The mitotic kinase Aurora--a promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα(+) breast cancer cells.

In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα(+) breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα(+) breast cancer patients resistant to conventional endocrine therapy.

Single-walled carbon nanotube-induced mitotic disruption.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Induction of aneuploidy by single-walled carbon nanotubes.

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.

Impaired p53 function leads to centrosome amplification, acquired ERalpha phenotypic heterogeneity and distant metastases in breast cancer MCF-7 xenografts.

In this study, we establish an MCF-7 xenograft model that mimics the progression of human breast carcinomas typified by loss of p53 integrity, development of centrosome amplification, acquired estrogen receptor (ERalpha) heterogeneity, overexpression of Mdm2 and metastatic spread from the primary tumor to distant organs. MCF-7 cells with abrogated p53 function (vMCF-7(Dnp53)) maintained nuclear ERalpha expression and normal centrosome characteristics in vitro. However, following mitogen stimulation, they developed centrosome amplification and a higher frequency of aberrant mitotic spindles. Centrosome amplification was dependent on cdk2/cyclin activity since treatment with the small molecule inhibitor SU9516 suppressed centriole reduplication. In contrast to the parental MCF-7 cells, when introduced into nude mice as xenografts, tumors derived from the vMCF-7(DNp53) cell line developed a strikingly altered phenotype characterized by increased tumor growth, higher tumor histopathology grade, centrosome amplification, loss of nuclear ERalpha expression, increased expression of Mdm-2 oncoprotein and resistance to the antiestrogen tamoxifen. Importantly, while MCF-7 xenografts did not develop distant metastases, primary tumors derived from vMCF-7(DNp53) cells gave rise to lung metastases. Taken together, these observations indicate that abrogation of p53 function and consequent deregulation of the G1/S cell cycle transition leads to centrosome amplification responsible for breast cancer progression.

Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos.

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes after in vitro fertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result of de novo synthesis at the onset of differentiation of the pluripotent blastomeres.

Changes in the centrin and microtubule cytoskeletons after metaphase arrest of the Chlamydomonas reinhardtii met1 mutant.

The temperature-sensitive conditional met1 Chlamydomonas reinhardtii mutant arrests in metaphase at the restrictive temperature (33 degrees C) with an intact spindle and high cell division kinase levels. In this study, met1 was investigated with respect to changes in the microtubule and centrin-based cytoskeletons after arrest at 33 degrees C. Immunofluorescence microscopy revealed that, initially on arrest, the microtubule spindle and centrin-based cytoskeleton appeared as previously reported for wild-type metaphase cells; crescent-shaped spindles were seen with two brightly labelled centrin foci at each spindle pole in the basal body region at the cell surface. Observation of met1 held at the restrictive temperature reveals spindles can detach from one spindle pole and chromosomes eventually detach from the spindles. Moreover, a pseudo-anaphase event of spindle and nucleus elongation occurs in the absence of chromosome separation. Electron microscopy confirms that cytokinesis is initiated, the nuclei maintain a crescent shape but are distended and multiple pyrenoids are detected, suggesting chloroplast division also continues. Interestingly, prolamellar-like bodies usually present in etioplasts of dark-grown plants appear at the nuclear envelope. These results are discussed in relation to the coordination of division events in Chlamydomonas reinhardtii and the loss of viability in arrested cells of this mutant.

Ontogeny of Phex/PHEX protein expression in mouse embryo and subcellular localization in osteoblasts.

PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X-linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.

GFP-centrin as a marker for centriole dynamics in the human breast cancer cell line MCF-7.

Centrosome duplication plays an important role in genomic stability through bipolar spindle formation and equal chromosome segregation during mitosis. Defects in centrosome duplication and centrosome amplification correlate with aggressive tumors and aneuploidy. Cyclin-dependent cell cycle regulators play a key role in signaling centrosome duplication and the tumor suppressor genes p53, BRCA1 and BRCA2 are suspected to function at mitotic checkpoints that monitor centrosome duplication. The relationship between loss of hormone dependence in breast cancer, and signaling of centrosome duplication in tumor progression is not known. We have developed a MCF-7 cell line expressing GFP-centrin that allows direct visualization of centriole duplication during the cell cycle in living cells. GFP-centrin is expressed and selectively incorporated into the structure of both centrioles making them clearly visible in living cells. Our studies demonstrate three important aspects of recombinant GFP-centrin incorporation into centrioles. 1) GFP-centrin transfected cells grow normally in culture and show no adverse effect associated with GFP-centrin expression; 2) newly duplicated centrioles incorporate centrin during their genesis; and 3) GFP-centrin incorporation into centrioles does not grossly affect cell cycle progression, or centrosome function.

Methods for the analysis of centrosome reproduction in cancer cells.

The assay described here allows a direct comparison of centrosome function (i.e., MT nucleation capacity) between normal and tumor tissues. It can be applied to samples such as human tissues in which the materials are limited. The assay is rapid and uses equipment commonly available. Comparison of the ability of individual centrosomes to nucleate microtubules within the context of tissues can provide novel insight into the disease process itself. In the example shown here, tumor tissues nucleate significantly greater numbers of microtubules from single or amplified centrosomes in comparison to normal tissue. The increased microtubule nucleation capacity from multiple centrosomes seen in tumors may be related to the increased frequency of mitotic aberrations and to the loss of cell and tissue architecture that is seen in cancer. This assay can also be used to characterize the microtubule nucleation capacity of normal tissues, during development and aging, and in disease states other than cancer where microtubule dynamics may play an important role.

The contribution of epigenetic changes to abnormal centrosomes and genomic instability in breast cancer.

The centrosome is the major microtubule organizing center of the cell and as such it plays an important role in cytoskeletal organization and in the formation of the bipolar mitotic spindle. Centrosome defects, characterized by abnormal size, number, and microtubule nucleation capacity, are distinguishing features of most high grade breast tumors and have been implicated as a possible cause for the loss of tissue architecture and the origin of mitotic abnormalities seen in solid tumors in general. Centrosome defects arise through uncoupling of centriole duplication and the cell cycle as a result of either genetic alterations or through physical or chemical perturbation of centrosome function. Centrosomes manifest unique epigenetic properties whereby positional or structural information can be propagated through somatic cell lineages by way of nongenetic pathways. Because aberrant centrosome function can result in chromosomal instability, these properties may have important implications for the origin of malignant breast tumors.

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication.

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.

Characterization of the X-linked murine centrin Cetn2 gene.

A multi-gene family (Cetn1, Cetn2, and Cetn3) encodes the calcium-binding protein, centrin, in the mouse. This work characterizes the Cetn2 gene. Structurally, Cetn2 consists of five exons and four introns, and contains a classical TATA-less promoter. Cetn2 has two alternate transcription start sites, and a single length 3' untranslated region. Fluorescence in situ hybridization demonstrates that Cetn2 is an X-linked gene whose alleles replicate asynchronously during S-phase. Cetn2 encodes a 172 amino acid protein, with a predicted molecular mass of 19,795 Da (pI=4.71), that contains all of the defining characteristics of centrin. Northern blot analysis indicates that Cetn2 is ubiquitously expressed in the tissues of adult mice. RT-PCR shows that Cetn2 and Cetn3, but not Cetn1, are expressed in NIH 3T3 cells. Immunofluorescence microscopy demonstrates that mouse centrin 2 protein localizes to the region immediately surrounding the centrioles in the centrosome of NIH 3T3 cells.

GFP-centrin as a marker for centriole dynamics in living cells.

A long-standing puzzle in cell biology is the question of how cells generate one and only one new centrosome in each cell cycle and what is the role of the centriole pair in this process. In this study, the introduction of GFP-centrin into cultured cells allows direct visualization of centriole behavior in living cells and in real time. Using this method, centriole dynamics can be observed throughout the cell cycle and following a variety of experimental treatments. Our studies demonstrate that the biogenesis of new centrioles from individual members of a preexisting centriole pair is asynchronous: the older centriole initiates assembly of a new daughter centriole before the younger centriole initiates assembly of its daughter.

Altered centrosome structure is associated with abnormal mitoses in human breast tumors.

Centrosomes are the major microtubule organizing center in mammalian cells and establish the spindle poles during mitosis. Centrosome defects have been implicated in disease and tumor progression and have been associated with nullizygosity of the p53 tumor suppressor gene. In the present ultrastructural analysis of 31 human breast tumors, we found that centrosomes of most tumors had significant alterations compared to centrosomes of normal breast tissue. These alterations in included 1) supernumerary centrioles, 2) excess pericentriolar material, 3) disrupted centriole barrel structure, 4) unincorporated microtubule complexes, 5) centrioles of unusual length, 6) centrioles functioning as ciliary basal bodies, and 7) mispositioned centrosomes. These alterations are associated with changes in cell polarity, changes in cell and tissue differentiation, and chromosome missegregation through multipolar mitoses. Significantly, the presence of excess pericentriolar material was associated with the highest frequency of abnormal mitoses. Centrosome abnormalities may confer a mutator phenotype to tumors, occasionally yielding cells with a selective advantage that emerge and thrive, thus leading the tumor to a more aggressive state.

Centrosomes and cancer.

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.

Testis-specific murine centrin, Cetn1: genomic characterization and evidence for retroposition of a gene encoding a centrosome protein.

Centrin is a centrosome component in species from yeast to humans. Here, the mouse centrin 1 gene (Cetn1) is analyzed with respect to its genomic structure, chromosome localization, tissue-specific expression, and phylogenetic relationship to the other mouse centrin genes and their human orthologs. Cetn1 is an intronless gene located on chromosome 18A2 that encodes a 172-amino-acid protein with a predicted molecular mass of 19,696 Da (pI 4.61) and all of the structural features common to centrin. Cetn1 possesses the sequence features of an expressed retroposon: the gene lacks introns, the open reading frame is not interrupted by stop codons, and the coding region is flanked by a pair of direct repeats. Reverse transcriptase-polymerase chain reaction and Northern blot analysis demonstrate that Cetn1 expression is limited exclusively to the testis in adult male mice. Cetn1 expression is first seen in the neonatal testis at 14 days postpartum, reaching adult levels by day 17. These observations provide new insight into the regulation, function, and evolutionary history of centrin in higher eukaryotes.

Microtubule nucleating capacity of centrosomes in tissue sections.

We used a novel adaptation of methods for microtubule polymerization in vitro to assess the MTOC activity of centrosomes in frozen-sectioned tissues. Remarkably, centrosomes of tissue sections retain the ability to nucleate microtubules even after several years of storage as frozen tissue blocks. Adaptations of these methods allow accurate counts of microtubules from individual cells and the quantitative estimation the MTOC activity of the intact tissue. These methods can be utilized to characterize MTOC activity in normal and diseased tissues and in particular tissues at different stages of development. (J Histochem Cytochem 47:1265-1273, 1999)

Biparental inheritance of gamma-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm.

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents gamma-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. gamma-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal gamma-tubulin. Recruitment of maternal gamma-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm "priming" induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

Centriole and centrin degeneration during mouse spermiogenesis.

Centrosome reduction during mouse spermiogenesis has been studied by immunofluorescent microscopy using anticentrin antibody (20H5) and TEM. Centrin is detected as two spots in round spermatids, corresponding to a pair of centrioles. In elongating spermatids, centrin spots colocalize with the centrioles in the neck region, while the perinuclear ring from which manchette microtubules arise, does not label with the antibody 20H5. The proximal centriole of the elongating spermatids develops a prominent adjunct, which assembles an aster of microtubules. TEM studies after immunogold labeling revealed that centrin is associated with the distal and the proximal centrioles, but not with the adjunct. Centrin labeling in the neck region diminishes after spermiation stage, although it is not completely lost from all testicular sperm. Mature epididymal sperm do not display centrin labeling. Mouse sperm lose both distal and proximal centrioles at maturity. Loss of centrin staining appears to correlate with the degeneration of centrioles during mouse spermiogenesis.