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.

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.

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.

Mannose-binding lectin has a direct deleterious effect on ischemic brain microvascular endothelial cells.

Mannose-binding lectin (MBL), an initiator of the lectin pathway, is detrimental in ischemic stroke. MBL deposition on the ischemic endothelium indicates the beginning of its actions, but downstream mechanisms are not clear yet.We investigated MBL interactions with the ischemic endothelium by exposing human brain microvascular endothelial cells (hBMECs) to protocols of ischemia. Cells were exposed to hypoxia or oxygen-glucose deprivation (OGD), and re-oxygenated with human serum (HS) or recombinant MBL (rhMBL). Hypoxic hBMECs re-oxygenated with HS showed increased complement system activation (C3c deposition, +59%) and MBL deposition (+93%) than normoxic cells. Super-resolution microscopy showed MBL internalization in hypoxic cells and altered cytoskeletal organization, indicating a potential MBL action on the endothelial structure. To isolate MBL effect, hBMECs were re-oxygenated with rhMBL after hypoxia/OGD. In both conditions, MBL reduced viability (hypoxia: -25%, OGD: -34%) compared to conditions without MBL, showing a direct toxic effect. Ischemic cells also showed greater MBL deposition (hypoxia: +143%, OGD: +126%) than normoxic cells. These results were confirmed with primary hBMECs exposed to OGD (increased MBL-induced cell death: +226%, and MBL deposition: +104%). The present findings demonstrate that MBL can exert a direct deleterious effect on ischemic brain endothelial cells in vitro, independently from complement activation.

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.

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.

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.

RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells.

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 RARα. PML-RAR is responsible for the differentiation block of the leukemic blast. Besides PML-RAR, two endogenous RARα proteins are present in APL blasts, i.e. RARα1 and RARα2. We developed different cell populations characterized by PML-RAR, RARα2 and RARα1 knock-down in the APL-derived NB4 cell-line. Unexpectedly, silencing of PML-RAR and RARα2 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 RARα2 causes also similar perturbations in the whole genome gene-expression profiles of vehicle and ATRA treated NB4 cells. Unlike PML-RAR and RARα2, RARα1 knock-down blocks ATRA-dependent induction of several granulocytic differentiation markers. Many of the effects on myeloid differentiation are confirmed by over-expression of RARα2 in NB4 cells. RARα2 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 RARα1, PML-RAR and RARα2 exert opposite effects on APL-cell differentiation. These contrasting actions may be related to the fact that both PML-RAR and RARα2 interact with and inhibit the transcriptional activity of RARα1. 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 RARα1.

Breast cancer-secreted miR-939 downregulates VE-cadherin and destroys the barrier function of endothelial monolayers.

Exosomes-secreted microRNAs play an important role in metastatic spread. During this process breast cancer cells acquire the ability to transmigrate through blood vessels by inducing changes in the endothelial barrier. We focused on miR-939 that is predicted to target VE-cadherin, a component of adherens junction involved in vessel permeability. By in silico analysis miR-939 was found highly expressed in the basal-like tumor subtypes and in our cohort of 63 triple-negative breast cancers (TNBCs) its expression significantly interacted with lymph node status in predicting disease-free survival probability. We demonstrated, in vitro, that miR-939 directly targets VE-cadherin leading to an increase in HUVECs monolayer permeability. MDA-MB-231 cells transfected with a miR-939 mimic, released miR-939 in exosomes that, once internalized in endothelial cells, favored trans-endothelial migration of MDA-MB-231-GFP cells by the disruption of the endothelial barrier. Notably, when up taken in endothelial cells exosomes caused VE-cadherin down-regulation specifically through miR-939 as we demonstrated by inhibiting miR-939 expression in exosomes-releasing TNBC cells. Together, our data indentify an extracellular pro-tumorigenic role for tumor-derived, exosome-associated miR-939 that can explain its association with worse prognosis in TNBCs.

All-trans-retinoic Acid Modulates the Plasticity and Inhibits the Motility of Breast Cancer Cells: ROLE OF NOTCH1 AND TRANSFORMING GROWTH FACTOR (TGFß).

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-ß1. 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-ß1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFß contributes to the anti-migratory effect of ATRA. The retinoid switches TGFß 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.

MicroRNA networks regulated by all-trans retinoic acid and Lapatinib control the growth, survival and motility of breast cancer cells.

SKBR3-cells, characterized by ERBB2/RARA co-amplification, represent a subgroup of HER2+ breast-cancers sensitive to all-trans retinoic acid (ATRA) and Lapatinib. In this model, the two agents alone or in combination modulate the expression of 174 microRNAs (miRs). These miRs and predicted target-transcripts are organized in four interconnected modules (Module-1 to -4). Module-1 and Module-3 consist of ATRA/Lapatinib up-regulated and potentially anti-oncogenic miRs, while Module-2 contains ATRA/Lapatinib down-regulated and potentially pro-oncogenic miRs. Consistent with this, the expression levels of Module-1/-3 and Module-2 miRs are higher and lower, respectively, in normal mammary tissues relative to ductal-carcinoma-in-situ, invasive-ductal-carcinoma and metastases. This indicates associations between tumor-progression and the expression profiles of Module-1 to -3 miRs. Similar associations are observed with tumor proliferation-scores, staging, size and overall-survival using TCGA (The Cancer Genome Atlas) data. Forced expression of Module-1 miRs, (miR-29a-3p; miR-874-3p) inhibit SKBR3-cell growth and Module-3 miRs (miR-575; miR-1225-5p) reduce growth and motility. Module-2 miRs (miR-125a; miR-193; miR-210) increase SKBR3 cell growth, survival and motility. Some of these effects are of general significance, being replicated in other breast cancer cell lines representing the heterogeneity of this disease. Finally, our study demonstrates that HIPK2-kinase and the PLCXD1-phospholipase-C are novel targets of miR-193a-5p/miR-210-3p and miR-575/miR-1225-5p, respectively.

Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression.

Forty-two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all-trans retinoic acid (ATRA) sensitivity. Luminal and ER(+) (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA-MB157 and HCC-1599) are highly sensitive to the retinoid. Sensitivity of HCC-1599 cells is confirmed in xenotransplanted mice. Short-term tissue-slice cultures of surgical samples validate the cell-line results and support the concept that a high proportion of Luminal/ER(+) carcinomas are ATRA sensitive, while triple-negative (Basal) and HER2-positive tumors tend to be retinoid resistant. Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA-sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over-expression sensitizes retinoid-resistant MDA-MB453 cells to ATRA anti-proliferative action. Conversely, silencing of RARα in retinoid-sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short-term tissue cultures of Luminal/ER(+) and triple-negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid-based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes.

Retinoids and breast cancer: from basic studies to the clinic and back again.

All-trans retinoic acid (ATRA) is the most important active metabolite of vitamin A controlling segmentation in the developing organism and the homeostasis of various tissues in the adult. ATRA as well as natural and synthetic derivatives, collectively known as retinoids, are also promising agents in the treatment and chemoprevention of different types of neoplasia including breast cancer. The major aim of the present article is to review the basic knowledge acquired on the anti-tumor activity of classic retinoids, like ATRA, in mammary tumors, focusing on the underlying cellular and molecular mechanisms and the determinants of retinoid sensitivity/resistance. In the first part, an analysis of the large number of pre-clinical studies available is provided, stressing the point that this has resulted in a limited number of clinical trials. This is followed by an overview of the knowledge acquired on the role played by the retinoid nuclear receptors in the anti-tumor responses triggered by retinoids. The body of the article emphasizes the potential of ATRA and derivatives in modulating and in being influenced by some of the most relevant cellular pathways involved in the growth and progression of breast cancer. We review the studies centering on the cross-talk between retinoids and some of the growth-factor pathways which control the homeostasis of the mammary tumor cell. In addition, we consider the cross-talk with relevant intra-cellular second messenger pathways. The information provided lays the foundation for the development of rational and retinoid-based therapeutic strategies to be used for the management of breast cancer.

Human axonal survival of motor neuron (a-SMN) protein stimulates axon growth, cell motility, C-C motif ligand 2 (CCL2), and insulin-like growth factor-1 (IGF1) production.

Spinal muscular atrophy is a fatal genetic disease of motoneurons due to loss of full-length survival of motor neuron protein, the main product of the disease gene SMN1. Axonal SMN (a-SMN) is an alternatively spliced isoform of SMN1, generated by retention of intron 3. To study a-SMN function, we generated cellular clones for the expression of the protein in mouse motoneuron-like NSC34 cells. The model was instrumental in providing evidence that a-SMN decreases cell growth and plays an important role in the processes of axon growth and cellular motility. In our conditions, low levels of a-SMN expression were sufficient to trigger the observed biological effects, which were not modified by further increasing the amounts of the expressed protein. Differential transcriptome analysis led to the identification of novel a-SMN-regulated factors, i.e. the transcripts coding for the two chemokines, C-C motif ligands 2 and 7 (CCL2 and CCL7), as well as the neuronal and myotrophic factor, insulin-like growth factor-1 (IGF1). a-SMN-dependent induction of CCL2 and IGF1 mRNAs resulted in increased intracellular levels and secretion of the respective protein products. Induction of CCL2 contributes to the a-SMN effects, mediating part of the action on axon growth and random cell motility, as indicated by chemokine knockdown and re-addition studies. Our results shed new light on a-SMN function and the underlying molecular mechanisms. The data provide a rational framework to understand the role of a-SMN deficiency in the etiopathogenesis of spinal muscular atrophy.

Induction of miR-21 by retinoic acid in estrogen receptor-positive breast carcinoma cells: biological correlates and molecular targets.

Retinoids are promising agents for the treatment/prevention of breast carcinoma. We examined the role of microRNAs in mediating the effects of all-trans-retinoic acid (ATRA), which suppresses the proliferation of estrogen receptor-positive (ERα(+)) breast carcinoma cells, such as MCF-7, but not estrogen receptor-negative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ERα(+) cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of reducing cell motility. In ERα(+) cells, retinoid-dependent induction of miR-21 is due to increased transcription of the MIR21 gene via ligand-dependent activation of the nuclear retinoid receptor, RARα. RARα is part of the transcription complex present in the 5'-flanking region of the MIR21 gene. The receptor binds to two functional retinoic acid-responsive elements mapping upstream of the transcription initiation site. Silencing of miR-21 enhances ATRA-dependent growth inhibition and senescence while reverting suppression of cell motility afforded by the retinoid. Up-regulation of miR-21 results in retinoid-dependent inhibition of the established target, maspin. Knockdown and overexpression of maspin in MCF-7 cells indicates that the protein is involved in ATRA-induced growth inhibition and contributes to the ATRA-dependent anti-motility responses. Integration between whole genome analysis of genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells, prediction of miR-21 regulated genes, and functional studies led to the identification of three novel direct miR-21 targets: the pro-inflammatory cytokine IL1B, the adhesion molecule ICAM-1 and PLAT, the tissue-type plasminogen activator. Evidence for ICAM-1 involvement in retinoid-dependent inhibition of MCF-7 cell motility is provided.

Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities.

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However, the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g., the brain) is higher than for treatment of anemia. Notably, a dose-dependent risk of adverse effects has been associated with rhEPO administration, especially in high-risk groups, including polycythemia-hyperviscosity syndrome, hypertension, and vascular thrombosis. Of note, several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity, primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover, we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here, we demonstrate that rhEPO administration in the rat increases systemic blood pressure, reduces regional renal blood flow, and increases platelet counts and procoagulant activities. In contrast, carbamylated rhEPO, a heteromeric receptor-specific ligand that is fully tissue protective, increases renal blood flow, promotes sodium excretion, reduces injury-induced elevation in procoagulant activity, and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands, which appear to elicit fewer adverse effects, may be especially useful in clinical settings for tissue protection.