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

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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.
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