972. Inhibition of TGF-β by RNA Interference To Modulate Breast Cancer Metastasis

Abstract

Transforming Growth Factor-|[beta]| (TGF-|[beta]|) has been shown to perform a dual function as both a tumor suppressor in early stage carcinogenesis and a pro-metastatic factor in late-stage tumor progression. Early studies have shown that inhibition of TGF-|[beta]| signaling pathway can alter various aspects of tumor metastasis such as migration, invasion, angiogenesis and immune suppression. While these methods have shown modest success, several limitations prevent the wide spread use of these inhibitors as an anti-metastatic therapy. RNA interference (RNAi) has emerged as an effective tool for gene specific silencing in vitro and in vivo. The central hypothesis of the proposed studies is that adeno-associated virus (AAV) mediated deliver of siRNA sequences targeting TGF-|[beta]|1 in MDA- MB 435 breast cancer cells can alter the metastatic phenotype at defined time-points of tumor progression and/or act as an adjuvant to primary chemotherapies. Several siRNA target sequences have been screened to determine silencing capacity and two specific targets, siRNA4 and siRNA7 showed |[sim]|90% inhibition of TGF|[beta]|1 transcripts by Northern blot and at least a 5-6 fold reduction by RT-PCR analysis. The two siRNA sequences, along with a scrambled sequence, were cloned into a tet-inducible expression vector and used to generate a stable cell line that constitutively expresses this construct. This cell line is being using in various in vitro assays such as migration, invasion and gene expression, to determine the effects of TGF-|[beta]| silencing. This cell line will later be used in an orthotopic breast cancer model to determine the exact time-point at which TGF-|[beta]| silencing is most effective. A second set of studies are proceeding in parallel to determine the transduction of AAV serotypes 1-6 both in vitro and in vivo. Initial in vitro data has shown that AAV2 and AAV6 show transduction efficiencies of 40% |[ndash]| 60% in MDA-MB 435 and MDA-MB 231 cells. In vivo transduction studies, of the primary tumor mass, are ongoing using AAV-|[beta]|gal, Ad-GFP and AAV-GFP. Ongoing studies will determine the effects of inhibition on SMAD-independent signaling pathways that can play a role in detachment, migration, immune-suppression and angiogenesis. This inducible siRNA will allow better insight into the TGF-|[beta]| |[ldquo]|switch|[rdquo]| from tumor suppressor to pro-metastatic factor along disease progression and possible development of a TGF-|[beta]| targeted anti-cancer therapy. If promising, this therapy may also be used in combination with current chemotherapies to provide a synergistic effect against breast cancer metastasis.