MicroRNA-200 family modulation in distinct breast cancer phenotypes.

PLoS One. 2012;7(10):e47709.

Castilla MÁ, Díaz-Martín J, Sarrió D, Romero-Pérez L, López-García MÁ, Vieites B, Biscuola M, Ramiro-Fuentes S, Isacke CM, Palacios J.

Department of Pathology, Instituto de Biomedicina de Sevilla-CSIC-Universidad de Sevilla, Hospital Universitario Virgen del Rocío, Seville, Spain.

Abstract

The epithelial to mesenchymal transition (EMT) contributes to tumor invasion and metastasis in a variety of cancer types. In human breast cancer, gene expression studies have determined that basal-B/claudin-low and metaplastic cancers exhibit EMT-related characteristics, but the molecular mechanisms underlying this observation are unknown. As the family of miR-200 microRNAs has been shown to regulate EMT in normal tissues and cancer, here we evaluated whether the expression of the miR-200 family (miR-200f) and their epigenetic state correlate with EMT features in human breast carcinomas. We analyzed by qRT-PCR the expression of miR-200f members and various EMT-transcriptional inducers in a series of 70 breast cancers comprising an array of phenotypic subtypes: estrogen receptor positive (ER+), HER2 positive (HER2+), and triple negative (TN), including a subset of metaplastic breast carcinomas (MBCs) with sarcomatous (homologous or heterologous) differentiation. No MBCs with squamous differentiation were included. The DNA methylation status of miR-200f loci in tumor samples were inspected using Sequenom MassArray® MALDI-TOF platform. We also used two non-tumorigenic breast basal cell lines that spontaneously undergo EMT to study the modulation of miR-200f expression during EMT in vitro. We demonstrate that miR-200f is strongly decreased in MBCs compared with other cancer types. TN and HER2+ breast cancers also exhibited lower miR-200f expression than ER+ tumors. Significantly, the decreased miR-200f expression found in MBCs is accompanied by an increase in the expression levels of EMT-transcriptional inducers, and hypermethylation of the miR-200c-141 locus. Similar to tumor samples, we demonstrated that downregulation of miR-200f and hypermethylation of the miR-200c-141 locus, together with upregulation of EMT-transcriptional inducers also occur in an in vitro cellular model of spontaneous EMT. Thus, the expression and methylation status of miR-200f could be used as hypothetical biomarkers to assess the occurrence of EMT in breast cancer.

PMID: 23112837

 

Supplement:

Breast cancer constitutes a heterogeneous disease encompassing a large variety of entities with different clinical features. During the last decade gene expression profiling studies have established a molecular taxonomy of breast cancer defining four intrinsic molecular groups with clinical relevance: luminal A, luminal B, HER2+, and basal-like. Luminal subtypes are defined by a gene expression signature partially resembling that of the luminal epithelial layer of the mammary gland, with positive expression of estrogen (ER) and/or progesterone (PR) receptors. HER2 tumors exhibit overexpression of the HER2 receptor and neighboring genes. Basal-like tumors are defined by the expression of basal/myoepithelial markers and generally lack the expression of ER, PR and HER2. Importantly, this molecular classification predicts treatment response and prognosis: Tumors classified into the basal-like and HER2 groups exhibit more aggressive clinical behavior compared to carcinomas with luminal gene expression signatures.

EMT and the reverse process MET (mesenchymal-epithelial transition) are fundamental processes in developmental morphogenesis and have been proposed to play a key role in cancer development. Epithelial cells undergoing EMT lose cell-cell interactions and other epithelial traits while acquiring a migratory and invasive phenotype. This has led to the notion that EMT is an important event for tumor invasion and metastasis. In human breast cancer, we have previously reported that basal-like tumors and metaplastic breast carcinomas (MBCs) might undergo EMT-like processes. MBCs, a specific subset of triple-negative tumors (TN, HER2-/ER-/PR-), represent extreme examples of phenotypic plasticity that may be related to the high aggressiveness and the characteristic metastatic spreading of these tumors.

Functional E-cadherin loss is a hallmark of EMT. Several E-cadherin repressors, function as EMT-transcriptional inducers and their overexpression in cancer cells associate to EMT features and generally aggressive tumors. However, these transcription repressors are not the only EMT drivers. In the past few years several studies reported on the involvement of microRNAs (miRNAs) in controlling EMT. miRNAs are small non-coding RNAs (~23 nt) that silence gene expression by targeting specific mRNAs. Members of the miR-200 family (miR-200f) have been shown to be major regulators of EMT through the targeted silencing of the EMT-transcriptional inducers ZEB1 and ZEB2, which in turn transcriptionally repress miR-200f in a double-negative feedback loop. In addition, miR-200f opposes EMT by directly targeting genes involved in motility and invasion. Therefore, expression of miR-200f in normal and cancer cells promotes the maintenance of an epithelial phenotype. We have observed that TN and specially MBCs breast cancers exhibit lower level expression of miR-200f compared to ER+ tumors. Using an in vitro model of EMT and the analysis in human breast cancer samples we have shown that the strong decrease of miR-200f expression levels is accompanied by upregulation of EMT-transcriptional inducers and hypermethylation of miR-200c-141 locus, thus supporting the functional association between EMT and miR-200f regulation in breast cancer. Therefore, these data indicate that expression and methylation status of miR-200f could be used as a hypothetical biomarker to assess the occurrence of EMT in breast cancer both in vitro and in vivo.

Juan Díaz Martín

 

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