Phosphatidylinositol 3-Kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin-1 depletion in breast cancer cells

Abstract

BackgroundFlotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of flotillins in breast cancer.MethodsHuman adenocarcinoma MCF7 breast cancer cells were stably depleted of flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant flotillin proteins.ResultsWe here show that stable knockdown of flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression.ConclusionsThis study demonstrates that flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon flotillin knockdown which may even facilitate cancer cell growth and proliferation.

A novel role for flotillin‐1 in H‐Ras‐regulated breast cancer aggressiveness

“…Inconsistent with our findings, it was reported that flotillin‐1 knockdown significantly reduced both EGFR and EGFR phosphorylation (Y1173) in HeLa cells . Of note, flotillin‐1 depletion caused an increase in EGFR and its activation in MCF‐7 cells . We speculate that the function of flotillin‐1 in EGFR expression and activation may be dependent on cell type context.…”

“…38 Of note, flotillin-1 depletion caused an increase in EGFR and its activation in MCF-7 cells. 39 We speculate that the function of flotillin-1 in EGFR expression and activation may be dependent on cell type context. Further studies need to be conducted to elucidate the underlying mechanisms for the functional significance of flotillin-1.…”

OFD1 and Flotillins Are Integral Components of a Ciliary Signaling Protein Complex Organized by Polycystins in Renal Epithelia and Odontoblasts

“…However, in breast cancer cells with mutant PIK3CA, a well-known downstream target of EGFR signaling, knockdown of flotillin-1 causes an upregulation of EGFR and hyperactivation of MAPK signaling. These data indicate that the flotillins have dual roles in enabling receptor tyrosine kinase activation and downstream signaling, as well as in restraining signaling components such as PIK3CA in an inactive state [49]. The importance of ciliary organization of EGFR signaling is further reinforced by recent studies showing that when cilia are ablated, EGFR mediated activation of apical calcium channels is increased 64-fold [50].…”

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

“…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]…”