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 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...
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