Abstract
SummaryBackground. 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.
Highlights
Uterine leiomyosarcoma is a subtype of soft tissue sarcoma and arises in the smooth muscle of the uterus
A marker of aggressive disease, and increased rDNA transcription have been reported in uterine leiomyosarcoma [13]. These studies suggest that inhibition of Polymerase I (Pol I) transcription may be an effective new therapeutic strategy for the treatment of uterine leiomyosarcoma. To investigate this hypothesis, (i) we analysed the genetic profile of the human uterine leiomyosarcoma cell line, SK-UT-1, to identify key driver mutations linked to Pol I activity and to establish the cell line as a representative in vitro model of this rare cancer type, developing a robust and suitable model system for testing novel therapies, and (ii), we evaluated the effects of the Pol I transcription inhibitor, CX-5461 [14,15,16], against this cell line
RNA (500 ng) was DNase treated, incubated in T100TM Thermocycler (Bio-Rad, CA, USA) using the settings detailed in Supplementary Table 3. cDNA synthesis was performed using SuperScriptTM IV Reverse Transcriptase (Invitrogen, Carlsbad, CA), Hexamer Random Primers (Promega, Madison, WI), and dNTPs (Invitrogen, Carlsbad, CA) with incubation in a T100TM Thermocycler
Summary
Uterine leiomyosarcoma is a subtype of soft tissue sarcoma and arises in the smooth muscle of the uterus. Surgical resection and chemotherapy are used as the mainstay for uterine leiomyosarcoma management, the curative effects are limited [3,4,5]. Loss of heterozygosity at loci containing the tumour suppressor genes, tumour protein 53 (TP53), retinoblastoma 1 (RB1), phosphatase and tensin homolog (PTEN) and cyclindependent kinase inhibitor protein (CDKN2A), in uterine leiomyosarcoma confers chemotherapy resistance [6, 7]. There is an urgent need, to identify new targets and effective therapeutic strategies to improve the outcomes of this aggressive disease. One of the classic hallmarks, is associated with elevated rates of protein synthesis and ribosome biogenesis (RiBi) [8].
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