Search

FRA1 (FOSL1) is a transcription factor and a member of the activator protein-1 superfamily. FRA1 is expressed in most tissues at low levels, and its expression is robustly induced in response to extracellular signals, leading to downstream cellular processes. However, abnormal FRA1 overexpression has been reported in various pathological states, including tumor progression and inflammation. To date, the molecular effects of FRA1 overexpression are still not understood. Therefore, the aim of this study was to investigate the transcriptional and functional effects of FRA1 overexpression using the CGL1 human hybrid cell line. FRA1-overexpressing CGL1 cells were generated using stably integrated CRISPR-mediated transcriptional activation, resulting in a 2–3 fold increase in FRA1 mRNA and protein levels. RNA-sequencing identified 298 differentially expressed genes with FRA1 overexpression. Gene ontology analysis showed numerous molecular networks enriched with FRA1 overexpression, including transcription-factor binding, regulation of the extracellular matrix and adhesion, and a variety of signaling processes, including protein kinase activity and chemokine signaling. In addition, cell functional assays demonstrated reduced cell adherence to fibronectin and collagen with FRA1 overexpression and altered cell cycle progression. Taken together, this study unravels the transcriptional response mediated by FRA1 overexpression and establishes the role of FRA1 in adhesion and cell cycle progression. [ABSTRACT FROM AUTHOR]

Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity. [ABSTRACT FROM AUTHOR]

Simple Summary: For the first time, we demonstrated that the significant decrease in p63/p73 expression together with the absence of functional p53 could underlie an increase in the fraction of polyploid cells, transformation rates, and the glycolytic NAD(P)H production in multifraction X-ray radiation exposure (MFR)-surviving cancer cells, providing conditions for radioresistance associated with epithelial–mesenchymal transition (EMT)-like process activation. During radiation therapy (RT), the treatment dose, fractionation, and dose limits for organs at risk (OARs) do not change between patients and are still prescribed mainly based on the Tumor, Node, Metastasis (TNM) stage, performance status, and comorbidities, taking no account of the tumor biology. Our data once again emphasize that non-small cell lung cancer (NSCLC) therapy approaches should become more personalized according to RT regimen, tumor histology, and molecular status of critical proteins. Radiotherapy is a primary treatment modality for patients with unresectable non-small cell lung cancer (NSCLC). Tumor heterogeneity still poses the central question of cancer radioresistance, whether the presence of a particular cell population inside a tumor undergoing a selective outgrowth during radio- and chemotherapy give rise to metastasis and tumor recurrence. In this study, we examined the impact of two different multifraction X-ray radiation exposure (MFR) regimens, fraction dose escalation (FDE) in the split course and the conventional hypofractionation (HF), on the phenotypic and molecular signatures of four MFR-surviving NSCLC cell sublines derived from parental A549 (p53 wild-type) and H1299 (p53-null) cells, namely A549FR/A549HR, H1299FR/H1299HR cells. We demonstrate that sublines surviving different MFR regimens in a total dose of 60 Gy significantly diverge in their molecular traits related to irradiation regimen and p53 status. The observed changes regarding radiosensitivity, transformation, proliferation, metabolic activity, partial epithelial-to-mesenchymal transition (EMT) program activation and 1D confined migratory behavior (wound healing). For the first time, we demonstrated that MFR exposure led to the significant decrease in the expression of p63 and p73, the p53-family members, in p53null cells, which correlated with the increase in cell polyploidy. We could not find significant differences in FRA1 expression between parental cells and their sublines that survived after any MFR regimen regardless of p53 status. In our study, the FDE regimen probably causes partial EMT program activation in MFR-survived NSCLC cells through either Vimentin upregulation in p53null or an aberrant N-cadherin upregulation in p53wt cells. The HF regimen likely less influences the EMT activation irrespectively of the p53 status of MFR-survived NSCLC cells. Our data highlight that both MFR regimens caused overall higher cell transformation of p53null H1299FR and H1299HR cells than their parental H1299 cells. Moreover, our results indicate that the FDE regimen raised the radioresistance and transformation of MFR-surviving NSCLC cells irrespectively of their p53 status, though the HF regimen demonstrated a similar effect on p53null NSCLC cells only. Our data once again emphasize that NSCLC therapy approaches should become more personalized according to radiation therapy (RT) regimen, tumor histology, and molecular status of critical proteins. [ABSTRACT FROM AUTHOR]