Loss of CHFR in Human Mammary Epithelial Cells Causes Genomic Instability by Disrupting the Mitotic Spindle Assembly Checkpoint

Abstract

CHFR is an E3 ubiquitin ligase and an early mitotic checkpoint protein implicated in many cancers and in the maintenance of genomic stability. To analyze the role of CHFR in genomic stability, by siRNA, we decreased its expression in genomically stable MCF10A cells. Lowered CHFR expression quickly led to increased aneuploidy due to many mitotic defects. First, we confirmed that CHFR interacts with the mitotic kinase Aurora A to regulate its expression. Furthermore, we found that decreased CHFR led to disorganized multipolar mitotic spindles. This was supported by the finding that CHFR interacts with alpha-tubulin and can regulate its ubiquitination in response to nocodazole and the amount of acetylated alpha-tubulin, a component of the mitotic spindle. Finally, we found a novel CHFR interacting protein, the spindle checkpoint protein MAD2. Decreased CHFR expression resulted in the mislocalization of both MAD2 and BUBR1 during mitosis and impaired MAD2/CDC20 complex formation. Further evidence of a compromised spindle checkpoint was the presence of misaligned metaphase chromosomes, lagging anaphase chromosomes, and defective cytokinesis in CHFR knockdown cells. Importantly, our results suggest a novel role for CHFR regulating chromosome segregation where decreased expression, as seen in cancer cells, contributes to genomic instability by impairing the spindle assembly checkpoint.

Connections between CHFR, the cell cycle, and chemosensitivity: Are they critical in cancer?

“…5 This work established CHFR as a component of the so-called "antephase" checkpoint which monitors microtubule-dependent events between prophase and metaphase. 5 More recent studies have also implicated CHFR in the spindle assembly checkpoint between metaphase and anaphase, 4,6,7 which can also be triggered by microtubule targeting drugs. Given this, it is hypothesized that CHFR functions in two distinct cellular checkpoints during mitosis where CHFR functions to halt the cell cycle in response to microtubule damage or spindle defects, ensuring proper chromosome segregation and cell division.…”

“…8,9 Reduction of endogenous CHFR in cell lines resulted in onset of tumor-like phenotypes such as increased mitotic index, growth rate, invasiveness, aneuploidy, motility and soft agar colony formation. 7 In cancer cell lines that express little or no CHFR, restoration of CHFR expression reduced phenotypes including mitotic index, invasiveness, motility and growth rate.…”

“…Cell lines in which CHFR expression has been inhibited show reduced survival and increased apoptosis when treated with microtubule-targeting drugs. 7,20,21 Therefore, CHFR represents an important marker for sensitivity to microtubulespecific chemotherapeutics.…”

“…12 Similar phenotypes are seen when CHFR expression is reduced, which increases AurA levels in the cell. 4,7 MAD2 (Mitotic Arrest Deficient 2), a protein that is recruited to unattached kinetochores to act in the spindle-assembly checkpoint during mitosis, has recently been shown to bind to CHFR protein. 7,13 Loss of CHFR results in the mislocalization of MAD2 and BUBR1, both of which bind to kinetochores and are critical for the spindle assembly checkpoint.…”

Epithelial Ovarian Cancer

“…CHFR has multiple functions in checkpoints during mitosis, such as regulation of the G2/M transition by its inherent ubiquitin ligase activity and targeting of key proteins, such as AURA, to the proteasome [29][30][31][32]. Nevertheless, a better understanding of the multiple signaling pathways associated with ovarian tumorigenesis is needed in order to identify new ways to target signaling pathways in EOC and in this way increase the efficiency of ovarian cancer treatment and minimize recurrent disease.…”