2n or not 2n: Aneuploidy, polyploidy and chromosomal instability in primary and tumor cells.

Mitotic defects leading to aneuploidy have been recognized as a hallmark of tumor cells for over 100 years. Current data indicate that ∼85% of human cancers have missegregated chromosomes to become aneuploid. Some maintain a stable aneuploid karyotype, while others consistently missegregate chromosomes over multiple divisions due to chromosomal instability (CIN). Both aneuploidy and CIN serve as markers of poor prognosis in diverse human cancers. Despite this, aneuploidy is generally incompatible with viability during development, and some aneuploid karyotypes cause a proliferative disadvantage in somatic cells. In vivo, the intentional introduction of aneuploidy can promote tumors, suppress them, or do neither. Here, we summarize current knowledge of the effects of aneuploidy and CIN on proliferation and cell death in nontransformed cells, as well as on tumor promotion, suppression, and prognosis.

Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons.

The mitotic checkpoint is the major cell cycle checkpoint acting during mitosis to prevent aneuploidy and chromosomal instability, which are hallmarks of tumor cells. Reduced expression of the mitotic checkpoint component Mad1 causes aneuploidy and promotes tumors in mice [Iwanaga Y, et al. (2007) Cancer Res 67:160-166]. However, the prevalence and consequences of Mad1 overexpression are currently unclear. Here we show that Mad1 is frequently overexpressed in human cancers and that Mad1 up-regulation is a marker of poor prognosis. Overexpression of Mad1 causes aneuploidy and chromosomal instability through weakening mitotic checkpoint signaling caused by mislocalization of the Mad1 binding partner Mad2. Cells overexpressing Mad1 are resistant to microtubule poisons, including currently used chemotherapeutic agents. These results suggest that levels of Mad1 must be tightly regulated to prevent aneuploidy and transformation and that Mad1 up-regulation may promote tumors and cause resistance to current therapies.

Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability.

Increased Aurora B protein expression, which is common in cancers, is expected to increase Aurora B kinase activity, yielding elevated phosphorylation of Aurora B substrates. In contrast, here we show that elevated expression of Aurora B reduces phosphorylation of six different Aurora B substrates across three species and causes defects consistent with Aurora B inhibition. Complexes of Aurora B and its binding partner INCENP autophosphorylate in trans to achieve full Aurora B activation. Increased expression of Aurora B mislocalizes INCENP, reducing the local concentration of Aurora B:INCENP complexes at the inner centromere/kinetochore. Co-expression of INCENP rescues Aurora B kinase activity and mitotic defects caused by elevated Aurora B. However, INCENP expression is not elevated in concert with Aurora B in breast cancer, and increased expression of Aurora B causes resistance rather than hypersensitivity to Aurora B inhibitors. Thus, increased Aurora B expression reduces, rather than increases, Aurora B kinase activity.

High rates of chromosome missegregation suppress tumor progression but do not inhibit tumor initiation.

Aneuploidy, an abnormal chromosome number that deviates from a multiple of the haploid, has been recognized as a common feature of cancers for >100 yr. Previously, we showed that the rate of chromosome missegregation/chromosomal instability (CIN) determines the effect of aneuploidy on tumors; whereas low rates of CIN are weakly tumor promoting, higher rates of CIN cause cell death and tumor suppression. However, whether high CIN inhibits tumor initiation or suppresses the growth and progression of already initiated tumors remained unclear. We tested this using the Apc(Min/+) mouse intestinal tumor model, in which effects on tumor initiation versus progression can be discriminated. Apc(Min/+) cells exhibit low CIN, and we generated high CIN by reducing expression of the kinesin-like mitotic motor protein CENP-E. CENP-E(+/-);Apc(Min/+) doubly heterozygous cells had higher rates of chromosome missegregation than singly heterozygous cells, resulting in increased cell death and a substantial reduction in tumor progression compared with Apc(Min/+) animals. Intestinal organoid studies confirmed that high CIN does not inhibit tumor cell initiation but does inhibit subsequent cell growth. These findings support the conclusion that increasing the rate of chromosome missegregation could serve as a successful chemotherapeutic strategy.

The ARF tumor suppressor prevents chromosomal instability and ensures mitotic checkpoint fidelity through regulation of Aurora B.

The ARF tumor suppressor is part of the CDKN2A locus and is mutated or undetectable in numerous cancers. The best-characterized role for ARF is in stabilizing p53 in response to cellular stress. However, ARF has tumor suppressive functions outside this pathway that have not been fully defined. Primary mouse embryonic fibroblasts (MEFs) lacking the ARF tumor suppressor contain abnormal numbers of chromosomes. However, no role for ARF in cell division has previously been proposed. Here we demonstrate a novel, p53-independent role for ARF in the mitotic checkpoint. Consistent with this, loss of ARF results in aneuploidy in vitro and in vivo. ARF(-/-) MEFs exhibit mitotic defects including misaligned and lagging chromosomes, multipolar spindles, and increased tetraploidy. ARF(-/-) cells exhibit overexpression of Mad2, BubR1, and Aurora B, but only overexpression of Aurora B phenocopies mitotic defects observed in ARF(-/-) MEFs. Restoring Aurora B to near-normal levels rescues mitotic phenotypes in cells lacking ARF. Our results define an unexpected role for ARF in chromosome segregation and mitotic checkpoint function. They further establish maintenance of chromosomal stability as one of the additional tumor-suppressive functions of ARF and offer a molecular explanation for the common up-regulation of Aurora B in human cancers.