Targeting V600EB-Raf and Akt3 Using Nanoliposomal-Small Interfering RNA Inhibits Cutaneous Melanocytic Lesion Development

Abstract

Most events promoting early melanoma development are yet to be identified but deregulation of the B-Raf and Akt3 signaling cascades are important regulators of this process. Approximately 90% of normal moles and ~60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation (V600EB-Raf), leading to 10X higher enzyme activity and constitutive activation of the MAP kinase pathway. Furthermore, ~70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting V600EB-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound mediated approach has been developed for delivering siRNA specifically targeting V600EB-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting V600EB-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight 4-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of V600EB-Raf and Akt3 led to a cooperatively acting ~65% decrease in early or invasive cutaneous melanoma compared to inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting V600EB-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas.

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

“…Historically, researchers have relied on physicochemical methods to facilitate epidermal penetration and cellular delivery of negatively charged nucleic acids (11,(30)(31)(32)(33). However, these methods require high concentrations of free siRNAs, can have unacceptable toxicity in in vivo models, and are limited by the instability of conventional siRNAs.…”

“…Although the small treated areas showed a marked reduction in skin thickening, the pain and the impracticality associated with these injections made the approach untenable. Other mechanical approaches, such as ultrasound and laser, have been used to facilitate penetration through the mouse stratum corneum and drive siRNA into skin, but require specialized equipment (30,31,33), limit the area of delivery, and can potentially harm the skin. Recent investigations have demonstrated uptake of siRNAs and DNAs into the epidermis in mice with no stratum corneum (e.g., open wounds or mucosae) or with atopic dermatitis, an inflammatory skin disorder with an ineffective epidermal barrier (33,34).…”

Integrating BRAF/MEK inhibitors into combination therapy for melanoma

“…Other studies have shown that PI3K and MEK inhibitors synergise to reduce growth and survival of melanoma cell lines grown under 3D organotypic cell culture conditions Meier et al, 2007). There is also evidence that siRNA knockdown of AKT3 and BRAF V600E leads to the enhanced inhibition of melanoma xenograft growth in nude mice Tran et al, 2008). …”

The Akt isoforms, their unique functions and potential as anticancer therapeutic targets

“…Furthermore, the increase in Akt3 activity in their melanoma model was associated with both Akt3 gene amplification and mutations in PTEN (104). Subsequently, data from the Robertson group (42,105) demonstrated a direct link between tumor progression in melanoma cells with Akt3-dependent phosphorylation of B-RAF.…”