Abstract C17: Role of miR-135b in gemcitabine sensitivity for metastatic breast cancer patients
Authors: Tessari Anna , Palmieri Dario , Nigita Giovanni , Veneziano Dario , Cresta Sara , Paolini Biagio , Cona Maria Silvia , Vargo Taylor , Reese Erika , Sheetz Tyler , Coppola Vincenzo , De Braud Filippo , Croce Carlo M. 
Published: May 2016

Background: MicroRNAs are a class of small non-coding RNAs that modulate gene expression at post-transcriptional level, resulting crucial in many physiological and pathological processes. Their involvement in cancer initiation and tumor progression has been clearly established, as well as their linkage with chemoresistance. Gemcitabine is a nucleoside analog used in the treatment of metastatic breast cancer (MBC), characterized by a favorable safety profile. Only a small percentage of pts show a strong and prolonged response to this drug, thus suggesting the need of predictive biomarkers. Here, we aimed to identify microRNAs whose expression could be related to gemcitabine sensitivity in MBC.

Methods: 24 MBC pts were treated with gemcitabine as single-agent therapy from 1999 to 2011 at Fondazione IRCCS Istituto Nazionale Tumori (Milan). They were selected and classified by the response to the treatment (11 gemcitabine sensitive, stable or partial response after &gt;24 weeks of treatment, and 13 gemcitabine resistant, progressive disease within 16 weeks from the first drug administration). MicroRNA expression profiling was performed by Nanostring nCounter Technology using RNA from FFPE metastatic lesions. Biological normalization was executed to correct for differences in sample abundances. Each sample was normalized to the geometric mean of the top 100 most highly expressed microRNAs. Student's t test was used to calculate statistical significances of pairwise comparisons. The nCounter analysis was validated on a wider population of 50 MBC pts (17 sensitive and 33 resistant) by qRT-PCR.

The correlation between microRNA expression levels and gemcitabine half maximal inhibitory concentration (IC50) was evaluated using different BC cell lines. Finally, gemcitabine sensitivity was assessed on these cell lines by cell survival assays following exogenous modulation of microRNA expression levels.

Results: Median age at diagnosis was 47 yrs (range 21-74 yrs). Hormone receptors were positive in thirty-one pts (62%), while Her2 in 15 cases (30%). Soft tissues were the most frequent site of metastasis (84%), followed by bone (64%), liver (36%), lung (34%) and CNS (16%). Sixteen pts (32%) had local recurrences. Median number of chemotherapies and endocrine therapies received prior to gemcitabine was 3 (range 1-6) and 2 (range 1-5) respectively.

From the nCounter analysis, a subset of 20 microRNAs was significantly deregulated (p&lt;0.001) between sensitive and resistant pts. Among them, the three most relevant (miR-135b, miR-146b and miR-155) were selected for further investigation. RT-PCR validation confirmed microRNA expression profiling data on the wider cohort of 50 pts (p&lt;0.05). In vitro studies confirmed the correlation between miR-135b levels and gemcitabine sensitivity in different BC cell lines. Moreover, the exogenous upregulation of this microRNA increased the response to the drug.

Conclusions: We identified miR-135b, miR-146b and miR-155 as potentially predictive for gemcitabine sensitivity in MBC pts. In particular, miR-135b appears to be causally involved in BC cells response to gemcitabine. These results pave the way to a better understanding of the molecular mechanisms underlying gemcitabine resistance in BC, and may have a clinical impact to identify those patients expected to obtain strong and prolonged benefits from this well-tolerated treatment.

Citation Format: Anna Tessari, Dario Palmieri, Giovanni Nigita, Dario Veneziano, Sara Cresta, Biagio Paolini, Maria Silvia Cona, Taylor Vargo, Erika Reese, Tyler Sheetz, Vincenzo Coppola, Filippo De Braud, Carlo M. Croce. Role of miR-135b in gemcitabine sensitivity for metastatic breast cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C17.