Centrosome hypertrophy in human breast tumors: Implications for genomic stability and cell polarity

RESULTS

A terminal duct from normal inactive breast tissue, consisting of distinctly polarized cuboidal epithelial cells and surrounding myoepithelial cells, is illustrated in Fig. 1A. Centrosome position was determined for normal ductal epithelial cells by using indirect immunofluorescence staining of the centrosomal protein centrin (Fig. 1B). In normal breast tissues, centrin labeling is restricted to the pair of centrioles located above the nucleus, very near the luminal membrane, in each cuboidal epithelial cell (see quantitative analysis below). Centrioles of all normal breast tissue cell types, including epithelial cells, myoepithelial cells, fibroblasts, and vascular endothelial cells, showed comparable levels of centrin immunostaining. Likewise, regions of normal tissue adjacent to breast tumors showed normal centrosome staining patterns.

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Figure 1

Centrosome number and size in normal breast duct epithelia (A–C) and adenocarcinoma cells (D–F). Hematoxylin and eosin-stained paraffin sections of a normal human breast duct (A) and a breast adenocarcinoma (D). (B) Confocal image stack of a normal breast duct stained for centrioles with anticentrin mAb 20H5 (fluorescein isothiocyanate secondary antibody) and for nuclear DNA with propidium iodide. Approximately 20 pairs of centrioles are located apical to the nuclei of epithelial cells that line this normal duct. (C) Binary processed image showing the volume of centrin labeling for the same normal epithelial image stack shown in B, from which a portion of the data in Table 1 was derived. (E) Confocal image stack of a breast adenocarcinoma stained as above. Many large centrin-staining spots mark the location of abnormal centrosomes in the tumor tissue. (F) Binary processed image showing the volume of centrin labeling for the same tumor image stack shown in E, from which a portion of the data in Table 1 was derived. (Bar = 20 μm.)

In contrast, individual tumor cells of invasive adenocarcinomas displayed a characteristic loss of structural differentiation, a high nuclear-to-cytoplasmic ratio, and nuclear pleomorphism (Fig. 1D). Tumor cell centrosomes were present in greater numbers, and they were located chaotically within the cytoplasm compared with normal cells (Fig. 1E). Moreover, centrin localization in tumor cells was not restricted to the centrioles as it is in normal epithelial tissues (compare normal Fig. 1 B–C and tumor Fig. 1 E–F); in tumor cells, centrin staining also was seen as an extensive labeling of pericentriolar material.

Quantitative analysis demonstrated significant differences in centrosome number and volume between normal and tumor cells (Table 1). Results of a detailed analysis of three normal breast tissue specimens and five high grade breast adenocarcinomas are presented here. The mean volume for centrin- staining spots in normal epithelial cells is 0.013 μm³, which is the volume of a centriole calculated from its physical dimensions [π × (0.1 μm)² × 0.4 μm = 0.013 μm³]. In contrast, the mean volume of centrin-stained spots analyzed in five separate tumor samples ranged from 0.092 to 0.138 μm³ (overall mean for all five tumor samples = 0.114 μm³), ≈7–10× the value observed for centrosomes in normal cells. On a per nucleus basis, the differences were even larger, with over 40 times the centrosome volume in tumor compared with normal cells. These volume differences in centrin staining for normal vs. tumor cells are significant [Wilcoxon rank sum test (30), P < 0.005]. Normal cells contain a mean number of 1.5 centrin-stained spots (corresponding to centrioles) per cell; in contrast, tumor cells contain two-to-four times this number of significantly larger centrin-stained spots per cell (Table 1A). Centrin staining of tumor centrosomes is, therefore, more pervasive than expected for staining of individual centrioles and is likely to represent labeling of excess pericentriolar material observed by electron microscopic examination (see below and refs. 10, 18, and 19).

Staining of a second well characterized centrosome marker, γ-tubulin (Fig. 2 A and C), also demonstrates abnormal centrosome size and number in tumor cells. Furthermore, in double-labeled preparations, centrin and γ-tubulin staining nearly coincide (Fig. 2 B–D), suggesting that excessive centrosome size may be the result of general accumulation of centrosomal proteins in tumor cells.

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Figure 2

Immunofluorescence of γ-tubulin in tumor centrosomes. (A) In this overview of a breast tumor cryosection, γ-tubulin appears as many large spots scattered randomly throughout the tumor, similar to the distribution of centrin in Fig. 1E. Double labeling (B–D) of tumor centrosomes by using antibodies to centrin (B, red) and γ-tubulin (C, green) show virtual coincidence of signal (D, yellow). (Bar = 20 μm.)

Centrosomes of normal and tumor cells also were examined by transmission electron microscopy (Fig. 3). Normal epithelial cells had two apically positioned centrioles with sparse electron-dense pericentriolar material (Fig. 3 A–B). In contrast, tumor cell centrosomes are characterized by multiple centrioles (range = 2–12) surrounded by abundant densely staining pericentriolar material (Fig. 3 C–E).

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Figure 3

Normal and tumor centrosome ultrastructure. Electron micrographs of thin sections of normal epithelial cell centrosomes (A–B) and centrosomes of tumor cells (C–E). (A) A normal epithelial cell centrosome illustrating the orientation of the pair of centriole cylinders and sparse pericentriolar material surrounding them. (B) A centrosome of another normal breast epithelial cell illustrating two centriole profiles and sparse pericentriolar material. The three tumor cell centrosomes illustrated in C–E show large accumulations of densely staining pericentriolar material with numerous centrioles and procentrioles. (E) A section of a tumor centrosome that includes five centriole and procentriole profiles (arrowheads). (Bar = 0.5 μm.)

Quantitative analysis of electron micrographs demonstrated that tumor cells have larger numbers of centrioles than do normal cells; the number of centriole profiles seen in an individual ≈900 Å-thick section of normal cells ranged from 0–2 per section, whereas the range seen in individual tumor cell sections of the same thickness is 0–5 per section (Fig. 3E; Table 1B). Although a single section can reveal the two centrioles present in normal cells, it is unlikely that a single section will reveal all of the centrioles within a given tumor cell. We have observed up to 12 centrioles in individual tumor cells by examining serial sections, whereas in normal controls >2 centrioles were never observed (Table 1B; data not shown). Tumor centrosomes, therefore, were characterized by a larger number of centrioles and an excess of electron-dense pericentriolar material.

Centrosomes in tumor cells also displayed anomalous phosphorylation of centrosomal proteins. A polyclonal antibody, αHPC, was raised against the carboxyl-terminal 13 amino acids of human centrin in which the serine representing residue 170 was phosphorylated. Western blot analysis showed that αHPC identifies exclusively phosphorylated, but not nonphosphorylated, centrin (Fig. 4A, compare gel lanes a, a′ and b, b′). αHPC also recognized phosphocentrin in whole cell extracts of human breast tumors (Fig. 4A, compare gel lanes c and d). Indirect immunofluorescence using αHPC demonstrated that normal breast epithelia (Fig. 4B) stain sparsely, if at all, whereas for five tumor specimens that we examined, >80% of the tumor cells showed intense centrosome staining (Fig. 4C). Indirect immunofluorescence using αHPC revealed bright staining of spindle poles in mitotic cells of the normal immortal breast epithelial cell line MCF10A (25) (Fig. 4D), whereas interphase MCF10A centrosomes did not stain (Fig. 4E). Taken together, these results indicate that, in normal breast epithelial cells, centrin was normally phosphorylated only during mitosis and not during interphase. In contrast, in breast adenocarcinoma cells, centrin is phosphorylated at inappropriate times during the cell cycle and phosphocentrin accumulates in the unusually large tumor centrosomes.

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Figure 4

Inappropriate phosphorylation of breast tumor centrosomes. (A) Specificity of αHPC antibodies for phosphocentrin. Western blot analysis of phosphorylated and nonphosphorylated bacterially expressed recombinant centrin (A, gel lanes a′ and b′) and autoradiography of the same gel lanes (A, gel lanes a and b). Comparison of the Western blot analysis (A, gel lane a′) by using mAb 20H5 that reacts with centrin regardless of phosphorylation status and the corresponding autoradiogram (A, gel lane a) demonstrates that the slower migrating centrin band is phosphorylated. Western blot analysis (A, gel lane b′) by using αHPC antibody to detect phosphorylated centrin demonstrates reactivity only with the slower migrating form of centrin and no reaction with the nonphosphorylated form. Analysis of whole cell extracts demonstrates that centrin is phosphorylated in breast tumor tissue (A, compare gel lanes c and d). (B) A section of a normal breast duct labeled with αHPC antibody demonstrates only a background level of staining in normal epithelial cells. (C) A section of a breast adenocarcinoma labeled with αHPC antibody demonstrates that centrosomes stain intensely, indicating that centrin is phosphorylated in tumor cells. (D–E) Immunofluorescence of the normal breast epithelial cell line MCF10A stained with αHPC antibody for phosphocentrin and propidium iodide for DNA. The spindle poles of a mitotic cell react strongly with αHPC antibody (D), whereas interphase cells show no labeling of their centrosomes (E). Tumor centrosome and mitotic MCF10A cell staining with αHPC are completely eliminated with a fivefold excess of competing phosphopeptide (data not shown). (Bar = 20 μm.)

Finally, a simple functional assay was developed to assess the capacity of centrosomes of frozen-sectioned tissues to support microtubule nucleation. Fig. 5A illustrates the microtubule nucleating capacity of a normal ductal epithelial cell wherein the apical centrosome nucleated 12–13 microtubules that grew from a single focused MTOC. Fig. 5 B–C shows individual cells from touch preparations of normal breast tissues. Normal cells typically nucleate <50 microtubules in this assay (range 0–52; Table 1C). In contrast, tumor cells contain multiple MTOCs scattered around the periphery of the nucleus that nucleate significantly larger numbers of microtubules (range 0–270; Fig. 5 D–F; Table 1C). Fig. 5D illustrates a section of a breast tumor where individual centrosomes of most of the cells presented nucleated many microtubules, and Fig. 5 E–F show touch preparations of cells from the same tumor. The capacity of interphase tumor cell MTOCs to nucleate an unusually large number of microtubules is characteristic for most tumor cells in a given preparation. As defined by the microtubule nucleation assay described here, tumor cells with multiple MTOCs also have multiple centrin-staining centrosomes.

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Figure 5

Tumor centrosomes nucleate more microtubules than do normal epithelial cell centrosomes. (A) A section of normal breast tissue after incubation with Xenopus egg extract and staining for microtubules. (D) A section of a breast tumor illustrating nucleation of large numbers of microtubules by many individual adenocarcinoma cell centrosomes. Touch preparations were analyzed for the purpose of making accurate counts of microtubules nucleated by normal breast epithelial cells (B–C) and by tumor cells (E–F). Normal breast epithelial cells nucleate <45 microtubules that originate from a single, distinctly focused MTOC (B–C). In contrast, tumor cells nucleate many more microtubules that originate from multiple large MTOCs (E–F). Microtubules are stained with antitubulin antibodies, and nuclei are stained by using propidium iodide. Data from 50 normal and 50 tumor cells are summarized in Table 1C. (Bar = 20 μm.)

DISCUSSION

Human breast tumor cells show striking and characteristic abnormalities of several centrosome properties, including excess accumulation of pericentriolar material, supernumerary centrioles, and inappropriate phosphorylation of centrosomal proteins. In addition, tumor centrosomes show functional abnormalities characterized by increased numbers of MTOCs that nucleate unusually large microtubule arrays. Together, these defects in centrosome organization and behavior constitute a condition that we define here as “centrosome hypertrophy”. Centrosome hypertrophy is characteristic for 34 of 35 high grade breast adenocarcinoma specimens we have surveyed to date. A careful analysis of nearly 200 additional breast specimens, including ductal carcinoma in situ, lobular carcinoma in situ, malignant breast tumors of lower histological grade, and fibrocystic disease tissue is currently underway.

In the study presented here, two well characterized centrosomal markers [centrin, and γ-tubulin (31, 32)] demonstrated centrosome hypertrophy in human breast tumor centrosomes. Given the molecular complexity of the centrosome, it is likely that centrosome hypertrophy observed in breast tumor cells involves both accumulation of many centrosomal proteins and their assembly into new centrioles and associated pericentriolar material. Centrosome hypertrophy may result from an increase in centrosome protein expression or a decrease in turnover of centrosome components and/or in recruitment of centrosome precursors from cytoplasmic pools. Centrosome hypertrophy also is likely to be related to perturbation of tumor cell cycle regulatory pathways involved in centrosome duplication (1, 22, 33–35). Anomalous phosphorylation of centrosome proteins in breast tumor cells may be a manifestation of this disregulation of cell cycle controls. Centrosome abnormalities induced by tumor promoters and experimental evidence linking the tumor suppressor p53 to regulation of centrosome dynamics suggest a relationship between centrosome defects and tumor progression (22–24, 33, 36, 37). Although p53 mutation status has yet to be determined for the tumors analyzed in the studies presented here, preliminary experiments suggest that there is not a strict correlation between p53 immunohistochemistry labeling index and centrosome hypertrophy.

Neoplastic transformation is a multistep process that involves activation of protooncogenes, inactivation of tumor suppressor genes, and disregulation of cell cycle checkpoints (38–39). Although the order of these genetic changes may be important for development of certain tumors [for example, colorectal tumors (38)], this importance is not clear for tumors in which progression is less well defined, as is the case for neoplastic transformation of breast epithelia (40). There are at least two functional consequences of centrosome hypertrophy that may play important roles during neoplastic transformation and tumor progression. First, aberrant centrosome duplication may adversely affect maintenance of cell polarity in interphase cells because cytoplasmic architecture and directional vesicular trafficking may be disorganized in a cell with multiple MTOCs. High grade tumors are characterized by the loss of cellular organization, particularly with regard to orientation of adjacent cells relative to one another and to normal cell polarity (together termed “anaplasia”) (41). Second, centrosome hypertrophy may increase the incidence of multipolar mitoses, which lead to chromosomal segregation abnormalities and aneuploidy. Aneuploidy is the predominant class of genomic instability found in colorectal, breast, and perhaps solid tumors in general (42–46). Acquisition of such centrosome defects could affect tumor progression by increasing the likelihood of developing further genetic changes that lead to cellular disregulation and metastasis.

These observations have important clinical implications. Given the relationship of centrosome function to cell polarity and to maintenance of genomic integrity, the degree and nature of centrosomal defects may have predictive value with regard to patient prognosis. Tumor cell centrosomes also may present new targets for chemoprevention and development of novel therapeutics.

Finally, the observations presented here are a tribute to Theodor Boveri’s prescient depth of understanding of the biology of the centrosome and its relationship to cancer. Ten years after Boveri’s death, Maynard Metcalf wrote, “Boveri was the greatest cytologist of his generation … a man so keen, so careful and so cautious that any least suggestion from him deserves most thorough consideration. Boveri’s work should be the starting point for any studies of causes, inheritance or cure of cancer” (47).