Abstract 5333: Identification of miRNA targets in axillary breast cancer metastases.
Authors: Gill Mandeep , Marian Catalin , Torresan Clarissa , Ribeiro Enilze , Cavalli Iglenir J. , Gusev Yuriy , Li Xi (James) , Cavalli Luciane R. 
Published: August 2014

Introduction: MicroRNAs (miRNAs) are a class of non-coding endogenous RNA molecules that play a role in the invasion and metastatic breast cancer process. Correlation of miRNA alterations and lymph node (LN) breast cancer metastasis status are described, however most of the data are in advanced metastatic cancer cell lines and/or mouse mammary models. There are limited miRNA analysis that are conducted directly in axillary LN specimens and to our knowledge, there are no analysis performed specifically in sentinel lymph nodes (SLN), the first axillary metastatic site. Therefore our main objective is to identify the miRNA profile and its corresponding miRNA targets in paired samples of primary breast tumors (PBT) and SLN metastatic lesions. We hypothesize that these changes will represent the ones that occur in early stages of the axillary breast cancer metastatic process, before distant metastasis occurs. Methods: RNA was isolated from microdissected 50μm tumor sections of formalin-fixed paraffin-embedded from 30 paired samples of PBT and SLN metastasis. MiRNA profiling was performed using the Nanostring miRNA system and significantly differentially expressed miRNAs (with at least 2 fold difference and p≤0.05) between the lesions were calculated by the comparative Ct method (ΔΔCt). The miRNA data was integrated with previous array-CGH data from the same cases, to select for targets with concomitant copy number and miRNA expression alterations. Combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems were performed to identify predicted target functions. Results: The miRNA profiling of the paired PBT and SLN metastasis, revealed 25 miRNAs significantly differently expressed between these lesions. Among the significant miRNAs observed, were the miR-24-3p, miR-221-3p and miR-99a-5p, which are involved in epithelial-mesenchymal transition (EMT) of metastatic cells. Using miRBase and MiRDB target prediction databases we identified 4691 target genes of deregulated miRNAs, that were reduced to 41 after integration with DNA copy number data from the same cases. Ingenuity pathway analysis on these predicted targets function, identified 73 canonical pathways, including the cyclin D1 cell cycle regulation, p53 and cAMP-mediated signaling. Conclusions: The miRNA profiling in our set of paired PBT and SLN metastasis revealed significant differently expressed miRNAs between these lesions. The selection of the most relevant miRNA targets in the pathways identified with a role in tumor invasion/progression are underway and will be evaluated for its co-expression with the corresponding miRNAs. Finally, the direct functional validation of these targets, by in vitro and in vivo experimental models, will allow us to identify the most relevant ones that are mechanistically involved in the initial stages of axillary breast cancer metastases.

Citation Format: Mandeep Gill, Catalin Marian, Clarissa Torresan, Enilze Ribeiro, Iglenir J. Cavalli, Yuriy Gusev, Xi (James) Li, Luciane R. Cavalli. Identification of miRNA targets in axillary breast cancer metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2013-5333