Cellular and micro-environmental responses influencing the antitumor activity of all-trans retinoic acid in breast cancer.

All-trans retinoic acid (ATRA) is the most relevant and functionally active metabolite of Vitamin-A. From a therapeutic standpoint, ATRA is the first example of pharmacological agent exerting its anti-tumor activity via a cell differentiating action. In the clinics, ATRA is used in the treatment of Acute Promyelocytic Leukemia, a rare form of myeloid leukemia with unprecedented therapeutic results. The extraordinary effectiveness of ATRA in the treatment of Acute Promyelocytic Leukemia patients has raised interest in evaluating the potential of this natural retinoid in the treatment of other types of neoplasias, with particular reference to solid tumors.The present article provides an overview of the available pre-clinical and clinical studies focussing on ATRA as a therapeutic agent in the context of breast cancer from a holistic point of view. In detail, we focus on the direct effects of ATRA in breast cancer cells as well as the underlying molecular mechanisms of action. In addition, we summarize the available information on the action exerted by ATRA on the breast cancer micro-environment, an emerging determinant of the progression and invasive behaviour of solid tumors. In particular we discuss the recent evidences of ATRA activity on the immune system. Finally, we analyse and discuss the results obtained with the few ATRA-based clinical trials conducted in the context of breast cancer.

Anti-tumor activity of all-trans retinoic acid in gastric-cancer: gene-networks and molecular mechanisms.

BACKGROUND: Gastric-cancer is a heterogeneous type of neoplastic disease and it lacks appropriate therapeutic options. There is an urgent need for the development of innovative pharmacological strategies, particularly in consideration of the potential stratified/personalized treatment of this tumor. All-Trans Retinoic-acid (ATRA) is one of the active metabolites of vitamin-A. This natural compound is the first example of clinically approved cyto-differentiating agent, being used in the treatment of acute promyelocytic leukemia. ATRA may have significant therapeutic potential also in the context of solid tumors, including gastric-cancer. The present study provides pre-clinical evidence supporting the use of ATRA in the treatment of gastric-cancer using high-throughput approaches. METHODS: We evaluated the anti-proliferative action of ATRA in 27 gastric-cancer cell-lines and tissue-slice cultures from 13 gastric-cancer patients. We performed RNA-sequencing studies in 13 cell-lines exposed to ATRA. We used these and the gastric-cancer RNA-sequencing data of the TCGA/CCLE datasets to conduct multiple computational analyses. RESULTS: Profiling of our large panel of gastric-cancer cell-lines for their quantitative response to the anti-proliferative effects of ATRA indicate that approximately half of the cell-lines are characterized by sensitivity to the retinoid. The constitutive transcriptomic profiles of these cell-lines permitted the construction of a model consisting of 42 genes, whose expression correlates with ATRA-sensitivity.  The model predicts that 45% of the TCGA gastric-cancers are sensitive to ATRA. RNA-sequencing studies performed in retinoid-treated gastric-cancer cell-lines provide insights into the gene-networks underlying ATRA anti-tumor activity. In addition, our data demonstrate that ATRA exerts significant immune-modulatory effects, which seem to be largely controlled by IRF1 up-regulation. Finally, we provide evidence of a feed-back loop between IRF1 and DHRS3, another gene which is up-regulated by ATRA. CONCLUSIONS: ATRA is endowed with significant therapeutic potential in the stratified/personalized treatment gastric-cancer. Our data represent the fundaments for the design of clinical trials focusing on the use of ATRA in the personalized treatment of this heterogeneous tumor. Our gene-expression model will permit the development of a predictive tool for the selection of ATRA-sensitive gastric-cancer patients. The immune-regulatory responses activated by ATRA suggest that the retinoid and immune-checkpoint inhibitors constitute rational combinations for the management of gastric-cancer.

Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare multisystem genetic disorder which is caused by genetic defects involving the Nipped-B-like protein (NIPBL) gene in the majority of clinical cases (60-70%). Currently, there are no specific cures available for CdLS and clinical management is needed for life. Disease models are highly needed to find a cure. Among therapeutic possibilities are genome editing strategies based on CRISPR-Cas technology. METHODS: A comparative analysis was performed to test the most recent CRISPR-Cas technologies comprising base- and prime-editors which introduce modifications without DNA cleavages and compared with sequence substitution approaches through homology directed repair (HDR) induced by Cas9 nuclease activity. The HDR method that was found more efficient was applied to repair a CdLS-causing mutation in the NIPBL gene. Human-induced pluripotent stem cells (hiPSCs) derived from a CdLS patient carrying the c.5483G > A mutation in the NIPBL were modified through HDR to generate isogenic corrected clones. RESULTS: This study reports an efficient method to repair the NIPBL gene through HDR mediated by CRISPR-Cas and induced with a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. This sequence repair method allowed the generation of isogenic wild-type hiPSCs clones with regular karyotype and preserved pluripotency. CONCLUSIONS: CdLS cellular models were generated which will facilitate the investigation of the disease molecular determinants and the identification of therapeutic targets. In particular, the hiPSC-based cellular models offer the paramount advantage to study the tissue differentiation stages which are altered in the CdLS clinical development. Importantly, the hiPSCs that were generated are isogenic thus providing the most controlled experimental set up between wild-type and mutated conditions.

Role of cardiolipins, mitochondria, and autophagy in the differentiation process activated by all-trans retinoic acid in acute promyelocytic leukemia.

The role played by lipids in the process of granulocytic differentiation activated by all-trans retinoic acid (ATRA) in Acute-Promyelocytic-Leukemia (APL) blasts is unknown. The process of granulocytic differentiation activated by ATRA in APL blasts is recapitulated in the NB4 cell-line, which is characterized by expression of the pathogenic PML-RARα fusion protein. In the present study, we used the NB4 model to define the effects exerted by ATRA on lipid homeostasis. Using a high-throughput lipidomic approach, we demonstrate that exposure of the APL-derived NB4 cell-line to ATRA causes an early reduction in the amounts of cardiolipins, a major lipid component of the mitochondrial membranes. The decrease in the levels of cardiolipins results in a concomitant inhibition of mitochondrial activity. These ATRA-dependent effects are causally involved in the granulocytic maturation process. In fact, the ATRA-induced decrease of cardiolipins and the concomitant dysfunction of mitochondria precede the differentiation of retinoid-sensitive NB4 cells and the two phenomena are not observed in the retinoid-resistant NB4.306 counterparts. In addition, ethanolamine induced rescue of the mitochondrial dysfunction activated by cardiolipin deficiency inhibits ATRA-dependent granulocytic differentiation and induction of the associated autophagic process. The RNA-seq studies performed in parental NB4 cells and a NB4-derived cell population, characterized by silencing of the autophagy mediator, ATG5, provide insights into the mechanisms underlying the differentiating action of ATRA. The results indicate that ATRA causes a significant down-regulation of CRLS1 (Cardiolipin-synthase-1) and LPCAT1 (Lysophosphatidylcholine-Acyltransferase-1) mRNAs which code for two enzymes catalyzing the last steps of cardiolipin synthesis. ATRA-dependent down-regulation of CRLS1 and LPCAT1 mRNAs is functionally relevant, as it is accompanied by a significant decrease in the amounts of the corresponding proteins. Furthermore, the decrease in CRLS1 and LPCAT1 levels requires activation of the autophagic process, as down-regulation of the two proteins is blocked in ATG5-silenced NB4-shATG5 cells.

Involvement of aldehyde oxidase in the metabolism of aromatic and aliphatic aldehyde-odorants in the mouse olfactory epithelium.

Xenobiotic-metabolizing enzymes (XMEs) expressed in the olfactory epithelium (OE) are known to metabolize odorants. Aldehyde oxidase (AOX) recognizes a wide range of substrates among which are substrates with aldehyde groups. Some of these AOX substrates are odorants, such as benzaldehyde and n-octanal. One of the mouse AOX isoforms, namely AOX2 (mAOX2), was shown to be specifically expressed in mouse OE but its role to metabolize odorants in this tissue remains unexplored. In this study, we investigated the involvement of mouse AOX isoforms in the oxidative metabolism of aldehyde-odorants in the OE. Mouse OE extracts effectively metabolized aromatic and aliphatic aldehyde-odorants. Gene expression analysis revealed that not only mAOX2 but also the mAOX3 isoform is expressed in the OE. Furthermore, evaluation of inhibitory effects using the purified recombinant enzymes led us to identify specific inhibitors of each isoform, namely chlorpromazine, 17ß-estradiol, menadione, norharmane, and raloxifene. Using these specific inhibitors, we defined the contribution of mAOX2 and mAOX3 to the metabolism of aldehyde-odorants in the mouse OE. Taken together, these findings demonstrate that mAOX2 and mAOX3 are responsible for the oxidation of aromatic and aliphatic aldehyde-odorants in the mouse OE, implying their involvement in odor perception.

A DOCK1 Gene-Derived Circular RNA Is Highly Expressed in Luminal Mammary Tumours and Is Involved in the Epithelial Differentiation, Growth, and Motility of Breast Cancer Cells.

Circular RNAs are regulatory molecules involved in numerous cellular processes and may be involved in tumour growth and diffusion. Here, we define the expression of 15 selected circular RNAs, which may control the process of epithelial-to-mesenchymal transition, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of these tumours and consisting of three groups according to the mesenchymal/epithelial phenotype. A circular RNA from the DOCK1 gene (hsa_circ_0020397) shows low/undetectable levels in triple-negative mesenchymal cell lines, while its content is high in epithelial cell lines, independent of estrogen receptor or HER2 positivity. RNA-sequencing experiments performed on the triple-negative/mesenchymal MDA-MB-231 and MDA-MB-157 cell lines engineered to overexpress hsa_circ_0020397 demonstrate that the circRNA influences the expression of 110 common genes. Pathway analysis of these genes indicates that overexpression of the circular RNA differentiates the two mesenchymal cell lines along the epithelial pathway and increases cell-to-cell adhesion. This is accompanied by growth inhibition and a reduction in the random/directional motility of the cell lines. The upregulated AGR2, ENPP1, and PPP1R9A genes as well as the downregulated APOE, AQP3, CD99L2, and IGFBP4 genes show an opposite regulation by hsa_circ_0020397 silencing in luminal CAMA1 cells. The results provide novel insights into the role played by specific circular RNAs in the generation/progression of breast cancer.

OXER1 and RACK1-associated pathway: a promising drug target for breast cancer progression.

Recent data indicate that receptor for activated C kinase 1 (RACK1) is a putative prognostic marker and drug target in breast cancer (BC). High RACK1 expression is negatively associated with overall survival, as it seems to promote BC progression. In tumors, RACK1 expression is controlled by a complex balance between glucocorticoids and androgens. Given the fact that androgens and androgenic derivatives can inhibit BC cell proliferation and migration, the role of androgen signaling in regulating RACK1 transcription in mammary tumors is of pivotal interest. Here, we provide evidence that nandrolone (19-nortosterone) inhibits BC cell proliferation and migration by antagonizing the PI3K/Akt/NF-κB signaling pathway, which eventually results in RACK1 downregulation. We also show that nandrolone impairs the PI3K/Akt/NF-κB signaling pathway and decreases RACK1 expression via binding to the membrane-bound receptor, oxoeicosanoid receptor 1 (OXER1). High levels of OXER1 are observed in several BC cell lines and correlate with RACK1 expression and poor prognosis. Our data provide evidence on the role played by the OXER1-dependent intracellular pathway in BC progression and shed light on the mechanisms underlying membrane-dependent androgen effects on RACK1 regulation. Besides the mechanistic relevance, the results of the study are of interest from a translational prospective. In fact, they identify a new and actionable pathway to be used for the design of innovative and rational therapeutic strategies in the context of the personalized treatment of BC. In addition, they draw attention on nandrolone-based compounds that lack hormonal activity as potential anti-tumor agents.

Retinoic Acid Sensitivity of Triple-Negative Breast Cancer Cells Characterized by Constitutive Activation of the notch1 Pathway: The Role of Rarß.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks effective therapeutic options. In this study, we profile eighteen TNBC cell lines for their sensitivity to the anti-proliferative action of all-trans retinoic acid (ATRA). The only three cell lines (HCC-1599, MB-157 and MDA-MB-157) endowed with ATRA-sensitivity are characterized by genetic aberrations of the NOTCH1-gene, causing constitutive activation of the NOTCH1 γ-secretase product, N1ICD. N1ICD renders HCC-1599, MB-157 and MDA-MB-157 cells sensitive not only to ATRA, but also to γ-secretase inhibitors (DAPT; PF-03084014). Combinations of ATRA and γ-secretase inhibitors produce additive/synergistic effects in vitro and in vivo. RNA-sequencing studies of HCC-1599 and MB-157 cells exposed to ATRA and DAPT and ATRA+DAPT demonstrate that the two compounds act on common gene sets, some of which belong to the NOTCH1 pathway. ATRA inhibits the growth of HCC-1599, MB-157 and MDA-MB-157 cells via RARα, which up-regulates several retinoid target-genes, including RARß. RARß is a key determinant of ATRA anti-proliferative activity, as its silencing suppresses the effects exerted by the retinoid. In conclusion, we demonstrate that ATRA exerts a significant anti-tumor action only in TNBC cells showing constitutive NOTCH1 activation. Our results support the design of clinical trials involving combinations between ATRA and γ-secretase inhibitors for the treatment of this TNBC subtype.

All-Trans Retinoic Acid Stimulates Viral Mimicry, Interferon Responses and Antigen Presentation in Breast-Cancer Cells.

All-trans retinoic acid (ATRA), a recognized differentiating agent, has significant potential in the personalized/stratified treatment of breast cancer. The present study reports on the molecular mechanisms underlying the anti-tumor activity of ATRA in breast cancer. The work is based on transcriptomic experiments performed on ATRA-treated breast cancer cell-lines, short-term tissue cultures of patient-derived mammary-tumors and a xenograft model. ATRA upregulates gene networks involved in interferon-responses, immune-modulation and antigen-presentation in retinoid-sensitive cells and tumors characterized by poor immunogenicity. ATRA-dependent upregulation of these gene networks is caused by a viral mimicry process, involving the activation of endogenous retroviruses. ATRA induces a non-canonical type of viral mimicry, which results in increased expression of the IRF1 (Interferon Responsive Factor 1) transcription factor and the DTX3L (Deltex-E3-Ubiquitin-Ligase-3L) downstream effector. Functional knockdown studies indicate that IRF1 and DTX3L are part of a negative feedback loop controlling ATRA-dependent growth inhibition of breast cancer cells. The study is of relevance from a clinical/therapeutic perspective. In fact, ATRA stimulates processes controlling the sensitivity to immuno-modulatory drugs, such as immune-checkpoint-inhibitors. This suggests that ATRA and immunotherapeutic agents represent rational combinations for the personalized treatment of breast cancer. Remarkably, ATRA-sensitivity seems to be relatively high in immune-cold mammary tumors, which are generally resistant to immunotherapy.

Evolution, expression, and substrate specificities of aldehyde oxidase enzymes in eukaryotes.

Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.

Correction to: Role of mitochondria and cardiolipins in growth inhibition of breast cancer cells by retinoic acid.

In the original publication of this article [1], the images of Figs. 4 and 5 were exchanged and the legends of the two figures did not correspond due to a typesetting error.

Role of mitochondria and cardiolipins in growth inhibition of breast cancer cells by retinoic acid.

BACKGROUND: All-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied. METHODS: We used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and -resistant cell-lines. Bio-computing approaches were used to analyse the high-throughput lipidomic and transcriptomic data. RESULTS: ATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative-phosphorylation. ATRA reduces the amounts of cardiolipins and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells. CONCLUSIONS: The observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria.

Aldehyde oxidase at the crossroad of metabolism and preclinical screening.

Human AOX1 is a member of the mammalian aldehyde oxidase (AOX) family of enzymes and it is an emerging cytosolic enzyme involved in phase I drug-metabolism, bio-transforming a number of therapeutic agents and xenobiotics. The current trend in drug-development is to design molecules which are not recognized and inactivated by CYP450 monooxygenases, the main drug-metabolizing system, to generate novel therapeutic agents characterized by optimal pharmacokinetic and pharmacodynamic properties. Unfortunately, this has resulted in a substantial enrichment in molecules which are recognized and metabolized by AOXs. The observation has raised interest in the generation of tools capable of predicting AOX-dependent drug-metabolism of novel molecules during the early phases of drug development. Such tools are likely to reduce the number of failures occurring at the clinical and late phase of the drug development process. The current review describes different in silico, in vitro and in vivo methods for the prediction of AOX metabolizing ability and focuses on the existing drawbacks and challenges associated with these approaches.

Assessing Autophagy During Retinoid Treatment of Breast Cancer Cells.

Retinoids are derived from vitamin A through a multi-step process. Within a target cell, retinoids regulate gene expression by activating the retinoid acid receptors (RAR) and retinoid x receptors (RXR), which are ligand-dependent transcription factors. Besides its therapeutic use in dermatological disorders, all-trans retinoic acid (ATRA) is successfully utilized to treat acute promyelocytic leukemia (APL) patients. The use of ATRA in APL patients is the first example of clinically useful differentiation therapy. Therapeutic strategies aiming at cancer cell differentiation have great potential for solid tumors, including breast cancer. The few clinical studies conducted with ATRA in breast cancer are rather disappointing. However, these studies did not take into account the heterogeneity of the disease and were conducted on unselected cohorts of patients.We recently showed that ATRA treatment of breast cancer cells induces autophagy, a highly conserved process aiming at degrading and recycling superfluous or harmful cellular components. In addition, autophagy inhibition significantly increases the therapeutic activity of ATRA. This finding is of fundamental importance, since autophagy has a dual role in cancer. Whereas autophagy may be a protective mechanism during the initial phases of cancer development, it may support cancer cell survival in already established tumors. Furthermore, autophagy can lower or enhance therapeutic efficiency, depending on the tumor type and the anticancer agent considered. Therefore, it is important to investigate the role of autophagy in the context of specific tumors and therapeutic approaches. Accurate autophagy studies are challenging given the dynamic nature of the process and the difficulty of measuring the rate of autophagosome degradation (autophagic flux). In this chapter, we provide protocols for a careful assessment of the autophagic flux in ATRA treated 2D and 3D breast cancer cultures.

HER2-positive breast-cancer cell lines are sensitive to KDM5 inhibition: definition of a gene-expression model for the selection of sensitive cases.

Targeting of histone methylation has therapeutic potential in oncology. Here, we provide proof-of-principle that pharmacological inhibition of KDM5 histone-demethylases is a new strategy for the personalized treatment of HER2+ breast cancer. The anti-proliferative effects of the prototype of a new class of selective KDM5-inhibitors (KDM5-inh1) are evaluated in 40 cell lines, recapitulating the heterogeneity of breast cancer. This analysis demonstrates that HER2+ cells are particularly sensitive to KDM5 inhibition. The results are confirmed in an appropriate in vivo model with a close structural analog (KDM5-inh1A). RNA-seq data obtained in HER2+ BT-474 cells exposed to KDM5-Inh1 indicate that the compound alters expression of numerous genes downstream of the ERBB2 gene-product, HER2. In selected HER2-positive breast-cancer cells, we demonstrate synergistic interactions between KDM5-inh1 and HER2-targeting agents (trastuzumab and lapatinib). In addition, HER2+ cell lines with innate and acquired resistance to trastuzumab show sensitivity to KDM5-inh1. The levels of KDM5A/B/C proteins, which are selectively targeted by the agent, have no significant association with KDM5-inh1 responsiveness across our panel of breast-cancer cell lines, suggesting the existence of other determinants of sensitivity. Using RNA-seq data of the breast-cancer cell lines we generate a gene-expression model that is a robust predictor of KDM5-inh1 sensitivity. In a test set of breast cancers, this model predicts sensitivity to the compound in a large fraction of HER2+ tumors. In conclusion, KDM5 inhibition has potential in the treatment of HER2+ breast cancer and our gene-expression model can be developed into a diagnostic tool for the selection of patients.

BET proteins regulate homologous recombination-mediated DNA repair: BRCAness and implications for cancer therapy.

Bromodomain and Extra-Terminal (BET) proteins are historically involved in regulating gene expression and BRD4 was recently found to be involved in DNA damage regulation. Aims of our study were to assess BRD4 regulation in homologous recombination-mediated DNA repair and to explore novel clinical strategies through the combinations of the pharmacological induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer (TNBC) cells by means of BET inhibitors and compounds already available in clinic. Performing a dual approach (chromatin immunoprecipitation and RNA interference), the direct relationship between BRD4 and BRCA1/RAD51 expression was confirmed in TNBC cells. Moreover, BRD4 pharmacological inhibition using two BET inhibitors (JQ1 and GSK525762A) induced a dose-dependent reduction in BRCA1 and RAD51 levels and is able to hinder homologous recombination-mediated DNA damage repair, generating a BRCAness phenotype in TNBC cells. Furthermore, BET inhibition impaired the ability of TNBC cells to overcome the increase in DNA damage after platinum salts (i.e., CDDP) exposure, leading to massive cell death, and triggered synthetic lethality when combined with PARP inhibitors (i.e., AZD2281). Altogether, the present study confirms that BET proteins directly regulate the homologous recombination pathway and their inhibition induced a BRCAness phenotype in BRCA1 wild-type TNBC cells. Noteworthy, being this strategy based on drugs already available for human use, it is rapidly transferable and could potentially enable clinicians to exploit platinum salts and PARP inhibitors-based treatments in a wider population of TNBC patients and not just in a specific subgroup, after validating clinical trials.

S100A3 a partner protein regulating the stability/activity of RARα and PML-RARα in cellular models of breast/lung cancer and acute myeloid leukemia.

All trans-retinoic acid (ATRA) is used in the treatment of acute promyelocytic leukemia (APL) and it is a promising agent also in solid tumors. The pharmacological activity of ATRA is mediated by the ligand-activated RAR and RXR transcription factors. In the present study, we define the basal and ATRA dependent RARα interactome in a RARα-overexpressing breast cancer cellular model, identifying 28 nuclear proteins. We focus our attention on the S100A3 calcium-binding protein, which interacts with RARα constitutively. In ATRA-sensitive breast cancer cells, S100A3 binds to RARα in basal conditions and binding is reduced by the retinoid. The interaction of S100A3 with RARα is direct and in lung cancer, APL and acute-myeloid-leukemia (AML) cells. In APL, S100A3 interacts not only with RARα, but also with PML-RARα. The interaction surface maps to the RARα ligand-binding domain, where the I396 residue plays a crucial role. Binding of S100A3 to RARα/PML-RARα controls the constitutive and ATRA-dependent degradation of these receptors. S100A3 knockdown decreases the amounts of RARα in breast- and lung cancer cells, inducing resistance to ATRA-dependent anti-proliferative/differentiating effects. Conversely, S100A3 knockdown in PML-RARα+ APL and PML-RARα- AML cells reduces the amounts of RARα/PML-RARα and increases basal and ATRA-induced differentiation. In this cellular context, opposite effects on RARα/PML-RARα levels and ATRA-induced differentiation are observed upon S100A3 overexpression. Our results provide new insights into the molecular mechanisms controlling RARα activity and have practical implications, as S100A3 represents a novel target for rational drug combinations aimed at potentiating the activity of ATRA.

Massive NGS data analysis reveals hundreds of potential novel gene fusions in human cell lines.

Background: Gene fusions derive from chromosomal rearrangements. The resulting chimeric transcripts are often endowed with oncogenic potential. Furthermore, they serve as diagnostic tools for the clinical classification of cancer subgroups with different prognosis and, in some cases, they can provide specific drug targets. To date, many efforts have been carried out to study gene fusion events occurring in tumor samples. In recent years, the availability of a comprehensive next-generation sequencing dataset for all existing human tumor cell lines has provided the opportunity to further investigate these data in order to identify novel and still uncharacterized gene fusion events. Results: In our work, we have extensively reanalyzed 935 paired-end RNA-sequencing experiments downloaded from the Cancer Cell Line Encyclopedia repository, aiming at addressing novel putative cell-line specific gene fusion events in human malignancies. The bioinformatics analysis has been performed by the execution of four gene fusion detection algorithms. The results have been further prioritized by running a Bayesian classifier that makes an in silico validation. The collection of fusion events supported by all of the predictive software results in a robust set of ∼1,700 in silico predicted novel candidates suitable for downstream analyses. Given the huge amount of data and information produced, computational results have been systematized in a database named LiGeA. The database can be browsed through a dynamic and interactive web portal, further integrated with validated data from other well-known repositories. Taking advantage of the intuitive query forms, the users can easily access, navigate, filter, and select the putative gene fusions for further validations and studies. They can also find suitable experimental models for a given fusion of interest. Conclusions: We believe that the LiGeA resource can represent not only the first compendium of both known and putative novel gene fusion events in the catalog of all of the human malignant cell lines but it can also become a handy starting point for wet-lab biologists who wish to investigate novel cancer biomarkers and specific drug targets.