Abstract 4790: Overexpression of miR-3151 leads to direct deregulation of the TP53 pathway and is associated with BRAF mutations in malignant melanoma
Authors: Lankenau Malori , Patel Ravi , Markowitz Joseph , Carson William E. , Chapelle Albert de la , Eisfeld Ann-Kathrin 
Published: May 2015

Background: The BRAF gene is the most frequently mutated gene in malignant melanoma (MM). When mutated, it is associated with a more aggressive disease phenotype. Recently, intronic microRNA miR-3151 was identified in intron 1 of BAALC via deep-sequencing of MM samples. BAALC is reported to be the most upregulated gene in BRAF mutated melanoma. In acute myeloid leukemia, both high miR-3151 and BAALC are associated with poor survival. In addition, miR-3151 has direct leukemogenic activity via direct deregulation of TP53.

Aims: To decipher miR-3151's role in MM carcinogenesis and to elucidate its relation with BRAF mutations.

Methods/Results: miR-3151 and TP53 expression levels were determined in MM patients (n=20). Indeed, patients with high miR-3151 had lower TP53 expression. To prove direct downregulation of TP53 by miR-3151, we stably expressed miR-3151 and antagomiR-3151 in MM cell lines (Mel-39, A375, MeWo). RT-PCR and Western blots confirmed downregulation of TP53 by forced miR-3151 expression and upregulation of TP53 by antagomiR-3151. Increased levels of miR-3151 led to increased cell proliferation and decreased apoptosis by miR-3151, whereas antagomiR-3151 reduced cell growth and increased apoptosis. Concurrent Annexin/PI-staining revealed a reduction of cells in S and G2 phase in response to antagomiR-3151. Next, we determined the BRAF mutation status in the set of 20 MM patients and tested for a possible association with miR-3151 expression. BRAF mutated (BRAFmut) patients had a 5-fold higher miR-3151 expression compared to BRAF wild-type (BRAFwt) patients. To test whether BRAF mutations directly influence miR-3151, we performed si-mediated knock-down in BRAFmut cell lines (A375, Mel-39) and introduced the BRAF mutation into the BRAFwt cell line (MeWo) and then determined miR-3151 and TP53 expression. Silencing BRAF decreased miR-3151 expression and consequently increased TP53 expression, while introducing the BRAF mutation increased miR-3151 expression and decreased TP53 when compared to scramble control. Finally, as pharmacologic inhibition of aberrantly activated BRAF is a promising treatment approach for BRAFmut MM and as we had just identified miR-3151 as a downstream target of BRAF, we tested whether simultaneous knock-down of miR-3151 would add to the known effects of the BRAF inhibitor Zelboraf. The presence of antagomiR-3151 reduced the dose of Zelboraf required to achieve cell death by 60%.

Summary / Conclusions: miR-3151 deregulates TP53 in MM patients, thereby increasing the proliferation of MM cell lines. AntagomiR-3151 reduced MM cell growth and increased apoptosis in vitro. High miR-3151 expression is associated with the presence of BRAF mutations in MM patients, which directly increases miR-3151 expression. Simultaneous treatment of BRAFmut cell lines with the BRAF inhibitor Zelboraf and antagomiR-3151 significantly reduced the IC50 of Zelboraf.

Citation Format: Malori Lankenau, Ravi Patel, Joseph Markowitz, William E. Carson, Albert de la Chapelle, Ann-Kathrin Eisfeld. Overexpression of miR-3151 leads to direct deregulation of the TP53 pathway and is associated with BRAF mutations in malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4790. doi:10.1158/1538-7445.AM2014-4790