FOXE1 Gene Dosage Affects Thyroid Cancer Histology and Differentiation In Vivo.

The transcription factor Forkhead box E1 (FOXE1) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele (FOXE1+/-) were crossed with a BRAFV600E-inducible cancer model to develop thyroid cancer in either a FOXE1+/+ or FOXE1+/- genetic background. In FOXE1+/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer.

Insight into Nephrocan Function in Mouse Endoderm Patterning.

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5-11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn-/- mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

Exploring the Molecular Crosstalk between Pancreatic Bud and Mesenchyme in Embryogenesis: Novel Signals Involved.

Pancreatic organogenesis is a multistep process that requires the cooperation of several signaling pathways. In this context, the role of pancreatic mesenchyme is important to define the epithelium development; nevertheless, the precise space-temporal signaling activation still needs to be clarified. This study reports a dissection of the pancreatic embryogenesis, highlighting the molecular network surrounding the epithelium-mesenchyme interaction. To investigate this crosstalk, pancreatic epithelium and surrounding mesenchyme, at embryonic day 10.5, were collected through laser capture microdissection (LCM) and characterized based on their global gene expression. We performed a bioinformatic analysis to hypothesize crosstalk interactions, validating the most promising genes and verifying the precise localization of their expression in the compartments, by RNA in situ hybridization (ISH). Our analyses pointed out also the c-Met gene, a very well-known factor involved in stimulating motility, morphogenesis, and organ regeneration. We also highlighted the potential crosstalk between Versican (Vcan) and Syndecan4 (Sdc4) since these genes are involved in pancreatic tissue repair, strengthening the concept that the same signaling pathways required during pancreatic embryogenesis are also involved in tissue repair. This finding leads to novel strategies for obtaining functional pancreatic stem cells for cell replacement therapies.

Mice lacking microRNAs in Pax8-expressing cells develop hypothyroidism and end-stage renal failure.

BACKGROUND: Non-coding RNAs have gained increasing attention during the last decade. The first large group of non-coding RNAs to be characterized systematically starting at the beginning of the 21st century were small oligonucleotides--the so-called microRNAs (miRNAs). By now we have learnt that microRNAs are indispensable for most biological processes including organogenesis and maintenance of organ structure and function. The role of microRNAs has been studied extensively in the development of a number of organs, so far most studies focussed on e.g. the heart or the brain whilst the role of microRNAs in the development and maintenance of complex epithelial organs is less well understood. Furthermore most analyses regarding microRNA function in epithelial organs employed conditional knockout mouse models of the RNAse III Dicer to abrogate microRNA biogenesis. However, there is increasing evidence for Dicer to have multiple functions independent from microRNA maturation. Therefore Dicer independent models are needed to gain further insight into the complex biology of miRNA dependent processes. RESULTS: Here we analyze the contribution of microRNA-dependent transcriptional control in Pax8-expressing epithelial cells. Pax8 is a transcription factor that is crucial to the development of epithelial organs. The miRNA machinery was disrupted by crossing conditional DiGeorge syndrome critical region 8 (Dgcr8) fl/fl mice to Pax8Cre mice. The Dgcr8/Drosha complex processes pri-miRNAs in the nucleus before they are exported as pre-miRNAs for further maturation by Dicer in the cytoplasm. Dgcr8 fl/fl; Pax8Cre+ knockout mice died prematurely, developed massive hypothyroidism and end stage renal disease due to a loss of miRNAs in Pax8 expressing tissue. CONCLUSION: Pax8Cre-mediated conditional loss of DiGeorge syndrome critical region 8 (Dgcr8), an essential component of the nuclear machinery that is required for microRNA biogenesis, resulted in severe hypothyroidism, massively reduced body weight and ultimately led to renal failure and death of the animals. These data provide further insight into the importance of miRNAs in organ homeostasis using a Dicer independent model.

Loss of CDKL5 impairs survival and dendritic growth of newborn neurons by altering AKT/GSK-3ß signaling.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in a neurodevelopmental disorder characterized by early-onset intractable seizures, severe developmental delay, intellectual disability, and Rett's syndrome-like features. Since the physiological functions of CDKL5 still need to be elucidated, in the current study we took advantage of a new Cdkl5 knockout (KO) mouse model in order to shed light on the role of this gene in brain development. We mainly focused on the hippocampal dentate gyrus, a region that largely develops postnatally and plays a key role in learning and memory. Looking at the process of neurogenesis, we found a higher proliferation rate of neural precursors in Cdkl5 KO mice in comparison with wild type mice. However, there was an increase in apoptotic cell death of postmitotic granule neuron precursors, with a reduction in total number of granule cells. Looking at dendritic development, we found that in Cdkl5 KO mice the newly-generated granule cells exhibited a severe dendritic hypotrophy. In parallel, these neurodevelopmental defects were associated with impairment of hippocampus-dependent memory. Looking at the mechanisms whereby CDKL5 exerts its functions, we identified a central role of the AKT/GSK-3ß signaling pathway. Overall our findings highlight a critical role of CDKL5 in the fundamental processes of brain development, namely neuronal precursor proliferation, survival and maturation. This evidence lays the basis for a better understanding of the neurological phenotype in patients carrying mutations in the CDKL5 gene.

Gene expression profiling at early organogenesis reveals both common and diverse mechanisms in foregut patterning.

The thyroid and lungs originate as neighboring bud shaped outgrowths from the midline of the embryonic foregut. When and how organ specific programs regulate development into structures of distinct shapes, positions and functions is incompletely understood. To characterize, at least in part, the genetic basis of these events, we have employed laser capture microdissection and microarray analysis to define gene expression in the mouse thyroid and lung primordia at E10.5. By comparing the transcriptome of each bud to that of the whole embryo as well as to each other, we broadly describe the genes that are preferentially expressed in each developing organ as well as those with an enriched expression common to both. The results thus obtained provide a valuable resource for further analysis of genes previously unrecognized to participate in thyroid and lung morphogenesis and to discover organ specific as well as common developmental mechanisms. As an initial step in this direction we describe a regulatory pathway involving the anti-apoptotic gene Bcl2 that controls cell survival in early thyroid development.

In vivo role of different domains and of phosphorylation in the transcription factor Nkx2-1.

BACKGROUND: The transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation. RESULTS: Mouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1. CONCLUSIONS: The pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors.

BAZ1B is a candidate gene responsible for hypothyroidism in Williams syndrome.

Williams syndrome (WS) is a rare neurodevelopmental disorder associated to a hemizygous deletion of 28 genes located on chromosome 7q11.23. WS affected subjects frequently suffer from several endocrine abnormalities including hypothyroidism due to defects in thyroid morphology. To date, several genes involved in thyroid dysgenesis have been identified, nonetheless, none of them is located in the 7q11.23 region. Thus, the hypothyroidism-linked molecular features in WS are not yet known. In this study we focused on one of the WS deleted gene, BAZ1B, demonstrating that its downregulation in thyroid cells leads to cell viability and survival decrement. Taking together, our results show that BAZ1B could be the mainly responsible for thyroid defects observed in some of WS patients and that these alterations are activated by PTEN-mediated mechanisms.

Double knock-out of Hmga1 and Hipk2 genes causes perinatal death associated to respiratory distress and thyroid abnormalities in mice.

The serine-threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) modulates important cellular functions during development, acting as a signal integrator of a wide variety of stress signals, and as a regulator of transcription factors and cofactors. We have previously demonstrated that HIPK2 binds and phosphorylates High-Mobility Group A1 (HMGA1), an architectural chromatinic protein ubiquitously expressed in embryonic tissues, decreasing its binding affinity to DNA. To better define the functional role of HIPK2 and HMGA1 interaction in vivo, we generated mice in which both genes are disrupted. About 50% of these Hmga1/Hipk2 double knock-out (DKO) mice die within 12 h of life (P1) for respiratory failure. The DKO mice present an altered lung morphology, likely owing to a drastic reduction in the expression of surfactant proteins, that are required for lung development. Consistently, we report that both HMGA1 and HIPK2 proteins positively regulate the transcriptional activity of the genes encoding the surfactant proteins. Moreover, these mice display an altered expression of thyroid differentiation markers, reasonably because of a drastic reduction in the expression of the thyroid-specific transcription factors PAX8 and FOXE1, which we demonstrate here to be positively regulated by HMGA1 and HIPK2. Therefore, these data indicate a critical role of HIPK2/HMGA1 cooperation in lung and thyroid development and function, suggesting the potential involvement of their impairment in the pathogenesis of human lung and thyroid diseases.

A ceRNA Circuitry Involving the Long Noncoding RNA Klhl14-AS, Pax8, and Bcl2 Drives Thyroid Carcinogenesis.

Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology. SIGNIFICANCE: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.

TRPV2 Calcium Channel Gene Expression and Outcomes in Gastric Cancer Patients: A Clinically Relevant Association.

Gastric cancer (GC) is characterized by poor efficacy and the modest clinical impact of current therapies. Apoptosis evasion represents a causative factor for treatment failure in GC as in other cancers. Since intracellular calcium homeostasis regulation has been found to be associated with apoptosis resistance, the aberrant expression of intracellular calcium regulator genes (CaRGs) could have a prognostic value in GC patients. We analyzed the association of the expression levels of 98 CaRGs with prognosis by the log-rank test in a collection of 1524 GC samples from four gene expression profiling datasets. We also evaluated differential gene expression in comparison with normal stomach tissue, and then we crossed results with tissue microarrays from the Human Protein Atlas. Among the investigated CaRGs, patients with high levels of TRPV2 expression were characterized by a shorter overall survival. TRPV2 expression was found to increase according to tumor stage. Both mRNA and protein levels were significantly higher in tumor than normal stomach samples. TRPV2 was also associated with poor prognosis in the Lauren's intestinal type GC and in patients treated with adjuvant therapy. Overall, we highlighted the relevance of TRPV2 not only as a prognostic biomarker but also as a potential therapeutic target to improve GC treatment efficacy.

Gastric Normal Adjacent Mucosa Versus Healthy and Cancer Tissues: Distinctive Transcriptomic Profiles and Biological Features.

Gastric cancer (GC) is a leading cause of cancer-related deaths in the world. Molecular heterogeneity is a major determinant for the clinical outcomes and an exhaustive tumor classification is currently missing. Histologically normal tissue adjacent to the tumor (NAT) is commonly used as a control in cancer studies, nevertheless a recently published paper described the unique characteristics of the NAT in several tumor types. Little is known about the global gene expression profile of gastric NAT (gNAT) which could be an effective tool for a more realistic definition of GC molecular signature. Here, we integrated data of 512 samples from the Genotype-Tissue Expression project (GETx) and The Cancer Genome Atlas (TCGA) to analyze the transcriptome of healthy gastric tissues, gNAT, and GC samples. We validated TCGA-GETx data mining through inHouse gNAT and GC expression dataset. Differential gene expression together with pathway enrichment analyses, indeed, led to different results when using the gNAT or the healthy tissue as control. Based on our analyses, gNAT showed a peculiar gene signature and biological features, like the estrogen receptor pathways activation, suggesting a molecular behavior partially different from both healthy and GC tissues. Therefore, using gNAT as healthy control tissue in the characterization of tumor associated biological processes and pathways could lead to suboptimal results.

Rescue of prepulse inhibition deficit and brain mitochondrial dysfunction by pharmacological stimulation of the central serotonin receptor 7 in a mouse model of CDKL5 Deficiency Disorder.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause CDKL5 Deficiency Disorder (CDD), a rare neurodevelopmental syndrome characterized by severe behavioural and physiological symptoms. No cure is available for CDD. CDKL5 is a kinase that is abundantly expressed in the brain and plays a critical role in neurodevelopmental processes, such as neuronal morphogenesis and plasticity. This study provides the first characterization of the neurobehavioural phenotype of 1 year old Cdkl5-null mice and demonstrates that stimulation of the serotonin receptor 7 (5-HT7R) with the agonist molecule LP-211 (0.25 mg/kg once/day for 7 days) partially rescues the abnormal phenotype and brain molecular alterations in Cdkl5-null male mice. In particular, LP-211 treatment completely normalizes the prepulse inhibition defects observed in Cdkl5-null mice and, at a molecular level, restores the abnormal cortical phosphorylation of rpS6, a downstream target of mTOR and S6 kinase, which plays a direct role in regulating protein synthesis. Moreover, we demonstrate for the first time that mitochondria show prominent functional abnormalities in Cdkl5-null mouse brains that can be restored by pharmacological stimulation of brain 5-HT7R.

NFE2-Related Transcription Factor 2 Coordinates Antioxidant Defense with Thyroglobulin Production and Iodination in the Thyroid Gland.

BACKGROUND: The thyroid gland has a special relationship with oxidative stress. While generation of oxidative substances is part of normal iodide metabolism during thyroid hormone synthesis, the gland must also defend itself against excessive oxidation in order to maintain normal function. Antioxidant and detoxification enzymes aid thyroid cells to maintain homeostasis by ameliorating oxidative insults, including during exposure to excess iodide, but the factors that coordinate their expression with the cellular redox status are not known. The antioxidant response system comprising the ubiquitously expressed NFE2-related transcription factor 2 (Nrf2) and its redox-sensitive cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) defends tissues against oxidative stress, thereby protecting against pathologies that relate to DNA, protein, and/or lipid oxidative damage. Thus, it was hypothesized that Nrf2 should also have important roles in maintaining thyroid homeostasis. METHODS: Ubiquitous and thyroid-specific male C57BL6J Nrf2 knockout (Nrf2-KO) mice were studied. Plasma and thyroids were harvested for evaluation of thyroid function tests by radioimmunoassays and of gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Nrf2-KO and Keap1-KO clones of the PCCL3 rat thyroid follicular cell line were generated using CRISPR/Cas9 technology and were used for gene and protein expression studies. Software-predicted Nrf2 binding sites on the thyroglobulin enhancer were validated by site-directed in vitro mutagenesis and chromatin immunoprecipitation. RESULTS: The study shows that Nrf2 mediates antioxidant transcriptional responses in thyroid cells and protects the thyroid from oxidation induced by iodide overload. Surprisingly, it was also found that Nrf2 has a dramatic impact on both the basal abundance and the thyrotropin-inducible intrathyroidal abundance of thyroglobulin (Tg), the precursor protein of thyroid hormones. This effect is mediated by cell-autonomous regulation of Tg gene expression by Nrf2 via its direct binding to two evolutionarily conserved antioxidant response elements in an upstream enhancer. Yet, despite upregulating Tg levels, Nrf2 limits Tg iodination both under basal conditions and in response to excess iodide. CONCLUSIONS: Nrf2 exerts pleiotropic roles in the thyroid gland to couple cell stress defense mechanisms to iodide metabolism and the thyroid hormone synthesis machinery, both under basal conditions and in response to excess iodide.

Conditional inactivation of the E-cadherin gene in thyroid follicular cells affects gland development but does not impair junction formation.

We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.

Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells.

MicroRNA-125a-5p (miR-125a) is a vertebrate homolog of lin-4, the first discovered microRNA, and plays a fundamental role in embryo development by downregulating Lin-28 protein. MiR-125a is also expressed in differentiated cells where it generally acts as an antiproliferative factor by targeting membrane receptors or intracellular transductors of mitogenic signals. MiR-125a expression is downregulated in several tumors, including hepatocellular carcinoma (HCC) where it targets sirtuin-7, matrix metalloproteinase-11, VEGF-A, Zbtb7a, and c-Raf. In this study, we have isolated the transcription promoter of human miR-125a and characterized its activity in HCC cells. It is a TATA-less Pol II promoter provided with an initiator element and a downstream promoter element, located 3939 bp upstream the genomic sequence of the miRNA. The activity of the promoter is increased by the transcription factor NF-kB, a master regulator of inflammatory response, and miR-125a itself was found to strengthen this activation through inhibition of TNFAIP3, a negative regulator of NF-kB. This finding contributes to explain the increased levels of miR-125a observed in the liver of patients with chronic hepatitis B.

Tissue- and Cell Type-Specific Expression of the Long Noncoding RNA Klhl14-AS in Mouse.

lncRNAs are acquiring increasing relevance as regulators in a wide spectrum of biological processes. The extreme heterogeneity in the mechanisms of action of these molecules, however, makes them very difficult to study, especially regarding their molecular function. A novel lncRNA has been recently identified as the most enriched transcript in mouse developing thyroid. Due to its genomic localization antisense to the protein-encoding Klhl14 gene, we named it Klhl14-AS. In this paper, we highlight that mouse Klhl14-AS produces at least five splicing variants, some of which have not been previously described. Klhl14-AS is expressed with a peculiar pattern, characterized by diverse relative abundance of its isoforms in different mouse tissues. We examine the whole expression level of Klhl14-AS in a panel of adult mouse tissues, showing that it is expressed in the thyroid, lung, kidney, testis, ovary, brain, and spleen, although at different levels. In situ hybridization analysis reveals that, in the context of each organ, Klhl14-AS shows a cell type-specific expression. Interestingly, databases report a similar expression profile for human Klhl14-AS. Our observations suggest that this lncRNA could play cell type-specific roles in several organs and pave the way for functional characterization of this gene in appropriate biological contexts.

Lack of Cdkl5 Disrupts the Organization of Excitatory and Inhibitory Synapses and Parvalbumin Interneurons in the Primary Visual Cortex.

Cyclin-dependent kinase-like 5 (CDKL5) mutations are found in severe neurodevelopmental disorders, including the Hanefeld variant of Rett syndrome (RTT; CDKL5 disorder). CDKL5 loss-of-function murine models recapitulate pathological signs of the human disease, such as visual attention deficits and reduced visual acuity. Here we investigated the cellular and synaptic substrates of visual defects by studying the organization of the primary visual cortex (V1) of Cdkl5-/y mice. We found a severe reduction of c-Fos expression in V1 of Cdkl5-/y mutants, suggesting circuit hypoactivity. Glutamatergic presynaptic structures were increased, but postsynaptic PSD-95 and Homer were significantly downregulated in CDKL5 mutants. Interneurons expressing parvalbumin, but not other types of interneuron, had a higher density in mutant V1, and were hyperconnected with pyramidal neurons. Finally, the developmental trajectory of pavalbumin-containing cells was also affected in Cdkl5-/y mice, as revealed by fainter appearance perineuronal nets at the closure of the critical period (CP). The present data reveal an overall disruption of V1 cellular and synaptic organization that may cause a shift in the excitation/inhibition balance likely to underlie the visual deficits characteristic of CDKL5 disorder. Moreover, ablation of CDKL5 is likely to tamper with the mechanisms underlying experience-dependent refinement of cortical circuits during the CP of development.

"Stockpile" of Slight Transcriptomic Changes Determines the Indirect Genotoxicity of Low-Dose BPA in Thyroid Cells.

Epidemiological and experimental data highlighted the thyroid-disrupting activity of bisphenol A (BPA). Although pivotal to identify the mechanisms of toxicity, direct low-dose BPA effects on thyrocytes have not been assessed. Here, we report the results of microarray experiments revealing that the transcriptome reacts dynamically to low-dose BPA exposure, adapting the changes in gene expression to the exposure duration. The response involves many genes, enriching specific pathways and biological functions mainly cell death/proliferation or DNA repair. Their expression is only slightly altered but, since they enrich specific pathways, this results in major effects as shown here for transcripts involved in the DNA repair pathway. Indeed, even though no phenotypic changes are induced by the treatment, we show that the exposure to BPA impairs the cell response to further stressors. We experimentally verify that prolonged exposure to low doses of BPA results in a delayed response to UV-C-induced DNA damage, due to impairment of p21-Tp53 axis, with the BPA-treated cells more prone to cell death and DNA damage accumulation. The present findings shed light on a possible mechanism by which BPA, not able to directly cause genetic damage at environmental dose, may exert an indirect genotoxic activity.