2319

FLOT2

flotillin 2;FLOT2;flotillin 2

Flotillin 2 (epidermal surface antigen 1)|epidermal surface antigen|flotillin-2|membrane component chromosome 17 surface marker 1|membrane component, chromosome 17, surface marker 1 (35kD protein identified by monoclonal antibody ECS-1)

17q11-q12

protein-coding

Flotillin microdomains, specialized lipid raft domains in cell membranes, serve as physical platforms for many different molecules important in crucial intracellular signaling pathways. Flotillin-2 (Flot2), together with flotillin-1, is a marker for lipid raft microdomains distinct from caveolar lipid rafts, and has been implicated in the progression of cancer and metastasis formation. Based largely on studies in xenograft models, flotillin-2 has been implicated in the progression of multiple types of human tumors, including breast cancer. In our studies, we identified flotillin-2 as highly amplified in a high-throughput comparative genomic hybridization screen of human breast cancer cell lines and breast tumor samples. Short hairpin RNA-mediated reduction of flotillin-2 protein levels significantly reduced the tumorigenicity and metastatic capability of a human breast cancer cell line in vivo. We generated mice deficient for flotillin-2 and also found a reduction of flotillin-1 protein levels and complete absence of flotillin-specific membrane microdomains in these mice. To examine the role of Flot2 in mammary tumorigenesis and lung metastasis, we used an in vivo molecular genetics approach, crossing a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with gene-targeted Flot2(-/-) mice. Flotillin-2 deficiency lead to a striking reduction in the number of lung metastasis observed, but had no influence on primary tumor formation in this model. Our results indicate, using a novel in vivo animal model approach, that Flot2 is an important regulator of mammary tumor-derived lung metastasis.

Flotillin 2 (flot-2) is a highly conserved protein isolated from caveolae/lipid raft domains that tether growth factor receptors linked to signal transduction pathways. Flot-2 protein and mRNA were increased in tumorigenic and metastatic melanoma cell lines in vitro, and the immunostaining intensity increased substantially across a tissue array of melanocytic lesions. Flot-2 transfection transformed SB2 melanoma cells from nontumorigenic, nonmetastatic to highly tumorigenic and metastatic in a nude mouse xenograft model. SB2 cells stably transfected with the flot-2 cDNA (SB2-flot)-2 cells proliferated faster in the absence of serum, and their migration through Matrigel was additionally enhanced by thrombin. When SB2-flot-2 cells were compared with SB2-vector-control cells on a cancer gene pathway array, SB2-flot-2 cells had increased expression of protease activated receptor 1 (PAR-1) mRNA, a transmembrane, G-protein-coupled receptor involved in melanoma progression. PAR-1 and flot-2 were coimmunoprecipitated from SB2-flot-2 cells. Up-regulation of PAR-1 was additionally confirmed in SB2-flot-2 cells and melanoma cell lines. SB2-flot-2 cells transfected with flot-2-specific small-interfering RNAs made substantially less flot-2 and PAR-1 mRNA. In conclusion, flot-2 overexpression is associated with melanoma progression, with increased PAR-1 expression, and with transformation of SB2 melanoma cells to a highly metastatic line. Flot-2 binds to PAR-1, a known upstream mediator of major signal transduction pathways implicated in cell growth and metastasis, and may thereby influence tumor progression.

Autotaxin, a lysophospholipase D producing lysophosphatidic acid, augments invasive and metastatic potential of tumor cells. Current investigations have focused on understanding the molecular mechanisms by which autotaxin regulates the expression of a major mediator of tumor invasion and metastasis, urokinase-type plasminogen activator (uPA) in human A2058 melanoma cells. Autotaxin induced uPA expression in a dose-dependent manner that was inhibited by pharmacological inhibitors for Gi (pertussis toxin), phosphoinositide 3-kinase (PI3K, LY294002), Akt inhibitor (AktI), proteosome activity and IkappaB phosphorylation (pyrrolidine dithiocarbamate), and by a dominant negative mutant (DN) of Akt. Autotaxin phosphorylated Akt and induced the translocation of nuclear [corrected] factor-kappaB (NF-kappaB) to the nucleus that were inhibited by AktI or by overexpressing DN-Akt. Consistently, green fluorescence protein-tagged p65 of NF-kappaB accumulated in the nucleus by autotaxin that was abrogated when the cells were transfected with DN-Akt. Moreover, autotaxin increased the DNA binding ability of NF-kappaB and promoter activity of uPA. Collectively, these data strongly suggest autotaxin induces uPA expression via the Gi-PI3K-Akt-NF-kappaB signaling pathway that might be critical for autotaxin-induced tumor cell invasion and metastasis.