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Biomarker; Hormone receptor-positive; Breast cancer Biomarcador; Cáncer de mama; Receptor hormonal positivo Biomarcador; Càncer de mama; Receptor hormonal positiu CDK4/6 inhibitors benefit a minority of patients who receive them in the breast cancer adjuvant setting. p27Kip1 is a protein that inhibits CDK/Cyclin complexes. We hypothesized that single-nucleotide polymorphisms that impaired p27Kip1 function could render patients refractory to endocrine therapy but responsive to CDK4/6 inhibitors, narrowing the patient subpopulation that requires CDK4/6 inhibitors. We found that the p27Kip1 V109G single-nucleotide polymorphism is homozygous in approximately 15% of hormone-positive breast cancer patients. Polymorphic patients experience rapid failure in response to endocrine monotherapy compared with wild-type or heterozygous patients in the first-line metastatic setting (progression-free survival: 92 vs 485 days, P

Background: CDK4/6 inhibitors benefit a limited percentage of hormone receptor-positive breast cancer (HRPBC) patients in the adjuvant setting: according to the MonarchE study, from all patients treated with the endocrine plus CDK4/6 inhibitor combination, 84% were adequately treated with endocrine therapy alone, ~5% experienced benefit from the combination, and 11% were not rescued from relapse by abemaciclib. Given the side effects and the cost, biomarkers to guide treatment decisions in this setting are appealing. We found that the p27Kip1 V109G SNP was enriched in HRPBC patients experiencing relapse despite endocrine treatment. p27Kip1 binds to cyclins and CDKs, restraining cells from cycling by inhibiting the formation of CDK/cyclin complexes and their kinase activity, resulting in less phosphorylation of Rb. A functionally impaired p27Kip1 could render tumor cells insensitive to endocrine therapy, while being rescued by CDK4/6 inhibitors. Thus, this SNP could narrow down the patient population that requires adjuvant CDK4/6 inhibitors. Methods: Isogenic HRPBC cell lines, wild-type or polymorphic homozygous for the p27Kip1 V109G SNP were generated with CRISPR-Cas9. Cell cycle and cell viability were assessed with BRDU incorporation and colony assays. Immunoprecipitation coupled with western blot (WB) was used to measure the formation of CDK/Cyclin complexes; Rb phosphorylation was assessed by WB. An in vitro kinase assay was set up to measure the CDK4 activity of p27Kip1/CDK/Cyclin complexes. Patients (n=115) with metastatic, HRPBC receiving endocrine monotherapy or in combination with CDK4/6 inhibitors were genotyped for the p27Kip1 V109G SNP, and PFS by genotype and therapy compared with the Kaplan-Meier method. All statistical tests were two-sided. Results: three isogenic polymorphic clones were generated from the wild-type T47-D hormone-positive cell line. The three clones were resistant to hormonal deprivation compared to wild-type cells. The relative plating efficiency (RPE) in the colony assays of the polymorphic clones exposed to hormonal deprivation compared to that of deprived T47-D cells was 550% (clone C1), 165% (clone E1) and 100% (Clone F5); P< 0.005. The three clones were also resistant to fulvestrant (Fulv) (300%, 170% and 180%, respectively); P< 0.005. Cell cycle (positive BRDU cells) decreased ~3 fold in wild type cells (18% to 6.5%) when exposed to hormonal deprivation or Fulv, but remained unaltered in the polymorphic clones. However, when palbociclib was added to hormonal deprivation or Fulv, the effects in RPE increased and were similar in polymorphic clones and parental cells (>5% RPE compared to vehicle, both in polymorphic and wild-type cells). The p27Kip1 V109G SNP was found in homozygosity in ~15% of metastatic HRPBC patients. When patients received endocrine monotherapy in the first-line setting, polymorphic patients experience rapid failure (N=51) compared to wild-type/heterozygous patients (4.3 vs. 21.1 months; P < 0.0001). However, when patients received hormonal plus CDK4/6 inhibitors, the differences disappeared (18.3 vs. 24.3 months; P=0.85). Mechanistically, we observed that the formation of CDK2/CyclinA, CDK2/CyclinE and CDK4/Cyclin D1 complexes was >200% higher in polymorphic than in wild-type cells (P< 0.05). Regarding CDK4 kinase activity of p27Kip1/CDK/Cyclin complexes, as opposed to wild-type p27Kip1, p27Kip1 V109G was unable to suppress the kinase activity of CDK4 in presence of Fulv or hormonal deprivation. However, palbociclib was able to fully suppress CDK4 kinase activity regardless of the p27Kip1 genotype. Conclusion: Germline p27Kip1 genotyping can constitute a tool for treatment selection: whereas wild-type patients are adequately treated with endocrine monotherapy, polymorphic patients are inherently resistant, but are rescued with CDK4/6 inhibitors. Thus, hormonal+CDK4/6 inhibitor combos could be reserved for the polymorphic patients. Citation Format: Miguel Quintela-Fandino, Silvana Mouron, Maria J. Bueno, Manuel Muñoz, Raul Torres, Sandra Rodriguez, Rodrigo Sánchez-Bayona, Luis Manso, Jorge Silva, Marcos Malumbres. p27Kip1 V109G single-nucleotide polymorphism (SNP): pinpointing the hormone-receptor positive breast cancer subpopulation that requires CDK4/6 inhibitors in addition to endocrine therapy. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-28.

Mitochondria have a major impact on virtually all processes linked to oncogenesis. Thus, mitochondrial metabolism inhibition has emerged as a promising anticancer strategy. In this review, we discuss the anticancer potential of mitochondrial inhibitors, with particular focus on metformin, in the context of more effective, targeted therapeutic modalities, and diagnostic strategies for cancer patients. Metformin has gained interest as an antitumor agent. However, promising results have not been translated into remarkable advances in the clinical practice. Recent findings emphasize the need of providing a metabolic context in which mitochondrial inhibitors may elicit its anticancerous effects. In addition, mitochondria are critical regulators in orchestrating immune responses. Thus, the immunomodulatory effect of mitochondrial inhibitors should also be taken into account to optimize its clinical use. Targeting mitochondrial metabolic network represents a promising therapeutic strategy in cancer. However, there is a need to define the metabolic context in which mitochondrial inhibitors are more effective, as well as how the cross-talk between many immunological functions and mitochondrial functionality may be exploited for a therapeutic benefit in cancer patients.

Preclinical evidence indicates the potential of targeting mitochondrial respiration as a therapeutic strategy. We previously demonstrated that mitochondrial inhibitors' efficacy was restricted to a metabolic context in which mitochondrial respiration was the predominant energy source, a situation achievable by inducing vascular normalization/hypoxia correction with antiangiogenics. Using molecular imaging, we showed how the same antiangiogenic agent may display different normalizing properties in patients with the same tumor type. This is of key importance, since patients experiencing normalization seem to get more benefit from standard chemotherapy combinations, and also could be eligible for combination with antimitochondrial agents. This scenario emphasizes the need for monitoring vascular normalization in order to optimize the use of antiangiogenics. We have also proposed a method to evaluate anti-mitochondrial agents' pharmacodynamics; despite promising accuracy in animal studies the clinical results were inconclusive, highlighting the need for research in this field. Regarding patients that respond to antiangiogenics increasing vessel abnormality, in this case an immunosuppressive tumor microenvironment is generated. Whether anti-mitochondrial agents can positively modulate the activity of T effector cell subpopulations remains an area of active research. Our research sheds light on the importance of refining the use of antiangiogenics, highlighting the relevance of tracing vascular normalization as a potential biomarker for antiangiogenics to assist patient-tailored medicine and exploring the role of mitochondrial inhibitors in the context of vascular normalization and correction of hypoxia. Sí

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations, Background FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear. Methods FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied. Results FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P, MQF is a recipient of the following grants: AES - PI16/00354 and AES – PI19/00454 funded by the ISCIII (Instituto de Salud Carlos III, Ministry of Science and Innovation, Spain) and co-funded by the European Regional Development Fund (ERDF); 2017/BMD3733 - Call for Coordinated Research Groups from Madrid Region - Madrid Regional Government - ERDF funds. RC and MJB are co-recipients of an AES – PI17/01865 grant funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF). RC is a recipient of funding PIE15/00068, PI20/01458, and PT17/0017/0007 by the ISCIII. This study was partially funded by Bayer Inc. CRIS Contra el Cancer Foundation contributed with a generous donation to this study

Full differentiation potential along with self‐renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already‐established murine and human PSCs. Short exposure to miR‐203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human–mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR‐203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.

SUMMARYFull differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression ofde novoDNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies., M.Q.F. is a recipient of the following grants: FIS PI13/00430 and FIS PI16/00354 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF) and AECC Scientific Foundation (Beca de Retorno 2010). R.C. is a recipient of the following grants: FIS PI11/00832 and FIS PI14/00726 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF), II14/00009 and PIE15/00068 from the Ministerio de Sanidad, Spain. N.S.C. is a recipient of an NIH grant (5R35CA197532). O.Y.T. is a recipient of the grants BFU2014-57466 from the Ministerio de Economía y Competitividad (MINECO). J.P.B. is funded by MINECO (SAF2016-78114-R), Instituto de Salud Carlos III (RD12/0043/0021), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA, and Fundación Ramón Areces. This study was partially supported by the generous donations from Fundación CRIS Contra el Cáncer and AVON Spain. We thank Drs. Erwin Wagner and Nabil Djouder for their critical review of the paper.

One proposed mechanism through which antiangiogenics exert their effect in epithelial malignancies is by improving the status of the aberrant vascular network and secondarily facilitating the delivery of concurrently administrated cytotoxic agents. During this process, known as vascular normalization, the oxygenation of the tumor is usually improved. Many mechanisms of resistance have been proposed to evade the action of this drug class elicited through this mechanism of action. However, a less explored mechanism of action is vascular choking, as increased hypoxia is thought to be associated with the inevitable progression of certain tumor-promotion features. Here we review the available evidence regarding decreased blood flow as a mechanism of action of antiangiogenics at the preclinical and clinical level. Similar to vascular normalization, there are also escape mechanisms against chronic hypoxia generated by treatment with antiangiogenics. Among other compensatory responses, chronic hypoxia is related with the upregulation of lipidic anabolism. Therefore, we focus on how fatty acid synthase, a key player in this response, can be targeted to delay acquired resistance against antiangiogenics, including experimental data from our group. This effect seems to be specific to those cases in which the antiangiogenic treatment induces a hypoxic response, but not in models where the antiangiogenic agent induced normalizing effects. Whether antiangiogenics induce vascular normalization or a hypoxic environment seems to be tractable with a noninvasive PET-tracer: 18F-fluoromisonidazole PET.

Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors downregulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic, and phosphoproteomic study revealed that this metabolic switch is mediated by downregulation of HIF1α and AKT and upregulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, leading to metabolic synthetic lethality. Our study uncovers mechanistic insights in the process of tumor resistance to TKIs and may have clinical applicability. M.Q.-F. is a recipient of the following grants: FIS PI10/0288 and FIS PI13/00430 from the Ministry of Health (Spain), 2010-BECA-RETORNO from theAECC Scientific Foundation, and donations from the Rosae Foundation andAvon Espan ̃a S.A.U. I.Z. is a recipient of La Caixa-CNIO PhD 2011 fellowship.M.Q.-F. received research funds from Boehringer-Ingelheim and Novartis. Sí

Inappropriate activation of the GLI/hedgehog (GLI/Hh) signalling occurs in several human cancers, including haematological neoplasms. However, little is known about its relevance in precursor T-cell lymphoblastic lymphomas (T-LBL) development. Moreover, the mechanisms whereby GLI/Hh signalling is activated in haematological malignancies are not fully clear. Here, we show that the gene Smoothened (SMO), the only non-redundant gene of this pathway, is up-regulated in mouse and human T-LBL. Interestingly, down-regulation of micro-RNAs mmu-miR-30a and mmu-miR-141 as well as hsa-miR-193b clearly contributes to enhance the expression of this gene in mouse and human lymphomas and, subsequently, to activate the GLI/Hh signalling. Activation of the GLI/Hh signalling in T-LBL promotes cell survival and proliferation, since inhibition of the pathway using short-hairpin-RNA-mediated SMO knockdown, or cyclopamine as a specific antagonist, significantly reduces these cellular processes. These findings suggest that sustained SMO up-regulation may contribute to T-LBL development and advocate the use of specific SMO inhibitors or microRNAs-based therapies as an attractive possibility to treat an important subset of T-LBL.

Cdc14 is a dual-specific phosphatase with relevant functions during mitotic exit in yeast. The relevance of vertebrate Cdc14 phosphatases is not well understood due to the presence of two paralogs, Cdc14A and Cdc14B, and their dispensability for cell cycle progression. Here, we report that overexpression of mammalian Cdc14B, but not Cdc14A, leads to dramatic changes in morphology and malignant transformation of normal murine fibroblasts. Cdc14B disrupts the cytoskeletal F-actin organization with loss of actin stress fibers and vinculin adhesions in a phosphatase-dependent manner. These morphological changes are associated to cellular transformation, as Cdc14B-overexpressing cells display anchorage-independent growth and are able to form tumors in vivo. These alterations are similar to those induced by Ras oncogenes ,and both Cdc14B and H-RasV12 lead to similar changes in the transcriptional profile of transformed cells. Pharmacologic inhibition of the Ras-Mek pathway rescues these defects. These data suggest that Cdc14B, but not Cdc14A, is one of the few phosphatases that display oncogenic activity in mammals and point to the Ras-MAP kinase pathway as a major effector pathway during oncogenic transformation by Cdc14B.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis and proliferation. Several miRNAs display defective expression patterns in human tumors with the consequent alteration of target oncogene or tumor suppressor genes. Many of these miRNAs modulate the major proliferation pathways through direct interaction with critical regulators such as RAS, PI3K/ PTEN or ABL, as well as members of the retinoblastoma pathway, Cyclin-CDK complexes or cell cycle inhibitors of the INK4 or Cip/ Kip families. A complex interplay between miRNAs and MYC or E2F family members also exists to modulate cell cycle-dependent transcription during normal or tumoral proliferation. The ability of miRNAs to modulate these proliferation pathways may have relevant implications not only in physiological or developmental processes but also in tumor progression or cancer therapy.

A new residue analytical method for the confirmation and quantification of avermectin residues in food is described in this article. This method allows a fast analysis for the determination of avermectin residues, abamectin (ABM), ivermectin (IVM), emamectin benzoate (EMA) and doramectin (DOR) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Separation was performed using a short column of 1.8 microm particle size. The hybrid quadrupole/linear ion trap (QqQ(LIT)) system via the linearly accelerating (LINAC) high-pressure collision cell, allows the MS detection in multiple reaction monitoring (MRM) mode operating in fast scan acquisition times. The effect of reduced dwell times on mass spectral quality and sensitivity is evaluated in this study. For quantitative purposes, the influence of dwell time on S/N ratio and peak area was observed. ABM, IVM, EMA and DOR show an increased trend of peak area and S/N ratio, when dwell times are of 50 ms against 10-20 ms, suited when the number of compounds to be analyzed is higher. The sensitivity achieved by using the LC-MS/MS system is enough for the confirmation of avermectin residues in the selected commodities (salmon muscle and pepper) at trace concentration levels (sub-microg/kg and microg/kg) and therefore a sample pre-concentration step was not necessary. The instrumental limits of quantification (ILQ) are in the range of 0.15-5 ppb. Samples were extracted by solid-liquid extraction (SLE) procedure using acetonitrile, and cleaned-up using alumina. The average recoveries obtained were acceptable (80-95%). The calibration curves were linear over the working range from ILQs to 500 microg/kg. For the quantitative analysis, matrix-matched calibration and dilution of SLE extracts was proven as reliable alternative to compensate matrix-effects and for its feasible application in routine analysis.

The mammalian genome contains several hundred microRNAs that regulate gene expression through modulation of target mRNAs. Here, we report a fragile chromosomal region lost in specific hematopoietic malignancies. This 7 Mb region encodes about 12% of all genomic microRNAs, including miR-203. This microRNA is additionally hypermethylated in several hematopoietic tumors, including chronic myelogenous leukemias and some acute lymphoblastic leukemias. A putative miR-203 target, ABL1, is specifically activated in these hematopoietic malignancies in some cases as a BCR-ABL1 fusion protein (Philadelphia chromosome). Re-expression of miR-203 reduces ABL1 and BCR-ABL1 fusion protein levels and inhibits tumor cell proliferation in an ABL1-dependent manner. Thus, miR-203 functions as a tumor suppressor, and re-expression of this microRNA might have therapeutic benefits in specific hematopoietic malignancies.

Background : Given our preclinical data showing synergy between dovitinib and paclitaxel in preclinical models we conducted this phase I trial aiming to define the recommended phase II-dose (RP2D) on the basis of toxicity and pharmacodynamic criteria while searching for genetic variants that could sensitize patients to the regimen under study. Patients and methods A 3+3 escalation schedule was adopted. Seriated FGF23 and dovitinib and paclitaxel pharmacokinetic profiles were determined along a single-agent dovitinib “priming-phase” followed by a dovitinib + paclitaxel combination phase. RECIST 1.1 criteria and NCI CTCAE V.4.0 were used. In fresh pre-treatment tumor biopsy samples, FGFR1, 2 and 3 amplifications were revealed by FISH probes; 32 missense variants were genotyped in tumors and peripheral blood mononuclear cells with Taqman genotyping assays (FGFR1-3 and RET). Constructs encoding for wild-type and variant genes associated with clinical benefit were transfected into HEK-293 cells for preclinical experiments checking constitutive activation and dovitinib sensitivity of the variants. Results twelve patients were recruited in three dose-levels. At level 1B (200 mg dovitinib 5-days-on/2-days-off plus 60 mg/m 2-week of paclitaxel) more than 50% FGF23 upregulation was observed and no dose-limiting-toxicities (DLTs) occurred. The most frequent toxicities were asthenia, neutropenia, nausea/vomiting and transaminitis. Two patients with progressive disease prior to trial inclusion achieved prolonged disease stabilization. Both had the germline variant G2071A in the RET gene, which led to constitutive activation of the protein product and Y-905 phosphorylation, both in transfectants and in patients with the alteration. This variant was sensitive to dovitinib; in addition both patients experienced progression upon medication withdrawal. Conclusions Level 1B was the RP2D as it provided adequate pharmacodynamic exposure to dovitinib. The G2071A germline variant act as a genetic modifier that renders different tumors sensitive to dovitinib.

Many mammalian transcripts contain target sites for multiple miRNAs although it is not clear to what extent miRNAs may coordinately regulate single genes. We have mapped the interactions between downregulated miRNAs and overexpressed target protein-coding genes in murine and human lymphomas. Myc, one of the hallmark oncogenes in these lymphomas, stands out as the upregulated gene with the highest number of genetic interactions with downregulated miRNAs in mouse lymphomas. The regulation of Myc by several of these miRNAs is confirmed by cellular and reporter assays. The same approach indentifies MYC and multiple Myc targets as a preferential target of downregulated miRNAs in human Burkitt's lymphoma, a pathology characterized by translocated MYC oncogenes. These results indicate that several miRNAs must be coordinately downregulated in order to enhance critical oncogenes such as Myc. Some of these Myc-targeting miRNAs are repressed by Myc, suggesting that these tumors are a consequence of the unbalanced activity of Myc versus miRNAs., This work was supported by grants from the Association for International Cancer Research (AICR #08-0188), Fundación Ramón Areces, Fundación Mutua Madrileña Automovilista (to M.M.), and the MICINN (SAF2009-11426 to J.F.P.; SAF2007-64571 to I.P.C.; and SAF2009-07973 to M.M.). The CBM/UAM group is supported by the Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER) network from the MICINN. The Bioinformatics Unit of the CNIO is supported by the Spanish National Bioinformatics Institute (Genoma España). The Cell Division and Cancer Group of the CNIO is supported by the OncoCycle Programme (Comunidad de Madrid, S-BIO-0283-2006), the OncoBIO Consolider-Ingenio 2010 Programme (MICINN, CSD2007-00017) and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° [HEALTH-F5-2010-241548]

Transcription of microRNAs (miRNAs) is thought to be regulated similarly to that of protein-coding genes. However, how miRNAs are regulated during the cell division cycle is not well understood. We have analyzed the transcription profiles of miRNAs in response to mitogenic stimulation in primary fibroblasts. About 33% of the miRNAs expressed in these cells are induced upon exit from quiescence. Many of these miRNAs are specifically induced by E2F1 or E2F3 during the G(1)/S transition and are repressed in E2F1/3-knockout cells. At least four miRNA clusters, let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25, are direct targets of E2F1 and E2F3 during G(1)/S and are repressed in E2F1/3-null cells. Interestingly, these miRNAs do not contribute to E2F-dependent entry into S phase but rather inhibit the G(1)/S transition by targeting multiple cell cycle regulators and E2F targets. In fact, E2F1 expression results in a significant increase in S-phase entry and DNA damage in the absence of these microRNAs. Thus, E2F-induced miRNAs contribute to limiting the cellular responses to E2F activation, thus preventing replicative stress. Given the known function of E2F of inducing other oncogenic miRNAs, control of miRNAs by E2F is likely to play multiple roles in cell proliferation and in proliferative diseases such as cancer.

In this work the risk posed to seawater organisms, predators and humans is assessed, as a consequence of exposure to 12 organic micro-pollutants, namely metronidazole, trimethoprim, erythromycin, simazine, flumequine, carbaryl, atrazine, diuron, terbutryn, irgarol, diphenyl sulphone (DPS) and 2-thiocyanomethylthiobenzothiazole (TCMTB). The risk assessment study is based on a 1-year monitoring study at a Spanish marine fish farm, involving passive sampling techniques. The results showed that the risk threshold for irgarol concerning seawater organisms is exceeded. On the other hand, the risk to predators and especially humans through consumption of fish is very low, due to the low bioconcentration potential of the substances assessed.