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Deregulated expression of microRNAs has been associated with angiogenesis. Studying the miRNome of locally advanced breast tumors we unsuspectedly found a dramatically repression of miR-204, a small non-coding RNA with no previous involvement in tumor angiogenesis. Downregulation of miR-204 was confirmed in an independent cohort of patients and breast cancer cell lines. Gain-of-function analysis indicates that ectopic expression of miR-204 impairs cell proliferation, anchorage-independent growth, migration, invasion, and the formation of 3D capillary networks in vitro. Likewise, in vivo vascularization and angiogenesis were suppressed by miR-204 in a nu/nu mice model. Genome-wide profiling of MDA-MB-231 cells expressing miR-204 revealed changes in the expression of hundred cancer-related genes. Of these, we focused on the study of pro-angiogenic ANGPT1 and TGFβR2. Functional analysis using luciferase reporter and rescue assays confirmed that ANGPT1 and TGFβR2 are novel effectors downstream of miR-204. Accordingly, an inverse correlation between miR-204 and ANGPT1/TGFβR2 expression was found in breast tumors. Knockdown of TGFβR2, but not ANGPT1, impairs cell proliferation and migration whereas inhibition of both genes inhibits angiogenesis. Taken altogether, our findings reveal a novel role for miR-204/ANGPT1/TGFβR2 axis in tumor angiogenesis. We propose that therapeutic manipulation of miR-204 levels may represent a promising approach in breast cancer.

microRNAs are small non-coding RNAs of ~22 nucleotides that function at post-transcriptional level as negative regulators of gene expression. Aberrant expression of microRNAs could promote uncontrolled proliferation, migration and invasion of human cancer cells. In this study, we analyzed the expression of microRNA-18b (miR-18b) in breast cancer cell lines and in a set of clinical specimens. Our results showed that miR-18b was upregulated in four out of five breast cancer cell lines and also in breast tumors. In order to identify potential gene targets, we carried out transcriptional profiling of MDA-MB-231 breast cancer cells that ectopically expressed miR-18b. Our results showed that 263 genes were significantly modulated in miR-18b-deficient cells (fold change >1.5; P≤0.05). We found that knock-down of miR-18b induced the upregulation of 55 olfactory receptor (OR) genes and nine genes (NLRP7, KLK3, OLFM3, POSTN, MAGED4B, KIR3DL3, CRX, SEMG1 and CEACAM5) with key roles in cell migration and metastasis. Consistently, we found that ectopic inhibition of miR-18b suppressed the migration of two breast cancer cell models in vitro. In conclusion, we have uncovered genes directly or indirectly modulated by miR-18b which may represent potential therapeutic targets in breast cancer. Our data also pointed out a role of miR-18b in migration of breast cancer cells.

microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value = 0.05, Fold Change = 2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The expression of 14 microRNAs was replicated in an independent set of 55 tumors. Bioinformatic analysis of mRNA targets of the altered miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process which are important in breast carcinogenesis are affected by the altered microRNA expression, including signaling through MAP kinases and TP53 pathways, as well as biological processes like cell death and communication, focal adhesion and ERBB2-ERBB3 signaling. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described.