Stress-induced transcription factors and their role in RNA disruption in ovarian cancer cells

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.