Stress-induced transcription factors and their role in RNA disruption in ovarian cancer cells
Date
2021-05-20
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Abstract
When cancer patients respond positively to chemotherapy treatment, their tumour
ribosomal RNA (rRNA) is degraded, which has been termed RNA Disruption. The RNA
disruption assay (RDA) has been developed to quantify RNA disruption as an RNA disruption
index (RDI), which is the mass ratio of degraded to intact rRNAs. The underlying mechanism(s)
for RNA disruption remain unclear. RNA disruption occurs in vivo and in vitro in response to
various cellular stressors, including many structurally and mechanistically distinct chemotherapy
drugs. Therefore, there must be a common pathway or mechanism occurring in each of these
cases. Transcription factors are good upstream targets to begin elucidating a potential pathway or
mechanism for RNA disruption, since they are major regulators of gene expression. The goal of
this study was to: (i) identify transcription factors that are activated in response to treatment with
agents known to induce RNA disruption, (ii) create enriched cytoplasmic and nuclear extracts
from treated and untreated A2780 ovarian tumour cells, (iii) assess the relationship between
stress-induced RNA disruption and activation/altered localization of stress-induced transcription
factors in relation to RNA disruption. A2780 cells were treated with docetaxel or left untreated
as a control for 8 hours, and nuclear extracts from these cells were prepared. The activation of a
wide variety of known transcription factors in these extracts were analyzed using transcription
factor activation profiling plate arrays. The activation was measured based on their ability to
bind specific DNA sequences resulting in luminescence. Using a variety of criteria ranging from
statistical significance to relevance to stress in the literature, a few transcription factors of
interest were chosen: TFEB, TFE3, Nrf2, and YY1. Cytoplasmic and nuclear extracts were then
made from A2780 cells after treatment with doxorubicin, docetaxel, thapsigargin, and starvation
for 0, 2, 16, or 24 hours. The amount of RNA disruption was then assessed by RDA 72 hours
after treatment with the same stressors. Treatment with doxorubicin, docetaxel, thapsigargin, and
starvation resulted in significant change in RDI compared to control cells. Nrf2 was found
localized in the nucleus with or without treatment. Thus, Nrf2 appeared to be constitutively
activated in A2780 ovarian cancer cells, perhaps due to its role in cell survival. This study
corroborates the results of another study (Investigating the Relationship Between the Activation
of Transcription Factor Nrf-2 and Ribosomal RNA Degradation Upon Exposure to Various
Cellular Stressors, Carly Zulich, 2020) and eliminates Nrf2 as a potential transcription factor
associated with the activation of RNA disruption. Contrary to expected findings, there appear to
be higher levels of TFEB and TFE3 in both the cytoplasm and nucleus of the untreated and
DMSO vehicle control than cells treated with chemotherapy or other cellular stressors. Future
studies could investigate the potential of other transcription factors as targets for elucidating the
mechanism(s) for RNA disruption.
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Keywords
Transcription factor, Ovarian cancer, YY1, TFEB, TFE3, Nrf2, RNA disruption, Docetaxel, Doxorubicin, Thapsigargin, Starvation