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

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.

Influence of the microenvironment on modulation of the host response by typhoid toxin.

Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

Immunohisto(cyto)chemistry: an old time classic tool driving modern oncological therapies.

In the era of precision medicine immunohistochemistry (IHC) and immunocytochemistry (ICC) share some of the highlights in personalized treatment. Survival data obtained from clinical trials shape the cut-offs and IHC scoring that serve as recommendations for patient selection both for targeted and conventional therapies. Assessment of Estrogen and Progesterone Receptors along with HER2 status has been among the first approved immunostaining assays revolutionizing breast cancer treatment. Similarly, ALK positivity predicts the efficacy of ALK inhibitors in patients with non-small cell lung cancer (NSCLC). In recent years, Programmed Death Ligand 1 (PD-L1) IHC assays have been approved as companion or complimentary diagnostic tools predicting the response to checkpoint inhibitors. Anti-PD-L1 and anti-PD-1 monoclonal antibodies have inaugurated a new period in the treatment of advanced cancers, but the path to approval of these biomarkers is filled with immunohistochemical challenges. The latter brings to the fore the significance of molecular pathology as a hub between basic and clinical research. Besides, novel markers are translated into routine practice, suggesting that we are at the beginning of a new exciting period. Unraveling the molecular mechanisms involved in cellular homeostasis unfolds biomarkers with greater specificity and sensitivity. The introduction of GL13 (SenTraGor®) for the detection of senescent cells in archival material, the implementation of key players of stress response pathways and the development of compounds detecting common mutant P53 isoforms in dictating oncological treatments are paradigms for precision oncology.