Targeting the oncogene LSF with either the small molecule inhibitor FQI1 or siRNA causes mitotic delays with unaligned chromosomes, resulting in cell death or senescence

Abstract

Background: The oncogene LSF (encoded by TFCP2) has been proposed as a novel therapeutic target for multiple cancers. LSF overexpression in patient tumors correlates with poor prognosis in particular for both hepatocellular carcinoma and colorectal cancer. The limited treatment outcomes for these diseases and disappointing clinical results, in particular, for hepatocellular carcinoma in molecularly targeted therapies targeting cellular receptors and kinases, underscore the need for molecularly targeting novel mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models, with no observable toxicity. Methods: To understand how the LSF inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Cell proliferation and cell cycle progression were analyzed, using HeLa cells as a model cancer cell line responsive to FQI1. Cell cycle progression was studied either by time lapse microscopy or by bulk synchronization of cell populations to ensure accuracy in interpretation of the outcomes. In order to test for biological specificity of targeting LSF by FQI1, results were compared after treatment with either FQI1 or siRNA targeting LSF. Results: Highly similar cellular phenotypes are observed upon treatments with FQI1 and siRNA targeting LSF. Along with similar effects on two cellular biomarkers, inhibition of LSF activity by either mechanism induced a strong delay or arrest prior to metaphase as cells progressed through mitosis, with condensed, but unaligned, chromosomes. This mitotic disruption in both cases resulted in improper cellular division leading to multiple outcomes: multinucleation, apoptosis, and cellular senescence.

Factor quinolinone inhibitors alter cell morphology and motility by destabilizing interphase microtubules

“…In addition, cell blebbing was observed in the presence of FQI1 (see Supplementary Movies 1 and 2 ), which is consistent with activation of the myosin II pathway via the observed phosphorylation of MLC2. Since FQI1 can cause cells to undergo mitotic arrest 17 , 18 , which also results in cells “rounding-up”, although not as quickly, the cell cycle stage of cells exhibiting this compact phenotype was examined by cellular DNA profiling. By this analysis, there was no apparent shift in cell cycle phases upon such a short incubation with FQI1 (Fig.…”

“…To test whether the novel phenotypic effects in FH-B and RPE cells are related to the previously described activities of FQI1 15 , 17 , we tested whether these phenotypes were replicated using a recently developed FQI family member, FQI2-34. FQI2-34 is more potent than FQI1, exhibiting 17-fold higher efficacy than FQI1 in inhibiting proliferation of FH-B cells (see Supplementary Fig.…”

“…This underscores the need to understand not only how cancer cells respond to FQIs, but also how normal cells respond. Exploring the mechanism behind FQI1’s anti-proliferative activity in cancer cells revealed that FQI1 causes mitotic arrest with disrupted spindles and condensed, but unaligned chromosomes in vivo, as does siRNA specifically targeting LSF 17 , 18 , which can result in apoptosis or senescence 15 , 17 . Because FQI1 treatment disrupts mitotic microtubules and LSF also directly and specifically interacts with α-tubulin 19 , we hypothesized that FQI1 would also impact microtubule-associated processes beyond mitosis.…”

TFCP2 Overcomes Senescence by Cooperating With SREBP2 to Activate Cholesterol Synthesis in Pancreatic Cancer

“…Regulation of TFCP2 expression in liver cancer and expression of treatment-resistant and senescence-related gene have already been reported ( 27 ). Treatment of Hela cells with TFCP2 inhibitor FQI1 leads to polynuclear, apoptotic, and senescent Hela cells ( 22 ). These existing findings, plus our finding, further demonstrate the regulatory effect of TFCP2 on cell senescence.…”

“…It has been reported that treatment of glioma cells with HMGCR inhibitor statins can induce cell senescence ( 21 ). Currently, studies show that TFCP2 knockdown can induce the mitotic delay and senescence of Hela cells ( 22 ), but the mechanism is unclear.…”