Christopher J. Wraight

Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides.

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.

The Role of Growth Hormone, Insulin-Like Growth Factor and Somatostatin in Diabetic Retinopathy

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are implicated in the aberrant cell growth and pathological neovascularization that characterises proliferative diabetic retinopathy. While serum levels of IGF-I are reported to be either high or low in diabetic patients, there is evidence that local tissue levels of IGF-I may be more relevant to diabetic retinal pathology. IGF-I and IGF binding proteins (IGFBP) are expressed throughout the retina in vascular, neuronal and glial cells, and are altered in response to hyperglycaemia and hypoxia. Further support for a pathological role for local IGF-I comes from studies showing IGF-I to be increased in the vitreal fluids of patients with proliferative diabetic retinopathy. IGF-I may exert its cell growth promoting properties by stimulating a number of pathways including protein-kinase B (Akt), nuclear factor kB (NF-kappaB)/AP-1 and hypoxic-inducible factor-1alpha (HIF-1alpha). In addition, other growth factors may participate in IGF-I induced cell growth including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and fibroblast growth factor (FGF). The importance of the GH/IGF system in diabetic retinopathy and retinal neovascularization has been highlighted by the use of agents that inhibit the system. GH receptor antagonists, GH receptor antisense oligonucleotides, somatostatin analogues and receptor neutralising antibodies to IGF-I reduce hypoxic-induced retinal neovascularization. These approaches may also prove to have benefits for improving vascular patency and vision in patients with diabetic retinopathy.

Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in human keratinocytes is regulated by EGF and TGF?1

Insulin‐like growth factor‐I (IGF‐I) is essential for normal epidermal homeostasis; however, the role of IGF binding proteins (IGFBPs), regulators of IGF action, remains unclear. Here we examine the regulation of human keratinocyte‐produced IGFBPs by epidermal growth factor (EGF), transforming growth factor beta 1 (TGFβ1), and IGF‐I, growth factors known to be active in skin. In the absence of added growth factors, IGFBP‐3 was the major binding protein secreted into the medium by primary keratinocytes. Addition of EGF or TGFβ1 to keratinocyte cultures resulted in a significant decrease in IGFBP‐3 abundance in conditioned medium when compared with control, untreated cells. Specifically, EGF (50 ng/ml) and TGFβ1 (50 ng/ml) reduced IGFBP‐3 abundance to 15 ± 6% and 22 ± 9%, respectively. Using Northern blot analysis, we found EGF and TGFβ1 (50 ng/ml) to reduce IGFBP‐3 mRNA levels in keratinocytes to 51 ± 12% and 50 ± 38%, respectively, when compared with control, untreated cells. Treatment with IGF‐I or its analogue des(1‐3)IGF‐I did not lead to any consistent change in IGFBP‐3 abundance. However, both IGF‐I and des(1‐3)IGF‐I at 100 ng/ml led to a modest increase in IGFBP‐3 mRNA levels in keratinocytes, suggesting posttranscriptional regulation of IGFBP‐3 abundance. We propose that local modulation of IGFBP‐3 abundance may represent another level of regulation of growth factor action in the epidermis, where EGF and TGFβ1 and possibly other local growth factors specifically regulate the availability of IGF‐I to its keratinocyte receptors. J. Cell. Physiol. 179:201–207, 1999.

Antisense oligonucleotides in cutaneous therapy.

Antisense oligonucleotides have been the subject of intense interest as research tools to elucidate the functions of gene products and as therapeutic agents. Initially, their mode of action was poorly understood and the biological effects of oligonucleotides were often misinterpreted. However, research into these gene-based inhibitors of cellular action recently has succeeded in realising their exciting potential, particularly as novel therapeutic agents. An emerging application of this technology is in cutaneous therapy. The demand for more effective dermatological drugs will ensure further development of antisense strategies in skin, with key issues being drug delivery, therapeutic target selection, and clinical applicability.

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

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.

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/cm2 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.

Abstract 5376: Antisense oligonucleotide targeting insulin-like growth factor-1 receptor (IGF-1R) enhances paclitaxel sensitivity in a castrate-resistant and paclitaxel-resistant prostate cancer model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC While prostate cancer (PCa) initially responds to androgen deprivation therapy (ADT), castrate-resistant disease (CRPC) relapse is inevitable. Altered expression of insulin-like growth factor (IGF) axis components have been consistently found in PCa and associated with adaptive growth and survival signaling implicated in CRPC progression. These activities are mediated by activation of the IGF-I receptor (IGF-IR). While CRPC patients treated with taxanes (paclitaxel or docetaxel) alone or in combination with estramustine phosphate or predonisone show a survival benefit, development of chemotherapeutic resistance eventually occurs. Therefore novel strategies targeting the molecular basis of ADT and chemotherapy resistance are required. In this study, we assess the potency and anti-cancer activity of a 2′-MOE-modified antisense oligonucleotide (ASO) targeting human IGF-IR, ATL1101, on the CRPC cell line, PC3 and a paclitaxel-resistant androgen-independent PC3-derived cell line, PtxR PC3, in vitro and in vivo. PtxR PC3 cells were established by culturing in step-wise increased drug concentrations. IGF-IR mRNA and protein expression in ATL1101- and control oligonucleotides (ODN)-treated PtxR PC3 cells were measured by QT-PCR and immunoblotting. The effect of IGF-1R ASO and paclitaxel combination therapy on parental and PtxR PC3 cell growth and apoptosis in vitro was examined by crystal violet assay and flow cytometry. In vitro combination index (CI) was calculated by Calcusyn® softoware. For xenograft studies, parental and PtxR PC-3 cells were inoculated s.c. in the flanks of athymic male nude mice and tumor volume kinetics were compared for mice treated with paclitaxel injected i.v. and either ATL1101 or control ODN injected i.p. In vitro, the IC50 for paclitaxel in PtxR PC3 was 20-fold higher than that in parental cells. PtxR PC3 also show significant cross-resistance to docetaxel and mitoxantrone. We observed equivalent dose- and sequence-specific suppression of IGF-IR mRNA and protein expression, and comparable decreased proliferation and increased apoptosis in ATL1101-treated parental and PtxR PC3 cells in vitro. Combination of ATL1101 with paclitaxel showed CI values below 1 at the IC50, IC75, IC90, suggesting the drug interactions resulted in synergy. In vivo, ATL1101 significantly increased sensitivity of PtxR PC3 tumors to combination therapy with paclitaxel as compared to control ODN in murine xenografts. This study reports the first preclinical proof-of-principle data that this novel IGF-IR ASO suppresses growth of paclitaxel resistant PC tumors, synergistically enhancing paclitaxel sensitivity in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5376.