast cancer patients by real time quantitative PCR and immune-histology. We showed that upregulation of endogenous Rab27B mRNA and protein correlated with lymph node metastasis, tumor grade and positive ER-status.