Purpose: MicroRNA (miRNA) expression alterations have been described in cancer. Many cancers are characterized by areas of hypoxia, enhanced hypoxia-inducible factor (HIF) levels, and increased expression of hypoxically regulated genes, all of which correlate with patient outcome. We examined hypoxia-induced miRNA expression changes to identify markers of survival in breast cancer. Experimental Design: We used microarrays to analyze miRNA expression changes induced by hypoxia in MCF7 breast cancer cell lines and validated results by quantitative-PCR (Q-PCR). Small interfering RNA against HIF-1a and HIF-2a, and RCC4 cells transfected with the von Hippel-Lindau (VHL) protein were used to investigate HIF dependency of miRNA expression. miRNA Q-PCR assays were done on 219 early breast cancer samples with long-term follow-up. Correlation of expression with clinical variables was done using Pearson and Spearman's rank tests, univariate, and Cox multivariate analysis. Results: hsa-miR-210 induction was the most significant change under hypoxia by microarray analysis (3.4-fold, P < 0.001). hsa-miR-210 expression changes were validated by Q-PCR and detected in other cancer cell lines. Using small interfering RNAs and RCC4 cells transfected with VHL, we showed that the regulation by hypoxia of hsa-miR-210 was mediated by the HIF-1a/VHL transcriptional system but not HIF-2a. hsa-miR-210 expression levels in breast cancer samples correlated directly with a hypoxia score based on the expression of 99 genes. hsa-miR-210 expression levels showed an inverse correlation with disease-free and overall survival, significant in both univariate and multivariate analyses. Conclusions: We show that hsa-miR-210 overexpression is induced by hypoxia in a HIF-1aâ ndVHL-dependent fashion and its expression levels in breast cancer samples are an independent prognostic factor.Hypoxia in cancer appears as a consequence of the growth of a malignant tumor but can also act to promote tumor development. Hypoxic conditions in solid malignancies may confer resistance to conventional therapies and are associated with a poorer prognosis (1 -3). The exposure of cells to hypoxia leads to the coordinated regulation of many genes. The protein products of these genes have a wide variety of critical roles in processes such as energy metabolism, angiogenesis, growth, and apoptosis. Studies of the mechanisms underlying the regulation of such genes have implicated a central role for the transcription factor hypoxia-inducible factor (HIF), which exists as a heterodimer of an a and a h subunit (4). In the presence of oxygen, HIF-a molecules undergo prolyl hydroxylation which is catalyzed by three homologous 2-oxoglutarate -dependent dioxygenases, PHD1, PHD2, and PHD3. The von HippelLindau (VHL) protein recognizes and binds to two specific hydroxyprolyl residues in HIF-1a and HIF-2a, and facilitates ubiquitination leading to rapid proteasomal degradation. Further oxygen-regulated control of HIF-a is achieved by another dioxygenase (FIH-1), which catalyze...

AbstractmicroRNA expression profiling plays an emerging role in cancer classification and identification of therapeutic strategies. In this study, we have evaluated the benefits of a joint microRNA-mRNA analysis in breast cancer.Matched mRNA and microRNA global expression profiling was conducted in a well-annotated cohort of 207 cases with complete 10-year follow-up. Penalized Cox regression including microRNA expression, mRNA expression, and clinical covariates was used to identify microRNAs associated with distant relapse-free survival (DRFS) that provide independent prognostic information, and are not simply surrogates of previously identified prognostic covariates. Penalized regression was chosen to prevent overfitting. Furthermore, microRNA-mRNA relationships were explored by global expression analysis, and exploited to validate results in several published cohorts (n ¼ 592 with DRFS, n ¼ 1,050 with recurrence-free survival).Four microRNAs were independently associated with DRFS in estrogen receptor (ER)-positive (3 novel and 1 known; miR-128a) and 6 in ER-negative (5 novel and 1 known; miR-210) cases. Of the latter, miR-342, -27b, and -150 were prognostic also in triple receptor-negative tumors. Coordinated expression of predicted target genes and prognostic microRNAs strengthened these results, most significantly for miR-210, -128a, and -27b, whose targets were prognostic in meta-analysis of several cohorts. In addition, miR-210 and -128a showed coordinated expression with their cognate pri-microRNAs, which were themselves prognostic in independent cohorts.Our integrated microRNA-mRNA global profiling approach has identified microRNAs independently associated with prognosis in breast cancer. Furthermore, it has validated known and predicted microRNA-target interactions, and elucidated their association with key pathways that could represent novel therapeutic targets. Cancer Res; 71(17); 5635-45. Ó2011 AACR.

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