Targeted NF1 cancer therapeutics with multiple modes of action: small molecule hormone-like agents resembling the natural anticancer metabolite, 2-methoxyoestradiol

Abstract

Background: Both the number and size of tumours in NF1 patients increase in response to the rise in steroid hormones seen at puberty and during pregnancy. The size of tumours decreases after delivery, suggesting that hormone-targeting therapy might provide a viable new NF1 treatment approach. Our earlier studies demonstrated that human NF1 tumour cell lines either went through apoptosis or ceased growth in the presence of 2-methoxyoestradiol (2ME2), a naturally occurring anticancer metabolite of 17-β estradiol. Previous reports of treatment with sulfamoylated steroidal and non-steroidal derivatives of 2ME2 showed promising reductions in tumour burden in hormone-responsive cancers other than NF1. Here we present the first studies indicating that 2ME2 derivatives could also provide an avenue for treating NF1, for which few treatment options are available.

Methods: STX3451, (2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), a non-steroidal sulphamate analogue of 2ME2, was tested in dose-dependent studies of malignant and benign NF1 human tumour cell lines and cell lines with variable controlled neurofibromin expression. The mechanisms of action of STX3451 were also analysed.

Results: We found that STX3451-induced apoptosis in human malignant peripheral nerve sheath tumour (MPNST) cell lines, even in the presence of elevated oestrogen and progesterone. It inhibits both PI3 kinase and mTOR signalling pathways. It disrupts actin- and microtubule-based cytoskeletal structures in cell lines derived from human MPNSTs and in cells derived from benign plexiform neurofibromas. STX3451 selectively kills MPNST-derived cells, but also halts growth of other tumour-derived NF1 cell lines.

Conclusion: STX3451 provides a new approach for inducing cell death and lowering tumour burden in NF1 and other hormone-responsive cancers with limited treatment options.

Figure 1

Cell proliferation in response to various specified doses of analogues of 2ME2 analogues and non-steroidal sulphamate derivatives as well as hormones. (A) Chemical structures of STX2484, STX2895, and STX3451. Three different concentrations (0.1 μM, 0.3 μM, and 0.6 μM) of the compounds were used to test proliferation and viability of human NF1 tumour-derived cell lines. (B) ST88 human MPNST cells; (C) PNF human benign peripheral nerve sheath tumour cells. STX140; STX2484; STX243; STX2895; STX641; STX3451; DMSO vehicle. (D) S462, HEK293, and U2OS cells were treated with 0.1 μM, 0.3 μM of STX3451, or DMSO vehicle. (E) Cell proliferation in response to hormones and STX3451. Oestrogen (1 μM), progesterone (1 μM), or both were used, alone or with STX3451 (0.3 μM). DMSO and ethanol vehicles; 0.3 μM STX3451; progesterone; progesterone with STX3451; oestrogen; oestrogen with STX3451; oestrogen and progesterone; oestrogen, progesterone, and STX3451.

Figure 2

STX3451 treatment caused cytoskeletal structural changes, focal adhesion plaque alterations and apoptotic cell death in ST88 cells. (A) Cells were stained with anti-α-tubulin antibody for microtubules, phalloidin for actin filaments, caspase-3 for apoptotic cells, DAPI for nuclei, and phospho-paxillin for focal adhesion sites. Scale bar, 20 μm. (B) Percentage of caspase-3-positive cells DMSO and STX3451-treated cells. (C) Percentage of abnormal nuclei in both treatments. Statistical significance: *P<0.05; ***P<0.005.

Figure 3

STX3451 treatment caused cytoskeletal structural changes, focal adhesion plaque changes but not apoptotic cell death in PNF cells after 48 h treatment. (A) α-tubulin, phalloidin, caspase-3, DAPI, and phospho-paxillin staining shows both microtubules and microfilaments were affected, especially after 48 h. Scale bar, 50 μm. (B) Percentage of caspase-3-positive cells DMSO and STX3451-treated cells. (C) Percentage of abnormal nuclei in both treatments. Statistical significance: *P<0.05.

Figure 4

STX3451 reduced levels of pAKT Ser473 and Thr308, but had less effect than PI3K (wortmannin) or mTOR (KU0063794) inhibitors. Both wortmannin and KU0063794 were used at 1 μM. (A) Western blot of proteins extracted from ST88 cells treated with different conditions. (B and C) Graphical representation (n=3) with statistical significance *P<0.05 and **P<0.01. (D) Levels of activated AKT messenger in MPNST tumour cell line in vitro treated with vehicle or STX3451. Both attached and floating cells were collected after 24 h treatment and cell lysates obtained. (E) Results were then quantified: DMSO attached cells, STX attached cells, STX floating cells. n=4, with statistical significance *P<0.05. STX: STX3451; w- wortmannin; KU: KU0063794; (F) Schematic indicating where inhibitors act in the PI3K-AKT pathway.

Figure 5

Cytoskeletal structural changes in ST88 and PNF cells after 48 h treatment with wortmannin, KU0063794, and colchicine, alone or with STX3451. Wortmannin and KU0063794 were at 1 μM, colchicine at 2.5 μM. Scale bar, 20 μm for ST88; 50 μm for PNF.

Figure 6

Treatment of STX3451 caused shrinkage of and cell emigration from ST88 but not PNF spheroids. STX3451 was at 150 nM, Wortmannin and KU0063794 were at 0.5 μM. Treatments were 96 h. Scale bar, 200 μm. (B) Percentage of average diameters compared with that before treatment of ST88 cells. **P<0.01; ***P<0.001. (C) Percentage of average diameters compared with that before treatment of PNF cells.