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Abstract Aspirin and its active metabolite salicylate have emerged as promising agents for the chemoprevention of colorectal cancer (CRC). Moreover, aspirin suppresses the progression of established CRCs. However, the underlying molecular mechanisms are not completely understood. Here we found that salicylate induces the expression of the miR-34a and miR-34b/c genes, which encode tumor suppressive microRNAs, in a p53-independent manner. Salicylate activated AMPK, thereby activating NRF2, which directly induced miR-34a/b/c expression via ARE motifs. In addition, salicylate suppressed c-MYC, a known repressor of NRF2-mediated transactivation, via activating AMPK. The suppression of c-MYC by salicylate was necessary for NRF2-mediated activation of miR-34a/b/c. Inactivation of miR-34a/b/c largely abrogated the inhibitory effects of salicylate on migration, invasion and metastasis formation by CRC cells. In the future, aspirin and its derivates may be used therapeutically to activate miR-34a and miR-34b/c in tumors that have lost p53.

Members of the microRNA-34/miR-34 family are induced by the p53 tumor suppressor and themselves possess tumor suppressive properties, as they inhibit the translation of mRNAs that encode proteins involved in processes, such as proliferation, migration, invasion, and metastasis. Here we performed a comprehensive integrative meta-analysis of multiple computational and experimental miR-34 related datasets and developed tools to identify and characterize novel miR-34 targets. A miR-34 target probability score was generated for every mRNA to estimate the likelihood of representing a miR-34 target. Experimentally validated miR-34 targets were strongly enriched among mRNAs with the highest scores providing a proof of principle for our analysis. We integrated the results from the meta-analysis in a user-friendly METAmiR34TARGET website (www.metamir34target.com/) that allows to graphically represent the meta-analysis results for every mRNA. Moreover, the website harbors a screen function, which allows to select multiple miR-34-related criteria/analyses and cut-off values to facilitate the stringent and comprehensive prediction of relevant miR-34 targets in expression data obtained from cell lines and tumors/tissues. Furthermore, information on more than 200 miR-34 target mRNAs, that have been experimentally validated so far, has been integrated in the web-tool. The website and datasets provided here should facilitate further investigation into the mechanisms of tumor suppression by the p53/miR-34 connection and identification of potential cancer drug targets.

Abstract Background AP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells. Methods A CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, β-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APC min/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity. Results Inactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APC min/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner. Conclusions In summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation. Graphical abstract

Rational and long-term planning and sustainable mineral resources management is of strategic importance in Europe’s efforts to secure the self-supply of mineral raw materials. European mineral data is organized and accessible within the pan-European Minerals Intelligence Network. Most EU countries are part of this network, while the West Balkan region presents a gap in this regard. A common West Balkan mineral register needs to be established in order to close the gap and bring the area closer to the EU market. Including the region into the network would provide new opportunities to local industry and improve mineral management at the national and regional level. In this context, the Geological Survey of Slovenia is working in cooperation with numerous partners and stakeholders in different projects within the framework of European initiatives and programs. We collected and properly organized relevant data on minerals in the region according to the INSPIRE Directive. This paper describes the creation of the West Balkan Mineral Register of primary raw materials, and its content.

Abnormalities in bone development in humans and non-humans can lead to impaired physical and psychological health; however, evidence is lacking regarding the role of individual psychosocial factors in the development of poor bone conditions. Addressing this lack of knowledge, we used low-productive laying hens (n = 93) and assessed behavioral responses to an open-field test [at 17, 18, 29, 33 weeks of age (wa)], an aerial predator test (at 39 wa), and a social reinstatement test (at 42 wa). Bone condition was assessed using a palpation technique on five occasions (at 16, 29, 33, 45, 58 wa), with half of the hens experiencing damage (deviations, fractures, or both) at 29 wa and all hens by 58 wa. Corticosterone (CORT) concentration in feathers (at 16, 33, 58 wa) and body weight (at 23, 47, 58 wa) were also investigated. We hypothesized that lighter birds (at 23 wa) with higher CORT (at 16 wa) and open field-induced fear collected before the onset of lay (at 17 and 18 wa) are associated with a worse bone condition when in lay. We also hypothesized that those birds with more damage at the peak of laying (at 29 wa) would be lighter at 47 and 58 wa and more fearful by showing higher open field-induced (at 29 and 33 wa) and predator-induced fear responses, however, acting less socially toward conspecifics. These hens were also expected to have higher CORT (at 33 and 58 wa). Our results show no association between open-field fear level and fear behavior, CORT concentration, or body weight on the one hand (all measured before starting to lay) and bone damage at 29 wa on the other. When in lay, bone damage was associated with more pecking and less crossing zones when faced with an open-field situation at 29 wa and improved sociality at 42 wa. This study provides the first evidence of a relationship of bone health with fear, sociality, and stress response. When in poor bone condition, our hens had enhanced psychological stress measured by fear behavior reactivity but not physiological stress measured as feather CORT concentration.

Abstract Epithelial-mesenchymal transition (EMT) plays an important role in tumor invasion and metastasis. A comprehensive, bioinformatics analysis of CCLE and TCGA datasets of seven tumor types allowed us to identify a novel pan-cancer EMT-associated gene expression signature consisting of 16 epithelial and 4 mesenchymal state-associated mRNAs. Among the identified epithelial cell state-associated factors, down-regulation of the RBM47 (RNA binding motif protein 47) mRNA displayed the most significant association with metastasis and poor survival in multiple cohorts of colorectal cancer (CRC) patients. Moreover, decreased RBM47 protein expression was associated with metastasis in a cohort of primary CRCs. RBM47 was directly suppressed during EMT induced by IL6-activated STAT3 or ectopic SNAIL and SLUG expression via conserved binding motifs of these factors within the RBM47 promoter. Moreover, RNAi-mediated down-regulation of RBM47 in CRC lines resulted in increased cell migration, invasion and metastases formation. As demonstrated by the example of RBM47, the EMT-associated signature characterized here allows to identify biomarkers for predicting clinical outcome of CRC and presumably other cancer entities. In addition, our functional analysis of RBM47 shows that the down-regulation of RBM47 during CRC progression may promote EMT and metastasis.

The second pillar of the “Raw materials initiative − meeting our critical needs for growth and jobs in Europe” praises the intention of safeguarding mineral deposits from sterilisation. In the near future, each Member State may protect access to mineral deposits of public importance (MDoPI) in accordance with its national legislation framework. Every Member State should identify its own mineral deposits of public importance to be incorporated into spatial plans. A list of suggested MDoPIs has been created for Slovenia in terms of safeguarding the access to deposits and sustainable mineral supply in the future. The survey of potential MDoPIs is described, and a map of suggested mineral areas is created. 30 areas/deposits have been selected and designated on the national level. On the local/regional level, up to 50 extraction sites were suggested as aggregates supply centres (quarries of crushed stones and gravel pits). Less than 0.5 % of the national territory might be dedicated to future mineral safeguarding. This paper presents the proposed list of potential safeguarded areas of MDoPIs in Slovenia and the MDoPIs map. The list is not closed or fially accomplished, but it is a dynamic system. It will be adopted referring to new geological research results as well as social needs. Nineteen countries are participating in the MINATURA2020 project (Horizon2020 program), and seven of them were selected as case studies (United Kingdom, Italy, Sweden, Portugal, Hungary, Poland, and Slovenia) regarding the differences in the territorial size, types of mineral endowment, and national mineral policies. The project provides an opportunity to engage with the issue and begin the safeguarding process on the national level.