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Supplementary Fig. S3 from Atypical retinoids ST1926 and CD437 are S-phase-specific agents causing DNA double-strand breaks: significance for the cytotoxic and antiproliferative activity

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (γ-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. [Mol Cancer Ther 2008;7(9):2941–54]

Supplementary Methods from Inhibition of the Peptidyl-Prolyl-Isomerase Pin1 Enhances the Responses of Acute Myeloid Leukemia Cells to Retinoic Acid via Stabilization of RARα and PML-RARα

Supplementary Table 1 from Inhibition of the Peptidyl-Prolyl-Isomerase Pin1 Enhances the Responses of Acute Myeloid Leukemia Cells to Retinoic Acid via Stabilization of RARα and PML-RARα

Supplementary Figure Legends 1-9 from Inhibition of the Peptidyl-Prolyl-Isomerase Pin1 Enhances the Responses of Acute Myeloid Leukemia Cells to Retinoic Acid via Stabilization of RARα and PML-RARα

Supplementary Figures 6-9 from Inhibition of the Peptidyl-Prolyl-Isomerase Pin1 Enhances the Responses of Acute Myeloid Leukemia Cells to Retinoic Acid via Stabilization of RARα and PML-RARα

Supplementary Figures 1-5 from Inhibition of the Peptidyl-Prolyl-Isomerase Pin1 Enhances the Responses of Acute Myeloid Leukemia Cells to Retinoic Acid via Stabilization of RARα and PML-RARα

The peptidyl-prolyl-isomerase Pin1 interacts with phosphorylated proteins, altering their conformation. The retinoic acid receptor RARα and the acute-promyelocytic-leukemia–specific counterpart PML-RARα directly interact with Pin1. Overexpression of Pin1 inhibits ligand-dependent activation of RARα and PML-RARα. Inhibition is relieved by Pin1-targeted short interfering RNAs and by pharmacologic inhibition of the catalytic activity of the protein. Mutants of Pin1 catalytically inactive or defective for client-protein–binding activity are incapable of inhibiting ligand-dependent RARα transcriptional activity. Functional inhibition of RARα and PML-RARα by Pin1 correlates with degradation of the nuclear receptors via the proteasome-dependent pathway. In the acute myelogenous leukemia cell lines HL-60 and NB4, Pin1 interacts with RARα in a constitutive fashion. Suppression of Pin1 by a specific short hairpin RNA in HL-60 or NB4 cells stabilizes RARα and PML-RARα, resulting in increased sensitivity to the cytodifferentiating and antiproliferative activities of all-trans retinoic acid. Treatment of the two cell lines and freshly isolated acute myelogenous leukemia blasts (M1 to M4) with ATRA and a pharmacologic inhibitor of Pin1 causes similar effects. Our results add a further layer of complexity to the regulation of nuclear retinoic acid receptors and suggest that Pin1 represents an important target for strategies aimed at increasing the therapeutic index of retinoids. [Cancer Res 2009;69(3):1016–26

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.

All-trans retinoic acid (ATRA) is used in the treatment of Acute Promyelocytic Leukemia (APL) with exceptional results, inducing long-lasting remissions in 78% of the patients. In previous papers (Bolis M et al., Ann Oncol. 2017, 28:611; Centritto F et al., EMBO Mol Med. 2015, 7:950) we demonstrated the therapeutic potential of ATRA in the personalized treatment of breast cancer. The present study is aimed at providing pre-clinical evidence supporting the use of pharmacological strategies based on ATRA in the management of stomach cancer, a very heterogeneous type of tumor, which lacks effective therapeutic options other than surgery. To define the sensitivity of gastric cancer to the anti-proliferative action of ATRA, we used 27 cell-lines, recapitulating the heterogeneity of the tumor. Fourteen of these cell-lines are characterized by a G-DIF phenotype, while the remainders present with a G-INT phenotype. Each cell-line was exposed to increasing concentrations of ATRA and the growth-inhibitory effects of the retinoid were evaluated at 3, 6, and 9 days. The 27 cell-lines were ranked for their sensitivity to the retinoid with the “ATRA-score”, a continuous/quantitative index, which we developed. The results indicate that the cell-lines can be divided into 3 separate groups characterized by high, intermediate and low sensitivity to ATRA. No significant correlation between ATRA-sensitivity and the G-DIF or G-INT phenotype is observed in our panel of cell-lines. To support the results of the cell-lines, we evaluated the anti-proliferative effects exerted by ATRA on short-term tissue slice cultures of primary tumors derived from 13 cases of stomach cancer, which we classified as G-DIF or G-INT following determination of the basal gene-expression profiles with the use of RNA-sequencing studies. The results obtained with the short-term tissue culture model confirm the data generated with the cell-lines. The whole genome RNA-sequencing studies performed in the cell-lines and short-term tissue cultures in basal conditions allowed us to define a model consisting of 42 genes whose expression is quantitatively correlated with ATRA-sensitivity. The model was used to predict the sensitivity of gastric cancer cases to ATRA using the TCGA dataset. The results obtained indicate that approximately 60% of the gastric cancer cases are predicted to be characterized by high/intermediate sensitivity to ATRA, regardless of the G-DIF or G-INT phenotype. In conclusion, our results support the concept that ATRA-based therapeutic strategies are likely to be beneficial in the majority of stomach cancer cases. In addition, we generate a gene-expression tool that may be used for the selection of ATRA-sensitive patients in the clinics. Citation Format: Mineko Terao, Luca Guarrera, Silvio Ken Garattini, Marika Foglia, Maurizio Gianni, Enrico Garattini. Sensitivity of gastric cancer cells to all-trans retinoic acid, a new agent in the personalized treatment of this heterogeneous tumor: Definition of a predictive gene-expression model that may be used for the selection of patients who may benefit from retinoid-based treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1597.

Nuclear receptors (NRs) are ligand-dependent transcription factors which are involved in the proliferation/development of normal and neoplastic cells. The family of NR genes consists of 48 elements and the endogenous ligands binding the majority of NRs are known. Indeed, it is generally assumed that NRs function as sensors for fat-soluble hormones, vitamins and dietary lipids. For instance, RARs (Retinoic Acid Receptors) bind to and are trans-activated by retinoic acids, the active metabolites of vitamin A, while ERs (Estrogen Receptors) and PR (Progesterone Receptor) interact with estradiol and progesterone, respectively. In spite of all the available information, there are at least 15 NRs (ONRs, Orphan Nuclear Receptors), which are still devoid of a recognized endogenous ligand. The dysregulation of NRs has been associated with different types of cancer. NRs act in hierarchical, integrated networks to promote tissue- and cell-specific functions. In breast cancer, the role of ERs and PR has been thoroughly investigated, while the function of other members of the NR family is largely unknown. In the present study, we define the expression levels of NR mRNAs in normal and breast cancer tissues, using public databases. The results obtained indicate that the majority of NRs are deregulated in mammary tumors. We focus our attention on ONRs, as many of them are differentially expressed in normal and tumor samples. To get functional information, we use the siRNA technology to assess the effects of ONR silencing on the growth/motility of breast cancer cells. To this purpose, we employ 40 cell lines, which are representative of the different tumor subtypes. We choose at least five cell lines among the ones with high, medium and low expression of the ONR under investigation. We transiently silence the selected NR with the transfection of at least 3 selective siRNAs. Cell proliferation is evaluated after 2, 4 and 6-days from the transfection of the siRNAs using the sulphorodamine assay. We identify NR2F6 as the only ONR whose silencing inhibits cell proliferation. In contrast, NR2F2 silencing increases cell proliferation. We confirm the results obtained on NR2F6 with the use of the CRISPR/CAS9 and the shRNA technologies. In line with our results, NR2F6 expression is higher, while NR2F2 expression is lower, in breast tumors than in normal tissue. Interestingly, the highest levels of NR2F6 protein are observed in ER- and in HER2-positive breast cancer cell-lines. By converse, Triple-Negative cells are endowed with the lowest levels of this ONR. To identify the gene-networks modulated by NR2F2 and NR2F6, we performed RNA-sequencing experiments in control and silenced breast cancer cells, which provide evidence on the underlying molecular mechanisms of action. In conclusion, our data indicate that NR2F2 and NR2F6 represent novel targets for the development of therapeutic strategies in the context of the personalized treatment of breast cancer. Citation Format: Gabriela Paroni, Maria Azzurra Caricasulo, Adriana Zanetti, Andrea Cavallaro, Luca Guarrera, Mineko Terao, Enrico Garattini. The role of orphan nuclear receptors in breast cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3074.

Simple Summary In the present study, we define the expression of 15 selected circular RNAs, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of tumors and consisting of three distinct groups according to the mesenchymal/epithelial phenotype. We identify circDOCK1-1 generated from the DOCK1 gene as an important regulator of the epithelial differentiation of mammary tumour cells. In addition, our high-throughput transcriptomic studies provide information on the genes that are regulated by circDOCK1-1, casting new light on the gene networks involved in the processes underlying the development of mammary tumours. Our results further indicate that circDOCK1-1 controls cell–cell adhesion and consequently random as well as directional motility. Besides their interest from a basic point of view, the data generated may be of translational relevance, as they suggest that strategies aimed at increasing the amounts or at stimulating the activity of circDOCK1-1 may be of significance for the treatment of a subset of triple-negative breast cancer patients. Abstract 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.

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.

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, 17β-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.

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 &gamma, secretase product, N1ICD. N1ICD renders HCC-1599, MB-157 and MDA-MB-157 cells sensitive not only to ATRA, but also to &gamma, secretase inhibitors (DAPT, PF-03084014). Combinations of ATRA and &gamma, 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 RAR&alpha, which up-regulates several retinoid target-genes, including RAR&beta, RAR&beta, 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 &gamma, secretase inhibitors for the treatment of this TNBC subtype.

Background: All-trans retinoic-acid (ATRA) is a promising agent in the personalized treatment/chemo-prevention of breast-cancer. Triple-negative breast-cancer (TNBC) accounts for 15-20% of all mammary tumours and share common features such as a high proliferation index and a basal-like gene expression signature. In spite of this, TNBC is very heterogeneous and lacks effective therapeutic strategies.Methods: We profile eighteen TNBC breast-cancer cell-lines for their sensitivity to the anti-proliferative action of ATRA. In addition, we perform RNA-sequencing studies in two of the most sensitive cell-lines exposed to ATRA, a γ-secretase inhibitor and combinations thereof. Results: The only three TNBC cell-lines (HCC-1599, MB-157 and MDA-MB-157) endowed with ATRA-sensitivity are characterized by constitutive activation of the NOTCH1 γ-secretase product, N1ICD and we identify the associated genetic aberrations of the NOTCH1-gene. N1ICD expression renders HCC-1599, MB-157 and MDA-MB-157 cells sensitive not only to ATRA, but also to γ-secretase inhibitors, like DAPT [N-(N-(3,5-difluorophenacetyl)-L-alanyl)-S-phenylglycine-t-butyl-ester] and PF-03084014. The anti-proliferative action of ATRA and γ-secretase inhibitors is complementary, as combinations of ATRA and DAPT or PF-03084014 cause synergistic effects. This synergism is confirmed in mouse xenografts of HCC-1599 cells. RNA-sequencing studies performed in HCC-1599 and MB-157 cells exposed to ATRA and 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 RARβ. RARβ induction is observed only in HCC-1599, MB-157 and MDA-MB-157 cells, as the other TNBC cell-lines lack ATRA-dependent stimulation of the retinoid-receptor. RARβ is a key determinant of ATRA anti-proliferative activity, as its silencing suppresses the effects exerted by the retinoid. Conclusions: We demonstrate that ATRA exerts a significant anti-tumor action in TNBC cells characterized by constitutive NOTCH1 activation. We show that ATRA enhances the anti-tumor activity of γ-secretase inhibitors in an additive/synergistic manner. We support the idea that ATRA anti-proliferative activity is mediated by the Retinoid-Acid-Receptor-β (RARβ). The present study represents the basis for the design of clinical trials on the efficacy of combinations between ATRA and γ-secretase inhibitors in the treatment of patients affected by a specific subtype of TNBC.

This work was supported by Deutsche Forschungsgemeinschaft Grant Le1171/8-3 (to S. L.). FCT-MCTES, research unit UCIBIO (UID/Multi/04378/2019). project PTDC/BBB-BEP/1185/2014 (to M. J. R.). Fondazione Italo Monzino and the Associazione per la Ricerca sul Cancro (AIRC) (to E. G.). 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. publishersversion published

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.

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.

Aldehyde oxidases are molybdenum and flavin dependent enzymes characterized by a very wide substrate specificity and performing diverse reactions that include oxidations (e.g., aldehydes and aza-heterocycles), hydrolysis of amide bonds, and reductions (e.g., nitro, S-oxides and N-oxides). Oxidation reactions and amide hydrolysis occur at the molybdenum site while the reductions are proposed to occur at the flavin site. AOX activity affects the metabolism of different drugs and xenobiotics, some of which designed to resist other liver metabolizing enzymes (e.g., cytochrome P450 monooxygenase isoenzymes), raising its importance in drug development. This work consists of a comprehensive overview on aldehyde oxidases, concerning the genetic evolution of AOX, its diversity among the human population, the crystal structures available, the known catalytic reactions and the consequences in pre-clinical pharmacokinetic and pharmacodynamic studies. Analysis of the different animal models generally used for pre-clinical trials and comparison between the human (hAOX1), mouse homologs as well as the related xanthine oxidase (XOR) are extensively considered. The data reviewed also include a systematic analysis of representative classes of molecules that are hAOX1 substrates as well as of typical and well characterized hAOX1 inhibitors. The considerations made on the basis of a structural and functional analysis are correlated with reported kinetic and metabolic data for typical classes of drugs, searching for potential structural determinants that may dictate substrate and/or inhibitor specificities.

As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug–drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O6-benzylguanine as substrates. 17β-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC50 values. We also evaluated the potential DDI between an AOX substrate (O6-benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0–24) of O6-benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context.

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.

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.

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.

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.

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) is the first example of targeted therapy. In fact, the oncogenic fusion-protein (PML-RAR) typical of this leukemia contains the retinoid-nuclear-receptor RARalpha. PML-RAR is responsible for the differentiation block of the leukemic blast. Besides PML-RAR, two endogenous RARalpha proteins are present in APL blasts, i.e. RARalpha1 and RARalpha2. We developed different cell populations characterized by PML-RAR, RARalpha2 and RARalpha1 knock-down in the APL-derived NB4 cell-line. Unexpectedly, silencing of PML-RAR and RARalpha2 results in similar increases in the constitutive expression of several granulocytic differentiation markers. This is accompanied by enhanced expression of the same granulocytic markers upon exposure of the NB4 blasts to ATRA. Silencing of PML-RAR and RARalpha2 causes also similar perturbations in the whole genome gene-expression profiles of vehicle and ATRA treated NB4 cells. Unlike PML-RAR and RARalpha2, RARalpha1 knock-down blocks ATRA-dependent induction of several granulocytic differentiation markers. Many of the effects on myeloid differentiation are confirmed by over-expression of RARalpha2 in NB4 cells. RARalpha2 action on myeloid differentiation does not require the presence of PML-RAR, as it is recapitulated also upon knock-down in PML-RAR-negative HL-60 cells. Thus, relative to RARalpha1, PML-RAR and RARalpha2 exert opposite effects on APL-cell differentiation. These contrasting actions may be related to the fact that both PML-RAR and RARalpha2 interact with and inhibit the transcriptional activity of RARalpha1. The interaction surface is located in the carboxy-terminal domain containing the D/E/F regions and it is influenced by phosphorylation of Ser-369 of RARalpha1.

Acute myeloid leukemia (AML) is a rare disease with an estimated incidence of 14 500 and 2500 cases/year in the USA and Italy, respectively. AML is subdivided into groups according to the presence/...

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.

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.

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.

// Enrico Garattini 1 , Marco Bolis 1 , Maurizio Gianni’ 1 , Gabriela Paroni 1 , Maddalena Fratelli 1 and Mineko Terao 1 1 Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy Correspondence to: Enrico Garattini, email: // Keywords : nuclear receptors, breast cancer, drug targets, chemo-prevention, treatment Received : November 27, 2015 Accepted : January 29, 2016 Published : February 15, 2016 Abstract Breast-cancer is heterogeneous and consists of various groups with different biological characteristics. Innovative pharmacological approaches accounting for this heterogeneity are needed. The forty eight human Nuclear-Hormone-Receptors are ligand-dependent transcription-factors and are classified into Endocrine-Receptors , Adopted-Orphan-Receptors ( Lipid-sensors and Enigmatic-Orphans ) and Orphan-receptors . Nuclear-Receptors represent ideal targets for the design/synthesis of pharmacological ligands. We provide an overview of the literature available on the expression and potential role played by Lipid-sensors, Enigmatic-Orphans and Orphan-Receptors in breast-cancer. The data are complemented by an analysis of the expression levels of each selected Nuclear-Receptor in the PAM50 breast-cancer groups, following re-elaboration of the data publicly available. The major aim is to support the idea that some of the Nuclear-Receptors represent largely unexploited therapeutic-targets in breast-cancer treatment/chemo-prevention. On the basis of our analysis, we conclude that the Lipid-Sensors , NR1C3, NR1H2 and NR1H3 are likely to be onco-suppressors in breast-cancer. The Enigmatic-Orphans , NR1F1 NR2A1 and NR3B3 as well as the Orphan-Receptors , NR0B1, NR0B2, NR1D1, NR2F1, NR2F2 and NR4A3 exert a similar action. These Nuclear-Receptors represent candidates for the development of therapeutic strategies aimed at increasing their expression or activating them in tumor cells. The group of Nuclear-Receptors endowed with potential oncogenic properties consists of the Lipid-Sensors , NR1C2 and NR1I2, the Enigmatic-Orphans , NR1F3, NR3B1 and NR5A2, as well as the Orphan-Receptors , NR2E1, NR2E3 and NR6A1. These oncogenic Nuclear-Receptors should be targeted with selective antagonists, reverse-agonists or agents/strategies capable of reducing their expression in breast-cancer cells.

All-trans-retinoic acid (ATRA) is a natural compound proposed for the treatment/chemoprevention of breast cancer. Increasing evidence indicates that aberrant regulation of epithelial-to-mesenchymal transition (EMT) is a determinant of the cancer cell invasive and metastatic behavior. The effects of ATRA on EMT are largely unknown. In HER2-positive SKBR3 and UACC812 cells, showing co-amplification of the ERBB2 and RARA genes, ATRA activates a RARα-dependent epithelial differentiation program. In SKBR3 cells, this causes the formation/reorganization of adherens and tight junctions. Epithelial differentiation and augmented cell-cell contacts underlie the anti-migratory action exerted by the retinoid in cells exposed to the EMT-inducing factors EGF and heregulin-β1. Down-regulation of NOTCH1, an emerging EMT modulator, is involved in the inhibition of motility by ATRA. Indeed, the retinoid blocks NOTCH1 up-regulation by EGF and/or heregulin-β1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFβ contributes to the anti-migratory effect of ATRA. The retinoid switches TGFβ from an EMT-inducing and pro-migratory determinant to an anti-migratory mediator. Inhibition of the NOTCH1 pathway not only plays a role in the anti-migratory action of ATRA; it is relevant also for the anti-proliferative activity of the retinoid in HCC1599 breast cancer cells, which are addicted to NOTCH1 for growth/viability. This effect is enhanced by the combination of ATRA and the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester, supporting the concept that the two compounds act at the transcriptional and post-translational levels along the NOTCH1 pathway.

While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress and cancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism. Using FoxO3A−/− cancer cells generated with the CRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear or mitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response to metabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin. Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cell homeostasis might help devise novel therapeutic strategies to selectively disable FoxO3A prosurvival activity.

A study recently published in Science demonstrates a strong correlation between the total number of stem cell divisions and the lifetime risk of cancer in various organs. The tumors considered are divided into two classes, that is, D (Deterministic) and R (Replicative). Stochastic factors presumably attributable to errors deriving from DNA replication are proposed to be at the basis of R-tumor frequency, leading to the conclusion that ‘bad luck' is a primary determinant of certain types of cancer. The present Second Opinion highlights potential problems associated with the hypothesis of the study, as some of these also apply to kidney cancer. The aim is to point out that chance is not a major cause of cancer incidence because it is not substantiated by the data available through epidemiological evidence. In particular, we highlight that differences in tumor incidence associated with sex and geographic areas are not in line with the ‘bad luck' hypothesis. Further aspects of tumor biology that do not entirely fit with the idea that stochastic events related to DNA replication of normal stem cells are the heterogeneity of the cancer cell phenotype and the heterogeneity of intra-organ localization, often observed in tumors originating from the same tissue. From a public health perspective, the points discussed here as well as the absence of data on prevalent tumors, like breast and prostate cancer, do not support the oversimplified message conveyed by the media that the majority of cancer cases cannot be prevented.