Antisense oligonucleotide targeting of insulin-like growth factor-1 receptor (IGF-1R) in prostate cancer.

OBJECTIVE: Altered expression of insulin-like growth factor receptor (IGF-1R) is associated with castrate-resistant prostate cancer (CRPC) progression. We hypothesize that increased expression and/or responsiveness of IGF-IR may promote disease progression. This study assesses ATL1101, a 2'-MOE-modified antisense oligonucleotide (ASO) targeting human IGF-IR, with regard to potency and anti-cancer activity in androgen-responsive (LNCaP) and -independent (PC3) prostate cancer cells in vitro and in vivo. METHODS: IGF-IR mRNA and protein expression was assessed in ATL1101- and control oligonucleotides (ODN)-treated prostate cancer cells by QT-PCR and immunoblotting. The effect of IGF-1R ASO on cell growth and apoptosis in vitro was examined by crystal violet assay, flow cytometry, and expression and activation state of downstream signaling targets was examined by immunoblotting. In vivo growth of subcutaneous xenografts was performed in nude mice treated with intraperitoneally administered ATL1101 or control ODN by measuring tumor volume of PC3 xenografts in intact mice, and tumor volume and serum prostate-specific antigen levels in castrated mice harboring LNCaP xenografts. RESULTS: We observed dose- and sequence-specific suppression of IGF-IR mRNA and protein expression in ATL1101-treated cells in vitro. Suppressed IGF-IR expression correlated with decreased proliferation and increased apoptosis of PC3 cells under standard culture conditions and of LNCaP cells under androgen-deprived culture conditions. ATL1101 suppressed PC3 tumor growth as a monotherapy and delayed CRPC progression of LNCaP xenografts. CONCLUSIONS: This study reports the first preclinical proof-of-principle data that this novel IGF-IR ASO selectively suppresses IGF-1R expression, suppresses growth of CRPC tumors, and delays CRPC progression in vitro and in vivo.

An antisense oligonucleotide targeting the growth hormone receptor inhibits neovascularization in a mouse model of retinopathy.

PURPOSE: We have demonstrated that a 2'-O-methoxyethyl modified antisense oligonucleotide against the mouse growth hormone (GH) receptor (GHr) reduces GH binding and serum insulin-like growth factor-1 in normal mice. We tested whether this systemically delivered antisense oligonucleotide could inhibit neovascularization in mice with oxygen induced retinopathy (OIR). METHODS: OIR was induced in C57BL/6 mice by housing them in 75% oxygen across postnatal days (P)7 to 12 followed by five days in room air. Shams were in room air from P0-17. GHr antisense oligonucleotide, ATL 227446, was administered by early (P7, 8, 9, 11, 13, 15, and 17) or late (P12-16) intervention at doses of 5, 10, 20, and 30 mg/kg. Other mice were treated with either vehicle (saline), the somatostatin analog octreotide (20 mg/kg/bi-daily), or control oligonucleotides ATL 261303 (at 20 mg/kg by late and early intervention) or ATL 260120 (at 20 and 30 mg/kg by early intervention only). Blood vessel profiles were counted in 3 mm paraffin sections of inner retina. RESULTS: OIR increased blood vessel profiles by 2.5 fold compared to shams. In OIR, early intervention GHr antisense oligonucleotide ATL 227446 reduced blood vessel profiles at higher doses including 10 mg/kg, and 30 mg/kg resulted in the greatest reduction (38%). In OIR, late intervention with all doses of GHr antisense oligonucleotide ATL 227446 reduced blood vessel profiles to a similar extent, and the highest dose resulted in a 26% reduction compared to OIR. Octreotide reduced blood vessel profiles in OIR mice by 26%. In OIR, ATL 261303 had no effect on blood vessel profiles, while 30 mg/kg ATL 260120 reduced blood vessel profiles by 18%. CONCLUSIONS: Systemically delivered antisense oligonucleotides directed against the GHr are a potential novel treatment for ocular neovascularization related disorders.

UV induced responses of the human epidermal IGF system: impaired anti-apoptotic effects of IGF-I in HaCaT keratinocytes.

The insulin-like growth factor receptor (IGF-IR) is critical in epidermal development and IGF binding protein-3 (IGFBP-3), a modulator of cellular activity with or without IGF-dependence, co-localises with epidermal IGF-IRs. We have investigated whether the greater UV susceptibility of a human keratinocyte cell line (HaCaT) in comparison to normal human keratinocytes (NHKs) may involve differences in the IGF system. At 24 h after UV (960 mJ/cm(2) UVB), in comparison to NHKs, HaCaT keratinocytes exhibited significantly higher levels of apoptosis, refractoriness to IGF-I treatment and reduced IGF-IR phosphorylation. Secreted, intact IGFBP-3 (38-42 kDa) and IGFBP-3 mRNA abundance were reduced in HaCaT keratinocytes, but not consistently altered in NHKs. Immunoreactive IGFBP-3 fragments (16-11 kDa) were detected in both UV-exposed cultures. These data suggest that an altered IGF system contributes to HaCaT keratinocyte UV susceptibility and that following UV insult the IGF system may enhance keratinocyte viability and contribute to a return to epidermal homeostasis.

Epidermal homeostasis: the role of the growth hormone and insulin-like growth factor systems.

GH and IGF-I and -II were first identified by their endocrine activity. Specifically, IGF-I was found to mediate the linear growth-promoting actions of GH. It is now evident that these two growth factor systems also exert widespread activity throughout the body and that their actions are not always interconnected. The literature highlights the importance of the GH and IGF systems in normal skin homeostasis, including dermal/epidermal cross-talk. GH activity, sometimes mediated via IGF-I, is primarily evident in the dermis, particularly affecting collagen synthesis. In contrast, IGF action is an important feature of the dermal and epidermal compartments, predominantly enhancing cell proliferation, survival, and migration. The locally expressed IGF binding proteins play significant and complex roles, primarily via modulation of IGF actions. Disturbances in GH and IGF signaling pathways are implicated in the pathophysiology of several skin perturbations, particularly those exhibiting epidermal hyperplasia (e.g., psoriasis, carcinomas). Additionally, many studies emphasize the potential use of both growth factors in the treatment of skin wounds; for example, burn patients. This overview concerns the role and mechanisms of action of the GH and IGF systems in skin and maintenance of epidermal integrity in both health and disease.

C-5 propyne-modified oligonucleotides penetrate the epidermis in psoriatic and not normal human skin after topical application.

We have previously shown that antisense oligonucleotides effectively reduced insulin-like growth factor I receptor expression in human psoriatic skin grafted on to nude mice when injected intradermally. We therefore investigated the penetration of C-5 propyne modified antisense oligonucleotides into human normal and psoriatic skin after topical administration. Oligonucleotide (37.5 microg; 250 microM) was applied in aqueous solution or 5% methylcellulose gel for 24 h, prior to live confocal microscopy and fluorescence microscopy of fixed sections. We found that oligonucleotide could penetrate through the stratum corneum of psoriatic but not normal human skin over large regions of the epidermis. The oligonucleotide was localized to the nucleus of large parakeratotic cells in the psoriatic skin as well as smaller basal and suprabasal keratinocytes. In normal human skin, oligonucleotide was confined to the stratum corneum, with little or no oligonucleotide apparent in the viable epidermis. Electrophoresis of oligonucleotide recovered from treated psoriatic and normal skin revealed that the oligonucleotide remained intact over the 24 h period. In summary, we found that C-5 propyne modified antisense oligonucleotides could reach the target cells (in this case basal keratinocytes) after topical administration to psoriatic but not normal skin.

Sequence dependence of C5-propynyl-dU,dC-phosphorothioate oligonucleotide inhibition of the human IGF-I receptor: mRNA, protein, and cell growth.

Human keratinocytes are highly responsive to mitogenic and antiapoptotic signaling by the insulin-like growth factor-I receptor (IGF-IR). IGF-IR hyperstimulation is a feature of hyperplastic skin conditions, making the IGF-IR an appealing target for antisense therapeutic intervention. In this study, we used a C5-propynyl-dU,dC-phosphorothioate oligo-2'-deoxyribonucleotide antisense 15-mer to the human IGF-IR mRNA, along with liposome transfection, to inhibit IGF-IR activity in a human keratinocyte cell line and demonstrated potent inhibition of cell growth despite the presence of serum. To investigate the sequence specificity of these effects and to establish the concentration range over which a purely antisense effect could be demonstrated, we introduced 1, 2, 4, 8, and 15 base mismatches into the oligonucleotide and analyzed changes in inhibitory efficacy. In the 10-30 nM concentration range, the introduction of 1 and 2 mismatches into the middle of the 15-mer only modestly affected inhibitory efficacy, whereas >4 mismatches profoundly reduced mRNA, protein, and growth-inhibitory effects. From these results, we conclude that (1) sequence-specific antisense inhibition of IGF-IR activity in keratinocytes is achievable, (2) potent anti-IGF-IR antisense inhibition can be achieved in vitro at concentrations as low as 10 nM, and (3) a sequence-dependent mechanism is likely to underpin the observed in vivo therapeutic effects (Wraight et al. Nat. Biotechnol. 2000;18:521) of these antisense oligonucleotides (AS-ODN) in cutaneous hyperplastic disorders, such as psoriasis.