Knockdown of FLOT1 Impairs Cell Proliferation and Tumorigenicity in Breast Cancer through Upregulation of FOXO3a

Abstract

Purpose: Lipid rafts, specialized domains in cell membranes, function as physical platforms for various molecules to coordinate a variety of signal transduction processes. Flotinllin-1 (FLOT1), a marker of lipid rafts, is involved in the progression of cancer, but the precise mechanism remains unclear. The aim of the present study was to examine the role of FLOT1 on the tumorigenesis of breast cancer cells and its clinical significance in progression of the disease.Experimental Design: FLOT1 expression was analyzed in 212 paraffin-embedded, archived clinical breast cancer samples by using immunohistochemistry (IHC). The effect of FLOT1 on cell proliferation and tumorigenesis was examined in vitro and in vivo. Western blotting and luciferase reporter analyses were carried out to identify the effects of downregulating FLOT1 on expression of cell cycle regulators and transcriptional activity of FOXO3a.Results: IHC analysis revealed high expression of FLOT1 in 129 of the 212 (60.8%) paraffin-embedded archived breast cancer specimens. The overall expression level of FLOT1 significantly correlated with clinical staging and poor patient survival of breast cancer. Strikingly, we found that silencing FLOT1 inhibited proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo, which was further shown to be mechanistically associated with suppression of Akt activity, enhanced transcriptional activity of FOXO3a, upregulation of cyclin-dependent kinase inhibitor p21Cip1 and p27 Kip1, and downregulation of the CDK regulator cyclin D1.Conclusions: FLOT1 plays an important role in promoting proliferation and tumorigenesis of human breast cancer and may represent a novel prognostic biomarker and therapeutic target for the disease.

Function of Flotillins in Receptor Tyrosine Kinase Signaling and Endocytosis: Role of Tyrosine Phosphorylation and Oligomerization

“…Knockdown of flotillin-1 also inhibited the proliferation of breast cancer cell lines by inducing a G1-S phase arrest of the cell cycle. In line with this, the expression of the cyclin dependent kinase (CDK) regulator cyclin D1 was reduced after flotillin-1 silencing, whereas that of the CDK inhibitors p21 Cip1 and p27 Kip1 was considerably increased (66), suggesting that flotillin-1 is an important regulator of the cell cycle. This result is strongly supported by our findings showing that in flotillin-1 knockdown cells, cyclin D1 expression after EGF stimulation is severely impaired (27).…”

“…Lin et al detected a high expression of flotillin-1 in the majority of the breast cancer specimens they studied (66). Furthermore, they revealed a significant correlation between the expression level of flotillin-1 and poor survival of the patients, suggesting that flotillins might prove useful as novel diagnostic markers for cancer.…”

“…One emerging important function of flotillins, especially of flotillin-1, is the regulation of membrane receptor signaling, e.g. through mitogen activated protein kinases (MAPK) (27,30,43,56,(63)(64)(65)(66)(67). Below, we have summarized the data on the function of flotillins in some of these processes.…”

Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase

“…Conversely, FOXO3a nuclear localization is associated with good prognosis in luminal-like breast cancer (Habashy et al 2011). Moreover, exogenous expression of FOXO3a inhibits tumor growth in vitro and in vivo in breast cancer (Hu et al 2004;Yang et al 2008;Lin et al 2011;Sisci et al 2013).…”

“…Another FOXO family member, FOXO1, as well as IkBKB also produced hydroxylation signals . We focused on FOXO3a because it consistently produced stronger hydroxylation signals than did either FOXO1 or IkBKB and because FOXO3a suppresses the proliferation of a variety of cell lineages, including mammary cells, which are known to be affected by EglN2 loss (Hu et al 2004;Yang et al 2008;Lin et al 2011). …”