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

BACKGROUND: Flotillin-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. METHODS: Human 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. RESULTS: We 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. CONCLUSIONS: This 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.

IgG against the Membrane-Proximal Portion of the Desmoglein 3 Ectodomain Induces Loss of Keratinocyte Adhesion, a Hallmark in Pemphigus Vulgaris.

Pemphigus vulgaris is a severe autoimmune blistering disease characterized by IgG autoantibodies (auto-abs) against the desmosomal adhesion molecules desmoglein (DSG) 3 and DSG1. Underlying mechanisms leading to blister formation upon binding of DSG-specific IgG auto-abs are not fully understood. Numerous studies showed the pathogenicity of IgG auto-ab binding to the aminoterminal region 1 (EC1) of the DSG3 ectodomain. However, auto-abs in pemphigus vulgaris are polyclonal, including IgG against both aminoterminal- and membrane-proximal epitopes of the DSG3 ectodomain. In this study, the pathogenicity of a previously uncharacterized murine monoclonal IgG antibody, 2G4, directed against the membrane-proximal region (EC5) of the DSG3 ectodomain was characterized and tested in various specificity and functionality assays. The results clearly show that 2G4 is capable of inhibiting intercellular keratinocyte adhesion and of inducing cellular DSG3 redistribution by activation of the p38MAPK signal transduction pathway. In this study, we provide evidence that an IgG auto-abs directed against the membrane-proximal region EC5 of DSG3 induces acantholysis, the hallmark in pemphigus vulgaris. These findings challenge the current concept that IgG auto-abs targeting the NH2-terminal portion of the DSG3 ectodomain are pathogenic only. Our study provides further aspects for a deeper understanding of desmosomal keratinocyte adhesion and improves our insight into the complex auto-ab‒induced blister formation in pemphigus vulgaris.

Loss of flotillin expression results in weakened desmosomal adhesion and Pemphigus vulgaris-like localisation of desmoglein-3 in human keratinocytes.

Desmosomes are adhesion plaques that mediate cell-cell adhesion in many tissues, including the epidermis, and generate mechanical resistance to tissues. The extracellular domains of desmosomal cadherin proteins, desmogleins and desmocollins, are required for the interaction with cadherins of the neighbouring cells, whereas their cytoplasmic tails associate with cytoplasmic proteins which mediate connection to intermediate filaments. Disruption of desmosomal adhesion by mutations, autoantibodies or bacterial toxins results in severe human disorders of e.g. the skin and the heart. Despite the vital role of desmosomes in various tissues, the details of their molecular assembly are not clear. We here show that the two members of the flotillin protein family directly interact with the cytoplasmic tails of desmogleins. Depletion of flotillins in human keratinocytes results in weakened desmosomal adhesion and reduced expression of desmoglein-3, most likely due to a reduction in the desmosomal pool due to increased turnover. In the absence of flotillins, desmoglein-3 shows an altered localisation pattern in the cell-cell junctions of keratinocytes, which is highly similar to the localisation observed upon treatment with pemphigus vulgaris autoantibodies. Thus, our data show that flotillins, which have previously been connected to the classical cadherins, are also of importance for the desmosomal cell adhesion.

Flotillins directly interact with γ-catenin and regulate epithelial cell-cell adhesion.

Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6-8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.