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.
Highlights
Flotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking
Expression of the EGF receptor is increased in flotillin-1 knockdown cells Breast cancer cells frequently exhibit an increased amount of the HER2/ErbB2 receptor protein that belongs to the epidermal growth factor receptor (EGFR) receptor family
EGFR was virtually undetectable in control shRNA MCF7 cells by antibody staining, we readily observed a plasma membrane associated staining in all flotillin knockdown cells, consistent with the increased expression (Figure 2E)
Summary
Flotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. PI3 kinases (PI3K) can be activated by e.g. growth factor signaling and mediate the activation of AKT, a protein kinase with numerous substrates that include the mechanistic target of rapamycin (mTOR) and some members of the Forkhead transcription factor family, e.g. FOXO3 [3,4,5,6]. In line with its importance in cell survival, PI3K is frequently mutated in various tumors, especially in breast, gastric and colorectal cancers [7,8]. The most frequently observed mutations in this protein in cancers are the H107R substitution in the kinase domain and E545K in the helical domain [8,9,10] Both mutation result in constitutive activation of PI3K/AKT signaling and contribute to cellular transformation [11,12]
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