Dynamics of the subcellular localization of RalBP1/RLIP through the cell cycle: the role of targeting signals and of protein-protein interactions.

The small G protein Ras regulates many cell processes, such as gene expression, proliferation, apoptosis, and cell differentiation. Its mutations are associated with one-third of all cancers. Ras functions are mediated, at least in part, by Ral proteins and their downstream effector the Ral-binding protein 1 (RalBP1). RalBP1 is involved in endocytosis and in regulating the dynamics of the actin cytoskeleton. It also regulates early development since it is required for the completion of gastrulation in Xenopus laevis. RalBP1 has also been reported to be the main transporter of glutathione electrophiles, and it is involved in multidrug resistance. Such a variety of functions could be explained by a differential regulation of RalBP1 localization. In this study, we have detected endogenous RalBP1 in the nucleus of interphasic cells. This nuclear targeting is mediated by nuclear localization sequences that map to the N-terminal third of the protein. Moreover, in X. laevis embryos, a C-terminal coiled-coil sequence mediates RalBP1 retention in the nucleus. We have also observed RalBP1 at the level of the actin cytoskeleton, a localization that depends on interaction of the protein with active Ral. During mitosis RalBP1 also associates with the mitotic spindle and the centrosome, a localization that could be negatively regulated by active Ral. Finally, we demonstrate the presence of post-transcriptional and post-translational isoforms of RalBP1 lacking the Ral-binding domain, which opens new possibilities for the existence of Ral-independent functions.

Acquired resistance to endocrine treatments is associated with tumor-specific molecular changes in patient-derived luminal breast cancer xenografts.

PURPOSE: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC. EXPERIMENTAL DESIGN: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays. RESULTS: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival. CONCLUSIONS: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314-25. ©2014 AACR.