Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf

Abstract

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitfspecific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

“…Moreover, EP has proven to be an efficient method for in vitro and in vivo delivery of siRNA/shRNA molecules into tumor cells resulting in successful silencing of reporter or therapeutic genes, including K-ras. 11,[33][34][35][36] Previous in vivo study performed on pancreatic tumors showed that electrotransfection of subcutaneous or orthotopic pancreatic tumors (Panc-1) with synthetic siRNA or plasmid DNA encoding shRNA directed against K-ras dramatically reduced the tumor growth. 11 Results obtained from pancreatic tumor model and our results are consistent with the observation that a single electrotransfection of colorectal tumors with pmiRNA-Kras resulted in growth delay of 8 days, whereas in the study by Rejiba et al 11 the growth delay of 15 days was achieved after two consecutive electrotransfections of pancreatic tumors with pshRNA-K-ras (time interval: 7 days).…”

“…As it has been already proven in previous studies, this limitation might be overcome by repeated administration of silencing molecules. 11,[36][37][38] The repetitive treatments might be important for the eradication of tumor cells that fail to be transfected in a single treatment. For repetitive therapies, it is important that gene delivery method is safe and that it can be applied locally, which are both undoubtedly the advantages of EP.…”

Microphthalmia-Associated Transcription Factor Controls the DNA Damage Response and a Lineage-Specific Senescence Program in Melanomas

“…Consistently, inhibition of MITF expression induces a G 0 -G 1 growth arrest of melanoma cells (5)(6)(7). MITF has been also involved in the control of melanoma survival through the control of BCL2 or BIRC7 (8,9), but MITF silencing only marginally causes apoptosis (8,10). In addition to apoptosis, senescence is another cellular failsafe program that counteracts excessive mitogenic signaling observed in cancer cells.…”

Microphthalmia-Associated Transcription Factor Is a Critical Transcriptional Regulator of Melanoma Inhibitor of Apoptosis in Melanomas

“…Loss of MITF function, either by siRNA knockdown or expression of a dominant negative allele, leads to reduced melanoma growth and decreased melanocyte cell viability (25,40). If regulation of ML-IAP expression by MITF is required for melanoma cell viability, then overexpression of ML-IAP from a constitutive promoter should be able to rescue MITF siRNA-induced cell death.…”