Rab4 interacts with the human P-glycoprotein and modulates its surface expression in multidrug resistant K562 cells Clotilde Ferra ´ndiz-Huertas, Asia Ferna ´ndez-Carvajal, and Antonio Ferrer-Montiel Instituto de Biologı ´a Molecular y Celular, Universidad Miguel Herna ´ndez, Av de la Universidad s/n, 03202 Elche, Alicante, Spain P-glycoprotein (P-gp) is a plasma membrane glycoprotein that has been signaled as a primary cause of multidrug resistance (MDR) in tumors. We performed a yeast 2-hybrid screen using the C-terminal domain of P-gp and identified 2 small GTPases involved in vesicular trafficking, Rab4 and Rab14, which complex with P-gp. The overexpression of GFP-Rab4, either transiently or stably, but not of Rab14, in K562ADR cells decreased the presence of P-gp in the cell surface. As a result, expression of this GTPase reduced the MDR phenotype of K562ADR cells, by augmenting the intracellular accumulation of daunomycin (DNM). This effect was mimicked by the constitutively active Rab4Q72L mutant, but not by the dominant negative Rab4S27N mutant. Rab4 regulated excocytotic P-gp trafficking to the plasma membrane from intracellular compartments, and this modulation required the interaction of both proteins and the GTPase activity. Noteworthy, K562ADR cells exhibited a significant reduction of Rab4 levels, but not of other Rab GTPases, as compared with the sensitive parental cell line, suggesting that the development of the MDR phenotype in these cells involves upregulation of P-gp and a concomitant downregulation of proteins that regulate its surface expression. Attenuation of endogenous Rab4 levels in K562ADR by RNA interference enhanced the expression of P-gp in the cell surface, and reduced the uptake of DNM. Accordingly, these findings substantiate the notion that modulation of the temporal and spatial distribution of P-gp in cancer cells may be a valid therapeutic strategy to alleviate the MDR phenotype, and signal to Rab4 as a potential target. P-glycoprotein (P-gp or ABCB1) is a glycosylated integral membrane protein that belongs to the ATP binding cassette (ABC) transporter family encoded in humans by the MDR1 gene. 1–3 P-gp is highly expressed in epithelial and endothelial cells such as those in the gastrointestinal track and the blood brain barrier. 4–6 The physiological role of P-gp is the extru- sion of a broad spectrum of structurally unrelated xenobiotics across cell membranes by acting as an energy-dependent drug-efflux pump. 1,7 In addition, this glycoprotein is overex- pressed in tumor cell membranes critically contributing to the manifestation of the multidrug resistance (MDR) pheno- type in human cancers. The expression of P-gp in tumors could be intrinsic or induced by antichemotherapy drugs. In vivo, P-gp expression is associated with poor overall prognosis and response of tumors to current chemotherapy, in part, because of the pro- tein capacity to extrude a broad range of compounds, includ- ing anthracyclines, vinca-alkaloids and taxanes. 8 Chemothera- peutically induced expression of P-gp has been well documented in tumors such as acute leukemia and small- lung cancer, breast and ovarian cancer, head and neck tumors, Kaposi sarcoma, and child neuroblastoma. 9 The cen- tral role of P-gp in clinical oncology has prompted the dis- covery and development of antagonists. 10–16 These agents are known as chemosensitizers or revertants of the MDR pheno- type. By antagonizing the active efflux of anticancer drugs, chemosensitizers promote an accumulation of these cytotoxic agents into tumor cells, thus augmenting the efficacy of the chemotherapeutic treatment. 9,17 Despite the important role played by P-gp in cellular physiology and in the manifestation of the MDR phenotype, limited information is available on cytosolic interacting part- ners that may modulate aspects of protein expression and function. Previous studies using the linker region of P-gp as a bait have shown the RING finger protein 2 (RNF2), an E3 ubiquitin ligase, as an interacting protein that regulates the cellular abundance of P-gp. 18 A complementary approach using overlapping peptides of this linker region identified a- and b-tubulin as proteins that directly bind to P-gp. 19 In addition, the interaction of P-gp with caveolin-1 has been documented. 20,21 Caveolin-1 appears to inhibit P-gp Key words: multidrug resistance, Rab proteins, ABC transporters, chemotherapy, protein trafficking, cancer Abbreviations: ADR: doxorubicin; C t -P-gp: C-terminal domain of P-gp; DNM: daunomycin; EE: early endosomes; MDR: multidrug resistance; P-gp: P-glycoprotein; Tf-TRITC: transferrin-TRITC Grant sponsor: Spanish Ministry of Science and Innovation; Grant numbers: BFU2009-08346, CSD2008-00005; Grant sponsor: Fundacio ´n La Marato ´ de TV3 DOI: 10.1002/ijc.25310 History: Received 24 Nov 2009; Accepted 18 Feb 2010; Online 5 Mar 2010 Correspondence to: Antonio Ferrer-Montiel, Instituto de Biologı ´a Molecular y Celular, Universidad Miguel Herna ´ndez, Av de la Universidad s/n, 03202 Elche, Alicante, Spain, Phone: þ34966658727, E-mail: aferrer@umh.es Cancer Therapy Int. J. Cancer: 128, 192–205 (2011) V C 2010 UICC International Journal of Cancer IJC