The anti-cancer compound, Factor Quinolinone Inhibitor 1, inhibits stable kinetochore-microtubule attachment during mitotic progression
Yunes, Sarah Ann
MetadataShow full item record
Factor Quinolinone Inhibitor 1 (FQI1), discovered as a small molecule inhibitor of the transcription factor LSF, causes cell death in many cancer cell lines and inhibits tumor growth in tumor xenografts and an endogenous hepatocellular carcinoma model in mice. Significantly, multiple animal studies have shown minimal to no toxicity after FQI1 treatment, making it a promising potential lead chemotherapeutic for multiple cancer types. In determining how FQI1 causes cancer cell death, it was previously shown that FQI1 treatment, like knockdown of LSF expression by siRNA, produced a mitotic arrest with condensed but unaligned chromosomes, but with no clearly observable transcriptional dysregulation. In this thesis, I establish that introducing FQI1 to cells already in mitosis induces a mitotic arrest in colorectal cancer cells, demonstrating that FQI1 inhibits mitotic processes directly while these processes are occurring. This mitotic arrest is characterized by defects in the mitotic spindle and limited connections of mitotic spindles to the kinetochores, as indicated by a dramatic decrease cold-stable microtubules in mitosis. Additionally, in a dose-dependent manner, FQI1 treatment resulted in supernumerary γ-tubulin-containing mitotic centrosomes and γ-tubulin-deficient aster-like bodies, indicating a defect in centrosome stability. As FQI1 is known to be a specific inhibitor of LSF, with its dose dependence for LSF inhibition directly proportional to its ability to inhibit cell proliferation, these findings suggested the novel hypothesis that LSF regulates mitosis through non-transcriptional mechanisms by interacting with key mitotic proteins required for proper spindle formation and metaphase alignment. By mass spectrometry, multiple proteins were identified that interact with biotinylated LSF in mitosis in a FQI1-sensitive manner, with several related to the formation and stability of the mitotic spindle. Proximity ligation assays validated endogenous LSF interactions with CKAP5, a processive microtubule polymerase that protects kinetochore microtubules from depolymerization, and MISP, a requirement for proper mitotic spindle positioning. However, in this assay these interactions were not demonstrably FQI1-sensitive. In conclusion, FQI1 treatment results in defects in kinetochore-microtubule attachment and centrosome stability, triggering a mitotic arrest. Combined with the target specificity of FQI1, this suggests the hypothesis that LSF is required for proper mitotic spindle formation through its protein interactions in mitosis.