BACKGROUND: Senescence 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. METHODS: In 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. RESULTS: 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 CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. CONCLUSIONS: Consequently, 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.

Biomedical Research Foundation of the Academy of Athens 4 Soranou Ephessiou St GR 11527 Athens Greece

CRUK MRC Institute for Radiation Oncology Department of Oncology University of Oxford Oxford OX3 7DQ UK

Department of Biology School of Medicine National and Kapodistrian University of Athens 75 Mikras Asias St GR 11527 Athens Greece Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas GR 70013 Heraklion Crete Greece

Department of Biostatistics and Translational Medicine Medical University of Lodz 15 Mazowiecka St 92 215 Lodz Poland

Department of Biostatistics and Translational Medicine Medical University of Lodz 15 Mazowiecka St 92 215 Lodz Poland Department of Radiation Oncology Dana Farber Cancer Institute 450 Brookline Ave Boston MA 02215 USA

Department of Cell Biology and Biophysics Faculty of Biology National and Kapodistrian University of Athens GR 15784 Athens Greece

Department of Immunology and Microbiology University of Copenhagen Blegdamsvej 3c DK 2200 Copenhagen Denmark

Department of Radiation Oncology Dana Farber Cancer Institute 450 Brookline Ave Boston MA 02215 USA Harvard Medical School 25 Shattuck St Boston MA 02115 USA

Genome Integrity Unit Danish Cancer Society Research Centre Strandboulevarden 49 DK 2100 Copenhagen Denmark Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Hněvotínská 1333 5 779 00 Olomouc Czech Republic Department of Medical Biochemistry and Biophysics Karolinska Institute Science for Life Laboratory Division of Translational Medicine and Chemical Biology SE 171 77 Stockholm Sweden

Molecular Carcinogenesis Group Department of Histology and Embryology School of Medicine National and Kapodistrian University of Athens 75 Mikras Asias St GR 11527 Athens Greece

Molecular Carcinogenesis Group Department of Histology and Embryology School of Medicine National and Kapodistrian University of Athens 75 Mikras Asias St GR 11527 Athens Greece Biomedical Research Foundation of the Academy of Athens 4 Soranou Ephessiou St GR 11527 Athens Greece Faculty of Biology Medicine and Health University of Manchester Manchester Academic Health Science Centre Wilmslow Road Manchester M20 4QL UK

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