The therapeutic potential of RNA Polymerase I transcription inhibitor, CX-5461, in uterine leiomyosarcoma

Abstract

Background: Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1.

Methods: SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis.

Results: Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription.

Conclusions: SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.

Keywords: CX-5461; RNA Polymerase I transcription inhibitor; SK-UT-1; Uterine leiomyosarcoma.

Fig. 1

Basal expression of key proteins on uterine leiomyosarcoma SK-UT-1 cells. Basal protein abundance of (a) p53, (b) c-Myc and (c) RB1 were evaluated by western blot analysis. Human fibroblasts (HF) were used as a control cell line (note: intervening lanes with non-relevant cell lines have been removed from the image). M is abbreviated for marker lane. Band intensities were normalized to β-actin. Statistical analysis was conducted using a Mann Whitney U test (Error bars represent mean ± SEM of n = 3–4 biological replicates)

Fig. 2

Anti-proliferation effects of CX-5461 on SK-UT-1 cells. (a) SK-UT-1 cells were treated with CX-5461, at the concentrations indicated, or vehicle (50 mM NaH₂PO₄ (pH4.5)) and assessed for proliferation via phase contrast microscopy and IncuCyte^® ZOOM Live Cell Imaging System (Error bars represent mean ± SEM of n = 3 biological triplicates). A dose–response curve (c), generated from data obtained in (b), with the 50% growth inhibitory concentration (GIC₅₀) of CX-5461 calculated using non-linear regression analysis

Fig. 3

Inhibitory effects of CX-5461 on rDNA transcription in SK-UT-1 cells (a) The effects of treatment with CX-5461 at varying concentrations, and vehicle control, for 1 h on rDNA transcription rate in SK-UT-1 cells, determined via qPCR with the result of the external transcribed spacer (ETS) normalised to the house-keeping gene, β2 microglobulin (β2M). Statistically significance differences between ETS-2 and β2M were determined using two-way ANOVA (Error bars represent mean ± SEM of n = 3) *p <0.05, **p <0.01, ***p <0.001. (b) The 50% rDNA transcription inhibitory concentration (tIC₅₀) of CX-5461 was calculated from (a) using non-linear regression analysis (Error bars represent mean ± SEM of n = 3 biological triplicates)

Fig. 4

Effects of CX-5461 on cell cycle progression in SK-UT-1 cells (a) Flow cytometry gating strategy for cell cycle analysis using BrdU and PI staining at 0 and 72 h following treatment with 30 nM CX-5461, or vehicle, is shown. (b) SK-UT-1 cells were treated with the GIC₅₀ CX-5461 dose (30 nM) for the times indicated, and stained with a anti BrdU specific antibody and PI for cell cycle analysis as in (a) (bars represent mean ± SD of n = 2 biological duplicates). *V-72 h: Vehicle for 72 h