Chromosome Segregation

Upregulation of the mitotic checkpoint gene Mad1

The mitotic checkpoint (also known as the spindle assembly checkpoint) is the major cell cycle checkpoint acting during mitosis to prevent chromosome missegregation. Previous work has demonstrated that reduction of mitotic checkpoint proteins, including Mad1, weakens checkpoint signaling, resulting in chromosome missegregation and aneuploidy. We found that elevating levels of Mad1 in cells that were previously chromosomally stable (Movie 1) also weakens mitotic checkpoint signaling, leading to chromosome missegregation (Movie 2). Mad1 plays an essential role in mitotic checkpoint signaling by recruiting its binding partner Mad2 to unattached kinetochores where it is converted into an active inhibitor of the Anaphase Promoting Complex/Cyclosome. Elevated levels of Mad1 saturate available binding sites and sequester Mad2 away from kinetochores, weakening mitotic checkpoint signaling.

These findings are clinically relevant, as Mad1 expression at both the mRNA and protein level is frequently elevated in human cancers. Moreover, patients with tumors expressing heightened levels of Mad1 mRNA exhibit decreased survival compared to those with lower levels of Mad1. These data suggest that upregulation of Mad1 is a clinically relevant mechanism of inducing aneuploidy in human tumors. (Ryan et al, PNAS, 2012)

In addition to its role in chromosome segregation during mitosis, our lab has identified two novel interphase roles of Mad1. First, we discovered that Mad1 localizes to the Golgi Apparatus during interphase. At the Golgi, Mad1 participates in a5 integrin secretion. Along with its binding partner b1 integrin, a5 integrin serves as a major cellular receptor for the extracellular matrix component fibronectin. This suggested that Mad1 levels influence cellular adhesion and motility. Consistent with the, Mad1 depletion delays attachment and migration on fibronectin coated surfaces, while Mad1 upregulation increases the rate of motility. Interestingly, this is the first localization and function of Mad1 to be identified that is independent of the Mad1 binding partner Mad2. This work suggests that interphase consequences of Mad1 upregulation may contribute to tumor phenotypes, potentially promoting metastasis (Wan et al, Curr Biol 2014).

We have also found that Mad1 upregulation destabilizes the tumor suppressor p53, further supporting the idea that interphase roles of upregulated Mad1 are influential in tumor initiation and progression. p53 normally only accumulates in response to cell stress, since it causes cell cycle arrest or death. The E3 ubiquitin ligase MDM2 normally ubiquitinates p53, leading to its degradation. In response to cell stress, such as DNA damage, PML escorts MDM2 into nucleoli, physically separating it from p53 and allowing p53 to accumulate. However, upregulated Mad1 displaces MDM2 from p53, permitting continued degradation of p53 even in the presence of DNA damage. A SUMO-interacting motif (SIM) in the C-terminal domain of Mad1 is essential for PML binding and destabilization of p53. Moreover, upregulation of wild type Mad1 is sufficient to promote orthotopic mammary tumors, and this is dependent on the Mad1 SIM. This demonstrates that an unexpected interphase role of this mitotic checkpoint protein plays a critical role in its tumor promoting activity (Wan et al, Nat Comm 2019).

Roles of cancer genes in chromosome segregation

Loss or mutation of multiple tumor suppressors, including ARF, Rb, PTEN, and APC results in chromosome missegregation and aneuploidy. The ARF tumor suppressor plays a well-characterized role in inhibiting MDM2 and stabilizing p53. We have identified a novel function of ARF in suppressing chromosome missegregation independent of p53. ARF null cells show a variety of mitotic defects consistent with chromosome missegregation including lagging chromosomes (Figure 1), as well as a weakened mitotic checkpoint and elevated expression of the mitotic kinase Aurora B. The increase in Aurora B expression, which is due to increased protein stability, is sufficient to account for the mitotic defects in ARF null cells, as overexpression of Aurora B phenocopies ARF loss and depleting Aurora B to near-normal levels in ARF null cells rescues the mitotic defects.  These results define an unexpected, p53-indepent role for ARF in chromosome segregation (Britigan et al MBoC 2014)