Flavopiridol causes cell cycle inhibition and demonstrates anti-cancer activity in anaplastic thyroid cancer models

Abstract

Anaplastic thyroid cancer (ATC) is a rare, but nearly uniformly fatal disease that is typically resistant to chemotherapy and radiation. Alternative strategies to target this cancer at a molecular level are necessary in order to improve dismal outcomes for ATC patients. We examined the effects of flavopiridol, a CDK inhibitor, in a panel of ATC cell lines. When cell lines were treated over a ten-point concentration range, CAL62, KMH2 and BHT-101 cell lines had a sub micromolar half-maximal inhibitory concentration, while no effect was seen in the non-cancerous cell line IMR-90. Flavopiridol treatment resulted in decreased levels of the cell cycle proteins CDK9 and MCL1, and induced cell cycle arrest. Flavopiridol also decreased the in vitro ability of ATC cells to form colonies and impeded migration using a transwell migration assay. In vivo, flavopiridol decreased tumor weight and tumor volume over time in a patient-derived xenograft model of ATC. Given the observed in vitro and in vivo activity, flavopiridol warrants further investigation for treatment of ATC.

Fig 1. Flavopiridol inhibits cell growth, clonogenic capabilities and migration in ATC cell lines.

(A) Flavopiridol was tested against 3 ATC cell lines, with 2 biological replicates per cell line. Cells were treated with flavopiridol for 48hrs and plate readings were completed after incubation with Presto Blue. Mean IC₅₀ values are shown ± standard deviation (indicated by the error bar). (B) Dose-response curve demonstrating the sensitivity of the lung fibroblast cell line, IMR-90, over 10 concentrations (0.06 to 32μM) of flavopiridol with 2 biological replicates. Mean values for each concentration are shown ± standard deviation as indicated by the error bars. (C) Clonogenic assays comparing untreated and treated (60 and 125nM flavopiridol) ATC cell lines (CAL62 and BHT-101) after 7 days. Colonies were counted and graphed for 3 representative fields from each well (3 technical replicates per cell line as indicated by error bars showing the standard deviation). The number of colonies were compared to the untreated control for each cell line. (D) Cell migration was quantified using fluorometric analysis. Cells (CAL62, BHT-101 and KMH2) were seeded into the top chamber containing serum-free media at a density of 5x10⁴ cells per chamber with either 60 or 125 nM flavopiridol (2 biological replicates per concentration tested as indicated by the error bars showing the standard deviation). Each chamber was then placed into a well containing media with serum. Cells which migrated through to serum-containing media after 24 hours were quantified using fluorometric detection as described using the standard protocol provided by the manufacturer. Fluorescence readings were completed using a microplate reader. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns = not significant, unpaired Student’s, two-tail t-test.

Fig 2. Flavopiridol treatment results in cell cycle arrest in vitro.

(A) Immunoblot of CAL62 and BHT-101 cells treated with flavopiridol for 24hrs. Cells were collected after drug treatment and whole cell lysates were prepared with 20 μg total protein loaded per well. (B) KMH2, CAL62 and BHT-101 cells were exposed to the vehicle control or flavopiridol (125nM) for 24hrs, with 3 biological replicates per cell line, prior to BrdU incorporation and labeling with propidium iodide. A minimum of 10,000 events was counted and the proportion of cells present in each phase of the cell cycle ± standard deviation is shown.

Fig 3. siRNA knockdown of CDK9 impairs ATC cell proliferation.

(A) Immunoblot (20 μg total protein) of CDK9 expression in KMH2 and CAL62 cell lines showing the siCT relative to the siCDK9 knockdown. (B) Changes in cell proliferation with siRNA-mediated knockdown with Lipofectamine® RNAiMAX Reagent of CDK9 (siCDK9; 10 μM) relative to the scrambled control (siCT; 10 μM) in KMH2 and CAL62 cells (3 technical replicates as indicated by the error bars showing the standard deviation). * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns = not significant, unpaired Student’s, two-tail t-test. (C) Dose-response curves of KMH2 and CAL62 cell lines comparing the siCT and siCDK9 cell lines treated with flavopiridol (0.06–4.00 μM) with their respective IC₅₀ values (3 technical replicates per dose as indicated by the error bars showing the standard deviation).

Fig 4. Flavopiridol inhibits tumor growth in a PDX model of ATC.

(A) Primary tumor was obtained from a 70-year old female (with informed consent) with diagnosed ATC stage T4N1M1. Five xenografts (per treatment) were generated and randomized to either daily flavopiridol (7.5 mg/kg/day) by intraperitoneal injection or a vehicle control (water). Mice were treated with flavopiridol for a period of 17 days (five days on, two days break in between) with the treatment beginning at 14 days post injection until controls reached endpoint at day 35. Standard deviations are indicated by error bars at each timepoint. (B) Mice were evaluated for tumor weight (g) comparing the vehicle control group to the flavopiridol treated group at endpoint. Standard deviations indicated by error bars at each timepoint. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns = not significant, unpaired Student’s, two-tail t-test. (C) Representative IHC sections of the vehicle and flavopiridol-treated specimens demonstrating H&E staining in the PDX model of ATC in addition to protein expression of CDK9 and MCL1. (D) Representative IHC sections of the vehicle and flavopiridol-treated specimens demonstrating Ki67 protein expression in the PDX model of ATC. Standard deviations indicated by error bars.