Proteome analysis of multidrug‐resistant, breast cancer–derived microparticles

Abstract

Cancer multidrug resistance (MDR) occurs when cancer cells evade the cytotoxic actions of chemotherapeutics through the active efflux of drugs from within the cells. Our group have previously demonstrated that multidrug-resistant breast cancer cells spontaneously shed microparticles (MPs) and that these MPs can transfer resistance to drug-responsive cells and confer MDR on those cells in as little as 4 h. Furthermore, we also showed that, unlike MPs derived from leukaemia cells, breast cancer–derived MPs display a tissue selectivity in the transfer of P-glycoprotein (P-gp), transferring the resistance protein only to malignant breast cells. This study aims to define the proteome of breast cancer–derived MPs in order to understand the differences in protein profiles between those shed from drug-resistant versus drug-sensitive breast cancer cells. In doing so, we detail the protein cargo required for the intercellular transfer of MDR to drug-sensitive recipient cells and the factors governing the transfer selectivity to malignant breast cells. We describe the first proteomic analysis of MPs derived from human breast cancer cells using SDS PAGE and liquid chromatography–tandem mass spectrometry (LC/MS/MS), in which we identify 120 unique proteins found only in drug-resistant, breast cancer–derived MPs. Our results demonstrate that the MP-mediated transfer of P-gp to recipient cells occurs alongside CD44; the Ezrin, Radixin and Moesin protein family (ERM); and cytoskeleton motor proteins within the MP cargo.

Membrane microparticles: shedding new light into cancer cell communication

“…These results suggest that these proteins are important for stabilizing Pgp in MPs (Pokharel et al 2014). Among the identified proteins, CD44, ezrin, radixin and moesin appeared to be involved in the resistance phenotype and metastasis (Donatello et al 2012).…”

“…Therefore, it has been suggested that ezrin is related to Pgp stabilization in MPs and incorporation by the drug-sensitive recipient cells (Brambilla et al 2012;Jaiswal et al 2013;Luciani et al 2002). Pokharel et al (2014) also observed CD73 loaded in the MPs derived from MDR breast cancer cells. Their data suggest that Pgp interacts with CD73 along with CD44 in MPs, which further suggests that CD73 may also participate in the MDR phenotype transfer from solid tumor cells to recipient cells (Pokharel et al 2014).…”

“…Among the identified proteins, CD44, ezrin, radixin and moesin appeared to be involved in the resistance phenotype and metastasis (Donatello et al 2012). Moreover, CD44 has been demonstrated to interact with Pgp and also to be associated with cell migration and invasiveness (Miletti-Gonzalez et al 2005), suggesting that the presence of CD44 in MPs may be required at least to facilitate the MDR phenotype transfer from solid tumor cells to drug-sensitive recipient cells (Pokharel et al 2014).…”

“…The presence of all of these proteins in MPs may facilitate the transfer and intracellular trafficking of Pgp. Probably, other membrane proteins are also involved in Pgp transfer to drug-sensitive cancer cells via MPs (Pokharel et al 2014). In addition, ezrin plays an important role in Pgp membrane insertion.…”

Advances in Studies of P-Glycoprotein and Its Expression Regulators

“…55) In particular, when Ezr and Msn are silenced by siRNA in parental cells (P-gp-recipient cells), the expression of P-gp is significantly increased by the co-incubation with SMVs compared to that in the case of control and Rdxsilenced parental cells.…”