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

ABSTRACT: 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.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.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.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.

SUBMITTER: Kurrle N

PROVIDER: S-EPMC4235219 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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

Kurrle Nina N Ockenga Wymke W Meister Melanie M Völlner Frauke F Kühne Sina S John Bincy A BA Banning Antje A Tikkanen Ritva R

BMC cancer 20131205

<h4>Background</h4>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.<h4>Methods</h4>Human adenocarcinoma MCF7 breast cancer cells were stably d ...[more]

PMID: 24304721

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Project description:Transforming growth factor-? (TGF-?) and epidermal growth factor (EGF) have critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-? in altering the response of breast cancer cells to EGF remains unknown. We show in this study that murine metastatic 4T1 breast cancer cells formed compact and dense spheroids when cultured under three-dimensional (3D) conditions, which was in sharp contrast to the branching phenotypes exhibited by their nonmetastatic counterparts. Using the human MCF10A series, we show that epithelial-type and nonmetastatic breast cancer cells were unable to invade to EGF, whereas their mesenchymal-type and metastatic counterparts readily invaded to EGF. Furthermore, EMT induced by TGF-? was sufficient to manifest dense spheroid morphologies, a phenotype that increased primary tumor exit and invasion to EGF. Post-EMT invasion to EGF was dependent on increased activation of EGF receptor (EGFR) and p38 mitogen-activated protein kinase, all of which could be abrogated either by pharmacologic (PF-562271) or by genetic (shRNA) targeting of focal adhesion kinase (FAK). Mechanistically, EMT induced by TGF-? increased cell-surface levels of EGFR and prevented its physical interaction with E-cadherin, leading instead to the formation of oncogenic signaling complexes with T?R-II. Elevated EGFR expression was sufficient to transform normal mammary epithelial cells, and to progress their 3D morphology from that of hollow acini to branched structures characteristic of nonmetastatic breast cancer cells. Importantly, we show that TGF-? stimulation of EMT enabled this EGFR-driven breast cancer model to abandon their inherent branching architecture and form large, undifferentiated masses that were hyperinvasive to EGF and showed increased pulmonary tumor growth upon tail vein injection. Finally, chemotherapeutic targeting of FAK was sufficient to revert the aggressive behaviors of these structures. Collectively, this investigation has identified a novel EMT-based approach to neutralize the oncogenic activities of EGF and TGF-? in aggressive and invasive forms of breast cancer.

Dataset Information

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

Publications

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

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