Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs

Abstract

Cellular uptake is limiting for the efficacy of many cytotoxic drugs used to treat cancer. Identifying endocytic mechanisms that can be modulated with targeted, clinically-relevant interventions is important to enhance the efficacy of various cancer drugs. We identify that flotillin-dependent endocytosis can be targeted and upregulated by ultrasound and microbubble (USMB) treatments to enhance uptake and efficacy of cancer drugs such as cisplatin. USMB involves targeted ultrasound following administration of encapsulated microbubbles, used clinically for enhanced ultrasound image contrast. USMB treatments robustly enhanced internalization of the molecular scaffold protein flotillin, as well as flotillin-dependent fluid-phase internalization, a phenomenon dependent on the protein palmitoyltransferase DHHC5 and the Src-family kinase Fyn. USMB treatment enhanced DNA damage and cell killing elicited by the cytotoxic agent cisplatin in a flotillin-dependent manner. Thus, flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery for cancer treatment.

Flotillins: At the Intersection of Protein S-Palmitoylation and Lipid-Mediated Signaling

“…Endocytosis of the entrapped fluid phase and plasma membrane proteins comes next, the preferred cargo of the flotillin-demarked vesicles being GPI-anchored proteins due to their raft localization ( Figure 2). Fekri et al [34] have pointed out that the USBM-induced endocytosis shares similarities with "lipid driven" MEND described by Hilgemann and coworkers [114]. Indeed, MEND also requires tyrosine phosphorylation of proteins and zDHHC5/2-dependent palmitoylation of cell surface proteins.…”

“…EGF receptor signaling leading to cell adhesion [8] EGF receptor clustering and phosphorylation, ERK1/2 and Akt phosphorylation; flotillins as MAP kinase scaffolding proteins [9] EGF receptor expression in breast cancer cells [10] activation of H-Ras in breast cancer cells [11] EGF receptor sorting and lysosomal degradation [12] TLR3 ligand internalization [13] TCR raft association and recycling [14][15][16][17] PrP [Ca +2 ] increase, Fyn and ERK1/2 activation, N-cadherin trafficking [18,19] IgE receptor [Ca +2 ] increase, phosphorylation of IgE receptor γ chain and ERK1/2 [20] insulin receptor insulin-induced glucose uptake via Glut4 transporter [21] Gq protein-coupled receptors p38 phosphorylation [22] not determined signaling leading to axon regeneration: Rho GTPase activation, formation of N-WASP-Arp3/cortactin complexes, p38, ERK1/2 and FAK kinase phosphorylation [23] IGF-1 receptor* IGF-1 receptor transport from endoplasmic reticulum to plasma membrane [24] integrins ERK2 and FAK kinase phosphorylation [25] ACTIN CYTOSKELETON REMODELING Affected processes filopodia formation [26] cell spreading [8] proper localization of Vav during T cell spreading [14] F-actin binding [27] axon regeneration [23] uropod formation in neutrophils [28] uropod formation in T cells, activation of ezrin/radix/moesin, localization of PIP5KIγ to uropod [29][30][31] formation of lamellipodia at the growth cone of neurons [32] cell adhesion and migration via α-actinin binding [25] ENDOCYTOSIS Cargo fluid phase (magnetic dextran, AF488-dextran) [33,34] GPI-anchored proteins [18,33,[35]…”

“…The possibility that S-palmitoylation of flotillin-2 could facilitate its oligomerization indicates an intriguing mode of modulation of flotillin functioning. The ability of flotillins to form hetero-oligomers has led to the proposal that those oligomers could serve as scaffolds for other protein complexes involved in cell signaling, endocytosis, protein sorting, and cell migration [17,28,29,34,62,84,85]. By shifting the balance between flotillin monomers and oligomers toward the latter, S-palmitoylation of flotillins could modulate these processes.…”

“…The data pointing to a dynamic nature and the nanoscale of membrane rafts often contrast with those on the cellular localization of flotillins. In a number of cell types, flotillins form submembrane patches up to 1 µm in diameter [33][34][35]. T lymphocytes and other nonadherent hematopoietic cells are an extreme example where flotillins cap one pole of the cell only [102].…”

“…Those studies also identified flotillin hetero-oligomerization as a factor determining this process [76,84]. Next, tyrosine phosphorylation and S-palmitoylation were added to the list of factors affecting the flotillin-mediated endocytosis [8,34,36]. Before discussing the role of S-palmitoylation in the internalization, the problem of the cargo of the flotillin-containing vesicles should be addressed.…”