The work presented here indicates that CHFR is extremely important for the maintenance of genomic stability in mammary epithelial cells. Our results support and help explain the previously published findings of aneuploidy in MEFs from Chfr
null mice and IHMEC lines [3,14
]. The observed chromosome rearrangements that we noted spectral karyotyping likely resulted from prolonged culture, and the disruption of DNA damage response genes secondary to the aneuploidy, which we have shown can develop within a few days after decreased CHFR expression. To the contrary, the presence of additional chromosomes with a numeric change in chromosome number, or aneuploidy, in cells treated with siRNA against CHFR
provides powerful evidence that CHFR is required for genomic stability through proper chromosome segregation during mitosis. Furthermore, the lack of chromosome breaks on the metaphase spreads from MCF10A cells transiently transfected with siRNA to decrease CHFR mRNA and protein suggested that CHFR might not participate directly in the DNA damage response induced by aphidicolin. This conclusion is supported by previous studies in which CHFR expression did not alter the DNA damage response after treatment with other genotoxic reagents [1,22
We were able to confirm the previously published finding that Chfr can regulate Aurora A expression in the mouse also holds true in humans [14
]. Aurora A is amplified and overexpressed in many cancers, including breast cancer, and overexpression in cultured human cells leads to transformation [20,23
]. In addition, a transgenic mouse overexpressing Aurora A in the mammary epithelium leads to tumor formation and genomic instability [24
]. The chromosome missegregation phenotypes in MCF10A:CHFR-siRNA cells were also highly reminiscent of MEFs that overexpress Aurora A [25
]. CHFR was recently characterized as a tumor suppressor and, as shown here, many of its genomic instability phenotypes resemble Aurora A overexpression; therefore, we propose that one major mechanism by which CHFR inhibits oncogenesis may be through its negative regulation of Aurora A [3,14
]. Novel drugs currently are being generated that target the Aurora kinases [26
]. Because decreased CHFR expression has been linked to sensitivity to microtubule-targeting drugs, future studies may find a synergistic effect when taxanes and Aurora kinase inhibitors are both used for treatment.
These findings also indicate that CHFR may play a role in regulating α-tubulin turnover or stability, especially after microtubule stress. This is the first clue as to how the “CHFR checkpoint” responds to microtubule poisons, although an unidentified signaling cascade is also likely to be involved in this checkpoint. The ubiquitination and possible degradation of α-tubulin may be necessary to remove those α/β-tubulin dimers that are targeted by microtubule poisons. In unstressed cells, CHFR may also be required for proper spindle formation because it seems to regulate the amount of acetylated α-tubulin, an important component of the mitotic spindle. Aurora A kinase is also required for proper spindle formation, supposedly through its positive regulation of a protein called HURP [27
]. HURP is required for both chromosome congression and alignment and for the polymerization and stabilization of microtubules during mitotic spindle formation. Therefore, the capacity of CHFR to control spindle formation may be through its upstream regulation of Aurora A, although it may also be due to CHFR's capability to ubiquitinate α-tubulin and control the amount of acetylated α-tubulin that is available for use during spindle assembly.
One of the characteristics of stabilized microtubules is the acetylation of α-tubulin on residue lysine 40. Acetylated α-tubulin is associated with decreased microtubule turnover and is localized to the mitotic spindle, centrosomes, and the mitotic midbody [28,29
]. An increase in acetylated α-tubulin, such as that observed here, would likely result in overstabilized microtubules that would hinder mitotic spindle movement or would prevent its proper formation. This may help to explain why CHFR-negative cells are more sensitive to taxanes. The cellular strain of the excess of overstabilized acetylated microtubules, combined with stress induced by microtubule poisons, may enable the cell to surpass a threshold of tolerable stress that would result in apoptosis. This hypothesis is supported by reports of a synergistic effect on both apoptotic response and microtubule stabilization, as indicated by acetylated α-tubulin, when endometrial cancer cells are treated with both the histone deacetylase inhibitor (HDI) trichostatin A and paclitaxel [30
]. Interestingly, some of the targets of HDIs are also tubulin deacetylase proteins, such as HDAC6 and SIRT2 [31,32
]. Recent studies also show that treating cells with HDIs down-regulates Aurora A expression [33
]. Future clinical studies may find that the synergistic effect between HDIs and taxanes may be different in CHFR-positive versus
CHFR-negative cancer cells.
The finding that CHFR knockdown results in increased amounts of acetylated α-tubulin is particularly interesting because another protein that has been found to initiate a “CHFR checkpoint-like” response to microtubule poisons is SIRT2, a tubulin and histone deacetylase [34
]. SIRT2 overexpression is a phenocopy of CHFR overexpression in regards to the regulation of mitotic entry and response to mitotic stress. Therefore, hypothetically, decreased SIRT2 expression should resemble decreased CHFR expression in both the response to mitotic stress and the amount of acetylated α-tubulin in the cell. Future studies should determine whether the increase in acetylated α-tubulin after decreased CHFR expression is due to SIRT2 or through the activation of Aurora A-regulated HURP.
We report here the identification of a novel CHFR interacting protein, MAD2. Although we found no evidence that MAD2 is a ubiquitination target of CHFR (data not shown), we noticed that when CHFR expression was drastically reduced and the cells were treated with nocodazole to induce the checkpoint response, there was a slight decrease in MAD2 protein levels (). This suggests that another ubiquitin ligase is possibly degrading MAD2 prematurely, or to a greater degree, in the absence of CHFR. Because decreased CHFR expression impairs MAD2/CDC20 complex formation, we hypothesize that, in this scenario, CDC20 is prematurely activating the APC complex to initiate anaphase and ubiquitinate target mitotic proteins, such as MAD2. Alternately, MAD2 expression itself may be down-regulated in nocodazole-treated cells with decreased CHFR expression. Further studies are warranted to clarify the mechanism(s) of impaired MAD2 function and expression in cells with decreased CHFR expression.
With an impaired spindle checkpoint, cells with decreased CHFR expression could enter anaphase without all of their chromosomes localized to the metaphase plate and the sister chromatids attached to the mitotic spindle, leading to the appearance of lagging chromosomes and unequal chromosome segregation among the two daughter cells, such as that reported here. One potential outcome of improper chromosome segregation is the abortion of cytokinesis, resulting in binucleated cells and tetraploidy, which was also observed in this work [21
]. Of interest, our work strongly agrees with previous findings that the yeast orthologs of CHFR, DMA1 and DMA2, also function in regulating the spindle checkpoint and cytokinesis [35,36
A recent report indicated that one isoform of CHFR
(Accession No. AF_170724; the same isoform was used in this article) contains a KEN box motif, which targets proteins to the CDH1-APC complex for degradation. This further supports a role for CHFR in regulatingmitosis and the spindle checkpoint [37
]. Although we found that a KEN box deletion mutant of CHFR did not impair CHFR/MAD2 coimmunoprecipitation (data not shown), further studies are warranted to determine whether the KEN box in CHFR is required for spindle checkpoint function and/or degradation by the CDH1-APC complex.
We also discovered that CHFR overexpression is toxic to many breast cell lines independent of the method of transfection or retroviral transduction (both transient and stable; data not shown), which is why HEK293 cells were used to express the Flag-tagged CHFR construct used in coimmunoprecipitation experiments. This suggests that CHFR expression must be tightly regulated—too much is toxic whereas too little causes genomic instability and tumorigenesis. This is reminiscent of other mitotic checkpoint proteins, such as MAD2, in that both too little and too much of the protein are deleterious [38
]. Recently, this finding was also reported in HCT116 and RKO colon cancer cell lines [39
]. Determining the mechanism(s) causing CHFR overexpression toxicity likely will answer many of the questions that remain about the function of CHFR.
These findings have led us to propose a model for how CHFR may regulate genomic instability and/or tumorigenesis (). We suggest that decreased or lost CHFR expression causes overexpression of Aurora A and both unmodified and acetylated α-tubulin, and the mislocalization of MAD2. Aurora A overexpression could lead to centrosome amplification, an impaired spindle checkpoint, and possibly defective mitotic spindle formation, leading to aneuploidy and impaired cytokinesis. The mislocalization of MAD2 also causes an impaired spindle checkpoint response. The increase in acetylated α-tubulin could cause stress on the mitotic spindle. Both pathways would lead to genomic instability, contributing to tumorigenesis. As indicated by the generality of this model, much research remains to elucidate the role of CHFR in regulating mitosis and genomic instability.
Figure 6 A proposed model of how CHFR regulates genomic instability. Decreased or lost CHFR expression causes Aurora A, α-tubulin, and acetylated α-tubulin overexpression and MAD2 mislocalization. The increase in acetylated α-tubulin occurs (more ...)
Many reports have indicated that CHFR plays an important role in carcinogenesis and has tumor-suppressive qualities. An abundance of evidence indicates that CHFR
mRNA expression is decreased in many cancer types, often due to promoter methylation (reviewed in Privette and Petty [40
]). In addition, a knockout mouse model and cell culture models of lost or decreased expression, respectively, indicate that CHFR has tumor-suppressive qualities [3,14
]. Additional evidence for CHFR's role in tumorigenesis is that it is important for cell cycle regulation, chemotherapeutic response to taxanes, and cellular proliferation [3,6,39
] (reviewed in Privette and Petty [40
]). Here, we present novel evidence for an additional tumor-suppressive function of CHFR—maintaining genomic stability by regulating the mitotic spindle assembly checkpoint. Cancer often develops in concert with the loss of cell cycle regulation and genomic instability; CHFR may function in both processes.