A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence

Abstract

BackgroundSenescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now.MethodsIn the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs.ResultsThis experimental setting has three advantages that are presented and discussed: i) it covers a “gap” in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics.ConclusionsConsequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4375-1) contains supplementary material, which is available to authorized users.

DNA replication stress: oncogenes in the spotlight

“…Interestingly, R-loop formation caused by CDC6 overexpression is preferentially observed within the nucleoli, consistent with the fact that CDC6 is important for transcriptional regulation of the highly repetitive heterochromatic ribosomal DNA (rDNA) (Huang et al, 2016). As a result of replication stress, CDC6 upregulation causes a number of structural and numerical chromosome aberrations in different models, with the majority of breakpoints located at CFS (Liontos et al, 2007;Sideridou et al, 2011;Komseli et al, 2018). As discussed before, it is important to consider that CDC6 upregulation may be a consequence of RAS or Cyclin E1 oncogene activation, leading to DNA rereplication, DDR activation, and genomic instability (Mailand and Diffley, 2005;Di Micco et al, 2006).…”

“…Besides increased origin firing and DNA rereplication, CDC6-induced replication stress can also occur through collision between replication and transcription machineries and formation of R-loops (Komseli et al, 2018). Interestingly, R-loop formation caused by CDC6 overexpression is preferentially observed within the nucleoli, consistent with the fact that CDC6 is important for transcriptional regulation of the highly repetitive heterochromatic ribosomal DNA (rDNA) (Huang et al, 2016).…”

Ageing, Cellular Senescence and Neurodegenerative Disease

“…SIRT6 belongs to the sirtuin protein family, whose function has been linked to longevity [ 68 ]. Micro-RNAs, a subclass of regulatory, non-coding RNAs that participate in regulation of cellular senescence may also be epigenetically modified [ 69 ]. Chromatic alterations such as senescence-associated heterochromatin formation (SAHF) may accompany cellular senescence in some settings with deactivation of proliferation-related genes [ 70 , 71 , 72 , 73 ].…”

Integrating the DNA damage and protein stress responses during cancer development and treatment

“…rRNA constitutes 80% of the total cellular RNA, making rDNA the highest‐transcribed locus of the genome. Obviously, the propensity for replication–transcription collisions and R‐loop formation is increased in rDNA loci, especially under conditions of oncogene‐induced RS . As a ‘precautionary measure’, the RFB* (supplementary material, Figure S4A), which is an intergenic rDNA site, is recognized as a site coordinating replication and transcription , whereas heterochromatin associated with the rDNA clusters acts as a ‘buffer zone’ against genotoxic conditions .…”