miR-223 Is a Coordinator of Breast Cancer Progression as Revealed by Bioinformatics Predictions

<div><p>MicroRNAs are single-stranded non-coding RNAs that simultaneously down-modulate the expression of multiple genes post-transcriptionally by binding to the 3′UTRs of target mRNAs. Here we used computational methods to predict microRNAs relevant in breast cancer progression. Specifically, we applied different microRNA target prediction algorithms to various groups of differentially expressed protein-coding genes obtained from four breast cancer datasets. Six potential candidates were identified, among them miR-223, previously described to be highly expressed in the tumor microenvironment and known to be actively transferred into breast cancer cells. To investigate the function of miR-223 in tumorigenesis and to define its molecular mechanism, we overexpressed miR-223 in breast cancer cells in a transient or stable manner. Alternatively we overexpressed miR-223 in mouse embryonic fibroblasts or HEK293 cells and used their conditioned medium to treat tumor cells. With both approaches, we obtained elevated levels of miR-223 in tumor cells and observed decreased migration, increased cell death in anoikis conditions and augmented sensitivity to chemotherapy but no effect on adhesion and proliferation. The analysis of miR-223 predicted targets revealed enrichment in cell death and survival-related genes and in pathways frequently altered in breast cancer. Among these genes, we showed that protein levels for STAT5A, ITGA3 and NRAS were modulated by miR-223. In addition, we proved that STAT5A is a direct miR-223 target and highlighted a possible correlation between miR-223 and STAT5A in migration and chemotherapy response. Our investigation revealed that a computational analysis of cancer gene expression datasets can be a relevant tool to identify microRNAs involved in cancer progression and that miR-223 has a prominent role in breast malignancy that could potentially be exploited therapeutically.</p></div

Six microRNA prediction occurrence in datasets according to [47].

<p>Six microRNA prediction occurrence in datasets according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084859#pone.0084859-Volinia2" target="_blank">[47]</a>.</p

miR-223 enhances anoikis and chemotherapy induced cell death.

<p>MDAMB231 cells were grown for 48(<b>A</b>) or in complete medium with Doxorubicin (DOXO) (<b>B</b>) or Paclitaxel (PTX) (<b>C</b>) after transient transfection with miR-223 or with unrelated miR precursors or their negative controls (pre-miR-223 or unrelated pre-miR or pre-control). Alternatively MDAMB231 were grown for 48 h in conditioned medium (CM) collected from HEK293 (HEK) cells stably transduced with pLemiR empty (empty) or miR-223 overexpression (miR-223) vectors. MDAMB231 cells were further transferred to regular medium without (Basal) or with PTX for 48 h and cell death was analyzed (<b>D</b>). To control chemotherapy-induced cell death, MDAMB231 cells were treated as in (C) in presence or absence of ZVAD, a caspase inhibitor (<b>E</b>). The percentage (%) dead cells displayed in histograms as mean±SEM (standard error mean) was evaluated by TMRM and AnnexinV-FITC or -APC stainings in a FACS analysis. At least three independent biological experiments were performed in duplicate. Duplicate means relative to three or more pooled biological experiments are shown and used for statistics. *P<0.05; **P<0.01; ***P<0.001.</p

miR-223 expression in MDAMB231 cells grown in Conditioned Medium from miR-223 overexpressing cells.

<p>miR-223 levels were measured in MDAMB231 cells grown in normal culture conditions (Growth medium) or in Conditioned Medium (CM) derived from Mouse Embryonic Fibroblasts (MEF) or Human Embryonic Kidney cells (HEK293-HEK) for 48 hours (<b>A</b>). Alternatively, CM was derived from the cells in (A) stably transduced with miR-223 overexpression (miR-223) lentiviral vectors (<b>B</b>). Results are presented as fold changes (mean±SD) relative to nomal growth conditions. Delta CTs were obtained after normalization on U6sno RNA level. SD = standard deviation; CT = threshold cycle number. Three biological experiments were performed, each with three technical triplicates. Statistics was performed on technical triplicates of one representative biological experiment.*P<0.05; **P<0.01; ***P<0.001.</p

Diseases and functions related to miR-223 predicted targets.

<p>Diseases and functions related to miR-223 predicted targets.</p

Prediction of miRs involved in breast cancer progression and their expression in cells.

<p>(<b>A</b>) Four public datasets of primary breast cancers were used to identify differentially expressed genes comparing patients with or without disease relapse, five years post-surgery (DFS = disease free survival). An hypergeometric test was applied to reveal microRNA seed enrichments, according to the predictions provided by at least two algorithms among TargetScan, Miranda, miRBase (MicroCosm Targets) and DIANA-microT and six miRs were identified. (<b>B–C</b>) Expression of the six predicted microRNAs in breast cancer cell lines (<b>B</b>), such as ER- highly aggressive, MDAMB231 and SUM149PT; ER+ non invasive, MCF7 and T47D, and stroma cells (<b>C</b>), such as monocytes (CD14), dendritic cells (DC, TNFα activated DC), activated T-cells (CD3/CD28), purified T-cells (CD8), hematopoietic stem cells (CD34) and mesenchymal cells (MSC). Results are presented in a log2 scale, as fold changes (mean±SD) relative to the delta CT mean of triplicates for each biological sample. Delta CTs were obtained after normalization on U6sno RNA level. SD = standard deviation; CT = threshold cycle number. TNFα = Tumor necrosis factor alpha.</p

Targeting Myeloid Differentiation Using Potent 2‑Hydroxypyrazolo[1,5‑<i>a</i>]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors

Human dihydroorotate dehydrogenase (<i>h</i>DHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. <i>h</i>DHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of <i>h</i>DHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo­[1,5-<i>a</i>]­pyridine, that has been designed starting from brequinar, one of the most potent <i>h</i>DHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound <b>4</b>, which shows brequinar-like <i>h</i>DHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound <b>4</b> also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of <i>h</i>DHODH inhibitors

Targeting Myeloid Differentiation Using Potent 2‑Hydroxypyrazolo[1,5‑<i>a</i>]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors

Human dihydroorotate dehydrogenase (<i>h</i>DHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. <i>h</i>DHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of <i>h</i>DHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo­[1,5-<i>a</i>]­pyridine, that has been designed starting from brequinar, one of the most potent <i>h</i>DHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound <b>4</b>, which shows brequinar-like <i>h</i>DHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound <b>4</b> also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of <i>h</i>DHODH inhibitors