The Role of CD44 and ERM Proteins in Expression and Functionality of P-glycoprotein in Breast Cancer Cells

Multidrug resistance (MDR) is often attributed to the over-expression of P-glycoprotein (P-gp), which prevents the accumulation of anticancer drugs within cells by virtue of its active drug efflux capacity. We have previously described the intercellular transfer of P-gp via extracellular vesicles (EVs) and proposed the involvement of a unique protein complex in regulating this process. In this paper, we investigate the role of these mediators in the regulation of P-gp functionality and hence the acquisition of MDR following cell to cell transfer. By sequentially silencing the FERM domain-binding proteins, Ezrin, Radixin and Moesin (ERM), as well as CD44, which we also report a selective packaging in breast cancer derived EVs, we have established a role for these proteins, in particular Radixin and CD44, in influencing the P-gp-mediated MDR in whole cells. We also report for the first time the role of ERM proteins in the vesicular transfer of functional P-gp. Specifically, we demonstrate that intercellular membrane insertion is dependent on Ezrin and Moesin, whilst P-gp functionality is governed by the integrity of all ERM proteins in the recipient cell. This study identifies these candidate proteins as potential new therapeutic targets in circumventing MDR clinically

A protein interactive complex governing multidrug resistance and tissue mechanical properties in cancer

University of Technology Sydney. Graduate School of Health.NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. The hardcopy may be available for consultation at the UTS Library.NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- Cancer multidrug resistance (MDR) is often attributed to the overexpression of a drug efflux transporter protein, P-glycoprotein (P-gp) that reduces the intracellular drug levels by virtue of its drug efflux capacity. Our group has previously shown that MDR cancer cells spontaneously shed microparticles (MPs) and that these MPs can transfer drug-resistance to drug-sensitive cells and confer MDR in as little as 4h. MPs are small membrane vesicles (0.1-1μm diameter) arising from membrane budding and are observed in most cell types during apoptosis or upon cell activation. We have reported that MPs derived from drug-resistant leukemia (VLB₁₀₀-MPs) cell lines transfer functional P-gp to both malignant and non-malignant cell lines. However, we have found that MPs derived from drug-resistant breast cancer (Dx-MPs) were more selective of recipient cell tissues. Dx-MPs selectively transfer this trait only to the drug-sensitive breast cancer cells examined within the same panel of cells. In defining the molecular basis governing this tissue selectivity, this study uses proteomic profiling and the comparative analysis of MPs isolated from Dx and VLB₁₀₀ cells. We identify 40 unique proteins exclusively in Dx-MPs relative to VLB₁₀₀-MPs including CD44, supporting the proposal that their presence may be required for the tissue selective transfer of P-gp. Additionally, we identified 177 proteins common to Dx-MPs and VLB₁₀₀-MPs. Included within this repertoire were the FERM domain proteins (F for 4.1 protein, Ezrin-Radixin-Moesin), which we found played a significant role in the vesicular transfer of functional P-gp. Specifically, we found that intercellular membrane insertion of P-gp is dependent on Ezrin and Moesin, whilst P-gp functionality is governed by the integrity of all ERM proteins in the MPs-recipient cells. We further observed that the vesicular transfer of P-gp mediated MDR via MPs was not only conferring MDR but also altering the biomechanical properties of recipient cells. Biomechanical properties, in particular, stiffness/elasticity are important factors contributing to the cancer cell function, motility, transformation, invasion and MDR. Therefore, this study expands on our previous findings and elucidates the rationale behind MP-mediated alteration of cellular elasticity. We demonstrate the alteration in cells and tumour core elasticity by sequentially silencing the protein complexes as well as performing the co-culture experiment with MDR-MPs in monolayer cells and tumor spheroids. These studies bring us closer to identifying the mediators regulating MDR in cancer and may be useful in developing targeted therapies for the circumvention of MDR clinically

Abstract 5114: The role of microvesicles on immune function in response to cancer

Cell to cell communication is vital for the co-ordination of physiological process and the regulation of an organism's phenotype. More recently communication via extracellular membrane vesicles has gained recognition. We first described a novel mechanism for the spread and dominance of multidrug resistance (MDR) and enhanced metastatic capacity in cancer via submicron microparticles (MPs). MPs are plasma membrane vesicles released spontaneously from various cell types, carrying bioactive material and are implicated in different physiological and pathophysiological processes. Through this communication apparatus, cancer cells can acquire and secure a survival advantage by various mechanisms. This study aims to examine a role of MPs in altering immune cell function in cancer.The effects of MPs isolated from human breast cancer cells were examined on antigen presenting cells (APC) in vitro. MP-mediated effects on cell phenotype and functionality was assessed by cytokine profiling and migration assay. We observed a cancer cell induced change in immune cell phenotype and functionality which have the potential to support a reduced global immune response in cancer. The elucidation of this pathway provides novel therapeutic strategies which can be exploited for the treatment of cancer

A novel mechanism governing the transcriptional regulation of ABC transporters in MDR cancer cells

P-glycoprotein (P-gp/ABCB1) and multidrug resistance-associated protein 1 (MRP1/ABCC1) are the main drug efflux transporters associated with treatment failure in cancer. Much attention has been focused on the molecular mechanisms regulating the expression of these transporters as a viable approach for identifying novel drug targets in circumventing cancer multidrug resistance (MDR) clinically. In this paper, we examine the role of miR-326 in the context of its intercellular transfer between cancer cells by extracellular membrane vesicles called microparticles (MPs). We observe that cellular suppression of ABCC1 by miR-326 is modulated by the presence of ABCB1 transcript. Specifically, we show that siRNA silencing of MP-transferred ABCB1 transcript reverses the knockdown effects of miRNA-326 on target MRP1/ABCC1 transcripts. We also demonstrate a dominance of ABCB1 transcripts when co-localized with ABCC1 transcripts, which is consistent with the facilitation of miR-326 function by ABCB1. This study identifies a novel pathway regulating the expression of ABC transporters and positions ABCB1 mRNA as a transcriptional regulator of other members of this superfamily in multidrug resistant cells through its actions on miRNAs