title: The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors.
abstract: The clinical benefit of selective BRAF inhibitor therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying the acquired resistance to a BRAF inhibitor by comparing the signaling pathways in the parental A375P cells and the resistant subline (A375P/Mdr). We demonstrate that MAPK re-activation does not contribute to the mechanism of resistance to UAI-201 of A375P/Mdr cells. The relative quantitative analysis using the 2(-ΔΔCt) method revealed that the BRAF inhibitor resistance observed in A375P/Mdr cells is not mediated through the overexpression of MDR proteins. In particular, we found that the expression of N-Ras was upregulated in BRAF inhibitor-resistant A375P/Mdr cells compared with A375P cells. In fact, siRNA-mediated N-Ras knockdown partially conferred UAI-201 sensitivity to A375P/Mdr cells, implying that N-Ras upregulation confers acquired resistance to BRAF inhibition. Notably, the flow cytometric analysis of the N-Ras-knockdown A375P/Mdr cells revealed that UAI-201 causes a significant accumulation of cells in the G 0/G 1 phase with a concomitant decrease in the number of cells in the S and G 2/M phases. However, platelet-derived growth factor receptor β (PDGFRβ) knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI-201, although a remarkable increase in PDGFRβ was observed in the A375P cells after UAI-201 treatment. Taken together, our results suggest that N-Ras is worth targeting to improve the therapeutic outcome of melanomas with acquired resistance to BRAF inhibitors.
authors.affiliation: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, 12-1 Songdo-dong, Yeonsu-gu, Incheon, 406-772, Republic of Korea.
authors.forename: Jun-Ho
authors.initials: JH
authors.lastname: Ahn
authors.forename: Michael
authors.initials: M
authors.lastname: Lee
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D015536:Down-Regulation; D055785:Gene Knockdown Techniques; D011905:Genes, ras; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors*; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D034741:RNA, Small Interfering; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf