TCEA3 Impairs Cancer Growth and Functions as a Tumor Suppressor

Abstract

TFIIS is a transcription elongation factor that has the ability to bind to RNA Polymerase II and enable the elongation of RNA polymerase II through transcriptional arrest sites. TCEA3 is one of three mammalian isoforms of TFIIS. It consists of three domains: the conserved N‐ and C‐terminal domains of TFIIS separated by a variable sequence linker region that is 50 amino acids longer than the other isoforms. TCEA3 is expressed in skeletal muscle and normal ovarian epithelial cells and known to be down regulated in ovarian cancer cells. Rhabdomyosarcoma (RMS) is a malignancy that arises from skeletal muscle precursors. It is the most common type of childhood soft tissue sarcoma and the fourth most common pediatric solid tumor. There are two major subtypes of rhabdomyosarcoma; embryonal (ERMS) and alveolar (ARMS).The aim of this study was to investigate the expression and function of TCEA3 in RMS and normal muscle.We found that TCEA3 is highly expressed in skeletal muscle cells at the level of both RNA and protein as assayed by quantitative real time PCR and western blots, respectively. Depletion of TCEA3 using shRNA constructs showed that TCEA3 promotes both cell proliferation and differentiation into muscle fibers. We found that the expression level of TCEA3 was significantly downregulated in RMS cells at the protein and RNA level when compared to normal skeletal muscle cells. RD and RH30 cell lines, representing both ERMS and ARMS respectively, were transfected with a TCEA3 expression plasmid to create stable cell lines and TCEA3 expression was confirmed at the level of both protein and RNA. Stably expressing TCEA3 cell lines were monitored for cell growth by a proliferation assay and we found that TCEA3 overexpression inhibited the proliferation rate of RMS cells. The scratch wound method was used to assay for cell mobility and migration in RMS cells and TCEA3 strongly impaired the migration of cells compared to RMS cells with a vector control. We also assayed for anchorage independent growth using a soft agar assay and these assays showed high contact inhibition and almost no clonal cell growth in TCEA3 overexpressing cells. A TUNEL assay was used to reveal apoptosis in TCEA3 overexpressed cells. Western blots assays using antibodies against the caspase protein family were used to investigate the apoptosis pathway induced by TCEA3 and we found that apoptotic signals were induced to a much higher degree in cells stably overexpressing TCEA3 compared to controls. Additionally, we found that cells stably expressing TCEA3 were highly sensitized to chemotherapeutic drugs compared to control. We next want to understand the effect of TCEA3 in additional cancer types by overexpressing TCEA3 in HeLa (cervical), PC3 (prostate), MDA‐MD‐231 (breast), and MCF7 (breast). For each cell line, the overexpression of TCEA3 was confirmed by RNA and protein analysis and we have found that the apoptotic marker, caspase 3, is activated in each of these cell lines. Taken together, our findings show that TCEA3 functions as a tumor suppressor in RMS and many additional cancers. TCEA3 impairs proliferation and induces apoptotic pathways in cancer cells. The present study suggests that activation of TCEA3 might serve as a novel therapeutic target in cancer.Support or Funding InformationNIH/NIAMS AR068622This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.