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.
Highlights
Senescence is a fundamental biological process implicated in various pathologies, including cancer
Komseli et al BMC Genomics (2018) 19:37 (Continued from previous page). This 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 Cell division cycle 6 (CDC6), implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics
The underlying principle for generating a CDC6-based non-malignant human epithelial model to study oncogene-induced senescence According to a carcinogenesis model we have proposed, activated oncogenes disrupt normal DNA replication provoking replication stress that in turn triggers the DNA damage response (DDR) pathway, promoting genomic instability and cancer development [36]
Summary
Senescence is a fundamental biological process implicated in various pathologies, including cancer. 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. Proteins represented the main workhorse of the cell This view has dramatically changed over the last years, as regulatory RNAs emerged as a versatile component of the molecular machinery interacting with both DNA and proteins “shaping” gene expression and protein function, respectively [1, 2]. Mounting evidence shows that this bidirectional interplay plays a vital role in a variety of cellular responses, such as senescence; the latter involved in the pathogenesis of various diseases, including cancer. Tumor development is mediated through a continuous “cross talk” between intra-cellular molecular pathways and intercellularly among cancer cells and the surrounding adjacent stroma [5]. In situ detection of biomolecules rises as a crucial necessity to interrogate the mechanistic aspects of carcinogenesis
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