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 (ER␣ ؉ ) breast carcinoma cells, such as MCF-7, but not estrogen receptornegative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ER␣ ؉ cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of reducing cell motility. In ER␣ ؉ 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, RAR␣. RAR␣ 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 ATRAinduced 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.All-trans-retinoic acid (ATRA) 2 and derivatives (retinoids) are promising agents in the treatment/chemoprevention of hematologic and other malignancies (1, 2), including breast carcinoma (3). Breast cancer is an heterogeneous group of tumors with variable response to therapeutic agents, including retinoids. Generally, breast carcinoma cells expressing estrogen receptor type ␣ (ER␣ ϩ ) are sensitive to, whereas the ER␣-negative (ER␣ Ϫ ) counterparts are refractory to, the antiproliferative activity of retinoids (1).MicroRNAs (miRNAs) are short RNAs controlling the stability of target mRNAs or their translation into protein products (4). They influence cell homeostasis and response to drugs (5, 6), modulating the activity of numerous target transcripts simultaneously, via binding to the 3Ј-untranslated region. Little is known about the effects of retinoids on miRNAs in breast carcinoma and/or other neoplasias (7).MCF-7 breast carcinoma cells are ER␣ ϩ , whereas the MDA-MB-231 counterparts are ER␣ Ϫ (8) (9). MCF-7 are sensitive, whereas MDA-MB-231 cells are refractory to the transcriptional and proliferative effects of E2. The pair of cell lines is a model (10 -16) for the association betw...

All-trans retinoic acid (ATRA), the only clinically available cyto-differentiating agent, has potential for the therapy/chemoprevention of breast carcinoma. Given the heterogeneous nature of this tumor, a rational use of ATRA and derivatives (retinoids) in the clinic requires the identification of patients that would benefit from retinoidbased protocols. Here, we demonstrate that 23-32% of the human ERBB2 þ breast cancers show coamplification of retinoic acid receptor alpha (RARA), encoding the retinoic acid receptor, RARa. This represents a novel subtype of breast cancer characterized by remarkable sensitivity to ATRA and RARa agonists, regardless of positivity to the estrogen receptor, a known modulator of retinoid sensitivity. In estrogen-receptor-negative cellular models showing coamplification of ERBB2 and RARA, simultaneous targeting of the corresponding gene products with combinations of lapatinib and ATRA causes synergistic growth inhibition, cyto-differentiation and apoptosis. This provides proof-of-principle that coamplification of ERBB2 and RARA can be exploited for the stratified and targeted therapy of a novel subtype of breast cancer patients, with an approach characterized by tumor cell selectivity and low predicted toxicity. The available cellular models were exploited to define the molecular mechanisms underlying the antitumor activity of combinations between lapatinib and ATRA. Global gene expression and functional approaches provide evidence for three components of the antiproliferative/apoptotic responses triggered by lapatinib þ ATRA. Induction of the retinoiddependent RARRES3 protein by ATRA stabilizes the effect of lapatinib inhibiting ERBB2 phosphorylation. Upregulation and activation of the transcription factor FOXO3A integrates ATRA-dependent transcriptional and lapatinib-dependent posttranscriptional signals, controlling the levels of effector proteins like the antiapoptotic factor, BIRC5. Stimulation of the TGFb pathway by ATRA mediates other components of the apoptotic process set in motion by simultaneous targeting of ERBB2 and RARa.

The peptidyl-prolyl-isomerase Pin1 interacts with phosphorylated proteins, altering their conformation. The retinoic acid receptor RARA and the acute-promyelocytic-leukemia-specific counterpart PML-RARA directly interact with Pin1. Overexpression of Pin1 inhibits ligand-dependent activation of RARA and PML-RARA. 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 RARA transcriptional activity. Functional inhibition of RARA and PML-RARA 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 RARA in a constitutive fashion. Suppression of Pin1 by a specific short hairpin RNA in HL-60 or NB4 cells stabilizes RARA and PML-RARA, 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]

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