Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs.

Mammosphere culture has been used widely for the enrichment of mammary epithelial stem cells and breast cancer stem cells (CSCs). Epithelial-to-mesenchymal transition (EMT) also induces stem cell features in normal and transformed mammary cells. We examined whether mammosphere culture conditions per se induced EMT in the epithelial MCF-7 breast cancer cell line. MCF-7 cells were cultured as mammospheres for 5 weeks, with dispersal and reseeding at the end of each week. This mammosphere culture induced a complete EMT by 3 weeks. Return of the cells to standard adherent culture conditions in serum-supplemented media generated a cell population (called MCF-7(M) cells), which displays a stable mesenchymal and CSC-like CD(44+)/CD(24-/low) phenotype. EMT was accompanied by a stable, marked increase in EMT-associated transcription factors and mesenchymal markers, and a decrease in epithelial markers and estrogen receptor α (ERα). MCF-7(M) cells showed increased motility, proliferation and chemoresistance in vitro, and produced larger tumors in immunodeficient mice with or without estrogen supplementation. MicroRNA analysis showed suppression of miR-200c, miR-203, and miR-205; and increases in miR-222 and miR-221. Antisense hairpin RNA inhibitor targeting miR-221 resulted in re-expression of ERα in MCF-7(M) cells. This study provides the first example of mammosphere culture conditions inducing EMT and of EMT regulating microRNAs that target ERα.

Yeast Bim1p promotes the G1-specific dynamics of microtubules.

Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. Our in vivo data provide evidence of a cell cycle-specific microtubule-binding protein that promotes microtubule dynamicity.

Calpain regulates actin remodeling during cell spreading.

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

Positioning of the mitotic spindle by a cortical-microtubule capture mechanism.

Correct positioning of the mitotic spindle is critical for cell division and development. Spindle positioning involves a search-and-capture mechanism whereby dynamic microtubules find and then interact with specific sites on the submembrane cortex. Genetic, biochemical, and imaging experiments suggest a mechanism for cortical-microtubule capture. Bim1p, located at microtubule distal ends, bound Kar9p, a protein associated with the daughter cell cortex. Bim1p is the yeast ortholog of human EB1, a binding partner for the adenomatous polyposis coli tumor suppressor. EB1 family proteins may have a general role in linking the microtubule cytoskeleton to cortical polarity determinants.

The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain.

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules.

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules. Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium.