Abstract 473: miR-135b mediates gemcitabine sensitivity in breast cancer cells by modulating epithelial-to-mesenchymal transition and mTOR-signaling

Background: We previously identified miR-135b as a possible biomarker for gemcitabine sensitivity in metastatic breast cancer patients. Here we aimed to better understand the role of this microRNA in breast cancer cells in order to identify new clinically relevant approaches to revert gemcitabine resistance. Methods: miR-135b KO MDA-MB-231 breast cancer cells were generated through CRISPR-Cas9 technology and tested for gemcitabine sensitivity by cell survival and apoptosis activation assays. Gene expression profile was performed by GeneChip® Human Transcriptome Array 2.0 (HTA 2.0 - Affymetrix). The One-Way between-subject ANOVA algorithm was used to calculate statistical significance of pairwise comparisons. The validation of different gene expression was assessed by qRT-PCR. The mesenchymal phenotype of miR-135b KO cells was tested by wound-healing and migration assay. mTOR pathway has been tested by Western blot analysis. Results: miR-135b KO MDA-MB-231 showed increased resistance to gemcitabine than WT cells. Gene expression profiling of miR-135b WT and KO cells revealed the upregulation of several genes, including miR-21 and gene involved in the epithelial-to-mesenchymal transition (EMT) in the absence of the microRNA. Functional enrichment analysis indicated the deregulation of multiple tumor-associated pathways, including focal adhesion, senescence and autophagy. Accordingly, miR-135b KO cells showed higher migrating properties and expression of mesenchymal genes. Moreover, miR-135b KO cells showed deregulation of the mTOR-signaling pathway upon starvation or treatment with gemcitabine. Based on these observations, we assessed whether inhibition of mTOR activation using a clinically approved inhibitor (everolimus) could enhance gemcitabine sensitivity in the absence of miR-135b. Our data indicate that blocking mTOR pathway could represent a valid approach to revert resistance to gemcitabine even in cancer cells with low levels of miR-135b. Conclusions: We identified several deregulated genes and pathways in the absence of miR-135b in breast cancer cells, in particular related to the EMT and mTOR pathway. These results confirm the role of miR-135b in breast cancer and pave the way for new studies for the combination of gemcitabine and mTOR inhibitors in a specific subset of metastatic breast cancer patients that could largely benefit from it. Citation Format: Anna Tessari, Meghan Pawlikowski, Kareesma Parbhoo, Olivia Willetts, Marianna Hernandez, Giovanni Nigita, Ashley Braddom, Joseph J. Mills, Dario Palmieri, Carlo M. Croce. miR-135b mediates gemcitabine sensitivity in breast cancer cells by modulating epithelial-to-mesenchymal transition and mTOR-signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 473.