Centrosome Amplification and the Origin of Chromosomal Instability in Breast Cancer

Jeffrey L. Salisbury

Introduction

Aneuploidy and chromosomal instability (CIN) are defming features of most aggressive breast cancers (BC). One consequence of CIN is a constantly changing genetic makeup of cancer cells - this in turn is a major driving force behind cancer cell heterogeneity, tumor progression, and acquisition of resistance to chemotherapeutics. How CIN arises in cancer and the mechanisms underlying this process have become a topical focus of cancer research. Yet it was nearly a century ago that Theodor Boveri first recognized that aneuploidy in cancer cells could arise through defects in the machinery for chromosomal segregation (1). Based on observations of abnormal chromosomal segregation in early sea urchin embryo development following dispermic fertilization and similarities to chromosomal anomalies seen in cancer, Boveri proposed that malignant tumors arise through defects that result in improper (1). At about this same time, Galeotti came to a similar conclusion from his studies on tumors (2). Despite these compelling arguments and a strident call to the medical research community in JAM by Maynard Metcalf a decade later (3) imploring that "Boveri's workshould be the startingpoint for a9studies of causes, inheritance or cure of cancer" it was not until near the end of the last century that investigations on human tumors and mouse models began to comborate Boveri's astute prescience (4-8). In this article, a review of centrosome structure and function, and the regulation of centrosome duplication in normal cells will be presented. Using recent studies on BC as an exemplary model, a discussion will follow on how deregulation of centrosome behavior can arise, result in centrosome amplification, and lead to CIN in cancer.

Centrosome Structure and Behavior in Normal Cells

The centrosome resides near the cell center (hence its name) and consists of a pair of and a surrounding matrix of (PCM) that anchor microtubule nucleation sites and consequently determines the number and organiza- Centrosome Amplification and Breast Cancer 107 tion of microtubules in interphase cells. Like chromosomes, double in number once in each in a process that is initiated with duplication. The centriole pair embodies an intrinsic counting mechanism that establishes the number of centrosome equivalents in the cell such that a pair of centrioles equals one, and two pair of centrioles equals two centrosome equivalents (9). Centrosomes increase in size through the recruitment of PCM and centrosomes of G2M cells show a dramatic increase in microtubule nucleating activity. At the time of cell division the two centrosomes (one residing at each spindle pole) organize microtubules of the bipolar mitotic spindle. In normal cells, spindle architecture is such that the two oppositely oriented half-spindle microtubule arrays (each arising from one of the two spindle poles) cast microtubules outward to engage and orient chromosomes so that sister chromatids face and can engage microtubules originating from opposite spindle poles. When all chromosomes are appropriately oriented and attached to microtubules originating from both spindle poles, the sister chromatids separate and move toward opposite poles by a molecular motor-driven process and dynamic shortening of their attached microtubules. Two new daughter cells form by cytokinesis and each inherit a complete complement of chromosomes along with one of the spindle poles that acts as the centrosome in the next cell cycle.

Centrosome Amplification in Cancer

Recent studies implicate centrosome abnormalities in the pathogenesis of cancer (4, 10-14). The term "centrosome amplification" refers to centrosomes that appear larger than normal, centrosomes that contain more than four centrioles, andlor when more than two centrosomes are present within a cell. In addition to these structural abnormalities, amplified centrosomes also show protein hyperphosphorylationand altered functional properties such as an increased microtubule nucleating capacity (4, 8, 15-17). Electron microscope studies revealed supernumerary centrioles in centrosomes of humans and animal model tumors, including leiomyosarcoma, neuroblastoma, glioma, and thymic carcinoid tumors (1 8-23). Systematic analyses of centrosomes in human breast carcinomas and a mouse model for prostate cancer revealed a range of abnormalities in centrosome structure including: excess number of centrioles, increased pericentriolar material, abnormal centriole orientation, and inverted polarity of centrosome location (5, 24). These structural centrosome abnormalities have been implicated as a potential cause of loss of cell and tissue architecture seen in cancer (i. e., anaplasia) through altered centrosome function in microtubule nucleation and organization, and to result in chromosome missegregation during as a consequence of multipolar spindle formation. 108 J.L. Salisbury

Correlation of Centrosome Amplification, Aneuploidy, and Chromosomal Instability A key question is whether or not centrosome amplification leads to CIN and aneuploidy or is a consequence of them; the proverbial chicken and egg riddle. Aneuploidy is characterized as the state of an abnormal karyotype, having gains andlor losses of whole chromosomes. Aneuploidy occurs early in the development of many tumor types, suggesting that it may play a role in both tumorigenesis and tumor progression. Indeed, aneuploidy is present in the great majority of malignant tumors, in contrast to benign tumors, which are most often diploid. Aneuploidy can be distinguished from the persistent generation of chromosomal variations, termed "CIN", which reflects the rate of change in karyotype (25). Quantitative analysis of CIN can be determined as the percent of cells with a chromosome number different from the modal chromosome number. Thus, tumors may show either "stable aneuploidy" (low CIN) or "unstable aneuploidy." Unstable karyotypes may lead to phenotypic heterogeneity in cancer, reflecting the persistent generation of new chromosomal variations (26,27). The development of aneuploidy may be a consequence of centrosome amplification, which can lead to the formation of multipolar spindles and miss- segregate sister chromatids during mitosis, and as a result to high CIN. CIN occur exclusively in aneuploid tumors and tumor-derived cell lines in contrast to diploid tumors, which contain centrosomes that are functionally and structurallynormal (4, 26,28). The degree of genomic instability in aneuploid tumors parallels the degree of centrosome abnormalities in cell lines fiom breast (29), pancreas (1 3), prostate (30), colon (28), and cervix tumors (31), from short-term culture of mouse mammary tumors (32), and from SV40 ST over-expressing fibroblasts (33). When tissues were examined, centrosome abnormalitieswere higher in high-grade prostate tumors (30) and high-grade cervical tumors (31) than in low-grade tumors. In prostate cancer, centrosome amplification has been implicated in the development of abnormal mitoses and CIN facilitating progression to advanced stages of the disease (30, 34, 35). Strong support for a direct mechanistic link between centrosome amplification and CIN is suggested by the significant linear correlation between centrosome amplification and the rate of change in karyotype (CIN) seen in human breast tumors (26). Although such correlation alone does not necessarily imply cause and effect, these observations have led many authors to propose the hypothesis that centrosome amplification is the primary cause of genomic instability observed in most tumors (13, 26, 31, 33). As discussed above, Boveri first recognized these features of cancer cells nearly a century ago and proposed that centrosome defects could lead to mitotic and subsequent chromosomal abnormalities (1). An alternative hypothesis has been proposed that CIN seen in cancer cells is caused by aneuploidy, that is that aneuploidy itself destabilizes the karyotype and thus initiates CIN leading to widespread heterogeneity in tumor cell phenotypes (36-39). Centrosome Amplification and Breast Cancer 109

Several independent lines of evidence support the proposition that centrosome abnormalities drive genomic instability. In a recent study of human breast tumors, all specimens of ductal carcinoma in-situ examined showed significant centrosome amplification, while aneuploidy is present, on average, suggesting that centrosome amplification is an early event that occurs prior to invasion in breast tumors (26). Furthermore, cells transfected to express the HPV E7 oncoprotein undergo centrosome amplification prior to developing nuclear morphology associated with aneuploidy (40,41). Finally, in a xenograft model of pancreatic cancer, metastatic foci showed a higher incidence of centrosome amplification than did the primary xenograft, and abnormal centrosome numbers were accompanied by a higher frequency of abnormal mitoses (42). Taken together, these studies suggest that centrosome amplification may be an early event in turnorigenesis that can drive CIN and lead to genotypic and phenotypic diversity of cells within a tumor.

Coordination of the DNA, Cell, and Centrosome Cycles in Normal Cells

Because the fidelity of equal segregation of sister chromatids into daughter cells depends on the bipolar nature of the mitotic spindle it is essential that cells maintain a strict linkage between the DNA, cell, and centrosome cycles. Cell cycle progression is governed by the location, activation and inactivation of the serinelthreonine cyclin-dependentprotein kinases (Cdks) (43). A direct role for the Cdks in regulating the mitotic activity of centrosomes was first suggested by the localization of cyclin B and Cdkl (p34cdc2)at the centrosome during mitosis, and fiom experiments implicating and B in the control of microtubule dynamics (44-47). Additional evidence pointed to a role for Cdk2 activity in linking centrosome duplication and the DNA cycle. Both processes are dependent on Cdk2 activation and are blocked by the Cdk2 inhibitors butyrolactone-I or roscovitine (48,49). In addition, centrosome duplication was arrested by protein inhibitors of Cdk2 (p2llwafl or p27), or by immuno-depletion of Cdk2 or , and centrosome duplication could be restored by excess purified cdk2/cyclin E (49- 5 1). Importantly, separation of the centriole pair, an early event in the centrosome duplication cycle, is dependent on Cdk2lcyclin E activity, suggesting that a Cdk- mediated phosphorylation event regulates centriole pair cohesion (5 1-54). Finally, DNA replication and centrosome duplication also depend on the phosphorylation status of retinoblastoma tumor suppressor Rb, which in turn governs the availability of the E2F transcription factor to promote S phase progression (55). Taken together these findings establish the mechanism by which the DNA and centrosome cycles are coordinated: both DNA replication and centrosome duplication are controlled by the Rb pathway, both processes depend on downstream transcriptional consequences of E2F activity, and both processes require Cdk2lcyclin activation. 110 J.L. Salisbury

Centrosome behavior and function during the cell cycle are also regulated by protein phosphorylation events in addition to those directly mediated by the Cdk- cyclins (56). At the onset of mitosis centrosome protein phosphorylation increases dramatically and then falls precipitously at the metaphaseJanaphase transition (57- 60). Several protein kinases and protein phosphatases have been identified that localize at centrosomes and affect the phosphorylation status of centrosome targets. Importantly, certain of these centrosome-associated kinases and their target substrates may become altered during the development of centrosome amplification in cancer. For example, over-expression of the breast tumor amplified kinase, BTAKlSTK15 (also known as Aurora A) can lead to centrosome amplification and CIN in breast epithelial cell lines and in mouse models for mammary tumorigenesis (8, 16, 61, 62). Studies on human tissues have also shown that inappropriate phosphorylation of centrosome proteins can serve as a sensitive marker for centrosome amplification in tumors (4). Deregulation of the Centrosome Cycle in Cancer GlJS and G2/M checkpoints enforce the orderly completion of cell cycle events, and when triggered, they inhibit the formation andlor activation of Cdks and thereby induce cell cycle arrest (63,64). Cell cycle checkpoints operate through the action tumor suppressor proteins and Rb, and their downstream activation target Cdk2. Interestingly both p53 and Cdk2 may also physically reside at the centrosome, albeit only transiently (65,66). The physical presence of key proteins involved in checkpoint control at the centrosome has led to the suggestion that the centrosome itself may provide an important structural context for coordinating cell cycle regulation (56,65-70). Centrosome abnormalities in cancer are correlated with loss of p53 function in carcinomas of the breast, head and neck, and prostate, and in neuroectodermal tumors (14, 34, 71). In tumors that retained wild-type p53, amplified centrosomes were frequently associated with overexpression of , which abrogates p53 function by promoting its degradation (71). Furthermore, gain-of-function p53 mutations and p53 null mice can result in deregulation of centrosome duplication leading to the generation of functionally amplified centrosomes and aberrant mitoses (72-74). Interestingly, in some cancers p53 mutations and cyclin E overexpression may act synergistically since together they increased the frequency of centrosome defects in cultured cells and in mouse models (75). Centrosome homeostasis is controlled at the GlJS and G2/M checkpoints (Figure 1) through transcriptional regulation by p53 of several downstream targets including the Cdk inhibitor p2lJwafl (76, 77). As discussed earlier, p2lJwafl blocks centrosome duplication through inhibition of Cdk2lcyclin E activity. This conclusion is supported by experiments in which anti-sense targeting of p2 llwafl in human cell lines resulted in endoreduplication and centrosome amplification (78). Centrosome Amplification and Breast Cancer 11 1

Figure 1. (a) Model illustrating the centrosome and DNA cycles in cells with normal and with defective checkpoint controls as discussed in the text. (b) Electron micrograph of an amplified centrosome with five centrioles from a human mammary tumor (courtesy of Wilma Lingle, Mayo Clinic).

Interestingly, while introduction of wild-type p53 into p53-'- mouse embryonic fibroblasts re-established centrosome homeostasis, overexpression of p2llwafl only partially restored control of centrosome duplication in p53-null fibroblasts, suggesting that alternative downstream p53 targets may also be involved in the regulation of centrosome homeostasis (79, 80). In this regard, GADD45, another downstream product of the p53 pathway, has been implicated in activation of the G2/M checkpoint and regulation of the centrosome cycle (81-83). Alternative mechanisms, independent of p53 function, may also lead to deregulation of centrosome homeostasis (25, 26, 84, 85). For example high-risk human papillomavirus (HPV) v-oncogenes, E6 and E7, have been implicated in the induction of centrosome amplification in human cell lines (86). HPV E6 and E7 interfere with centrosome homeostasis by targeting different pathways. Whereas E6 may operate through inactivation of p53 function resulting in accumulation of excess centrosomes by failure of the G2/M checkpoint leading to defects in cytokinesis, E7 may lead to centrosome amplification through inactivation of the Rb and G11S checkpoint resulting in abnormal centrosome duplication (86). In addition, as discussed above, centrosome amplification can be induced by over- expression of the centrosome kinase BTAKISTKl5 (8), mutations in the BRCAl and BRCA2 tumor suppressor genes (87-92) or by over-expression of the PCM structural protein pericentrin (30). Thus, centrosome defects and consequent genomic instability may result from inactivation of cell cycle checkpoints, inappropriate activation of key centrosome kinases, or alterations in structural proteins of the centrosome itself. These observations suggest several regulatory pathways operate in parallel to ensure linkage between the DNA, cell, and centrosome cycles. In the development of cancer, centrosome defects may result fiom an imbalance between negative and positive regulators that converge on GlIS and G2M checkpoints, or directly on components of the centrosome itself. 112 J.L. Salisbury

Centrosome Amplification as Potential Indicator of Tumor Aggressiveness

Can centrosome amplification be utilized as an indicator of tumor progression, the potential to develop aggressive tumor phenotypes, and to serve as a prognostic indicator of clinical outcome? Centrosome amplification is not only characteristic of aneuploid tumors in general, but also is more pronounced in advanced stage malignancies, in recurrent tumors, and in cell lines that show more aggressive malignant phenotypes in xenograph animal models (10, 11, 16, 29). These observations suggest that centrosome amplification might be useful in monitoring tumor progression and phenotypic diversity in cancer. In association with other established prognostic factors, centrosome amplification may be helpful in predicting outcomes and survival of patients with cancer. And finally, because the centrosome may serve as a structural context for the action of key cell cycle regulators, it may also represent a critical target for therapeutic intervention.

Acknowledgements

I have been fortunate to have many creative and talented individuals work in my laboratory on centrosome defects in cancer. I would especially like to acknowledge, Susan Barrett, Robert Busby, Lynn Cordes, Vivian Negron, Mark Sanders, Kelly Suino, and doctors Antonino D'Assoro and Wilma Lingle.

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