MicroRNA-3151 inactivates TP53 in BRAF-mutated human malignancies

Abstract

The B-Raf proto-oncogene serine/threonine kinase (BRAF) gene is the most frequently mutated gene in malignant melanoma (MM) and papillary thyroid cancer (PTC) and is causally involved in malignant cell transformation. Mutated BRAF is associated with an aggressive disease phenotype, thus making it a top candidate for targeted treatment strategies in MM and PTC. We show that BRAF mutations in both MM and PTC drive increased expression of oncomiR-3151, which is coactivated by the SP1/NF-κB complex. Knockdown of microRNA-3151 (miR-3151) with short hairpin RNAs reduces cell proliferation and increases apoptosis of MM and PTC cells. Using a targeted RNA sequencing approach, we mechanistically determined that miR-3151 directly targets TP53 and other members of the TP53 pathway. Reducing miR-3151's abundance increases TP53's mRNA and protein expression and favors its nuclear localization. Consequently, knockdown of miR-3151 also leads to caspase-3-dependent apoptosis. Simultaneous inhibition of aberrantly activated BRAF and knockdown of miR-3151 potentiates the effects of sole BRAF inhibition with the BRAF inhibitor vemurafenib and may provide a novel targeted therapeutic approach in BRAF-mutated MM and PTC patients. In conclusion, we identify miR-3151 as a previously unidentified player in MM and PTC pathogenesis, which is driven by BRAF-dependent and BRAF-independent mechanisms. Characterization of TP53 as a downstream effector of miR-3151 provides evidence for a causal link between BRAF mutations and TP53 inactivation.

Keywords: BRAF; PTC; TP53; melanoma; microRNA.

Fig. 1.

Effect of antagomiR-3151 on cell viability and discovery of miR-3151 downstream targets in MM. (A, Left) Viability of A375 and Mel-39 cells after forced miR-3151 expression or knockdown in chemiluminescent assays. Three experiments, bars, mean ± SD; *P < 0.05, **P < 0.005, two-tailed t test. In both A375 and Mel39 cells, overexpression of miR-3151 led to increased chemiluminescent activity and, thus, increased proliferation, whereas knockdown of miR-3151 led to decreased proliferation. (A, Middle) Example plots of annexin-V apoptosis assays and example histograms of propidium iodide cell cycle analysis. Green area is G₁ phase, yellow is S phase, and blue is G₂/M phase. Assays were performed in Mel-39 cells after miR-3151 knockdown. Infection with antagomiR-3151 led to an increase in the number of cells undergoing apoptosis and a decrease in cells undergoing cell division. (A, Right) Confocal microphotographs show A375 and Mel-39 cells transfected with scramble, miR-3151, or antagomiR-3151 stained for Caspase-3 (red). DAPI nuclear staining is shown in blue. In both cell lines, infection with antagomiR-3151 increased the expression of Caspase-3. (B) Heatmap of gene expression changes concordantly affected by forced miR-3151 expression or miR-3151 knockdown based on targeted RNA sequencing. Genes with concordant decreased expression upon miR-3151 overexpression and decreased expression after miR-3151 knockdown were considered potential direct targets of miR-3151. (C, Upper) Western blot with nuclear fractionation lysates to determine the relative nuclear (N) and cytoplasmic (C) abundance of TP53 in Mel-39 cells after manipulation of miR-3151 expression. Infection with antagomiR-3151 led to increased nuclear localization of TP53. (C, Lower) Confocal microphotographs show A375 and Mel-39 cells transfected with scramble, miR-3151, or antagomiR-3151 stained for TP53 (red). DAPI nuclear staining is shown in blue. When infected with antagomiR-3151, cells displayed increased nuclear localization of TP53. (D) TP53 expression in high miR-3151 and low miR-3151 expressing MM patients, *P < 0.05, two-tailed t test. Patients with high expression of miR-3151 had relatively lower TP53 expression compared with low miR-3151 expressers (as defined by median cut).

Fig. 2.

Regulation of miR-3151 by BRAF-dependent and BRAF-independent mechanisms. (A) Effects of BRAFmut on miR-3151 expression and TP53 expression in BRAFwt MeWo cells. Upon introduction of BRAFmut, cells had increased miR-3151 expression and decreased TP53 expression. Three experiments, bars, mean ± SD; *P < 0.05, two-tailed t test. (B) Effects of BRAF knockdown (si-BRAF) on miR-3151 expression and TP53 expression in BRAFmut A375 and Mel-39 cells. In both cell lines, when BRAF was silenced, miR-3151 expression decreased, whereas TP53 expression increased at both the RNA and protein levels. Three experiments, bars, mean ± SD; *P < 0.05, **P < 0.005, two-tailed t test. Inset shows an example immunoblot of TP53 protein expression after BRAF knockdown. (C) Effects of forced SP1 and NF-ĸB expression on miR-3151 expression in A375 and Mel-39 cells. A375 cells responded preferentially to SP1 transfection, whereas Mel-39 cells responded to NF-ĸB transfection with miR-3151 overexpression. Three experiments, bars, mean ± SD; *P < 0.05, **P < 0.005, two-tailed t test. (D) Visualization of SP1 binding to miR-3151s transcription start site (TSS-3151) by using EMSA. Nuclear extracts (NE) were used from A375 and Mel-39 cells, shifting performed with SP1-antibody (SP1-AB). The SP1/ NF-ĸB transactivating complex was bound to TSS-3151 in A375 and Mel-39 cells.

Fig. 3.

Downstream effects and upstream regulation of miR-3151 in PTC. (A) Endogenous miR-3151 (indicated by filled circles) and TP53 expression levels (indicated by open circles) in tumor samples from BRAFwt and BRAFmut PTC patients. Patients with high expression of miR-3151 had lower TP53 expression (P < 0.05, two-tailed Student’s t test). BRAFmut patients (indicated by red color) had higher expression of miR-3151 compared with BRAFwt patients (indicated by black color; P < 0.05, two-tailed Student’s t test). (B, Upper Left) miR-3151 expression achieved by stable overexpression with the lentiviral miR-3151 expression construct in KTC1 and BCPAP cell lines compared with scramble control. Three experiments, bars, mean ± SD; **P < 0.005, two-tailed t test. (B, Upper Right) Effects of forced miR-3151 expression on TP53 mRNA in KTC1 and BCPAP cells. Three experiments, bars, mean ± SD; *P < 0.05, two-tailed t test. (B, Lower) Effects of forced miR-3151 expression on TP53 protein levels in KTC1 and BCPAP cells. Forced expression of miR-3151 decreased TP53 expression at both the RNA and protein levels in both PTC cell lines. (C, Left) Confocal microphotographs show A375 and Mel-39 cells transfected with scramble, miR-3151, or antagomiR-3151 stained for TP53 or Caspase-3 (red). DAPI nuclear staining is shown in blue. Knockdown of miR-3151 led to increased expression of TP53 and Caspase-3 in PTC cells. (C, Right) Cell viability of KTC1 cells after knockdown of miR-3151, as measured by TiterGlo assay. Knockdown of miR-3151 led to decreased chemiluminescent activity and, therefore, a decreased proliferation rate. Three experiments, bars, mean ± SD; *P < 0.05, two-tailed t test. (D, Left) Effects of BRAF knockdown (si-BRAF) on miR-3151 expression and TP53 expression in KTC1 and BCPAP cells. Silencing BRAF in both cell lines decreased miR-3151 expression. Three experiments, bars, mean ± SD; *P < 0.05,**P < 0.005, two-tailed t test. (D, Right) Effects of SP1 and NF-ĸB on miR-3151 expression in KTC1 and BCPAP cells. Overexpression of NF-ĸB increased miR-3151 expression in both cell lines. Three experiments, bars, mean ± SD; *P < 0.05, **P < 0.005, two-tailed t test.

Fig. 4.

Effects of combined BRAFmut and miR-3151 inhibition on MM and PTC cells. (A) Schematic depiction of the proposed BRAF–miR-3151–TP53 axis. Targeting miR-3151 and BRAF through antagomiR-3151 and BRAF inhibitors, respectively, is a potential treatment strategy for both MM and PTC. (B) Effects of vemurafenib treatment on miR-3151 expression in Mel-39 (1.0 μM vemurafenib; Upper) and KTC1 cells (1.4 μM vemurafenib; Lower) compared with vehicle. Vemurafenib treatment led to decreased miR-3151 expression in both MM and PTC cells. Three experiments, bars, mean ± SD. (C) Representative images of the differential effect of vemurafenib treatment on Mel-39 and KTC1 cells after forced miR-3151 expression or knockdown. Overexpression of miR-3151 decreased the sensitivity to vemurafenib treatment, whereas knockdown of miR-3151 increased the sensitivity to vemurafenib treatment. (D) Changes in miR-3151 expression after development of vemurafenib resistance (A375 vem. res.) compared with the parental cell line (A375 vem. sens.). Vemurafenib resistant cells showed increased miR-3151 expression compared with vemurafenib sensitive cells. Resistant A375 cells were maintained in 2 μM vemurafenib. Three experiments, bars, mean ± SD; *P < 0.05, two-tailed t test.